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the absolute number of pertrochanteric fractures may increase in the years to come , along with the increase of population 's aging and osteoporosis , with absolute indication for surgery . a surgical intervention as the osteosynthesis of pertrochanteric fracture is a posttraumatic immune stimulus which contributes to the systemic inflammatory response syndrome representing the second hit for these patients after the first trauma . geriatric patients are particularly susceptible to profound inflammatory responses to trauma that , especially if exaggerated , drive mortality and morbidities including infectious complications . standard laboratory blood analyses are used as first - line tests to determine the preoperative diagnosis of infective / inflammatory status : the erythrocyte sedimentation rate , c - reactive protein ( crp ) serum level , and white blood - cell count play a substantial role as markers of inflammation . the extent of fracture and soft - tissue damage can be estimated by analysis of creatine kinase ( ck ) that is highly predictive with regard to long - term outcome after trauma . nevertheless these serum markers are not consistently reliable , as they are highly sensitive but less specific because they are affected by the age , the gender , and the medical comorbidities of the patient . the body 's response to trauma is a highly complex and heterogeneous sequence of events , and specific cytokine patterns , truly predictive of outcomes , are yet to be established . inflammatory cells that contribute to the clearance and the repair of necrotic tissue dominate the local response to injury . these cells release soluble molecules , mainly cytokines that also act on sites distant from the origin of their production while a systemic acute - phase response goes along the local inflammation . this is followed by a compensatory anti - inflammatory response to attenuate the proinflammatory state , and the balance between the pro- and anti - inflammatory responses determines the net outcome of the reaction . in major injury , disequilibrium between pro- and anti - inflammatory cytokines may start a generalized response that in turn may progress to a multiple organ dysfunction . literature evidence suggests that il-6 levels provide an estimate of not only local tissue trauma but also the subsequent systemic response to trauma . several studies have focused on the effect of inflammatory cytokines tnf- , il-6 , and il-1- produced predominantly by activated lymphocytes / mononuclear cells located in inflammatory loci including fracture sites or in bone marrow close to bone cells wherein bone remodeling proceeds . these cytokines are known to be powerful stimulators of bone resorption and to affect bone formation , through complex actions on dna and collagen synthesis in osteoblastic cells . in particular , while tnf- seems to have contrasting effects on bone formation through osteoblastic differentiation , il-6 has been shown to be associated with cartilage destruction in human rheumatoid arthritis , to regulate differentiation and apoptosis in preosteoblasts and to affect the mineralization of fracture callus in mice . in case of a major , but standardized , musculoskeletal injury like trochanteric fractures , the total hip replacement induced significant increments in serum levels of the proinflammatory cytokines in the postoperative course , at 6 hours and at 24 hours after surgery . minimally invasive methods for indirect reduction and fixation try to minimize the impact of the second inflammatory hit [ 11 , 12 ] . unfortunately , the expected benefit ( decreased additional tissue damage ) of these minimally invasive techniques has not been objectively measurable . recent developments in orthopaedic surgery indicate that pertrochanteric fractures can be successfully treated using advanced implants . dynamic hip screws ( dhss ) and proximal femoral nails ( pfns ) are commonly used and both produce good results [ 14 , 15 ] . encouraging reports tried to quantify the surgical trauma for pertrochanteric fractures treated by dhss , based on cytokines : a significant reduction of il-6 was found in patients undergoing fixation with dhs when comparing a minimally invasive to a more conventional surgical technique . accordingly , the aim of this study was to determine the second inflammatory hit in patients with fractures of the hip treated with the pfn antirotation ( pfna ) . this osteosynthesis method combines effectiveness in outcome , easiness of installation , and speed of intervention . the protocol was approved by the ethics committee at our institution . written informed consent was obtained from each patient enrolled in the study , according to the declaration of helsinki . the clinical data from 20 patients planned for pfna at the gemelli hospital of catholic university , department of orthopaedic sciences and traumatology , were collected and analyzed prospectively from november 2011 to september 2012 . inclusion criteria were pertrochanteric fracture of the femur ( 3.1 type - a according to the ao classification of fractures ) ( https://www.aofoundation.org , last visited 28/01/2015 ) , investigated by conventional hip rx in the anteroposterior projection and age > 65 years . patients on steroid therapy were excluded from the study because of the anti - inflammatory effects of these drugs . for each patient , the following was recorded : the age at surgery , the gender , the comorbidity , the preoperative waiting hours , and the operative time ( table 1 ) . all patients were operated for fracture reduction and fixation with intramedullary nail pfna , ( synthes , switzerland ) with percutaneous technique . this method involves closed reduction of the fracture by pulling the fractured limb on a special operating table and fluoroscopic control . the nail is inserted into the medullary canal of the proximal femur through a mini - incision of about 10 cm length at the level of greater trochanter . the fixing system involves the insertion of a blade along the femoral neck and up to the head and a screw passing through the two cortical of the femur and the distal portion of the nail . this was performed with a percutaneous technique , which is using two small incisions sufficient to the passage of the screws . samples of venous blood were obtained at 1 hour preoperatively and at 24 hours postoperatively . after centrifugation at 3000 rpm for 5 minutes sera were collected , divided in 3 aliquots for each sample , and stored at 80c until dosages were performed . they were analyzed anonymously . to check the inflammatory stimulus induced by the intervention blood levels of tnf- , il-6 , and crp the levels of interleukins tnf- and il-6 were measured by the use of commercial elisa kits ( tnf- , rd systems , minneapolis , mn ; il-6 , ebioscience , bender medsystems , vienna , austria ) . healthy donors ' control values were assumed according to guidelines of the companies , with an average of 15.6 pg / ml for tnf- and 5.8 pg / ml for il-6 . crp was measured by an immunometric test ( dimension vista , siemens medical solutions diagnostics gmbh , eschborn , germany ) normal value 3 plasma levels of creatine kinase ( ck ) were measured by absorption photometry ( cobas 8000 , roche diagnostics , switzerland ) , at admittance of the patients in the emergency room and 24 hours after surgery , as indicator of muscle necrosis ; normal value 190 ui / l . data were analyzed using student 's t - test ; p values < 0.05 were considered to be significant . there were 15 females and 5 males , with an average age of 85 ( sd 4.5 ) and 81 ( sd 10.8 ) years , respectively . the values of serum tnf- , il-6 , crp , and ck are shown in table 2 . the mean levels ( 1 sd ) of tnf- were 2.27 4.09 pg / ml preoperatively and 3.84 5.74 pg / ml after 24 hours postoperatively ; these differences were not statistically significant . the preoperative mean level of il-6 was 16.14 14.96 pg / ml , nearly threefold the value of normality though with a very wide range of variation , while the mean level after 24 hours postoperatively was 16.64 9.04 pg / ml : this difference was not statistically significant . before surgery , mg / l ) with a very wide range of variation , 63.08 33.72 the preoperative plasma ck was within the normal range ( 30190 ui / l ) for all patients , with a mean value of 84.35 44.81 there was a statistically significant increase during the first 24 postoperative hours ( with a mean value of 218.06 155.93 u / l , pre versus post p = 0.001 ) , in spite of a wide range of variation for ck . after 24 h after surgery , ck plasma levels of only eight patients greatly exceeded the normal range . the postoperative levels were positively significantly related to the surgical time length ( r = 0.3535 ) . figure 1 shows these results for the 16 patients of whom these data were available . fractures of the proximal femur and hip are relatively common injuries in adults and common source of morbidity and mortality among the elderly . incidence of fractures is increasing , which is not unexpected because the general life expectancy of the population has increased significantly during the past few decades . moreover , it provides for stable fixation , which allows early full mobilization of the patient . much research is now being conducted in order to understand the role of cytokines in the development of the inflammatory response following the surgical trauma . on this background , the aim of this study was to evaluate the second inflammatory hit , correlated to soft - tissue invasiveness , in 20 patients with fractures of the hip treated in our institution with pfna . to this purpose , we analyzed the changes in plasma levels of the inflammatory cytokines tnf- and il-6 , along with conventional systemic markers crp and ck . il-6 , a major proinflammatory cytokine , is mainly produced by monocytes and activated macrophages , even though other cells ( such as fibroblasts and myoblasts ) may also synthesize it [ 18 , 19 ] . surgery may instantly mediate il-6 release from these cells , or il-6 release may be induced by other locally released cytokines [ 20 , 21 ] . experimental data demonstrated that tnf- induces il-6 secretion in human myoblasts , the in vitro counterparts of the regenerating muscle cells that , following a muscle injury / trauma , develop from satellite cells , proliferate , fuse , and differentiate into new mature myofibers . furthermore , il-6 is principally responsible for activating the hepatic synthesis of crp , which has been considered the inflammatory biomarker of choice in orthopaedic surgery [ 23 , 24 ] . in our study , waiting time before surgery ranged from 99 h 41 to 14 h 33 ( average 45 h 30 ) , which is sufficient for cytokines to become detectable in serum . the total duration of surgery ranged from 17 to 71 minutes ( average 36 ) . we analyzed for tnf- , il-6 , and crp the patient sera collected 1 h before and 24 h after surgery . in the case of elevated cytokine concentrations at admission time , some authors reported that injured patients with high concentrations of il-6 and tnf- showed an increased risk of death . in our patients tnf- was not detectable before surgery , without significant changes in the postoperative 24 hours . in keeping with previous reports , tnf- is probably unuseful for the purpose of quantifying surgical tissue damage [ 16 , 25 ] ; in any case , the minimal surgical trauma induced by pfna was not sufficient to display an appreciable increase in serum levels of this cytokine . before surgery , il-6 was detectable in 16/20 patients with an average value that was nearly threefold the value of normality , without correlation with waiting time ; the maximum value was 45.9 pg / ml in a patient who had waited only 14 hours . however also for il-6 we did not find any statistically significant difference between the preoperative and the postoperative average value ( 16.14 14.96 versus 16.64 9.04 pg / ml ) , even with a very wide range of variation . since injured muscle tissue also produces il-6 , these data underline that the first hit ( trauma ) is able to cause an increase of il-6 , while osteosynthesis pfna method does not lead to a further soft tissue damage . other authors described il-6 production 1 hour after surgery , comparing conventional ( conv ) and minimally invasive ( mi ) dhs techniques : the preoperative levels of il-6 recorded showed no significant differences between the two groups , whereas postoperative values were on average higher in the dhs conv group ( 78.41 67.04 pg / ml ) compared with the dhs mi group ( 39.02 37.36 pg / ml ) . we selected the 24 hours ' time point instead of 1 hour after surgery because in our experience it represents the best time range to observe il-6 production peak following inflammatory stimuli [ 22 , 26 ] . crp is an acute phase protein that can be used as a marker for changes in the orthopedic postoperative inflammatory response and its levels may depend on the region of trauma . crp levels increase rapidly after surgery , peaking on day 2 , but already after 24 hours the increase starts to decline to plateau . we found that the crp values were increased , as measured 1 hour preoperatively and that there was a further increase of about 60% 24 hours after surgery ( from 63.08 33.72 to 104.46 38.69 although not significant , there was also a positive association trend between the values of crp and preoperative waiting hours ( data not shown ) . crp levels may also be normal preoperatively but consistently increase postoperatively with various types of fracture fixation . our results are in agreement with literature : hong and coworkers found similar results for crp analyzing serum soft - tissue marker differences after pfn fixation of stable pertrochanteric fractures in the elderly . in addition , many reports have confirmed ck as a possible indicator of soft tissue damage during surgery . ck changes in plasma have been reported as a result of a skeletal muscle injury and correlated with surgical incision length , approach , and operative time . a trend of ck increase was observed in elderly patients undergoing surgery for hip fracture with the peak values on day 1 postoperatively [ 29 , 30 ] . in our patients the preoperative plasma ck level was in the normal range with an average value of 84.35 44.81 ui / l but it increased significantly during the first 24 postoperative hours , reaching a mean value of 218.06 155.93 interestingly , only for 8/20 patients ck greatly exceeded the normal range with a very significant positive correlation to the surgery time , which may mean major muscle damage and inflammation . the mean ck value that we found 24 h postoperatively is in accordance with literature on minimally invasive hip surgery . to analyse and compare serum soft - tissue marker differences after the minimal invasive dhs and pfn fixation , hong and coworkers found that both methods showed similar patterns of change for ck and crp pre- and postoperatively ( up to 72 h ) without significative differences , suggesting that both approaches produce similar levels of soft - tissue damage . our results on pfn are similar to those reported by hong , even if the values are expressed in different units . in conclusion , in the treatment of pertrochanteric fracture , secretion of inflammatory markers is lower when minimally invasive techniques are used compared with traditional surgery . we confirm that , for the markers analyzed here , pfna has a low biomechanical - inflammatory profile that represents an advantage over other techniques . above all average time for the synthesis of a trochanteric fracture by a skilled operator is 25 minutes ( by incision to suture ) . the reduced surgical time together with the minimal surgical incision , not longer than 10 cm , represents the useful features of this method . further , closed reduction of the fracture performed under fluoroscopic control avoids a second incision at the level of the fracture . this allows a minimum of trauma to soft tissue , resulting in a lower inflammatory stimulus compared to methods that involve exposure of the fracture site .

pertrochanteric fractures are common injuries in adults and source of morbidity and mortality among the elderly . different surgical techniques were recommended for their treatment but undoubtedly they add an additional inflammatory trauma along the fracture itself . many attempts to quantify the degree of approach - related trauma are carried out through measurements of systemic inflammatory parameters . in this study we prospectively analyzed laboratory data of 20 patients over eighty with pertrochanteric fracture of the femur treated with proximal femoral nail antirotation ( pfna ) . this is an excellent device for osteosynthesis because it can be easily and quickly inserted by a mini - incision providing stable fixation and early full mobilization . serum tumor necrosis factor - alpha ( tnf- ) , interleukin-6 ( il-6 ) , c - reactive protein ( crp ) , and plasma creatin kinase ( ck ) were evaluated 1 hour preoperatively and 24 hours postoperatively . our results show that pfna did not induce significant increments in serum levels of inflammatory cytokines tnf- and il-6 ; crp was elevated preoperatively in correlation with waiting time for surgery ; crp and ck showed a significant increment in the first postoperatory day ; ck increment was correlated with surgical time length . we conclude that , for the markers we analyzed , pfna shows a low biomechanical - inflammatory profile that represents an advantage over other techniques .

forecasting cause - specific mortality can help estimate the future burden of diseases and can be used as fundamental data for developing public health policies . the number of deaths worldwide has been increasing ; it increased 13.5% from 46.5 million in 1990 to 52.8 million in 2010 , and the disease burden is expected to increase ( 1 ) . to reduce the disease burden , estimations of the future cause - specific mortality are needed to shape public policy and plan for expenditures . moreover , mortality projections are needed to address the increase in chronic diseases from population aging in korea . although the trend analysis of the leading causes of death has been conducted in the observed period 1983 - 2012 ( 2 ) , long - term future predictions of the leading causes of death up to 2032 have not been conducted in korea . the norwegian cancer registry developed nordpred software , which was an r - based program that relied on a modified apc ( age - period - cohort ) model ( 34 ) . instead of evaluating the cancer incidence and mortality , we adopted the general cause - specific mortality to the nordpred apc model . first , the cause - specific mortality in future periods ( 2013 - 2017 to 2028 - 2032 ) was estimated by reflecting the mortality trends in the observed periods 1983 - 1987 to 2008 - 2012 . second , the overall change in the death rates was analyzed with the following three components : the change in the risk , age structure and population size . the death and population data were obtained from statistics korea . the death data consisted of 12 major causes of death ( cancer , cerebrovascular diseases , heart diseases , suicide , liver diseases , diabetes mellitus , traffic accident , chronic lower respiratory diseases , pneumonia , hypertension diseases , pulmonary tuberculosis and alzheimer 's disease ) from 1983 to 2012 by sex and age group in korea ( 11 ) . the icd-10 codes ( international classification of diseases version 10 by world health organization ) of the 12 causes of death were shown in appendix 1 . from the who research , completeness and quality of the death registration data of each countries they grouped death registration data into high quality ( 23 countries ) , medium quality ( 55 countries ) and low quality ( 28 countries ) according to the criteria using the completeness and the ill - defined codes ( completeness was defined as the proportion of all deaths covered by the vital registration system ) . korea was included as medium quality group and has 89% completeness and 14% deaths coded to ill - defined codes ( 12 ) . the population data consisted of the observed ( 1983 - 2010 ) and estimated populations ( 2011 - 2032 ) by sex in korea . the future populations are estimated using the cohort component method from the population census provided by the statistics korea ( 13 ) . the death data were aggregated into 6 five - year periods ( 1983 - 1987 to 2008 - 2012 ) by sex and cause of deaths . the population data were aggregated into 10 five - year periods ( 1983 - 1987 to 2028 - 2032 ) by sex . the death data were aggregated into 17 five - year age groups ( 0 - 4 to 80 + ) by sex and cause of death . the population data were aggregated into the same age groups by sex . to forecast future mortality , we evaluated the general mortality data , instead of cancer mortality data , in nordpred ( modified apc model ) . the model was written as : rap = ( aa + d p + s pp + cc ) . ( 34 ) recommended , the power 5 function , instead of the logarithm function , was used as a link function to level off the exponential growth . if the trend had significant curvature ( s ) in the observed periods , the most recent 10 years instead of all observation periods were used to predict the future mortality . the drift parameters were empirically reduced by 0% , 25% , 50% , and 75% in the first , second , third , and fourth future periods , respectively . the lowest age groups in the model were determined by considering the number of deaths in each cell ( shown as appendix 1 ) . below the lowest age groups , the future mortality was estimated by using the average trend of the most recent 10 years . age - standardized rates were calculated using the segi world standard population with some modification of summing up the 80 - 84 and 85 + groups into the 80 + group ( 1415 ) . the prediction base ( number of periods ) was determined when fitting the model by considering the goodness of fit . the total change in the number of deaths consisted of the following three components : the change in the risk ( rate ) , the change in the age structure , and the change in the population size . nras was the number of deaths given a cancer risk r , an age structure a , and a population size s. nfff was the number of deaths given the future rate , the future age structure , and the future population size . nooo was the number of deaths given the observed rate , the observed age structure , and the observed population size . noff was the number of deaths given the observed rate , the future age structure , and the future population size . noof was the number of deaths given the observed rate , the observed age structure , and the future population size . the four changes were divided by nooo and were expressed as percentages ( % ) . the study protocol was approved by the institutional review board ( irb ) of kangwon national university ( irb no . the study protocol was approved by the institutional review board ( irb ) of kangwon national university ( irb no . table 1 summarized the observed ( 2008 - 2012 ) and predicted ( 2028 - 2032 ) numbers of deaths by cause of death , and the total change in the number of deaths was resolved into the change in the risk , age structure and population size from 2008 - 2012 to 2028 - 2032 . cr , crude rates ; asr , age - standardized rates with the world standard population ; apc , average percent change . per 100,000 person - years . for males , the number of deaths is expected to increase by 52.7% from 2008 - 2012 to 2028 - 2032 ( 707,538 to 1,080,552 deaths ) . the apc ( annual percent change ) of all deaths will be 2.1% over the 20 years from 2008 - 2012 to 2028 - 2032 . the overall change is due to the change in the risk ( -66.7% ) , age structure ( 119.4% ) and population size ( 4.4% ) . although the risk is predicted to decline from 2008 - 2012 to 2028 - 2032 , the overall number of deaths will increase because of the age structures , which make the largest contribution to the changes in the deaths . for females , 41.9% of all deaths are expected to increase from 2008 - 2012 to 2028 - 2032 ( 565,347 to 802,243 deaths ) . the apc of all deaths will be 1.8% during the 20 years from 2008 - 2012 to 2028 - 2032 . the overall change is due to the change in the risk ( -73.3% ) , age structure ( 115.2% ) and population size ( 6.4% ) . for both sexes , the trends in the mortality by the cause of death were similar to each other as follows : increasing pattern from 2008 - 2012 to 2028 - 2032 ( cancer , suicide , heart diseases , pneumonia , alzheimer s disease ) and decreasing pattern ( cerebrovascular diseases , liver diseases , diabetes mellitus , traffic accident , chronic lower respiratory diseases and pulmonary tuberculosis ) . hypertension diseases is expected to increase in males , while it is expected to decrease in females . for both sexes , cancer ranked first among the 12 causes of death in 2008 - 2012 , and 1 showed the projections of the crude rates and the age - standardized rates by major cause of death in the future period 2013 - 2032 based on the observed period 1983 - 2012 . for all deaths , the age - standardized rates in the world standard population are expected to decrease from 2008 - 2012 to 2028 - 2032 ( males : 447.6 to 307.0 [ -31.4% ] , females : 230.9 to 156.4 [ -32.3% ] per 100,000 person - years ) . however , the crude rates are expected to increase from 2008 - 2012 to 2028 - 2032 ( males : 570.7 to 834.7 [ 46.3% ] , females : 458.3 to 611.2 [ 33.4% ] per 100,000 person - years ) . the projection of the crude and age - standardized rates by the major causes of death in the future period 2013 - 2032 based on the observed period 1983 - 2012 . mc , male crude rates ; fc , female crude rates ; ma , male age - standardized rates ; fa , female age - standardized rates ( per 100,000 person - years ) . 2 showed the projections of the age - specific rates for the period 1983 - 2012 for all deaths on the basis of the observed period 2013 - 2032 . as the age group was older , the age - specific rates sharply increased , especially in the over 80 years of age group . the age - specific rates of all deaths in the past ( 1983 - 2012 ) and future periods ( 2013 - 2032 ) . 3 demonstrated that trends of the crude rates for the 12 major causes of death from 1983 - 1987 to 2028 - 2032 . the major causes of death in 2028 - 2032 will be cancer , cerebrovascular diseases , heart diseases , suicide , pneumonia and diabetes mellitus . the trends of the crude rates according to 13 major causes of death from 1983 - 1987 to 2028 - 2032 in both sexes . ca , cancer ; cvd , cerebrovascular diseases ; hd , heart diseases ; sui , suicide ; ld , liver diseases ; dm , diabetes mellitus ; ta , traffic accident ; clrd , chronic lower respiratory diseases ; pna , pneumonia ; htn , hypertension diseases ; tb , pulmonary tuberculosis ; alz , alzheimer 's disease . although the age - standardized rates are expected to decrease from 2008 - 2012 to 2028 - 2032 in both sexes , the total number of deaths is expected to increase in both sexes ( males : 52.7% , females : 41.9% ) . for both sexes , deaths from cancer , suicide , heart diseases , pneumonia and alzheimer s disease are predicted to increase from 2008 - 2012 to 2028 - 2032 . on the other hand , the number of deaths from cerebrovascular diseases , liver diseases , diabetes mellitus , traffic accident , chronic lower respiratory diseases and pulmonary tuberculosis are predicted to decline . additionally , cancer is expected to be the highest cause of death in both the 2008 - 2012 and 2028 - 2032 periods . furthermore , the largest contribution to the overall change in the number of deaths is the change in the population , especially the change in the age structures . in korea , the proportion of aged people is expected to increase in 2028 - 2032 compared with the past periods , 1983 - 1987 and 2008 - 2012 . the aged - child ratios ( percentages of people over 65 years old over those younger than 14 years old ) will increase in both sexes ( males : 10.3% in 1983 - 1987 , 53.5% in 2008 - 2013 and 168.1% in 2028 - 2032 ; females : 18.3% in 1983 - 1987 , 84.2% in 2008 - 2013 and 219.3% in 2028 - 2032 ) . comparing between korea and worldwide , the aged - child ratios of the aging population in korea are expected to be higher than the world standard population ( 22.6% ) in 2008 - 2013 and 2028 - 2032 . although the age - standardized rates are projected to decrease , the total number of deaths is projected to increase because of population aging in korea . worldwide in 2030 , ischemic heart diseases ( 13.4% of the total deaths ) , cerebrovascular diseases ( 10.6% ) , hiv / aids ( 8.9% ) , chronic obstructive pulmonary diseases ( copd ) ( 7.8% ) , lower respiratory infection ( 3.5% ) , lung cancer ( 3.1% ) and diabetes mellitus ( 3.0% ) are expected to be the leading causes of death ( 16 ) . in the comparison between worldwide and korea , cancer , cardiovascular diseases ( including heart diseases and cerebrovascular diseases ) , respiratory diseases and diabetes mellitus worldwide , ischemic heart disease and cerebrovascular disease were predicted to rank first and second , respectively , in 2002 and in 2030 . the rankings for the following causes of death are estimated to increase from 2002 to 2030 : hiv / aids , copd , lung cancer and diabetes mellitus . however , the rankings for the following causes of death are estimated to decrease : lower respiratory infection , perinatal condition , diarrheal disease and tuberculosis ( 16 ) . hiv / aids is projected to rank higher among the causes of death worldwide than korea because africa has had the highest rate of deaths from hiv / aids ( africa : 1,100,000 deaths and world : 1,500,000 deaths in 2013 from the who website ) . however , suicide is expected to rank higher in korea than worldwide due to the socioeconomic circumstances in korea . we evaluated the reasons for the increasing trends of several diseases ( cancer , cardiovascular diseases , suicide , pneumonia and alzheimer s disease ) and decreasing trends of other diseases ( liver diseases and pulmonary tuberculosis ) . the increasing numbers for various causes of death were significantly associated with the elderly and with the population aging in korea . cancer is estimated to be the leading cause of death among both men and women for the periods from 2008 - 2012 to 2028 - 2032 in korea . as the average life expectancy increases , cancer is more likely to occur because cancer mortality is higher in the older age groups than in the younger age groups ( 17 ) . cancer mortality might be influenced by the high prevalence of smoking among adult males in korea ( 1819 ) . smoking was responsible for 20.9% of cancer incident cases and for 32.9% of cancer deaths among adult males ( 20 ) . particularly , 71% of lung cancer deaths were attributable to smoking ( 20 ) . despite the prevalence of helicobacter pylori has been decreased , which is a well - demonstrated risk factor for gastric cancer and was responsible for 80.3% of non - cardia gastric cancers in males and 78.7% in females ( 21 ) . westernized diets ( red meat and processed meat ) , decreased physical activity , and increased prevalence of obesity were the risk factors for colorectal cancer in korea ( 22 ) . the number of deaths from cardiovascular diseases ( icd-10 : i00-i99 ) is estimated to increase 13.3% in males from 2008 - 2012 to 2028 - 2032 in korea . the increasing trend is likely because of the increased prevalence of risk factors , such as smoking , physical inactivity , obesity and high - fat diets ( 2324 ) . the prevalence of obesity in adult males has been increasing ( from 25.4% in 1998 to 36.7% in 2007 ) in korea ( 25 ) . additionally , the dietary habits and lifestyles have transitioned to the western style ( high fat content from red meat , high sugar and salt contents from processed foods ) in korea . from the who website , the age - standardized suicide rate of both sexes in korea ( 28.9 per 100,000 person - years ) was the second highest rate among the 171 countries in 2012 . we deduced that the high suicide rates were due to socioeconomic polarization and inequalities , social isolation and maladaptation to modern society in the setting of the rapid economic development in korea . the high suicide rates in the elderly have been a serious problem in korea ( 26 ) . the economically productive population ( 15 - 64 years ) has increased from 1983 - 1987 to 2008 - 2012 [ from 65.6% to 72.8% ] , and it will decrease sharply by 2028 - 2032 [ 63.1% ] . the children and infant dependency ratio ( 0 - 14 years / 15 - 64 years 100 ) has decreased from 1983 - 1987 to 2028 - 2032 [ from 46.0% to 20.0% ] , but the old age dependency ratio ( more than 65 years/15 - 64 years 100 ) has increased from 1983 - 1987 to 2028 - 2032 [ from 6.5% to 38.5% ] . overall , total dependency ratio ( sum of two ratios ) has decreased from 1983 - 1987 to 2008 - 2012 [ from 52.5% to 37.4% ] , which is expected to increase by 2028 - 2032 [ 58.5% ] . as population aging has progressed , the economically productive population is predicted to decrease and the dependency ratio is expected to increase . therefore , supporting expenses of the economically productive population and their social pressure are expected to increase , which will be the risk factors for suicide in korea . the number of deaths from pneumonia will sharply increase from 2008 - 2012 to 2028 - 2032 ( males : 319.9% , females : 274.1% ) . as population aging has been ongoing in korea , the number of deaths from pneumonia will increase because the incidence of pneumonia is higher in elderly people ( 27 ) and because the elderly are particularly vulnerable to the functional decline of organ systems , such as the respiratory , immune and digestive systems , as well as the comorbidity with age - associated diseases ( 28 ) . the number of deaths from liver diseases is predicted to decrease from 2008 - 2012 to 2028 - 2032 ( males : -48.1% , females : -33.1% ) . because hepatitis b virus ( hbv ) was a major risk factor for liver diseases such as hepatitis and cirrhosis , ( 29 ) decreasing future projections of deaths from liver diseases may be due to the decreasing trend of the seropositive prevalence for hepatitis b virus surface antigens ( hbsag ) ( 4.61% in 1998 and 2.98% in 2010 ) as well as the use of antiviral therapy for hbv in korea ( 30 ) . the number of deaths from alzheimer s disease will increase from 2008 - 2012 to 2028 - 2032 in korea ( males : 108.0% , females : 3.0% ) . the female population above 80 years has been approximately two times more than the male population from 1983 - 1987 to 2008 - 2012 , and this trend will persist up to 2028 - 2032 . therefore , female deaths due to alzheimer s disease will be more than male deaths because alzheimer s disease is frequently diagnosed among the aged people ( 31 ) . however , the number of deaths due to alzheimer s disease was relatively small compared to the number of deaths due to other diseases and mortality trends have drastically changed recently . the nordpred model using the power 5 function as a link function was very sensitive to the recent trends . we thought that the prediction results of alzheimer s disease had large rbs ( relative biases ) , and they should be interpreted carefully . a validation study for predicting mortality is needed in the future . in addition to the growing population of the aged people in korea , cerebrovascular disease , hypertension , type 2 diabetes , and obesity are the risk factors of alzheimer s disease . cerebrovascular diseases such as hemorrhagic or ischemic infarctions can directly affect the brain region involved in the memory function , which can cause an increase in the prevalence of alzheimer s disease . therefore , protein extravasation in the brain cells can increase , which can cause damage to the brain cells ( 32 ) . the number of deaths from pulmonary tuberculosis is projected to decrease from 2008 - 2012 to 2028 - 2032 ( males : -49.7% , females : -23.4% ) . this decreasing trend may be due to the development of an anti - tuberculosis drug and improvement of personal hygiene in korea . first , the predictions of the nordpred model were not performed well in some diseases . in a preliminary study , asr in 2008 - 2012 ( 5 years ) was predicted based on the period from 1983 - 1987 to 2003 - 2007 ( 25 years ) by sex and kinds of diseases . rbs were calculated between the estimated ( e ) and observed ( o ) asr as follows : rb=|e - o|/o100 . predictions of total deaths had better performance than those of disease - specific deaths because over - estimation and under - estimation of each disease - specific death were compensated by summing each death . rbs of all deaths were 3.6% in males and 0.1% in females , which were lower than those of the specific disease by sex . predicting mortality for some diseases having small numbers and recently drastic changes had large rbs than other diseases having large numbers and small recent changes . although the power 5 function as a link function was important for estimating the long - term projections , some diseases having small numbers were very sensitive to application of the model using the recent trends . second , prediction of mortality for the diseases except for cancer has not been performed yet . although the nordpred model was developed for projecting cancer incidence and mortality , we thought that it is applicable to estimating mortality for other diseases because of use of common age , periods and cohort variables ( apc model ) in the epidemiology . , modeling mortality was challenging than modeling the incidence ( 33 ) . despite the disadvantages , predictive performance of the nordpred model seemed to be good . a canadian group compared several apc models to estimate the long - term cancer incidence and mortality projections ( 34 ) . they evaluated differences between the projected and observed rates of each models to figure out which models more precisely predicted the rates . the nordpred model and the bayesian model performed better than the other models including gam ( generalized additive model ) and conventional apc model . particularly , the nordpred model using the power 5 function as a link function generally performed better than the model using the logarithm function . moller et al . ( 4 ) empirically compared the validity of the short and long - term projections among the various nordpred models by calculating the absolute median value of the differences between observed and predicted cases . generally , the nordpred model using the power 5 function more improved the predictions than the classical apc model by leveling off the overestimated trends of the apc model . our study was meaningful with respect to presenting the first picture of predicting the 20-year disease - specific mortality based on the 30-year data . under the circumstances of increasing medical expenditures of diseases such as cancer , cerebrovascular diseases , heart diseases and diabetes mellitus , a study predicting mortality can provide a valuable clue to develop policies and to allocate resources . icd-10 , version 10 of the international classification of diseases ( icd ) by the world health organization ( who ) .

forecasting cause - specific mortality can help estimate the future burden of diseases and provide a clue for preventing diseases . our objective was to forecast the mortality for causes of death in the future ( 2013 - 2032 ) based on the past trends ( 1983 - 2012 ) in korea . the death data consisted of 12 major causes of death from 1983 to 2012 and the population data consisted of the observed and estimated populations ( 1983 - 2032 ) in korea . the modified age - period - cohort model with an r - based program , nordpred software , was used to forecast future mortality . although the age - standardized rates for the world standard population for both sexes are expected to decrease from 2008 - 2012 to 2028 - 2032 ( males : -31.4% , females : -32.3% ) , the crude rates are expected to increase ( males : 46.3% , females : 33.4% ) . the total number of deaths is also estimated to increase ( males : 52.7% , females : 41.9% ) . additionally , the largest contribution to the overall change in deaths was the change in the age structures . several causes of death are projected to increase in both sexes ( cancer , suicide , heart diseases , pneumonia and alzheimer s disease ) , while others are projected to decrease ( cerebrovascular diseases , liver diseases , diabetes mellitus , traffic accidents , chronic lower respiratory diseases , and pulmonary tuberculosis ) . cancer is expected to be the highest cause of death for both the 2008 - 2012 and 2028 - 2032 time periods in korea . to reduce the disease burden , projections of the future cause - specific mortality should be used as fundamental data for developing public health policies .

animal models are widely and successfully used to reproduce important aspects of pathologies associated with brain disorders in many areas of neuroscience . even within a single neuropathological condition , there exists a wide diversity of models , each attempting to delineate a specific cause and/or potential treatment strategy for the condition in question . here , we present a novel animal model that has several favourable features that allow for examination of whether neuronal replacement in the hippocampus can reverse memory deficits caused by selective degeneration of hippocampal neurons . characterization of regenerative success in our simple model system should provide fundamental information about the required conditions for replacing neural circuitry in other brain regions more generally and ultimately should support a treatment strategy for disorders involving neuronal loss . the hippocampus , a key region of the medial temporal lobe , is a frequent target in many neurological diseases and most forms of dementia . it is well established that the hippocampus can degenerate in alzheimer 's disease , posttraumatic stress disorder , parkinson 's disease , epilepsy , and following acute trauma such as hypoxia and stroke , to mention only a few conditions . it is also clear that damage to the hippocampus accounts for many of the cognitive deficits observed in these diseases , particularly those concerned with long - term memory . in animal models of hippocampal neuronal loss , there is benefit from employing existing strategies for restoring functions . in none of the models , however , there is good evidence that lost cells are replaced and that the new cells take up normal positions in respect to connectivity . the grafting strategy attempts to replace lost tissue by placing donor embryonic or stem cells directly into the target region . in general , these approaches have success that is clearly limited [ 6 , 7 ] . in hippocampus , there is survival of a significant number of grafted cells , some evidence for integration into local circuitry , and at least partial reversal of some behavioural deficits . there is an increase in extracellular transmitter , which likely overcomes some of the deficits , but it is extremely unlikely , despite some synaptogenesis , that these transplanted cells have established correct pre- and postsynaptic connections that normalize information processing by the hippocampal network . even in instances of excellent graft survival with multiple transplant locations , there are clear persisting functional deficits [ 810 ] . it seems that normal information processing does not take place in hippocampal grafts [ 8 , 11 ] . there is some recent evidence that grafts of embryonic ca3 tissue promote expression of calbindin in existing hippocampal neurons after ca3 excitotoxic injury , and this might be beneficial for hippocampal excitability . furthermore , embryonic neuroblasts implanted into normal or pilocarpine - damaged hippocampal rats survive , establish some connections with the host , but remain in clumps , not likely restoring normal information processing . thus , regardless of approach , the objective of repairing damaged hpc circuits still seems to be beyond our reach . a new set of opportunities has opened up based upon the surprising discovery that in the adult brain , there are at least two pools of cells that continuously generate new neurons . one of these pools of neurogenic stem cells is centered on the subventricular zone of the lateral ventricular wall , and the other , a focus of our work , is located in the dentate subgranular zone of the hippocampus . this process of adult neurogenesis is known to occur in the hippocampus of adult humans . thus , our novel , and we believe promising , animal model involves upregulating hippocampal neurogenesis in order to repair the damaged circuitry and reverse associated cognitive impairments . therefore , a major advantage of our model is that the restoration of damaged circuitry occurs through manipulating an endogenous process . second , uniquely in the cortex , in adults , there is a steady addition of newborn granule cells to the hippocampal network . below , we describe and explain the experimental manipulation that produces a gradual and targeted loss of granule cells in the dentate gyrus subfield of the hippocampus of adult rats . specifically , we show histological findings that outline the time course and extent of this cell loss . next , we present evidence for the effects of this cell loss on the electrophysiological properties of the dentate gyrus and characterize its ensuing impact on behaviour . finally , we present a treatment strategy that takes advantage of adult dentate gyrus neurogenesis ; precisely , that upregulation of adult neurogenesis results in a significant repopulation of the dentate gyrus granule cell layer . our animal model utilizes adx to induce a gradual and specific loss of granule cells within the adult rat dentate gyrus . robert sloviter and colleagues first reported the loss of granule cells in the dentate gyrus resulting from adx in 1989 . they observed a selective loss of dentate gyrus granule cells in the hippocampus , three - to - four months after adx in adult rats . [ 15 , 16 ] showed that other hippocampal subfields were essentially unaffected by adx , demonstrating the specificity of cell loss after adx . moreover , cort replacement immediately after adx prevented cell loss from the granule cell layer . these findings suggest that the absence of cort associated with adx causes specific degeneration within the dentate gyrus while sparing other hippocampal regions . work by gould et al . confirmed the specificity of adx - induced cell loss , showing the presence of pyknotic cells in the hippocampal granule cell layer ( but not ca1 or ca3 ) of rats , three days after adx . subsequent investigation by woolley et al . demonstrated that specific activation of type 1 adrenal steroid receptors via aldosterone is also sufficient to prevent the loss of dentate gyrus granule cells associated with adx , providing identification of an initial trigger of the mechanism by which post - adx granule cell death occurs . woolley et al . also confirmed the specificity of cell death in the rat hippocampus , noting a significant increase in pyknotic cells in the granule cell layer of the dentate gyrus seven days after adx . using electron microscopy , sloviter et al . provided further evidence for apoptotic cell death in the dentate gyrus granule cell layer as a result of adx . they reported a number of morphological changes in dentate granule cells that are characteristic of apoptosis ( condensed nuclear chromatin , compaction of cytoplasm , blebbing ) . other markers of cell death have been employed to characterize granule cell loss after adx , including silver impregnation [ 19 , 20 ] , caspase-9 , and terminal dutp nick end labeling ( tunel , ) . although the aforementioned studies demonstrated adx - induced neural degeneration , none has provided a time course of the degeneration or described its progression . using the cell death marker fluoro - jade b , we examined both the onset and time course of hippocampal granule cell death after adx . specifically , fluoro - jade b expression within the dentate gyrus was assessed in rats that were sacrificed 17 days and 2 , 4 , 8 , or 23 weeks after adx . we found that as described previously [ 17 , 19 ] , cell death is evident by three days after complete removal of circulating cort . very little , if any , fluoro - jade positive cells were detected in rats sacrificed 1 or 2 days after adx , yet consistent labeling was observed in the rats that were sacrificed 3 or more days after . furthermore , granule cell death increased steadily from one to two weeks , peaking at the fourth week and remained high in week eight . moreover , cell death could still be observed 23 weeks after adx , but not to the same extent as in the previous time point . thus , for a period of up to at least 23 weeks , a large pool of granule cells die as a result of adx - induced cort depletion ( figure 1 ) . the continuing loss of hippocampal granule cells in the adult rat is a nice feature of this model , as it resembles slow neurodegenerative processes of neuronal loss experienced by individuals suffering from various forms of dementia , delayed hippocampal neuronal loss associated with stroke , as well as traumatic brain injury . the relatively slow loss of neurons as a result of adx is also congruent with many other neurodegenerative disorders including huntington 's and parkinson 's disease . the gradual loss of granule cells associated with chronic adx can lead to substantial damage in the dentate gyrus . after 10 weeks of adx we have found that approximately 25 percent of the volume of the granule cell layer is lost ( figure 2(c ) ; ) . these estimates were obtained by quantifying granule cell layer volume using the cavalieri volume estimator [ 3033 ] . this technique allows for a relatively quick and easy estimate of adx - induced damage in the dentate gyrus . a more refined estimate , however , can be obtained by quantifying the total number of remaining cells after adx using the optical fractionator technique . the latter revealed that 10 weeks of adx - induced cell death caused approximately a 50 percent reduction in dentate gyrus granule cells ( figure 2(d ) ; ) . not surprisingly , there is a strong and significant correlation between volume and total granule cell number in the dentate gyrus , suggesting that the decrease in volume after adx is at least in part due to the loss of granule cells . our findings are congruent with previous studies showing granule cell loss in the dentate gyrus after long - term adx [ 15 , 3436 ] . however , we advocate that the estimates that we present here are more accurate , as they relied on unbiased stereological approaches . in summary , adx and its resulting cort depletion causes prolonged cell death that is restricted to the granule cell layer of the dentate gyrus . a 10-week degeneration period results in noticeable damage in the dentate gyrus that can easily be quantified stereologically . as such , we have a slow neurodegeneration model that limits its damage to an area of the brain known for critical cognitive function , particularly memory . a fundamental question now arises : what are the in vivo effects of the selective degeneration of hippocampal granule cells ? we examined this question by investigating the electrophysiological properties of the dentate gyrus following adx - induced degeneration and more importantly its ensuing impact on behavior . to determine the functional effects of granule cell death in vivo , we utilized a battery of electrophysiological and behavioural indices . specifically , we performed input / output ( i / o ) curves on awake , freely behaving adx rats and recorded population spike amplitude and field excitatory post synaptic potentials ( fepsps ) . in addition , we used several behavioural tests ( morris water task , object / context mismatch , and open field activity ) to assess the effect of hippocampal granule cell loss on behaviour and cognition . our animal model requires that after the 10-week degeneration period , rats are provided with replacement cort . the administration of cort serves two purposes : ( 1 ) it prevents further adx - induced apoptosis and ( 2 ) allows us to determine the functional deficits strictly associated with the loss of granule cells and not the effects of cort depletion per se . indeed , any changes observed following adx could be due to complete removal of circulating cort and/or the ensuing loss of dentate gyrus granule cells . by administering cort , we mitigate the potential adverse effects of the hormone depletion and can more accurately assess the effects of granule cell degeneration specifically . thus , we administer a daily , oral dosage of cort ( 1 mg ) , after the 10-week degeneration period and throughout the remainder of our studies . this method of cort replacement has been demonstrated as effective at inducing a diurnal rhythm in adx rats that is similar to intact controls and in preventing adx - induced cell death , electrophysiology alterations , and behavioural deficits . with the exception of the report by sloviter et al . , much like post - adx reports of cell death [ 17 , 20 ] , electrophysiological studies have been performed at time points very soon after surgery . a number of experiments have reported alterations in granule cell electrophysiology , usually ranging from 3 [ 3739 ] to 10 days after adx . the short time course of these experiments means that little is known about the effects of chronic adx and the associated gradual loss of hippocampal granule cells on the electrophysiological properties of the dentate gyrus . pilot data collected from our laboratory shows that 10 weeks of adx - induced dentate gyrus granule cell layer degeneration is sufficient to produce deficits in electrophysiological function despite rats receiving replacement cort at the time of measurement . i / o curves were performed on awake , freely behaving control , and adx rats , and population spike amplitude and fepsps were recorded . we found a marked attenuation of the electrophysiological response of the dentate gyrus in chronic adx rats . specifically , we noticed a significantly lower fepsp slope and a decreased population spike in adx rats after 10 weeks of granule cell layer degeneration when compared to intact controls ( figure 3 ) . importantly , all adx rats received replacement cort starting one week prior to and during the time of data collection . thus , 10 weeks of cell death , that typically leads to a 50% decrease in cells in the granule cell layer of the dentate gyrus , cause substantial electrophysiological disturbances within the hippocampus . pivotal to our model , the adx - induced degeneration in the dentate gyrus leads to significant behavioural changes and cognitive deficits . more specifically , the loss of granule cells due to long - term adx results in cognitive deficits in at least two tasks that have been shown to depend on intact hippocampal function . using a moving platform version of the morris water task , we demonstrated that rats with dentate gyrus damage performed poorly relative to controls , replicating prior research that suggests damage focused on the dentate gyrus ( including adx ) produces deficits in spatial tasks [ 34 , 4143 ] . using a novel / familiar platform location paradigm we found that adx rats take longer to locate a hidden platform within the morris water task , regardless of whether it was in a familiar or novel location ( figure 4(a ) ) . this significant increase in time to find the platform is not explained by differences in swim speed between adx and intact control animals . of note is that during our investigation , all of our adx rats received replacement cort and still presented with spatial deficits . this finding adds to a body of evidence that suggests a loss of granule cells is sufficient to produce cognitive deficits . we have also reported behavioural deficits following 10 weeks of adx - induced cell degeneration in a spontaneous recognition task . prior investigation has shown that rats with damage limited to the hippocampus perform novel object preference tasks at levels similar to controls but display deficits when information about context or place becomes important for successful recognition . thus , we tested the ability of rats to recognize whether an object is associated with the context in which it was originally encountered . rats are presented with a pair of identical objects in one context ( context a ) , and after a short exploration period , they are presented with another pair in an alternate context ( context b ) . during this acquisition phase , rats are provided with equal exposure to the two object pairs , each pair housed in a different context . for the test , rats are returned to either of the contexts ( a or b ) , but now , an object from each pair is present . normally , rats will spend a greater proportion of their object investigation time with the mismatched object ( i.e. , the one never encountered in that context before ) . adx animals perform significantly worse than controls on this task and fail to show preferential investigation of the object that is mismatched ( figure 4(b ) ) . the deficits we observe in rats after long - term adx bear a family resemblance to those experienced by people displaying a loss of hippocampal function [ 4649 ] . spatial deficits as a result of hippocampal atrophy due to alzheimer 's disease and hippocampal damage associated with surgical resection have been reported in the virtual morris water task , the human analogue of the spatial task we employed to assess behaviour in our adx animals . show that human patients with surgery - induced unilateral hippocampal damage took significantly longer to locate a hidden platform and spent less time swimming in the correct quadrant during a probe trial in the virtual morris water task than their age - matched cohorts . the disruption in recognition memory by altering the context in which objects are presented has also been observed in human patients suffering from hippocampal damage , a finding that is congruent with our data , showing diminished novel object / context association in adx rats . pascalis and colleagues show that when the context in which an object is first presented ( familiarization ) and subsequently reintroduced ( test ) is changed , patients with hippocampal damage show a lack of object preference , despite intact object recognition when the background context remained constant . it is well established that the birth of new neurons in the brain continues throughout all of adulthood . the processes of proliferation , migration , and integration of new neurons in the adult brain are now well - described phenomena , both in the dentate gyrus and the subventricular zone . numerous modulators of the neurogenic process within the adult hippocampus have been discovered , ranging from the administration of pharmacological agents such as fluoxetine [ 53 , 54 ] , growth factors , exercise [ 56 , 57 ] , environmental enrichment [ 56 , 58 ] , to stress and stress hormones [ 5961 ] . gould and colleagues reported an increase in proliferation six days after adx as determined by an increase in the density of tritiated thymidine - labeled cells in the dentate gyrus of adx rats compared to controls . further studies have confirmed this phenomenon , finding increases in proliferation within the dentate gyrus at time points ranging from six to twelve days after adx [ 60 , 62 , 63 ] . all of the aforementioned studies assessing proliferation after adx have done so without cort replacement [ 59 , 60 , 62 , 63 ] . these studies demonstrate that proliferation continues in the adult dentate gyrus in the absence of cort , at least at relatively short time points after adx . it is well documented that adult born granule cells mature into functional units within the dentate gyrus , eventually becoming indistinguishable from their surrounding counterparts . as adx eliminates mature dentate gyrus granule cells it is not unreasonable to conclude that cort is necessary to ensure the survival of adult - born granule cells . our cort replacement strategy not only serves to prevent further granule cell layer degeneration but also ensures the survival of those hippocampal granule cells born in the adult adx rat . our animal model utilizes the phenomenon of adult neurogenesis in an attempt to repopulate the damaged dentate gyrus with adult - born granule cells . as such , it is necessary to demonstrate that neurogenesis continues in the damaged dentate gyrus and that it can be upregulated to a level sufficient to significantly replace lost neurons within the adult hippocampus . we have previously shown that neurogenesis continues in the adx brain at levels similar to adrenally intact controls . specifically , we report that the number of proliferating cells ( as determined by ki67 expression ) and migrating neuroblasts ( those cells expressing the microtubule - associated protein doublecortin ( dcx ) ) does not differ significantly between chronic adx and control rats . this is important because it shows that despite significant degeneration of the granule cell layer , the subgranular zone remains viable . indirect evidence from our 2007 study suggested that the treatment strategy we had employed ( eight weeks of daily fluoxetine administration ) transiently increased hippocampal neurogenesis . despite a lack of a significant difference in neurogenesis between adx rats and controls , there was a tendency for adx rats that were treated with fluoxetine to have a thicker dentate gyrus than those that were untreated . this suggests that fluoxetine may have induced a transient increase in neurogenesis , resulting in a thicker granule cell layer . we succeeded in substantially repopulating the dentate gyrus following 10 weeks of adx - induced degeneration by using a unique combination of enriched housing and wheel running . specifically , we housed our rats in a 24-hour alternating condition of group - housed environmental enrichment and individual access to a running wheel over a period of 6 weeks . it is believed that the effects of exercise and environmental enrichment on adult neurogenesis are dissociable . exercise ( in the form of wheel running ) has been shown to increase the proliferation of new cells , specifically targeting type-2 cells [ 51 , 57 ] . environmental enrichment can dramatically increase the number of new dentate gyrus granule cells but may not increase proliferation . enrichment may primarily increase the proportion of progenitors that are selected to survive to granule cell maturity [ 57 , 64 ] . the proliferation of hippocampal granule cells occurs in the adx rat ( with or without replacement cort ) , even at long time points . despite this , this suggests that it is the mature granule cells are critical for certain behaviours . simply increasing the proliferation of new cells is , therefore our combination of wheel running and enriched housing was selected to maximize the possibility that new cells in the damaged dentate would survive to a functional endpoint . we found that a significantly greater number of dcx positive cells were evident in adx rats that received the enriched environment and wheel running ( figures 5 and 6(a ) ) compared to those remaining in the home cage . this is consistent with extensive evidence suggesting that neurogenesis can be increased by enriched environmental housing [ 56 , 58 , 65 ] and exercise [ 66 , 67 ] . importantly , the number of dcx positive cells present in adx rats is similar to that which is observed in controls . despite the significant depletion of granule cells in the dentate gyrus caused by adx , hippocampal neurogenesis continues , providing further evidence that the subgranular zone remains viable after long - term adx . the most exciting finding , however , is that a combination of six weeks of environmental enrichment and wheel running increased the volume of the granule cell layer in adx rats compared to those that remained in the home cage ( figure 6 ) . hence , enhancing neurogenesis was successful in at least partially repopulating a damaged dentate gyrus in adult animals . the significantly higher levels of dcx in rats that received the enrichment / exercise treatment show that earlier stages of neurogenesis had indeed been upregulated . the increase in volume of the dentate gyrus in adx rats that experienced enrichment and exercise , a measure strongly correlated with granule cell number , suggests that the survival of adult born granule cells was also increased . although the volume of the hippocampal granule cell layer in treated adx rats did not reach control levels , the significant repopulation of cells suggests that treatments that upregulate adult neurogenesis may provide an effective strategy by which lost function may be restored . the high levels of dcx present in treated adx rats at the time of perfusion indicate that simply increasing the treatment period may be sufficient to completely repopulate the damaged dentate gyrus . taken at face value , our results differ from naylor et al . who report a transient increase in proliferation as a result of voluntary wheel running at nine , but not 24 days in rats . kempermann and gage also demonstrated that short - term but not long - term exposure to an enriched environment increased proliferation in mice , suggesting a similar transient phenomenon . by interleaving enrichment with exercise an alternate possibility is that switching between wheel running and a relatively novel , enriched environment every 24 hours may maintain a degree of novelty that has been suggested as responsible for increases in neurogenesis . importantly , the animal model we describe provides an excellent method by which to test treatment strategies targeting restoration of function after neuronal loss . preliminary evidence from our laboratory suggests that regeneration of the dentate gyrus granule cell layer via adult neurogenesis can restore impaired behaviour . after 10 weeks of granule cell layer degeneration due to adx , rats were placed on cort and administered a cocktail of growth factors ( fgf-2 and sonic hedgehog ) via an osmotic minipump for six weeks in an attempt to increase neurogenesis within the hippocampus . both fgf-2 [ 55 , 71 ] and sonic hedgehog [ 72 , 73 ] have been shown to increase neurogenesis in normal animals , specifically targeting early stages of the neurogenic process . during administration of growth factors , adx rats were group housed in an enriched environment , which has been shown to increase survival of adult born neurons in normal animals [ 56 , 57 ] . adx rats that received the neurogenic treatments presented with a greater hippocampal granule cell - layer volume compared to home cage housed adx rats given vehicle . furthermore , the volume of the hippocampal granule cell layer in treated adx rats did not differ from that of controls , indicating , at least by this measure , a complete regrowth of the previously degenerated hippocampal subfield . importantly , those adx rats that had an increased dentate gyrus granule cell - layer volume as a result of treatment with an enriched environment , fgf-2 , and sonic hedgehog performed significantly better that their adx cohorts receiving vehicle and did not differ from controls in a contextual discrimination task . this initial finding suggests a functional repopulation of the dentate gyrus granule cell layer in the adult animal , something that has not been reported before . although more work is required to confirm the results , this compelling finding suggests that functional recovery in the adult animal is a distinct possibility . our animal model utilizes adx to induce a progressive and specific loss of hippocampal granule cells followed by an upregulated neurogenic period to repopulate the damaged circuitry . to recap the essential features of the model , rats first undergo adx surgery and remain in their home cages for at least a 10-week degeneration period . the absence of cort associated with the removal of the adrenal glands leads to progressive degeneration , producing a 50% cell loss within the dentate gyrus granule cell layer after 10 weeks . second , the adx rats are placed on daily cort replacement to prevent further degeneration and allow new neurons to establish themselves within the damaged circuitry . in addition , a subset of adx rats is then exposed to neurogenic treatments in order to increase the endogenous levels of neurogenesis within the adult hippocampus . third , after the treatment period , rats are subjected to a battery of behavioural tasks and electrophysiological measures designed to evaluate hippocampal function . finally , the rats are perfused and the hippocampal granule cell layer is assessed using immunohistochemistry and stereological methods . the described animal model produces a specific and gradual degeneration of neurons within the adult hippocampus . the gradual loss of hippocampal neurons is a hallmark symptom of a number of pathological states , including traumatic brain injury [ 7476 ] , stroke [ 25 , 77 ] , and a number of dementias [ 24 , 46 ] . similar to patients that suffer hippocampal atrophy due to prolonged cell loss we show that rats with a long - term loss of hippocampal granule cells suffer from cognitive deficits in tasks that have been shown to depend upon intact hippocampal function . of note is that even after a significant degeneration of the granule cell layer , the stem cell niche within the subgranular zone remains a viable substrate for the birth of new neurons in the adult . we are the first to show that dentate gyrus neurogenesis continues at normal levels after chronic ( longer than 10 days ) adx in the adult rat . we are also the first to show that at much longer time points ( six - to - ten weeks after adx ) that in combination with cort replacement , methods that increase neurogenesis in the normal animal continue to do so in the damaged dentate gyrus . as in normal animals , we show that the stem - cell niche in the subgranular zone of chronic adx rats can be modulated . using a combination of exercise and environmental enrichment , we show that early stages of neurogenesis can be upregulated in the adx rat . a 24-hour alternating regimen of wheel running and enriched environment exposure resulted in a significant increase in the number of migrating neuroblasts , as indicated by the microtubule - associated protein dcx . this effect was similar to that which was observed in our controls , as well as to other reports of exercise - induced alterations in neurogenesis [ 66 , 67 ] . our observation provides further evidence of an intact neural stem - cell niche in the dentate gyrus of adx rats . this upregulation of neurogenesis resulted in a significant increase in the volume of the dentate gyrus of adx rats , relative to their home - cage adx cohorts . although at the six - week time point the volume of the granule cell layer in treated adx rats was still smaller than controls , the continuing high levels of dcx expression suggest that increasing the length of exposure to the combination of enrichment and exercise may suffice to restore the volume of the dentate gyrus granule cell layer to normal . the significant increase in dentate gyrus granule cell layer volume in the enriched adx rats provides indirect evidence that adult born neurons survive in the damaged dentate gyrus . we provide a method by which one form of intrinsic plasticity of the adult brain can be modulated in an attempt to induce functional recovery . here , we employ behavioural and pharmacological techniques that increase the birth and survival of new neurons in the hippocampus . we show that repopulation of the damaged dentate gyrus via adult - born neurons is a viable approach to induce functional recovery . treatment strategies employing transplantation of exogenous stem cells have proven to be relatively ineffective in alleviating function deficits [ 810 ] . a number of issues result from such methods , including the long - term survival of grafted cells and the inability to determine conclusively whether or not grafted cells functionally integrate into an existing neural network . here , we manipulate a naturally occurring system that continually results in the functional integration of new neurons into an existing network within the adult animal . the functional integration of new neurons in the adult animal provides a treatment strategy by which an increase in the number of new cells results in a repopulation of a previously damaged brain region . the effectiveness of treatment methods designed to induce repopulation of the granule cell layer and induce functional recovery can be assessed using the available tools and techniques discussed in the preceding paper . importantly , this demonstration of the loss of function associated with brain damage and the development of treatment strategies that result in functional recovery provides a model by which the required conditions for replacing neural circuitry in other brain regions may be explored .

long - term adrenalectomy ( adx ) results in an extensive and specific loss of dentate gyrus granule cells in the hippocampus of adult rats . this loss of granule cells extends over a period of weeks to months and ultimately results in cognitive deficits revealed in a number of tasks that depend on intact hippocampal function . the gradual nature of adx - induced cell death and the ensuing deficits in cognition resemble in some important respects a variety of pathological conditions in humans . here , we characterize behavioural and cellular processes , including adult neurogenesis , in the rat adx model . we also provide experimental evidence for a neurogenic treatment strategy by which the lost hippocampal cells may be replaced , with the goal of functional recovery in mind .

rat tracheal epithelial ( rte ) cells were cultured on membrane support with and without retinoic acid ( ra ) . in early ( 6-day - old ) cultures , the epithelium is a monolayer or bilayer of undifferentiated cells and secretes little mucuslike product either in the absence or presence of ra . in late ( 12- to 15-day - old ) cultures , the epithelium differentiates as a mucociliary epithelium in the presence of ra and as a squamous epithelium in the absence of ra . the purpose of our study was to determine whether a number of xenobiotic enzymes are expressed in these cultures and whether their expression depends on the state of differentiation . enzyme expression was characterized by electrophoresis and immunoblotting as a function of time in culture and phenotypic differentiation . cytochrome p450 1a1 was not expressed in freshly harvested rte cells . this isoenzyme was induced in rats by gavage with 2,3,7,8-tetrachlorodibenzo - p - dioxin ( tcdd ) or by exposure of early rte cell cultures to tcdd , provided ra was also added to the cultures . cytochrome p450 2b1 was observed in freshly isolated rte cells , but not in early or late rte cultures . in contrast , expression of nadph - cytochrome p450 reductase was decreased in early cultures , but was increased in well - differentiated cultures . flavin - containing monooxygenase was detected in lung tissue , but not in freshly harvested or cultured rte cells . glutathione s - transferase ( gst ) mu and pi were expressed in freshly harvested rte cells . gst pi was expressed in early and late cultures , whereas gst mu was expressed in late cultures , but could not be found in early cultured rte cells.(abstract truncated at 250 words)imagesfigure 1 . afigure 1 . bfigure 1 . cfigure 1 . dfigure 2.figure 3 . afigure 3 . bfigure 4.figure 5.figure 6.figure 7.figure 8 .

in the last 20 years , several research groups have explored treating conventionally untreatable epilepsies with delivery of aeds directly into epileptogenic tissue , the cortical subarachnoid space , or the cerebral ventricles [ 126 ] . this emerging strategy of intracranial pharmacotherapy uses some type of drug delivery device placed inside the cranium . this distinguishes it from both systemic pharmacotherapy , which delivers drugs into the brain through the gastrointestinal , dermal , and/or cardiovascular systems , and from intrathecal pharmacotherapy , which delivers drugs through the theca of the spinal cord . the present article reviews the diverse origins , present state , main challenges , and future prospects for intracranial pharmacotherapy . we focus on our efforts to develop a feedback - controlled intracranial drug delivery device , the subdural hnp , for neocortical epilepsies . historically , four major strategies have been used for the treatment of epilepsies . these are dietary and behavioral therapy , systemic pharmacology , and neurosurgery . dietary therapy and behavioral therapy , the overwhelming majority of drug refractory epilepsies ( dre ) can not be controlled with these strategies . neurosurgical interventions reduce or eliminate seizures by either removing the epileptogenic zone ( e.g. , temporal lobectomy , hemispherectomy , neocortical tissue resection ) or destroying the neural pathways of seizure propagation ( e.g. , by callosotomy , subpial resection ) . while these strategies can improve or cure many patients , both interventions are burdened with the risk of damaging normal neural tissue . however , during systemic aed intake the entire body is exposed to the compound , although the targeted epileptogenic zones occupy less than a thousandth of the body mass . indeed , a neocortical seizure focus with an average tissue volume of 7 cm is 10,000 less than the approx . 70,000 cm body volume of a 70 kg and 180 cm high person . the vagus nerve stimulator ( vns ) of cyberonics ( houston , tx ) , approved by fda for dre in 1997 , marked a new approach to epilepsy therapy . this device belongs to the family of neuroprostheses , which also includes the deep brain stimulator ( dbs ) [ 28 , 29 ] and the responsive neurostimulation ( rns ) system by neuropace ( mountain view , ca ) [ 3032 ] . intracranial pharmacotherapy is the product of the same intellectual wave that , departing from the conventional therapies , produced the vns and other brain stimulation devices , as well as the ideas that intracerebral gene transfer [ 33 , 34 ] , cell transplantation [ 35 , 36 ] , or local cooling might also be used to treat focal epilepsies . approximately 30% of the epilepsy patient population will not achieve complete remission of seizures with standard aed therapy [ 38 , 39 ] . this translates to about 600,000 people in our country and almost 15 million in the rest of the world with dre [ 8 , 40 , 41 ] . nearly a third of these patients suffer the severe condition of one or more seizures per month . many dre patients , especially those with mesial temporal lobe epilepsy ( mtle ) , are candidates for neurosurgical intervention . however , about 90% of patients with severe dre are unsuitable for surgical tissue resection / lesion [ 42 , 43 ] , because the seizure - generating regions ( a ) overlap primary sensory , primary motor , or language ( figure 1 ) areas , ( b ) occupy too large a tissue mass in one lobe or involve multiple foci which are multilobar and/or bihemispheric or ( c ) are nonlesional and difficult or impossible to localize . we estimate that there are about 140,000 dre patients in the us who might be considered as potential candidates for some form of nontraditional epilepsy treatment , including intracranial pharmacotherapy . the idea of treating epileptic seizures with drugs delivered directly into the brain is related to the paradigm shift in medicine that took place in the 1950s and 1960s , leading to the cardiac pacemaker , cochlear implant , and other implanted devices . thus , the pacemaker successfully implanted in the initial groups of patients could not be designed without the commercial availability of the transistor ; the vns could not be constructed without the microprocessor . the neurostimulators and other neuroprostheses designed to correct abnormal brain functions [ 4547 ] opened the eyes of the medical community to new possibilities in the treatment of neurological disorders . it has also become clear that with proper neurosurgical techniques and post - implantation care these devices cause no major damage in neural tissue , or at least such damage is not inherent to their use and does not carry substantially more risk than short - term intracranial electrode or catheter placement . the histopathology of brains of parkinson 's disease patients treated with dbs showed no differences in stimulated and nonstimulated tissues adjacent to the lead - track . in epilepsy clinical trials , no major side - effects were reported during the course of centromedian thalamic stimulation , just as no adverse stimulation - induced side effects were observed in epilepsy patients implanted with the rns device . thus , while these data obviously could not provide information on whether long - term intracranial drug applications would also be free of side - effects , they suggest that such interventions are not accompanied with prohibitive risks . neuropharmacological studies have shown that localized , intracerebral drug applications can modulate , prevent or stop epileptiform eeg and behavioral events . as early as 1970 , collins reported that muscimol , applied topically on the neocortical surface , blocked focal seizures induced by penicillin , bicuculline and picrotoxin , in rats . muscimol could also suppress audiogenic seizures if injected into the inferior colliculus , a structure later proven to be the generator site of sound - induced eeg seizures . piredda and gale showed in rats that local application of muscimol into the deep prepiriform cortex can temporarily eliminate epileptogenicity in this region , concluding that this area may also represent a site at which gaba agonists could function therapeutically to control epileptogenesis . in smith et al . 's paper describing the antiepileptic effect of lidocaine injected into the deep prepiriform cortex , they suggest that microchip and implantable pump technology should make it possible to construct a system that would predict onset of a seizure and then inactivate the neurons in the focus before they could initiate an ictal event . shortly after , eder et al . reported that cortically delivered diazepam can attenuate bicuculline - induced local epileptiform eeg spikes , again suggesting the possible role for aed perfusion directly on seizure focus as a therapy for intractable partial seizures . seizures usually originate in discrete epileptogenic zones ( figures 1(a ) and 1(b ) ) , while the rest of the brain may function normally until the electrophysiological seizure activity propagates to neighboring or even more distant structures . this has justified the search for ways to pharmacologically control cortical or subcortical epileptogenic zones , without the unnecessary and often harmful exposure of the body and the rest of the brain to drugs . advances in medical device manufacturing , neurostimulation research , intracerebral aed pharmacology and clinical epileptology were synthesized into specific engineering solutions for intracranial pharmacotherapy for focal epilepsy at the beginning of this decade . in 2000 , a microcomputer - controlled intracerebrally implanted drug delivery device , in which the timing and duration of drug deliveries are determined by the implanted brain tissue 's own electrical activity was described . also in 2000 , stein et al . published a study demonstrating the efficacy of an automated drug delivery system for focal epilepsy in rats , concluding that such therapy might avoid some of the problems inherent to systemic administration of antiepileptic drugs . in 2000 fischell et al . patented a responsive implantable system for the treatment of neurological disorders ( us patent # 6,134,474 ) , although these inventors focused on electrical stimulation and medication released into the cerebrospinal fluid of the human patient as the therapeutic interventions . the idea of focal methods of drug delivery tied to eeg activity was embraced by investigators at the national institutes of health and within a few years the development of intracranial pharmacotherapy for epilepsy has become the objective of several research teams in academia , in some cases closely collaborating with startup companies ( e.g. , sierra neuropharmaceuticals , medgenesis therapeutix , and others ) . the goal of treating focal epilepsy with intracranially delivered drugs is being pursued in diverse pathways . cortically implanted phenytoin ethylene - vinyl acetate ( evac ) controlled - release polymers have been demonstrated to reduce seizures in a cobalt - induced model of focal neocortical epilepsy . a second strategy aims to deliver aeds into the brain using a different approach : by utilizing fully implanted , responsive or nonresponsive , neuroprosthetic devices . these devices employ either intraparenchymal catheters or catheter / electrode units , or cannulas placed in the cerebral ventricles , or sealed , subdural fluid delivery / recording electrode units overlaying the neocortical epileptogenic zone(s ) . one promising technique for intraparenchymal drug administration into the seizure focus or foci , with or without electrophysiological recording capability , uses convection - enhanced delivery ( ced ) , which seeks to distribute a therapeutic agent homogeneously throughout clinically significant volumes of brain parenchyma . the relative safety of this method was shown in nonhuman primates , while its efficacy to reduce the severity of amygdala - kindled seizures in rats was demonstrated by gasior et al . , who administered n - type calcium blocker conotoxins into the amygdala via ced . however , this strategy addresses systemic , but not cns - related toxicity , at least with the devices and drug delivery protocols that have been tested in intracerebroventricular seizure - control studies . the anesthetic side - effect of intracerebroventricular pentobarbital administration in rats is a pentobarbital action that can be eliminated without decreasing its neocortical seizure - preventing potency by administering this compound transmeningeally into the cortex via a sealed device . this transmeningeal route offers another avenue for intracranial aed administration , with the assistance of the subdural / subarachnoid hnp device . the subdural hnp is a type of intracranial drug delivery device , which offers drug deliveries directly into epileptogenic brain tissue , via sealed , single or multiple , regularly flushed , subdural / subarachnoid units equipped with recording electrodes / sensors to provide feedback from the exposed neural tissue ( figure 2 ) . the basic concept and architecture of the device have been described [ 9 , 17 , 19 , 55 , 56 ] . its key distinguishing feature is the integration of both fluid exchange / drug delivery ports and recording electrodes / sensors into a silicone strip or grid that can be placed in the subarachnoid space ( figure 3 ) . thus , it is this subarachnoid space through which the device delivers aeds or other seizure - preventing therapeutic solutions into the underlying epileptogenic zone(s ) . consequently , the subdural hnp achieves pharmacological / therapeutic effects via drug diffusion through the cerebral arachnoid and pia maters , virtually eliminating the risk of damaging normal neocortical tissue . this transmeningeal pharmacotherapy is based on a known , albeit medically underutilized , physico - chemical property of the cerebral leptomeninges ; that is , their permeability to water - soluble molecules . this is why neurotransmitters released into the neocortical extracellular space can diffuse into a fluid collection device placed on the pia mater [ 57 , 58 ] . thus , water - soluble small molecules , like n - methyl - d - aspartate ( nmda ) or methylene blue , penetrate through these membranes into the underlying cerebral cortex and stay close to the delivery area ( figure 4 ) : findings consistent with prior autoradiographic studies . inclusion of recording electrodes ( and in the future neurochemical sensors ) in the subdural drug delivery unit gives potential for the device to execute three important functions . first , electrophysiological recordings can provide feed - back on the effects of the delivered drugs , so that their delivery parameters can be flexibly adjusted , eliminating the danger of applying too high , neurotoxic doses , while helping to avoid the application of too low , thus inefficient drug concentrations . second , these recordings can potentially provide information for the treating physician on the neurophysiological impact of the subdural implant , so that adverse reactions , if these occur , can be recognized and treated early . third , electrophysiological data acquisition may permit seizure prediction and seizure detection , thus allowing subdural drug delivery in a responsive , on - demand fashion , upon the occurrence of pre - seizure or seizure - onset signals . this set of three feed - back functions separates the subdural hnp from aed - releasing polymers , gene therapy and cell transplantation . multiple drug delivery / recording units , shaped either as strips or grids , can obviously also be used . thus , the device could apply treatment over large cortical areas , without the spatial limitations imposed upon devices using tissue - penetrating cannulas , catheters or tubes . this may allow the treatment of extended , multiple , and/or bilateral seizure foci , as well as diffusely distributed epileptogenic zones that are difficult to localize even with intracranial recordings . presently , muscimol is emerging as the choice of aed for the first generation subdural hnps [ 19 , 20 ] , because of the following reasons : ( a ) cortical seizure - preventing efficacy in low ( 1 mm ; figure 5 ) concentrations , ( b ) fast - developing ( ~30 seconds ) pharmacological action , ( c ) high water - solubility , ( d ) long - term ( ~4 month ) stability in solution , and ( e ) efficacy at neutral ph . another feature of the subdural hnp design is the sealing membrane around both the individual drug delivery ports ( figures 2 and 3 ) and the entire subdural unit . this prevents significant drug spillover to neighboring , normal cortical areas and limits systemic exposure to the administered aed . during pentobarbital administration into the neocortex through a sealed epidural cup ( as in rodents the thin , permeable dura mater allows transmeningeal drug application via such devices ) focal seizures can be readily prevented , while the rat remains awake . the lack of significant drug spillover into the csf or the rest of the cerebral cortex is also demonstrated in figure 5(a ) . it illustrates that if one side of the frontal cortex is pretreated with transmeningeal saline , while the contralateral site is simultaneously pre - treated with muscimol in the same way , subsequent application of ach into both cortical areas leads to focal seizures in the saline - treated but not the muscimol - treated side . the hnp minipump is a dual , fully implantable , and transcutaneously refillable , miniature peristaltic device ( figure 6 ) . its design and two fluid reservoirs allow the hnp to execute , in an alternating fashion , two functions . one reservoir is for aed delivery , while the other one is for delivering a flushing solution ( e.g. , saline or artificial csf ) or removing csf from the subarachnoid space to prevent pressure increase . in the experiment shown in figure 5(b ) , muscimol was delivered with this minipump into the cortical subarachnoid space of a freely moving rat : the apparatus was mounted on the head of the animal ( figure 6 ) . in addition to data generated in rats implanted with epidural drug delivery devices [ 1720 , 22 ] ( figures 46 ) , monkey and human studies also suggest the therapeutic viability of the subdural hnp . in anthropoid new world monkeys ( saimiri sciureus ) , muscimol delivery into the subarachnoid space prevented focal neocortical seizures . in patients with temporal lobe epilepsy ( n = 3 ) , in a neurosurgical setting prior to tissue resection , lidocaine ( an irb - approved compound for the hnp project at the time ) was applied to the pial surface overlaying the epileptogenic zone . within minutes , this local treatment markedly reduced the frequency of eeg spiking in the epileptogenic area . thus , seizure susceptibility in the primate cerebral cortex can be modulated by drugs delivered directly to the pial surface . the prospects of intracranial pharmacotherapy depend on how the intertwined scientific , engineering , clinical , and neurosurgical problems will be solved and the pertinent regulatory and commercial issues navigated . our initial aim is to investigate the most feasible simplest , nonresponsive version of the subdural hnp in phase i / ii clinical trials , after rat and monkey studies on safety and efficacy are completed . this can set the stage for testing the more complex , responsive hnp version [ 9 , 17 , 19 ] . the first main scientific challenge of hnp development is the thorough elaboration of the safety profile of the device . although subdural electrode grids and strips can be routinely kept over the neocortex for a few weeks with minimal or no complications for diagnostic purposes ( figure 1 ) , the safety of using the subdural unit of the hnp ( figure 3 ) for months or years to deliver drug solutions must be independently investigated . second , the potential of tolerance to the applied hnp drugs and the possibility of withdrawal seizures upon the cessation of this treatment must be explored . that tolerance to antiepileptic drugs , including those acting on the gaba system , can develop during their long - term use has been demonstrated by lscher and schmidt , while the possibility of withdrawal seizures after the cessation of continuous , long - term intracortical gaba administration was shown by brailowsky et al . . since muscimol is a gaba - a receptor agonist , clarifying the tolerance - inducing and withdrawal - seizure - inducing potency of this drug is essential . the third main scientific challenge is to understand the cellular and neurochemical effects , as well as pharmacokinetics , of transmeningeally delivered aeds , so that these drugs can be rationally used with intracranial devices . figure 7 illustrates the complexity of the likely effects and fate of muscimol molecules upon their delivery into the cerebral subdural/ subarachnoid space . mapping of the diverse cellular actions of this molecule , including its heterogeneous effects on postsynaptic , extrasynaptic and presynaptic gaba - a receptors [ 62 , 63 ] , as well as its clearance pattern in the neocortex , require a major research effort . but the furnished information , along with corresponding data for other transmeningeal aeds , will help to elaborate the optimal delivery conditions for seizure - controlling drugs delivered with the subdural hnp . the engineering challenges are related to the complexity of the hnp , as it involves both pharmacological and electrophysiological components . this complexity is the product of the goals of ( a ) delivering drugs into the epileptogenic zones in a feedback - controlled manner , requiring electrophysiological monitoring of the drug - exposed tissue , ( b ) providing information on the recording and drug delivery functions of the device to the treating physician , with the option of modifying these functional parameters , if needed , requiring the use of a bidirectional rf communication module , and ( c ) supplying this apparatus with sufficient electrical power for years , requiring the integration of a battery rechargeable transcutaneously via electromagnetic coupling . the drug delivery minipump ( figure 6 ) consumes about 8 mah energy , almost 10-times more than the rest of the device , and thus powering of the hnp needs a different engineering solution than what is adequate for the vns , dbs or rns . the complexity of the hnp as a seizure - preventing device mimics the evolutionary principle of implementing multiple ( neural , endocrine and immune ) control systems to maintain physiological functions and prevent disease . but complexity comes with a price , and this price for the subdural hnp is the increased likelihood of hardware errors : a risk less threatening for simpler drug delivery implants . yet the clinical challenges include the identification of the ideal candidates for intracranial pharmacotherapy and laying the groundwork for post - implantation patient care . within the population of focal dre patients who are not amenable to resective surgery the right subclass for the device needs to be further clarified . patients with nonlesional focal neocortical epilepsy , the most challenging group of surgical candidates , are likely candidates for subdural hnp treatment . but this is a heterogeneous group , including patients with frontal , parietal , extramesial temporal and occipital epileptogenic zones , some with involvement of mesial temporal lobe structures , and some with much less easily recognized influences from subcortical structures . the other challenge is to develop infrastructure for post - implantation care and identify protocols for ( a ) adjusting the right drug delivery parameters ( concentration , volume , frequency ) for the hnp , based on local recordings transmitted from the implant to the physician , ( b ) setting up the implant status indicators , and ( c ) confirming the integrity of the transcutaneous minipump - refilling and battery recharging modules . the main neurosurgical challenges are to determine ( a ) the optimal size and shape of the subdural / subarachnoid recording / drug delivery unit ( figure 3 ) , ( b ) the method for its safe placement over the epileptogenic zones in a way that assures fixed position , ( c ) the best technique to tunnel the wires and tubing from the hnp controller apparatus to the subdural implant , and ( d ) the optimal subcutaneous location for the minipump refilling ports and inner coil of the battery recharging circuit . whether the subdural hnp , along with other intracranial pharmacotherapy implants , will have a limited niche for the treatment of a quite specific class of patients or it may be useful for a larger patient population even beyond those afflicted by epilepsy , is difficult to predict . should the first generation of these devices prove to be safe and effective in the initial clinical trials , it will justify the development of the responsive version that could be used in patients with less frequent seizures . this device may well use microelectrodes to record multi - neuron activity , instead of conventional eeg electrodes , as monitoring cellular electrophysiological signals appears to be more suitable for early seizure detection / prediction and thus for activating aed delivery [ 6567 ] . newer hnps may also use a wide spectrum of drugs besides muscimol . since this device delivers drugs directly into the extracellular space , bypassing the blood - brain barrier ( bbb ) , it can use bbb - impermeable compounds , such as neuroactive peptides and proteins , to achieve therapeutic action . investigators in other fields of neurology may adapt intracranial pharmacotherapy for maximizing stroke recovery after neocortical infarcts , treating cortical tumors , or improving the cognitive functions in alzheimer 's disease . orrin devinsky and ruben i. kuzniecky are consultants and members of the board of directors at cortica , inc . , a company founded for the commercialization of the hnp .

intracranial pharmacotherapy is a novel strategy to treat drug refractory , localization - related epilepsies not amenable to resective surgery . the common feature of the method is the use of some type of antiepileptic drug ( aed ) delivery device placed inside the cranium to prevent or stop focal seizures . this distinguishes it from other nonconventional methods , such as intrathecal pharmacotherapy , electrical neurostimulation , gene therapy , cell transplantation , and local cooling . aed - delivery systems comprise drug releasing polymers and neuroprosthetic devices that can deliver aeds into the brain via intraparenchymal , ventricular , or transmeningeal routes . one such device is the subdural hybrid neuroprosthesis ( hnp ) , designed to deliver aeds , such as muscimol , into the subdural / subarachnoid space overlaying neocortical epileptogenic zones , with electrophysiological feedback from the treated tissue . the idea of intracranial pharmacotherapy and hnp treatment for epilepsy originated from multiple sources , including the advent of implanted medical devices , safety data for intracranial electrodes and catheters , evidence for the seizure - controlling efficacy of intracerebral aeds , and further understanding of the pathophysiology of focal epilepsy . successful introduction of intracranial pharmacotherapy into clinical practice depends on how the intertwined scientific , engineering , clinical , neurosurgical and regulatory challenges will be met to produce an effective and commercially viable device .

gestational diabetes mellitus ( gdm ) is the most common metabolic complications of pregnancy , and causes fetal mortality and morbidity ( 1 , 2 ) . gdm is a state of glucose intolerance with the onset or first recognition occurring during pregnancy ( 3 ) and approximately occurs in 2 - 5 % of all pregnancies ( 4 ) . offspring of mothers with gdm are at increased risk for diabetes and obesity ( 5 , 6 ) . the gestational diabetes prevalence is reported 1 - 3% in the united states , 10.9% in asian countries , 5.2% in europe ( 7 ) . a meta - analysis study indicated the prevalence of gdm ranged between 1.3% to 8.9% in different regions of iran ( 8) . diabetes is associated with long - term complications that affect almost every part of the body , often leading to blindness , cardiovascular disease and kidney failure and nerve damage ( 9 ) . long - term hyperglycemia causes irreversible pathological changes in the retina and leading to an increase in diabetic retinopathy ( 10 ) . the decrease of retina ganglionic cell layer thickness in diabetes type 1 indicates that the retinal layers are mostly influenced by the effects of diabetes ( 11 ) . diabetes - induced cell death has been observed in numerous retinal cell types such as endothelial cells and pericyte , neural retinal cells ( ganglion cells ) and retinal glial cells ( muller cells , astrocytes and microglia ) ( 12 ) . the muller cell is the principal glial cell of the vertebrate retina ; in the avascular retinae of many vertebrates ( including mammals ) it constitutes the only type of macroglial cells . muller cells are specialized radial glial cells which span the entire thickness of the retina and contact / ensheath all retinal neuronal somata and their processes . muller cells constitute an anatomical link between the retinal neurons and the compartments with which these need to exchange molecules , i. e. , the retinal blood vessels , the vitreous body and the subretinal space ( which , together with the retinal pigment epithelium ( rpe ) , constitutes the pathway to the choroidal blood vessels ) ( 13 ) . retinal glial cells , primarily muller glia change from quiescent to an injury - associated phenotype and express high levels of glial fibrillary acidic protein ( gfap ; a hallmark of glial cell activation ) in the human retina during early diabetes ( 14 ) . it has been reported that diabetes induces damage in avascular retinal neurons and muller glial cells ( 11 , 12 ) . in addition to microglial cells , there are two forms of neuron - supporting macroglial cells , astrocytes and muller ( radial glial ) cells ( 13 ) . to assess astrocyte change , labeling experiments were performed on control and diabetic retinas using antibodies to gfap a marker that labels retinal astrocytes but not muller cells ( 15 , 16 ) . muller cells in the mammalian retina normally express low levels of glial fibrillary acidic protein ( gfap ) ; however its expression is unregulated in response to the loss of retinal neurons . the change in expression of gfap is one of the earliest indicators of retinal damage and is correlated with the time course of disease ( 17 ) . gfap expression in the retinas of the diabetic rats was also detected in the end feet of the muller cells . in the retina of control rats , gfap expression was limited to astrocytes and was not detected in muller cells even at 40 weeks of follow - up . the expression of glial fibrillary acidic protein in muller cells was used as a cellular marker for retinal damage ( 18 ) . diabetes induces abnormalities in retinal muller cells , including increased expression of glial fibrillary acidic protein , reduction of glutamine syntheses and decreased function of glutamate transporter ( 19 ) . plasma cell membrane of retinal muller glial cell has an important function in regulation volume through outward water transport . recent studies indicated that retinal edema can be caused by swollen muller glial cells following cell injury and upregulation of gfap . therefore , this study was done to determine the effect of induced gestational diabetes on the expression gfap in muller cells of retinal layer in rat s offspring ( 20 ) . also , a study has shown that the uncontrolled gestational diabetes can reduces the number of ganglionic neurons and increase apoptotic ganglionic cells of retina layer in rat offspring ( 21 ) . regarding the important role of muller cell in supporting of neuronal retinal cell , this study was done to determine the effect of induced gestational diabetes on muller cells of retinal layer in rat s offspring . this experimental study was performed at the gorgan faculty of medicine , golestan university of medical sciences , gorgan , iran . guidelines on the care and use of laboratory animals and approval of the ethics committee of golestan university of medical sciences were obtained before the study . wistar rats , weighing 180 - 220 g ( 12 weeks old ) were used in this study . the animals were maintained in a climate - controlled room under a 12 hr alternating light / dark cycle , 20 c to 25 c temperature , and 50% to 55% relative humidity . after 2 weeks of acclimation to the diet and the environment , female wistar rats were placed with a proven breeder male overnight for breeding . vaginal smears were done the next morning to check for the presence of sperm . once sperm observed that day assigned as gestational day 0 ( gd0 ) . on day 1 of gestation , pregnant females randomly divided in two control and diabetic groups . six female rats in diabetic group were received 40 mg / kg / body weight of streptozotocin ( stz ) ( sigma , st louis , mo , usa ) dissolved in sterile saline solution ( 0.85% ) and control group ( six rats ) were received an equivalent volume normal saline intraperitoneally ( ip ) . blood glucose level of mothers ( both before mating and 72 hr after stz injection ) was obtained via tail vein and was measured with a glucometer ( accu - chek active glucometer , roche diagnostics , mannheim , germany ) ( 22 ) . the dams with blood glucose level 120 - 250 mg / dl were considered as gestational diabetes ( 21 , 23 ) . six offspring of gestational diabetic mothers and control mothers in 28 day after birth ( postnatal day 28 ) were randomly selected and were killed quickly with anesthesia . for light microscope preparations eyes were fixed in 10% neutral - buffered formalin and the tissue processing eyes sectioned at 6-micrometer thickness using a microtome ( microm hm 325 , germany ) . a photograph of sections was produced using an olympus bx51 microscope and a dp12 digital camera . the density of muller cells evaluated in 60000 m inner nucleus layer of eye and the thickness of inner retinal layer using olysia autobioreport software . immunocytochemical labeling to detect the muller cells was performed by monoclonal antibody anti gfap ( millipore corporation billerica , usa ) on eye coronal sections with 6 m thickness . in brief , deparaffinized sections were preincubated with citrate buffer and were washed for 9 min in 0.01 m phosphate - buffered saline ( pbs , ph 7.4 ) and treated with 0.3% hydrogen peroxide in 0.01 m pbs including 10% methanol . then , eye sections were incubated with antigfap ( 1:600 ) in a humidified chamber for 1 hr at room temperature . after rinse in 0.01 m pbs , the sections were incubated with the biotinylated secondary for 10 min and then with streptavidin hrp and rinsed in pbs . immuno - reactivity was visualized using 3,3 diaminobenzi - dine ( dab ; chromogen reagent ) for 30 min at room temperature . subsequently , the tissue specimen was counterstained with mayer s hematoxylin and mounted with entellan ( merck , usa ) . the eyes were removed and immersed in the fixative solution ( 250 ml of 4% paraformaldehyde , ph 7.4 at room temperature ) , overnight . a 400-m block of area retina was dissected and fixed in buffered 2.5% glutaraldehyde for an additional 48 hr . then the sections were washed in pbs solution and postfixed in 1% oso4 for 2 hr at room temperature . after that , they were put onto slices with resin and polymerized for 48 hr at 60 c . subsequently , 60 nm sections were cut and stained with 1% uranyl acetate and 2% lead citrate . immunocytochemical labeling to detect the muller cells was performed by monoclonal antibody anti gfap ( millipore corporation billerica , usa ) on eye coronal sections with 6 m thickness . in brief , deparaffinized sections were preincubated with citrate buffer and were washed for 9 min in 0.01 m phosphate - buffered saline ( pbs , ph 7.4 ) and treated with 0.3% hydrogen peroxide in 0.01 m pbs including 10% methanol . then , eye sections were incubated with antigfap ( 1:600 ) in a humidified chamber for 1 hr at room temperature . after rinse in 0.01 m pbs , the sections were incubated with the biotinylated secondary for 10 min and then with streptavidin hrp and rinsed in pbs . immuno - reactivity was visualized using 3,3 diaminobenzi - dine ( dab ; chromogen reagent ) for 30 min at room temperature . subsequently , the tissue specimen was counterstained with mayer s hematoxylin and mounted with entellan ( merck , usa ) . the eyes were removed and immersed in the fixative solution ( 250 ml of 4% paraformaldehyde , ph 7.4 at room temperature ) , overnight . a 400-m block of area retina was dissected and fixed in buffered 2.5% glutaraldehyde for an additional 48 hr . then the sections were washed in pbs solution and postfixed in 1% oso4 for 2 hr at room temperature . after that , they were put onto slices with resin and polymerized for 48 hr at 60 c . subsequently , 60 nm sections were cut and stained with 1% uranyl acetate and 2% lead citrate . the meansem of blood glucose concentrations before mating and 72 hr after stz injection were 100.422.1 and 211.606.30 mg / dl in diabetic dams . in control dams the meansem of blood glucose concentrations before mating and 72 hr after stz injection were 99.606.2 and 92.535.3 mg / dl , table 1 . the meansem of blood glucose level ( mg / dl ) in control and gestational diabetes dams in day 0 and day 3 after induction of diabetes the number of muller cell processes per 6000 m area of inner nucleus cell layer of retina were significantly increased in offspring of gestational diabetic mothers in comparison to controls ( p<0.05 ) figure 1 , table 2 . immunohistochemistry by monoclonal antibody anti gfap of retinal layers in postnatal day 28 of wistar rat . a ) it is prominent in muller cell inner processes in gestational diabetic retina , b ) control retina is gfap negative ( not shown ) . ( onl : outer nuclear layer , opl : outer plexiform layer , inl : inner nuclear layer , ipl : inner plexiform layer , gcl : ganglionic cell layer , 1000x , scale bar : 20 m ) the number of muller cell processes and the thickness of retinal layer in postnatal day 28 of gestational diabetic and controls rat offspring ( results were shown as meansem , the thickness of inl significantly increased from 26.360.93 m in the control group to 29.890.46 m in the experimental group ( p<0.001 ) and the thickness of retinal layer significantly increased from 173.313.8 mm in the control group to 182.522.5 m in the gestational diabetic group ( p<0.05 ) , table 2 . nuclear fragmentation and apoptotic bodies were observed in muller cell of gestational diabetic offspring whereas control group showed normal structure by electron microscope , figure 2 . electron micrograph , muller cells in the retina in postnatal day 28 ( p28 ) of wistar rat . the meansem of blood glucose concentrations before mating and 72 hr after stz injection were 100.422.1 and 211.606.30 mg / dl in diabetic dams . in control dams the meansem of blood glucose concentrations before mating and 72 hr after stz injection were 99.606.2 and 92.535.3 mg / dl , table 1 . the meansem of blood glucose level ( mg / dl ) in control and gestational diabetes dams in day 0 and day 3 after induction of diabetes the number of muller cell processes per 6000 m area of inner nucleus cell layer of retina were significantly increased in offspring of gestational diabetic mothers in comparison to controls ( p<0.05 ) figure 1 , table 2 . immunohistochemistry by monoclonal antibody anti gfap of retinal layers in postnatal day 28 of wistar rat . a ) it is prominent in muller cell inner processes in gestational diabetic retina , b ) control retina is gfap negative ( not shown ) . ( onl : outer nuclear layer , opl : outer plexiform layer , inl : inner nuclear layer , ipl : inner plexiform layer , gcl : ganglionic cell layer , 1000x , scale bar : 20 m ) the number of muller cell processes and the thickness of retinal layer in postnatal day 28 of gestational diabetic and controls rat offspring ( results were shown as meansem , the thickness of inl significantly increased from 26.360.93 m in the control group to 29.890.46 m in the experimental group ( p<0.001 ) and the thickness of retinal layer significantly increased from 173.313.8 mm in the control group to 182.522.5 m in the gestational diabetic group ( p<0.05 ) , table 2 . the number of muller cell processes per 6000 m area of inner nucleus cell layer of retina were significantly increased in offspring of gestational diabetic mothers in comparison to controls ( p<0.05 ) figure 1 , table 2 . immunohistochemistry by monoclonal antibody anti gfap of retinal layers in postnatal day 28 of wistar rat . a ) it is prominent in muller cell inner processes in gestational diabetic retina , b ) control retina is gfap negative ( not shown ) . ( onl : outer nuclear layer , opl : outer plexiform layer , inl : inner nuclear layer , ipl : inner plexiform layer , gcl : ganglionic cell layer , 1000x , scale bar : 20 m ) the number of muller cell processes and the thickness of retinal layer in postnatal day 28 of gestational diabetic and controls rat offspring ( results were shown as meansem , the thickness of inl significantly increased from 26.360.93 m in the control group to 29.890.46 m in the experimental group ( p<0.001 ) and the thickness of retinal layer significantly increased from 173.313.8 mm in the control group to 182.522.5 m in the gestational diabetic group ( p<0.05 ) , table 2 . nuclear fragmentation and apoptotic bodies were observed in muller cell of gestational diabetic offspring whereas control group showed normal structure by electron microscope , figure 2 . electron micrograph , muller cells in the retina in postnatal day 28 ( p28 ) of wistar rat . the focus of this article was the effect of gestational diabetes on retinal muller cells . muller cells are critically positioned between the vasculature and the neurons of the retina , has a important role in regulating the molecular composition of the retinal microenvironment ( 24 ) . the main clinical lesion that is caused by diabetes in the retina are those of blood vessels , but evidence is also mounting that neural and glial cells of the retina are affected early in both human and experimental diabetes ( 21 , 25 ) . the most prominent neuronal abnormality is apoptosis of cells whose size and location are consistent with ganglion cells ( 13 , 21 ) . the glial alterations explained to date relate to the pattern and level of expression of gfap . in diabetes , muller cells acquire prominent gfap immunoreactivity throughout the extension of their processes ( 26 ) . this study showed that uncontrolled gestational diabetes increased expression of glial fibrillary acidic protein gfap in the retinal muller cells and retinal layer thickness of rat offspring . our finding is similar to several studies including mizutani et al ( 1998 ) , ly et al ( 2011 ) , li et al ( 2001 ) and mancini et al ( 2013 ) , although these studies were done on diabetes type 1 . mizutani et al ( 1998 ) study on human eyes from certified eye banks through the national disease research interchange showed that the level of gfap was increased in the diabetic retinas ( 161106 densitometric units/g protein vs 5545 in the nondiabetic retinas , p= 0.03 ) ( 16 ) . also , li et al ( 2001 ) study in animal models using immunohistochemistry method by anti gfap reported in the retinas from control rats , gfap expression was limited to astrocyte . gfap expression in the retinas of the diabetic rats was detected in the end feet of the muller cells ( 27 ) . indeed , ly et al ( 2011 ) study on rats retina using immunohistochemistry method by anti gfap reported muller cell were labeled for the gfap after 4 and 6 weeks of diabetes . muller cells in central retina display increased gfap from 10 weeks of diabetes , whereas retinal occurs in the peripheral after 6 weeks of diabetes ( 28 ) . furthermore , mancini et al ( 2013 ) study on two groups of wistar rats injected with stz two days after birth , reported in the two diabetic groups increased retinal immunoreactivity of gfap in muller cells but in the nondiabetic group gfap expression was limited to astrocyte ( 29 ) . in our study , we observed increased gfap expression in stalk of muller cells in inner nuclear layer of retina . the cytological changes observed in muller cells in response to injury are accompanied by significant alteration in gene expression . whereas proteins such as gfap , glutamate / aspartate trans - porter ( glast ) are upregulatrd under phatological conditions . immunocytochemical and in situ hybridization studies have shown gfap is not expressed by muller cells in embryonic or adult mouse retina , gfap is integrated into muller cell cytoskeleton and turns over extremely slowly or not at all . in contrast , gfap mrna is transcribed for a limited time and the gene is subsequently turned off . the increase in gfap expression has been shown to be due to transcriptional activation of the gfap gene in muller cells . however , the cis and trans - activating factors that regulate gfap gene expression in muller cells have not been identified so far . cell transfection and gfap - lacz transgenic mice studies indicate that cis elements that stimulate gfap transcription in astrocytes and muller cells are different . there is some evidence that growth factors and cytokines are the signaling molecules involved in gfap induction . cellular mechanisms responsible for gfap expression or mitotic activity in muller cells : molecules emanating from degenerating photoreceptors , and cytokines released from retinal compartments or secreted by activated macrophages , might act on muller cells to induce gfap expression or mitotic activity ( 30 ) . also in this study nuclear fragmentation and apoptotic bodies were observed in muller cell of gestational diabetic offspring compared with normal structure control group in electron microscope images . this study is similar to kumar et al study they reported degenerated and swollen muller cell processes in tem ultramicrograph in diabetic group ( 31 ) . we concluded that the uncontrolled gestational diabetes can increase gfap expression and retinal layer thickness in muller cells of retina layer in rat offspring . we suggested the babies born from mothers with gestational diabetes must be screened for retinal damage and dysfunction .

objective(s):the muller cell is the principal glial cell of the vertebrate retina . the expression of glial fibrillary acidic protein ( gfap ) in the muller cells was used as a cellular marker for retinal damage . this study was done to evaluate the effect of gestational diabetes on retinal muller cells in rat s offspring.materials and methods : in this experimental study , 12 wistar rat dams were randomly allocated in control and diabetic groups . gestational diabetes was induced by 40 mg / kg / body weight of streptozotocin at the first day of gestation , intraperitoneally . dams in control group received an equivalent volume normal saline . eye of six offspring of each group were removed at postnatal day 28 ( p28 ) . the histopathological changes in retina were examined through h&e staining and ultrastructure transmission electron microscopy ( tem ) . the expression of gfap was examined using immunohisto - chemical staining of gfap in muller cells . photographs of retina were taken using olympus bx51 microscope and a digital camera dp12 and em leo906 ; zeiss , germany.results:in the control rat s offspring , gfap expression was not significant in muller cells . according to the optical microscope images , gfap expression was observed in the processes of the muller cell in the inner plexiform layer of retina in offspring of diabetic mothers . in tem technique , nuclear fragmentation and apoptotic bodies were observed in muller cell of diabetic offspring.conclusion:this study showed that the uncontrolled gestational diabetes can increase gfap expression in muller cells and retinal thickness of retinal layer in rat offspring s , therefore uncontrolled gestational can damage the muller cells .

transforming growth factor ( tgf ) is the prototype member of a highly evolutionary conserved secreted family of cytokines . the tgf family consists of tgf , activins , inhibins , nodals , anti - mullerian hormone ( amh ) and bone morphogenetic proteins ( bmps ) 1 , 2 . tgf family members affect various biological processes including cell proliferation , differentiation , migration , adhesion , apoptosis and extracellular matrix ( ecm ) production . consequently tgf members play crucial role in embryonic development , adult tissue homeostasis and in the pathogenesis of a variety of diseases such as cancer , autoimmune diseases , fibrosis and cardiovascular diseases . they all exert their cellular effects via heteromeric complexes of type i and type ii serine / threonine kinase receptors located at the plasmamembrane , and the smad transcription factors , which have a pivotal role in intracellular signaling 1 , 3 . tgf family members act in a highly contextual manner and can either stimulate or inhibit angiogenesis . genetic studies in mice and human have robustly confirmed the notion that tgf signaling is essential for regulation of vasculogenesis and angiogenesis 3 - 6 . inactivation of different components of tgf signaling in mice results in embryonic lethality mainly due to defects in the vascular system . mutations in tgf family genes or mutations , which lead to missregulated tgf signaling result in vascular pathologies , such as arteriovenous malformations ( avms ) , aneurysms , hypertension , atherosclerosis and cardiovascular disease 3 , 4 , 7 . cardiovascular disease is by far the largest chronic health problem causing significant morbidity , mortality and despite the advances in preventive and therapeutic strategies , it remains the leading cause of death worldwide . due to its role in the pathophysiology of cardiovascular disease , tgf has been considered as a potent therapeutic target in a wide spectrum of cardiovascular disorders . however , because of its multifunctional and context dependent action interference with tgf-dependent processes must be carefully designed and the effects of tgf signaling or its inhibition on the different cell types involved must be carefully studied . several recent reviews have discussed the role of tgf signaling in angiogenesis and tumor angiogenesis 4 , 5 , 8 . in the present review the prototypic family member tgf1 is secreted as an inactive latent precursor consisting of tgf1 and a latency associated peptide ( lap ) 1 , 2 . tgf gets activated by proteolytic cleavage of lap and ltbp by thrombospondin , plasmin , reactive oxygen species , acidic microenvironment , matrix metalloproteinases ( mmp2 and 9 ) and 6 integrin 3 . bmps appear to be secreted in an active form ; they are made in the cytoplasm as dimeric pro - protein complexes , which are cleaved by specific proteases . once secreted their bioavailability is regulated through reversible interactions with extracellular antagonists such as noggin , chordin , gremlin , cross - veinless 2 [ cv2 , also referred to as bmp endothelial cell precursor derived regulator ( bmper ) ] and matrix gla protein ( mgp ) . tgf family members elicit their cellular effects by inducing heterotetrameric complexes of type i and type ii serine / threonine kinase transmembrane receptors 1 ( fig . there are five type ii receptors : activin receptor type iia ( actriia ) , activin receptor type iib ( actriib ) , bmp type ii receptor ( bmprii ) , tgf type ii receptor ( trii ) and amh type ii receptor ( amhrii ) . there are seven type i receptors , termed activin receptor - like kinases ( alks ) , alk1- 7 1 . different ligands can bind to different combinations of type i and type ii receptors resulting in signal specificity . tgf signals in most cells via tgf type ii receptor ( trii ) and tri ( alk5 ) ; activins , inhibins and nodal signal via activin receptor type iia ( actriia ) and iib and alk4 ; bmps via bmp type ii receptor ( bmprii ) , actriis and alk1 , 2 , 3 and 6 1 ( fig . accessory receptors have been identified that regulate access of tgf family members to signaling receptors . for tgf , endoglin and betaglycan , and the inhibitory pseudoreceptor bmp and activin membrane - bound inhibitor homolog ( bambi ) have been best characterized . soluble forms of betaglycan and endoglin have been shown also to interfere with signaling by sequestering tgf family ligands from binding to signaling receptors . ligand binding to the constitutively active type ii receptor instigates the recruitment and phosphorylation of the type i receptor on specific serine and threonine residues in its intracellular juxtamembrane region . upon activation type i receptor recruits the receptor regulated ( r-)smad proteins . smads are intracellular signal transducers for the tgf superfamily and regulate transcription of target genes . smad proteins are classified into three groups the r - smad1 , -2 , -3 , -5 and -8 , common - mediator smads ( co - smad4 ) , and inhibitory smads ( i - smad6 and -7 ) 1 , 2 ( fig . activated type i receptors recruit and phosphorylate r - smads at two serine residues in their extreme carboxyl termini . alk4 and -5 ( and -7 ) mediate r - smad2 and -3 phosphorylation , while alk1 , -2 , -3 and -6 mediate phosphorylation of r - smad1 , -5 and -8 . tgf , activins , inhibins , nodal signal usually through smad2 and -3 1 , 2 . studies on endothelial cells ( ecs ) revealed that tgf can signal through two distinct type receptors , i.e. alk5 and the alk1 , resulting in activation of both smad2/3 and smad1/5/8 , respectively 9 . recently , it was demonstrated that tgf can signal via the smad1 pathway not only on ecs but also in tumor cells . endothelial cells ( paecs ) bmp9 was shown to induce phosphorylation of smad1/5 and smad2 via bmprii / actrii / alk1 . while activation of both bmpr - ii and actrii were required for smad1 phosphorylation , actrii mediated a greater proportion of phosphorylation on smad2 10 . activated r - smads interact with smad4 and the tri- or dimeric formed complexes can translocate to the nucleus , where , together with other transcription factors , co - activators and co - repressors , they regulate target gene expression . inhibitory ( i)- smads , i.e. smad6 and -7 , antagonize signaling by inhibiting r - smad activation either by competing with r - smads for type i receptor interaction and/or by recruiting specific ubiquitin ligases or phosphatases to the activated receptor complex , thereby targeting it for proteosomal degradation or dephosphorylation , respectively 1 , 2 , 11 . additionally i - smads can negatively regulate tgf family signaling via degradation of r - smads and through inhibition of r - smad - co - smad4 complex formation . smad7 inhibits signaling from all branches of the tgf superfamily , whereas smad6 selectively inhibits bmp signaling 1 , 2 , 11 . given the complexity and the variety of the tgf family responses , the basic smad signaling pathway is surprisingly simple . besides this highly conserved signaling core , tgf ligands can regulate the activity of a number of other signaling pathways ( non canonical pathways ) . tgf receptor activation results in activation of several other non - smad signaling pathways in a context dependent manner ( fig . non - smad signaling pathways can involve activation of tgf activated kinase ( tak)-1 , extracellular signal - regulated kinase ( erk ) , c - jun n - terminal kinase ( jnk ) and p38 mitogen activated protein ( map ) kinases , rho gtpases , pi3k - akt and traf6 pathway , which crosstalk with the smad pathways 12 . classical signaling cascades such as map kinases can influence the function and stability of smad proteins through different types of modifications such as phosphorylation , ubiquitination and sumoylation 2 . thus , cellular responses to tgf signaling result from the dynamic regulation of canonical and non - canonical cascades . genetic studies in mice and humans have provided evidence for the importance of components of the tgf signaling pathway in vascular morphogenesis and angiogenesis . deletion of components of the tgf pathway ( ligands , receptors and intracellular smad mediators ) in mice leads to embryonic lethality due to vascular abnormalities ( table 1 ) ( reviewed in 3 , 4 , 7 , 8) . numerous examples of divergent and contradictory responses of ecs to tgf have been reported . it is well accepted today that the tgf paradox is due to a number of factors which significantly alter how ecs respond to tgf : cellular density , tgf concentration , duration of treatment , presence of serum components , surrounding matrix , micro / macrovessel origin of ecs and also species derivation 3 , 4 , 47 , 48 . the inhibitory effects of tgf on ec migration and proliferation are mediated by the tgf/alk5/smad2/3 signaling pathway . in contrast , tgf signaling via the tgf/alk1/endoglin leads to proliferation and migration 9 , 49 . it was demonstrated that despite that the two pathways are opposed to each other , alk5 is required for efficient alk1 signaling and the ratio of the two receptors as well as endoglin expression determines the relative response to tgf 9 , 49 . interestingly endoglin can be shed off the ec membrane and result to a soluble form ( soleng ) . soleng was shown to interfere with angiogenesis and high levels of soleng contribute to ec dysfunction that is observed in preeclampsia . soleng inhibits vegf - induced angiogenesis , ec proliferation 50 - 52 as well as tumor growth . however , recent studies revealed that soleng specifically binds to bmp9 and 10 , suggesting that soleng may exerts its effects by interfering with bmp9/10 signaling in vivo 50 . it will be interesting to characterise the exact mechanism by which soleng interferes with vascular endothelial growth factor ( vegf)-induced angiogenesis . it can be hypothesized that soleng by scavenging tgf ligands affects the delicate balance required for angiogenesis . in addition to the control of tgf signaling by its classical receptors , additional receptor associated proteins have been identified for tgf in ecs . for example , vascular endothelial ( ve)-cadherin has been shown to recruit tgf- receptors i and ii into an active receptor complex and regulate tgf signaling 53 . moreover , the cytoplasmic domain of alk1 was demonstrated to bind casein kinase ( ck)2 , the regulatory subunit of protein kinase ck2 , which results in enhancement of alk1 signaling 54 . bmp2 , -4 , -6 and -7 have been shown to induce angiogenesis , ec proliferation and migration 3 , 4 , 8 . interestingly , although bmp4 was shown to induce capillary sprouting in a ec spheroid sprouting assay 55 , other reports have suggested that bmp4 inhibits basal and vegf induced angiogenesis in a human umbilical vein ec ( huvec)-fibroblast sprouting angiogenesis assay 56 . other bmps , such as bmp9 , have also inhibitory effects on ec activation , proliferation and angiogenesis 10 , 57 , 58 . however , bmp9 was also reported to induce angiogenesis in vivo and ec proliferation 59 . additionally , bmp9 in cooperation with tgf can induce tumor angiogenesis by inducing ec proliferation 60 . this bmp9 paradox needs to be further investigated and the factors and signaling molecules which determine the pro- or anti - angiogenic effect of bmp9 need to be characterized . the effects of bmps on ec function and angiogenesis can be modulated by expression of different bmp antagonists and regulators . it was shown initially that bmper interacts with bmps and when overexpressed antagonizes their activity 61 . in contrast , in a bmper - deficient zebrafish model bmper behaves as a bmp agonist . moreover , depletion of bmper from ecs results in reduced sprouting and bmper is required for the pro - angiogenic effects of bmp4 61 . these results suggest that bmper plays a pivotal role in fine - tuning bmp activity in angiogenesis . chl-1 was shown to antagonize the anti - angiogenic effects of bmp4 , and alters the homeostatic balance between bmp4 and vegf to synergize in driving retinal angiogenesis 56 . matrix gla protein ( mgp ) is expressed in ec and plays crucial role in ec function by affecting bmp , tgf and vegf signaling 62 . mgp inhibits bmp2 and bmp4 activity by direct interaction as determined by co - immunoprecipitation 62 , 63 , while potentiates tgf signaling . it was suggested that mgp induces vegf expression and angiogenesis by potentiating tgf signaling , thereby leading to increased vegf expression and angiogenesis . tgf signaling does not affect only proliferative and migratory ec responses , it also affects vascular permeability . inhibition of tgf signaling by the of tri / alk5 kinase chemical inhibitor sb431542 results in increased expression of the ec specific component claudin-5 and inhibits expression of adhesion molecules 64 . tgf is a known regulator of vegf in a variety of cell types , including epithelial cells 65 , macrophages 66 , osteoblasts 67 and perivascular cells 68 . thus , tgf can modulate ec barrier properties by influencing vegf synthesis in the capillary compartment . exogenous tgf can increases permeability in ec cultures 69 , 70 . however , inhibition of tgf signaling by neutralizing tgf antibody or by sb431542 in co - cultures of brain ecs and pericytes 71 or inhibition of tgf signaling by the sb431542 in retinal ecs and mural cell precursors co - cultures 72 leads to increased ec permeability . in addition , it was shown that tgf contributes to the maintenance of the ec barrier properties of the retinal microvasculature in vivo . systemic inhibition of tgf in adult mice led to increased vessel permeability as demonstrated by decreased association between the tight junction proteins zo-1 and occludin 72 . moreover , adenoviral infection with constitutively active alk5 ( caalk5 ) , but not caalk1 , induces ec retraction by decreasing ec permeability 73 . in addition in the ec - specific smad4 knockout mice it has been reported that ec permeability defects , due to impaired ec gap junction assembly , lead to impaired vessel maturation and embryonic lethality 74 . moreover , thrombospondin , an activator of latent tgf , could rescue the increased vascular permeability and impaired vascular maturation reported in akt1 knockout mice 75 . interestingly , inhibition of tgf signaling in vivo by pharmacological inhibition of alk5 resulted in enhanced delivery of nanoparticles to the tumor tissue extravascular space due to increased vessel leakiness 76 . in addition , soleng disrupts ec function and thus results in enhanced capillary permeability . taken together , these studies suggest that tgf signaling plays a critical role in ec function and as a result in vascular development and vascular function . tgf signaling besides operating as a rheostat that controls ec differentiation , viability and function , it plays also important role in smooth muscle cell ( smc ) differentiation and function . several studies provided evidence that smcs are a major cell type through which tgf affects vascular development and disease . tgf potentiates vascular smc ( vsmc ) differentiation by increasing the expression of -smooth muscle actin ( sma ) and smooth muscle myosin 78 , 79 . at low concentrations it promotes proliferation of smc , whereas , at high concentrations it inhibits smc proliferation and migration 80 - 82 . it was shown that tgf-induced expression of smc marker genes involves activation of protein kinase n ( pkn ) and p38 mapk pathways resulting in differentiation of smcs 83 . tgf1 regulates phenotype transition in smc by regulation of specific contractile proteins such as sma , sm22 and calponin 1 in different cells including 10t1/2 cells , neural crest cells and fibroblasts 84 - 86 . in the neural crest stem cell line monc-1 , rhoa modulates tgf1-induced smc differentiation 87 . in 10t1/2 cells , genetic studies in mice revealed that inactivation of critical tgf signaling molecules in all cells results in failure of vascular development which correlates with a lack of smc investment of nascent vessels 3 , 4 . in adult arteries , smcs express tgf receptors and appear to respond to tgf by synthesizing ecm that contributes to the growth of atherosclerotic and restenotic lesions 89 - 91 . lineage - specific ablation of tgf signaling in vsmcs resulted in alterations in vascular morphogenesis in mice , including impaired elastogenesis and vessel widening 92 . tgf actions on smc are both direct and indirect , mediated through downstream effects of tgf signaling in other cell types . for example , tgf acts directly on ecs , with consequent paracrine effects on smcs 93 , 94 . tgf could act directly on cells of the immune system , with effects on smc mediated through alterations in plasma cytokines . bmps were reported to regulate migration , proliferation , and apoptosis of vsmcs and to modulate the expression of smc phenotypic markers in vitro . interestingly , the effects of bmps on vsmcs vary , depending on the anatomic origin of the cells , their state of differentiation or dedifferentiation , and culture conditions . bmp2 , bmp4 and to a lesser extent bmp7 are reported to inhibit vsmc proliferation 95 - 99 . it was shown that bmp2 and bmp7-induced apoptosis of pulmonary smcs ( pasmcs ) 100 . it was also shown that bmp2 inhibits smc proliferation without stimulating ecm synthesis and that adenovirus - mediated transfer of the bmp2 gene inhibited injury - induced intimal hyperplasia suggesting the possibility of therapeutic application of bmp2 for the prevention of vascular proliferative disorders 97 . bmp7 inhibits tgf and platelet - derived growth factor ( pdgf)-bb - induced smc growth and stimulates the expression of smc - specific markers by inducing p21 and i - smad expression 101 , 102 . in addition , bmp7 exhibited anti - inflammatory activity by downregulating intercellular adhesion molecule-1 ( icam-1 ) expression 101 . these results suggest that bmp7 may be used in clinic in order to prevent vascular proliferative disorders and to maintain vascular integrity . in another study , bmp2 was shown to be expressed by human aortic smcs ( hasmc ) and to induce migration of hasmc in a concentration- and time - dependent manner . both bmp7 and bmp4 promote apoptotic cell death in human primary pasmcs by activating caspase-8 and caspase-9 . pasmcs expressing mutant forms of bmprii , identified in idiopathic pulmonary arterial hypertension ( ipah ) patients were resistant to bmp - mediated proapoptotic effects 103 . interestingly it was shown that pulmonary microvascular ecs ( pmvec ) secrete bmp4 in response to hypoxia and promote proliferation and migration of vsmcs in a bmp4-dependent fashion 95 . it was shown that bmp4 inhibited proliferation of pasmcs isolated from proximal pulmonary arteries , but stimulated proliferation of pasmcs from peripheral arteries , and conferred protection from apoptosis . although these differences were not caused by differential activation of bmp signaling pathways , the pro - proliferative effect of bmp4 on peripheral pasmcs was found to be p38mapk / erk - dependent 99 . the role of bmp signaling in pah will be discussed in more detail in following sessions . ec - mural cell interactions are important not only for the growth and maintenance of intact vessels but also for physiological vessel function . ec regulate the smcs contractile function by releasing a number of vasoactive soluble factors 104 . these factors stimulate either contraction or relaxation of mural cells and thus modulate capillary flow . nitric oxide ( no ) and prostacyclin ( pgi2 ) exert vasodilatory effects on smcs 105 . ec dysfunction , including decreased endothelial nitric - oxide synthase ( enos ) activity and loss of bioactive no , plays a prominent role in the development of pulmonary arterial hypertension ( pah ) 106 . on the other hand endothelin ( et)-1 is a potent vasoconstrictor which stimulates contraction of mural cells via et - a and et - b receptors and release of no from ecs via endothelial et - b receptors 107 . several studies have suggested that tgf plays an important role in the regulation of vascular tone and reactivity , since tgf signaling is involved in regulating the expression of both vasodilators and vasoconstrictors . it was demonstrated that tgf/alk5/smad3 pathway induces ec expression of et-1 , resulting in decreased ec migration and proliferation 108 , 109 . in addition tgf1 induces pgi2 production by ecs 110 , and increases ec levels of cycloxygenase-1 111 and prostacyclin - stimulating factor 112 , which induce synthesis of the vasodilatory molecule prostacyclin . in addition , iloprost , a prostacyclin analog induces increased tgf1 expression by smcs 113 , suggesting a potential feedback mechanism . tgf regulates also enos activity , the enzyme responsible for baseline no signaling in ecs 114 , 115 . in contrast , addition of a synthetic no donor diethylenetriamine nitric oxide adduct ( deta)/no to cultured human coronary smcs was shown to induce tgf mrna and protein expression in human coronary smooth muscle cells 116 . another study has demonstrated that aortas from no synthase deficient mice displayed enhanced basal tgf1 activity ; ecs from these animals showed increased smad phosphorylation and transcriptional activity . in the same study it was shown that no reduced the half - life of ectopically expressed smad2 by enhancing its ubiquitination . thus , no pathway in ecs interferes with tgf/smad2 signaling by directing the proteasomal degradation of activated smads and may act as a feedback mechanism to limit activation of no by tgf 117 . these results suggest that , since tgf signaling influences both vasodilators and vasoconstrictors , misregulation of tgf signaling may result in perturbation of the vessel tone . consistent with this notion it was shown that intravascular infusion of tgf in mice results in decreased vascular resistance and vasolidation . in addition , it was suggested that chronically increased levels of tgf1 in the brain of alzheimer leads to impaired constriction of cerebral vessels due to impaired et-1 mediated contractions 118 . overexpression of soleng in mice resulted in impaired peripheral microvessel blood flow response and collapsed retinal microvessels 72 , 119 . , soleng was found to inhibit tgf-mediated activation of enos , thereby affecting vascular tone 52 . similarly , mice heterozygous for endoglin have a severely vasoactive response as a result of decreased enos expression due to enos decreased half - life 115 . in addition eng+/ mice exhibit reduced matrigel - dependent capillary tube formation and delayed reperfusion in a hindlimb ischemia model 121 . moreover , it was shown that acetylcholine - induced vasorelaxation it was significantly increased in adult eng+/- vs. control vessels . these vasodilatory responses were inhibited by the nitric oxide synthase inhibitor n ( g)-nitro - l- arginine methyl ester ( l - name ) , suggesting enos dependence . in addition enos uncoupling was observed in lung tissues of adult , but not newborn , heterozygous mice and was associated with increased production of reactive o2 species ( ros ) in adult eng+/- vs. control lungs . these data suggest that enos - dependent h2o2 generation in eng+/- lungs accounts for the heightened pulmonary vasorelaxation 122 . in a recent study thalidomide induced vessel maturation by mural cell recruitment in eng - heterozygous mice and hereditary hemorrhagic telangiectasia ( hht ) patients , partly by inducing pdgf - b expression in ecs , further supporting the important role of endoglin in ec - smc interactions 123 . bmps were also shown to play important role in vessel tone regulation and vascular function . addition of exogenous bmp2 induces expression and activity of enos in intrapulmonary artery and pulmonary ec preparations 124 . in the same study it was shown that bmp2 heterozygous mice develop severe hypoxic pulmonary hypertension while bmp4 heterozygous mice are protected . this was shown to be due to decreased endothelial nitric oxide synthase ( enos ) expression and activity in the pulmonary vasculature of hypoxic bmp2+/- but not bmp4+/- mice . it was also shown that activation of bmprii promotes pulmonary artery ec ( paec ) survival , proliferation , and migration by activating enos 125 . interestingly functional deletion of the extracellular bmp modulator bmper results in reduced enos expression 126 . bmp4 infusion stimulated aortic nadph oxidase activity which led to endothelial dysfunction and subsequent impaired vasorelaxation and increased systolic blood pressure in mice 127 . addition of bmp4 in cultured human coronary arterial ecs exerts prooxidant , prohypertensive effects 128 . bmp4 treatment of mouse aortaes led to impaired endothelium - dependent relaxations , exaggerated endothelium - dependent contractions via cyclooxygenase-2 ( cox-2 ) upregulation in a reactive oxygen species ( ros ) dependent manner 129 . it has been also reported that increased pulmonary vascular remodelling in alk1+/ mice leads to signs of pah and it has been shown that this is associated with enos - dependent ros production and it can be averted by anti - oxidant treatment 130 . interestingly prostacyclin analogues were shown to enhance bmp induced smad1/5 phosphorylation by suppressing smad6 expression 131 . furthermore , prostacyclin analogues rescued the defects in smad1/5 phosphorylation in pasmcs from pah patients and inhibited smc proliferation and prevented pah progression in the monocrotaline rat model of pah 131 . the critical role of tgf signaling in vascular function was further verified by studies in human . genetic analysis revealed that mutations in genes of the tgf family or genes which affect tgf signaling function lead to vascular dysfunction and disease ( table 1 ) . mutations in the alk1 and endoglin genes lead to an autosomal dominant vascular dysplasia known as hht . hht type 1 is caused by mutations in endoglin 28 , 132 , whereas type 2 is due to mutations in alk1 23 , 24 . recently , it was reported that a subset of patients with juvenile polyposis ( jp ) , carrying mutations in the smad4 gene , can also develop hht 38 . interestingly targeted deletions of acvrl1 ( alk1 ) , tr1 ( alk5 ) , tr2 and eng in mice result in vascular abnormalities highly reminiscent of those described in patients with hht . the vascular abnormalities and fragile leaky vessels are due to abnormal ec proliferation and smc recruitment 3 . thalidomide treatment of hht patients was shown to enhance blood vessel stabilization and reduce nosebleed frequency 123 . moreover , it has been shown that vegf levels are elevated in skin telangiectatic lesions of hht patients and that anti - angiogenic drugs , such as thalidomide and bevacizumab ( anti - vegf antibody ) , are effective in treating gastrointestinal bleedings and liver avms , respectively 133 - 135 . endoglin can be shed from the cell surface by mmp14 and this result in a soluble form of endoglin 51 . excessive production of soleng by trophoblasts in placenta has been reported in patients affected by pre - eclampsia 52 , whether this is caused by excessive mmp14 activity remains to be shown . pre - eclampsia is characterized by ec dysfunction resulting in vasoconstriction , endo - organ ischemia and increased vascular permeability which leads to a raise in blood pressure , is a major cause of maternal , fetal , and neonatal mortality 52 . recent studies have shown that soleng can bind to bmp9 and bmp10 and inhibit their signaling 50 . although the exact mechanism is not known these results suggest that sol - endoglin may be used in anti - angiogenic therapeutic interventions . marfan syndrome ( mfs ) is a genetic disorder characterized by defects in the cardiovascular system and in aorta and heart valves due to increased tgf signaling activity . mfs is caused by mutations in the fibrillin gene 136 , 137 and affects the skeletal system , ocular system , cardiovascular system , pulmonary system as well as other organs . interestingly it was shown recently that mutations in trii associated with mfs2 and lds have differential effects on tgf-induced smad and erk signaling . it was suggested that the balance between tgf-induced smad and erk might be an important determinant of phenotypic severity in disorders related to different mutations in trii 139 . in a mouse model of mfs inhibition of tgf signaling by a tgf neutralizing antibody or the angiotensin ii type 1 receptor inhibitor losartan resulted in decreased aortic root dilatation , elastic fiber degeneration , smad2 activation , and reversal of skeletal myopathy 140 - 142 . these results further confirmed the contribution of excess tgf signaling to the vascular pathology of mfs . although the mechanism by which losartan protects against aortic aneurysm and reduces smad2 activation is not fully understood clinical studies have been initiated and losartan is now used in clinical trials with mfs 143 . recent observations demonstrated an essential role for the tgf non - smad signaling in aneurysm pathogenesis in mfs mice . it was demonstrated that tgf-induced erk1/2 activation contributes to aortic aneurysm progression and that antagonism of this pathway may be therapeutically useful 144 . the loeys - dietz ( lds ) syndrome is an aortic - aneurysm syndrome characterized by cardiovascular manifestations caused by inactivating mutations in the tgf type i or ii receptors 145 , 146 . although the molecular mechanism of lds is still poorly understood , lds is most likely related to tgf signaling defects . interestingly the aortae of patients with lds , who carry heterozygous inactivating mutations in either trii or tri display increased tgf-smad signaling and fibrosis 146 . recently , mutations in smad3 have been linked with a syndrome characterised by aneurysms , dissections and tortuosity throughout the arterial tree in association with mild craniofacial features and skeletal and cutaneous anomalies 35 . these findings implicate the tgf signaling pathway as a putative pharmacological target for the development of new treatment strategies for arterial wall anomalies and osteoarthritis . as discussed earlier tgf signaling is critical for vascular tone regulation and its perturbation leads to impaired blood flow . hypertension is a cardiovascular risk factor which contributes to the development of organ damage such as renal sclerosis , stroke , and coronary heart disease . recent studies have suggested that there is a link between increased levels of circulating tgf and hypertension 147 , 148 . it has been shown that emilin , an extracellular matrix protein , regulates tgf availability and that it is involved in the regulation of arterial diameter . genetic studies have shown that disruption of emilin-1 expression results in increased conversion of pro - tgf to the mature form and as a result increased tgf signaling 149 . this leads to a reduction in arterial lumen diameter with a resultant increase in vascular resistance and hypertension . it has been postulated that this is due to increased tgf signaling which leads to cytostasis of vsmcs and decreased arterial wall expansion . on the other hand , reduced arterial diameter and hypertension may be a secondary consequence of vascular remodelling in mice . this phenotype could be rescued genetically by disruption of one the tgf alleles and reduction of tgf levels 149 . interestingly , missregulation and reduction of tgf signaling in ecs , due to increased levels of soleng , results also in increased blood pressure in pre - eclampsia 52 . ipah is a disease characterized by constriction of the pre - capillary pulmonary arteries , which results in sustained pulmonary arterial pressure . pah is characterized by excessive proliferation of ecs and vsmcs 152 , 153 , increased vascular inflammation , increased vasoconstriction and reduced dilation . genetic studies in human revealed that loss of function mutations in bmp type ii receptor ( bmprii ) were linked with heritable pulmonary arterial hypertension ( hpah ) 26 , 154 - 157 . bmprii expression by ecs and vsmcs is significantly reduced not only in hpah cases with a bmprii mutation , but also in patients with secondary pah 158 - 160 . mice expressing a bmpr - ii mutant lacking c - terminal tail in the pulmonary smcs develop pulmonary vascular lesions similar to pah . interestingly it was shown that disruption of bmprii in pasmcs leads to reduced bmp2 and bmp4 signaling , while signaling by bmp6 and bmp7 is enhanced 161 . furthermore , it was shown that bmp4 is upregulated in hypoxia - induced pah and that heterozygous bmp4/lacz mice are protected from the development of hypoxia - induced pah , vsmc proliferation , and vascular remodelling . hypoxia induced pmvec to secrete bmp4 and promoted proliferation and migration of vsmcs in a bmp4-dependent manner 95 . in addition reduced bmp / smad signaling resulted in activation of the p38 mapk pathway which led in aberrant pasmc proliferation 99 , 162 , 163 . recently mutations in the downstream mediator smad8 , which is highly expressed in the pulmonary vasculature , were identified in patients with hpah 164 . this is surprising , since loss of smad8 function in canonical bmp signaling is largely compensated by smad1/5 . in the same study it was shown that smad8 mutations lead to decreased mir-21 levels and that mir-21 overexpression results in anti - proliferative effects in paec and pasmc . loss of smad8 function in mice results in abnormal vascular remodeling , increased vascular inflammation and pulmonary tumors 46 . interestingly , certain hht2 patients develop pah - like syndromes , suggesting that alk1 mutations can also be involved in pah 166 , 167 . abnormal tgf- signaling has been observed in the pulmonary vasculature of pah patients 96 , 168 . although tgf induced both smad2 and smad1/5 activation , only smad2 was shown to be important in tgf-induced apoptosis of pmvec . thus , imbalance of alk1 and alk5 may contribute to the development of pulmonary artery hypertension 169 . other studies provided evidence that increased tgf signaling participates in the pathogenesis of experimental pah . the development of monocrotaline - induced - pah was associated with increased vascular cell apoptosis and increased tgf signaling as documented by phosphorylated smad2 expression . inhibition of tgf signaling significantly attenuated the development of the pah and reduced pulmonary vascular remodeling 170 . although it is recognized that pah is characterized by increased number of smcs , this may not be only due to increased proliferation of smcs . it has been suggested that ecs may transdifferentiate into mesenchymal - like cells expressing sma , resulting thus in increased numbers of smc - like cells in vascular pathologies . this hypothesis is supported by the demonstration that transdifferentiation of pulmonary arteriolar ecs into smc - like cells occurs during hypoxia - induced pulmonary vascular remodeling and is regulated by myocardin 171 . this process during which ecs lose endothelial features , and acquire mesenchymal - like characteristics has been termed endothelial - to - mesenchymal transition ( endomt ) . for example , it was shown that endomt plays an important role in aortic and pulmonary artery development 172 , 173 . future studies will shed more light into the role of endomt in the pathology of pah and other vascular diseases . atherosclerosis is a chronic inflammatory disease of the arterial wall leading to regional ischemia and ischemic complications such as myocardial infarction and stroke , which greatly contribute to morbidity and mortality . atherosclerosis is characterised by immunological mechanisms leading to vascular inflammation and plaque formation 174 ( fig . tgf ligands , its receptors as well as smad proteins have been found to be expressed in fibro - fatty lesions and fibrous plaques 175 - 177 . consistent with such an expression profile , tgf has been found to affect the properties and function of all the cell types that are known to be present in atherosclerotic lesions such as ecs , smcs , monocytes / macrophages , platelets , t cells and myofibroblasts . it has been suggested originally that tgf exerts anti - atherogenic effects 178 thus having a protective role either by regulating vascular cell proliferation , plaque stability , immune system function or by its anti - inflammatory function 179 , 180 . interestingly , it was shown that there is an inverse relationship between serum tgf1 levels and the development of atherosclerosis and that concentration of active tgf1 is severely depressed in advanced atherosclerosis 181 - 183 . in addition , mutations of trii have been associated with atherosclerotic and restenotic vascular cells 184 . in contrast to the atheroprotective effects of tgf several studies have shown that atherogenic stimuli such as shear stress 185 , oxidized cholesterol 186 , angiotensin ii 187 or growth factors such as pdgf result in increased tgf expression 188 . at the same time increased tgf1 expression was found in atherosclerotic clinical specimens and increased levels of tgf1 were correlated with advanced atherosclerosis 189 , 190 ( fig . endoglin expression was shown to be upregulated in vsmc in human atherosclerotic plaques further supporting a role for endoglin in the maintenance of vascular integrity and in the response of the vessel wall to injury 89 , 191 . in addition it was shown that soleng levels are significantly higher in diabetic patients and in patients with hypertension and diabetes 192 . higher soleng levels were found in patients with diabetes who had retinopathy , in patients with diabetes who had a high probability of 10-year cardiovascular risk , and in patients with diabetes and hypertension who had three or more damaged target organs than in those with no organs affected 192 . changes in soleng plasma levels have been reported in other pathologies such as atherosclerosis and coronary artery disease 193 . also , changes of soleng plasma levels after an acute myocardial infarct are accurate predictors of acute mortality in these patients 194 . several studies suggested that bmps play also an important role in atherosclerosis by modulating ec inflammation and cell differentiation . it was shown that bmp4 is increased in response to the high fat diet and bmp2 and -4 have been implicated in proinflammatory effects in the endothelium 195 - 197 . furthermore , interference with the bmp pathway leads to reduced , while enhanced bmp activity results in increased atherosclerotic lesion formation in apolipoprotein ( apo)e knockout mice 195 - 197 . it was suggested that this might be due to the effects of bmp signaling on ec function . bmp2 and bmp4 have been implicated as mediators of endothelial inflammation in response to shear stress , oxidative stress and pro - inflammatory cytokines 198 , 199 . whether these effects are due to effects on mononuclear cell function is not known ( fig . although initially it was considered that vascular calcification is a passive process of accumulation of dying cells , later studies suggested that vascular calcification is a highly regulated process . several studies have provided evidence that vascular calcification is the result of an osteogenic process , which leads to mineralisation of the ecm and cell differentiation towards osteoblast - like lineages . it has recently become appreciated that under certain pathologic contexts and culture conditions , vsmcs can differentiate to acquire an osteogenic phenotype , and express lineage markers consistent with bone - forming cells as well as calcification - regulating proteins commonly found in bone 200 - 202 . considerable evidence suggests that tgf family members , particularly the bmps , are important modulators of vascular calcification 203 , 204 , probably by inducing ecm production that is amenable to mineralization and by favoring differentiation toward osteoblast - like lineages ( fig . is expressed in calcified human atherosclerotic plaques it was shown that bmps can direct osteogenic programming of vascular mesenchymal progenitors of the pericyte lineage 205 . later it was shown that bmp signaling as well as a variety of inflammatory stimuli can promote expression of osteoblast lineage markers such as alkaline phosphatase in cultured vsmcs 200 , 201 , 206 - 209 . treatment of smc with bmp2 enhanced vascular calcification via increased phosphate uptake and induction of osteogenic phenotype modulation in smc 210 . moreover , bmps may induce vsmc to express proteins , such as osteoprotegerin which may confer protection from vascular calcific and atheromatous disease 211 . recently , it was shown that inhibition of bmp signaling in apoe/ mice by transgene expression of mgp resulted in diminished smad1/5/8 signaling , as well as reduced inflammation , lesion formation , and calcification after fat feeding 197 . on the other hand mgp deficient apoe/ mice displayed enhanced smad1/5/8 signaling and extensive medial calcification as previously reported 197 . bmp2 was shown to be expressed in adventitial myofibroblasts in the aorta of fat - fed diabetic low - density lipoprotein receptor ( ldlr)/ mice and to induce an osteogenic phenotype in a subset of the myofibroblasts by inducing msx2 expression , which is associated with bone formation 212 . in diabetic rats interestingly , transgenic expression of mgp resulted in reduced bmp activity and diminished calcification in the entire media 213 . mgp , noggin and chordin are downregulated in dedifferentiated vsmcs , which would make them more susceptible to bmp action 195 . thus , the balance between endothelial bmps and their inhibitors may determine the health of the endothelium , and an efficient limitation of endothelial bmp activity may confer protection against atherosclerosis . tgf is present in calcified aortic valves and regulates vascular calcification and osteoblastic differentiation of vsmcs 214 , 215 . it has been proposed that during lesion formation enhanced bmp signaling may promote developmental conditions and trigger the differentiation of multipotential susceptible cells into the osteogenic lineage . it has been suggested that several cell populations might enter into an osteocondrogenic lineage reprogramming , however , the relative contribution of the respective cell population to local osteogenesis and vascular calcification in different arteriosclerotic disease states remains to be established . myofibroblasts present in the adventitia of diabetic and hyperlipidemic ldlr/ mice 212 may also acquire an osteogenic phenotype . pericytes have recently emerged as multipotent adult mesenchymal stem cells and it was shown that they have osteogenic potential 217 . bmps can direct osteogenic programming of vascular mesenchymal progenitors of the pericyte lineage ; however , the relative contribution of pericytes to osteogenic vascular calcification has yet to be clarified . it was proposed that vsmc can differentiate and acquire an osteogenic phenotype and this may directly contribute to medial calcific vascular disease . vsmc transdifferentiation was also shown to contribute to the vascular endochondral ossification mechanisms in mice 218 . it has been suggested recently that vascular ecs might enter into an osteocondrogenic lineage reprogramming 219 . several studies have suggested that ec plasticity allows ec to transdifferentiate into messenchymal cells during embryonic development 220 or as a response to chronic inflammation 221 , 222 . it was demonstrated that expression of a constitutively active form of the alk2 type i receptor in mice causes endomt ; ec cells acquire a stem cell - like phenotype which enables them to undergo osteogenic differentiation 219 . patients with fibrodysplasia ossificans progressiva are characterized by heterotopic calcification , caused by mutations in the alk2 gene . in addition , treatment of huvecs and human cutaneous microvascular ecs ( hcmecs ) with either tgf2 or bmp - resulted in induction of endomt , whereas bmp7 and vegf blocked it 219 . tgf has also been reported to induce endomt in ecs in vitro 223 - 226 . tgf treatment resulted in decreased expression of endothelial markers and while inducing mesenchymal marker expression as well as cytoskeletal rearrangements , which promoted cells to acquire a contractile phenotype 225 . the prototypic family member tgf1 is secreted as an inactive latent precursor consisting of tgf1 and a latency associated peptide ( lap ) 1 , 2 . tgf gets activated by proteolytic cleavage of lap and ltbp by thrombospondin , plasmin , reactive oxygen species , acidic microenvironment , matrix metalloproteinases ( mmp2 and 9 ) and 6 integrin 3 . bmps appear to be secreted in an active form ; they are made in the cytoplasm as dimeric pro - protein complexes , which are cleaved by specific proteases . once secreted their bioavailability is regulated through reversible interactions with extracellular antagonists such as noggin , chordin , gremlin , cross - veinless 2 [ cv2 , also referred to as bmp endothelial cell precursor derived regulator ( bmper ) ] and matrix gla protein ( mgp ) . tgf family members elicit their cellular effects by inducing heterotetrameric complexes of type i and type ii serine / threonine kinase transmembrane receptors 1 ( fig . there are five type ii receptors : activin receptor type iia ( actriia ) , activin receptor type iib ( actriib ) , bmp type ii receptor ( bmprii ) , tgf type ii receptor ( trii ) and amh type ii receptor ( amhrii ) . there are seven type i receptors , termed activin receptor - like kinases ( alks ) , alk1- 7 1 . different ligands can bind to different combinations of type i and type ii receptors resulting in signal specificity . tgf signals in most cells via tgf type ii receptor ( trii ) and tri ( alk5 ) ; activins , inhibins and nodal signal via activin receptor type iia ( actriia ) and iib and alk4 ; bmps via bmp type ii receptor ( bmprii ) , actriis and alk1 , 2 , 3 and 6 1 ( fig . accessory receptors have been identified that regulate access of tgf family members to signaling receptors . for tgf , endoglin and betaglycan , and the inhibitory pseudoreceptor bmp and activin membrane - bound inhibitor homolog ( bambi ) soluble forms of betaglycan and endoglin have been shown also to interfere with signaling by sequestering tgf family ligands from binding to signaling receptors . ligand binding to the constitutively active type ii receptor instigates the recruitment and phosphorylation of the type i receptor on specific serine and threonine residues in its intracellular juxtamembrane region . upon activation type i receptor recruits the receptor regulated ( r-)smad proteins . smads are intracellular signal transducers for the tgf superfamily and regulate transcription of target genes . smad proteins are classified into three groups the r - smad1 , -2 , -3 , -5 and -8 , common - mediator smads ( co - smad4 ) , and inhibitory smads ( i - smad6 and -7 ) 1 , 2 ( fig . activated type i receptors recruit and phosphorylate r - smads at two serine residues in their extreme carboxyl termini . alk4 and -5 ( and -7 ) mediate r - smad2 and -3 phosphorylation , while alk1 , -2 , -3 and -6 mediate phosphorylation of r - smad1 , -5 and -8 . tgf , activins , inhibins , nodal signal usually through smad2 and -3 1 , 2 . studies on endothelial cells ( ecs ) revealed that tgf can signal through two distinct type receptors , i.e. alk5 and the alk1 , resulting in activation of both smad2/3 and smad1/5/8 , respectively 9 . recently , it was demonstrated that tgf can signal via the smad1 pathway not only on ecs but also in tumor cells . bmps and amh signal through smad1 , -5 and -8 . in human pulmonary artery endothelial cells ( paecs ) bmp9 was shown to induce phosphorylation of smad1/5 and smad2 via bmprii / actrii / alk1 . while activation of both bmpr - ii and actrii were required for smad1 phosphorylation , actrii mediated a greater proportion of phosphorylation on smad2 10 . activated r - smads interact with smad4 and the tri- or dimeric formed complexes can translocate to the nucleus , where , together with other transcription factors , co - activators and co - repressors , they regulate target gene expression . inhibitory ( i)- smads , i.e. smad6 and -7 , antagonize signaling by inhibiting r - smad activation either by competing with r - smads for type i receptor interaction and/or by recruiting specific ubiquitin ligases or phosphatases to the activated receptor complex , thereby targeting it for proteosomal degradation or dephosphorylation , respectively 1 , 2 , 11 . additionally i - smads can negatively regulate tgf family signaling via degradation of r - smads and through inhibition of r - smad - co - smad4 complex formation . smad7 inhibits signaling from all branches of the tgf superfamily , whereas smad6 selectively inhibits bmp signaling 1 , 2 , 11 . given the complexity and the variety of the tgf family responses , the basic smad signaling pathway is surprisingly simple . besides this highly conserved signaling core , tgf ligands can regulate the activity of a number of other signaling pathways ( non canonical pathways ) . tgf receptor activation results in activation of several other non - smad signaling pathways in a context dependent manner ( fig . non - smad signaling pathways can involve activation of tgf activated kinase ( tak)-1 , extracellular signal - regulated kinase ( erk ) , c - jun n - terminal kinase ( jnk ) and p38 mitogen activated protein ( map ) kinases , rho gtpases , pi3k - akt and traf6 pathway , which crosstalk with the smad pathways 12 . classical signaling cascades such as map kinases can influence the function and stability of smad proteins through different types of modifications such as phosphorylation , ubiquitination and sumoylation 2 . thus , cellular responses to tgf signaling result from the dynamic regulation of canonical and non - canonical cascades . genetic studies in mice and humans have provided evidence for the importance of components of the tgf signaling pathway in vascular morphogenesis and angiogenesis . deletion of components of the tgf pathway ( ligands , receptors and intracellular smad mediators ) in mice leads to embryonic lethality due to vascular abnormalities ( table 1 ) ( reviewed in 3 , 4 , 7 , 8) . it is well accepted today that the tgf paradox is due to a number of factors which significantly alter how ecs respond to tgf : cellular density , tgf concentration , duration of treatment , presence of serum components , surrounding matrix , micro / macrovessel origin of ecs and also species derivation 3 , 4 , 47 , 48 . the inhibitory effects of tgf on ec migration and proliferation are mediated by the tgf/alk5/smad2/3 signaling pathway . in contrast , tgf signaling via the tgf/alk1/endoglin leads to proliferation and migration 9 , 49 . it was demonstrated that despite that the two pathways are opposed to each other , alk5 is required for efficient alk1 signaling and the ratio of the two receptors as well as endoglin expression determines the relative response to tgf 9 , 49 . interestingly endoglin can be shed off the ec membrane and result to a soluble form ( soleng ) . soleng was shown to interfere with angiogenesis and high levels of soleng contribute to ec dysfunction that is observed in preeclampsia . soleng inhibits vegf - induced angiogenesis , ec proliferation 50 - 52 as well as tumor growth . however , recent studies revealed that soleng specifically binds to bmp9 and 10 , suggesting that soleng may exerts its effects by interfering with bmp9/10 signaling in vivo 50 . it will be interesting to characterise the exact mechanism by which soleng interferes with vascular endothelial growth factor ( vegf)-induced angiogenesis . it can be hypothesized that soleng by scavenging tgf ligands affects the delicate balance required for angiogenesis . in addition to the control of tgf signaling by its classical receptors , additional receptor associated proteins have been identified for tgf in ecs . for example , vascular endothelial ( ve)-cadherin has been shown to recruit tgf- receptors i and ii into an active receptor complex and regulate tgf signaling 53 . moreover , the cytoplasmic domain of alk1 was demonstrated to bind casein kinase ( ck)2 , the regulatory subunit of protein kinase ck2 , which results in enhancement of alk1 signaling 54 . bmp2 , -4 , -6 and -7 have been shown to induce angiogenesis , ec proliferation and migration 3 , 4 , 8 . interestingly , although bmp4 was shown to induce capillary sprouting in a ec spheroid sprouting assay 55 , other reports have suggested that bmp4 inhibits basal and vegf induced angiogenesis in a human umbilical vein ec ( huvec)-fibroblast sprouting angiogenesis assay 56 . other bmps , such as bmp9 , have also inhibitory effects on ec activation , proliferation and angiogenesis 10 , 57 , 58 . however , bmp9 was also reported to induce angiogenesis in vivo and ec proliferation 59 . additionally , bmp9 in cooperation with tgf can induce tumor angiogenesis by inducing ec proliferation 60 . this bmp9 paradox needs to be further investigated and the factors and signaling molecules which determine the pro- or anti - angiogenic effect of bmp9 need to be characterized . the effects of bmps on ec function and angiogenesis can be modulated by expression of different bmp antagonists and regulators . it was shown initially that bmper interacts with bmps and when overexpressed antagonizes their activity 61 . in contrast , in a bmper - deficient zebrafish model bmper behaves as a bmp agonist . moreover , depletion of bmper from ecs results in reduced sprouting and bmper is required for the pro - angiogenic effects of bmp4 61 . these results suggest that bmper plays a pivotal role in fine - tuning bmp activity in angiogenesis . chl-1 was shown to antagonize the anti - angiogenic effects of bmp4 , and alters the homeostatic balance between bmp4 and vegf to synergize in driving retinal angiogenesis 56 . matrix gla protein ( mgp ) is expressed in ec and plays crucial role in ec function by affecting bmp , tgf and vegf signaling 62 . mgp inhibits bmp2 and bmp4 activity by direct interaction as determined by co - immunoprecipitation 62 , 63 , while potentiates tgf signaling . it was suggested that mgp induces vegf expression and angiogenesis by potentiating tgf signaling , thereby leading to increased vegf expression and angiogenesis . tgf signaling does not affect only proliferative and migratory ec responses , it also affects vascular permeability . inhibition of tgf signaling by the of tri / alk5 kinase chemical inhibitor sb431542 results in increased expression of the ec specific component claudin-5 and inhibits expression of adhesion molecules 64 . tgf is a known regulator of vegf in a variety of cell types , including epithelial cells 65 , macrophages 66 , osteoblasts 67 and perivascular cells 68 . thus , tgf can modulate ec barrier properties by influencing vegf synthesis in the capillary compartment . exogenous tgf can increases permeability in ec cultures 69 , 70 . however , inhibition of tgf signaling by neutralizing tgf antibody or by sb431542 in co - cultures of brain ecs and pericytes 71 or inhibition of tgf signaling by the sb431542 in retinal ecs and mural cell precursors co - cultures 72 leads to increased ec permeability . in addition , it was shown that tgf contributes to the maintenance of the ec barrier properties of the retinal microvasculature in vivo . systemic inhibition of tgf in adult mice led to increased vessel permeability as demonstrated by decreased association between the tight junction proteins zo-1 and occludin 72 . moreover , adenoviral infection with constitutively active alk5 ( caalk5 ) , but not caalk1 , induces ec retraction by decreasing ec permeability 73 . in addition in the ec - specific smad4 knockout mice it has been reported that ec permeability defects , due to impaired ec gap junction assembly , lead to impaired vessel maturation and embryonic lethality 74 . moreover , thrombospondin , an activator of latent tgf , could rescue the increased vascular permeability and impaired vascular maturation reported in akt1 knockout mice 75 . interestingly , inhibition of tgf signaling in vivo by pharmacological inhibition of alk5 resulted in enhanced delivery of nanoparticles to the tumor tissue extravascular space due to increased vessel leakiness 76 . in addition , soleng disrupts ec function and thus results in enhanced capillary permeability . taken together , these studies suggest that tgf signaling plays a critical role in ec function and as a result in vascular development and vascular function . tgf signaling besides operating as a rheostat that controls ec differentiation , viability and function , it plays also important role in smooth muscle cell ( smc ) differentiation and function . several studies provided evidence that smcs are a major cell type through which tgf affects vascular development and disease . tgf potentiates vascular smc ( vsmc ) differentiation by increasing the expression of -smooth muscle actin ( sma ) and smooth muscle myosin 78 , 79 . at low concentrations it promotes proliferation of smc , whereas , at high concentrations it inhibits smc proliferation and migration 80 - 82 . it was shown that tgf-induced expression of smc marker genes involves activation of protein kinase n ( pkn ) and p38 mapk pathways resulting in differentiation of smcs 83 . tgf1 regulates phenotype transition in smc by regulation of specific contractile proteins such as sma , sm22 and calponin 1 in different cells including 10t1/2 cells , neural crest cells and fibroblasts 84 - 86 . in the neural crest stem cell line monc-1 , rhoa modulates tgf1-induced smc differentiation 87 . in 10t1/2 cells , genetic studies in mice revealed that inactivation of critical tgf signaling molecules in all cells results in failure of vascular development which correlates with a lack of smc investment of nascent vessels 3 , 4 . in adult arteries , smcs express tgf receptors and appear to respond to tgf by synthesizing ecm that contributes to the growth of atherosclerotic and restenotic lesions 89 - 91 . lineage - specific ablation of tgf signaling in vsmcs resulted in alterations in vascular morphogenesis in mice , including impaired elastogenesis and vessel widening 92 . tgf actions on smc are both direct and indirect , mediated through downstream effects of tgf signaling in other cell types . for example , tgf acts directly on ecs , with consequent paracrine effects on smcs 93 , 94 . tgf could act directly on cells of the immune system , with effects on smc mediated through alterations in plasma cytokines . bmps were reported to regulate migration , proliferation , and apoptosis of vsmcs and to modulate the expression of smc phenotypic markers in vitro . interestingly , the effects of bmps on vsmcs vary , depending on the anatomic origin of the cells , their state of differentiation or dedifferentiation , and culture conditions . bmp2 , bmp4 and to a lesser extent bmp7 are reported to inhibit vsmc proliferation 95 - 99 . it was shown that bmp2 and bmp7-induced apoptosis of pulmonary smcs ( pasmcs ) 100 . it was also shown that bmp2 inhibits smc proliferation without stimulating ecm synthesis and that adenovirus - mediated transfer of the bmp2 gene inhibited injury - induced intimal hyperplasia suggesting the possibility of therapeutic application of bmp2 for the prevention of vascular proliferative disorders 97 . bmp7 inhibits tgf and platelet - derived growth factor ( pdgf)-bb - induced smc growth and stimulates the expression of smc - specific markers by inducing p21 and i - smad expression 101 , 102 . in addition , bmp7 exhibited anti - inflammatory activity by downregulating intercellular adhesion molecule-1 ( icam-1 ) expression 101 . these results suggest that bmp7 may be used in clinic in order to prevent vascular proliferative disorders and to maintain vascular integrity . in another study , bmp2 was shown to be expressed by human aortic smcs ( hasmc ) and to induce migration of hasmc in a concentration- and time - dependent manner . in contrast , bmp2 had no significant effect on hasmc proliferation 98 . both bmp7 and bmp4 promote apoptotic cell death in human primary pasmcs by activating caspase-8 and caspase-9 . pasmcs expressing mutant forms of bmprii , identified in idiopathic pulmonary arterial hypertension ( ipah ) patients were resistant to bmp - mediated proapoptotic effects 103 . interestingly it was shown that pulmonary microvascular ecs ( pmvec ) secrete bmp4 in response to hypoxia and promote proliferation and migration of vsmcs in a bmp4-dependent fashion 95 . it was shown that bmp4 inhibited proliferation of pasmcs isolated from proximal pulmonary arteries , but stimulated proliferation of pasmcs from peripheral arteries , and conferred protection from apoptosis . although these differences were not caused by differential activation of bmp signaling pathways , the pro - proliferative effect of bmp4 on peripheral pasmcs was found to be p38mapk / erk - dependent 99 . the role of bmp signaling in pah will be discussed in more detail in following sessions . ec - mural cell interactions are important not only for the growth and maintenance of intact vessels but also for physiological vessel function . ec regulate the smcs contractile function by releasing a number of vasoactive soluble factors 104 . these factors stimulate either contraction or relaxation of mural cells and thus modulate capillary flow . nitric oxide ( no ) and prostacyclin ( pgi2 ) exert vasodilatory effects on smcs 105 . ec dysfunction , including decreased endothelial nitric - oxide synthase ( enos ) activity and loss of bioactive no , plays a prominent role in the development of pulmonary arterial hypertension ( pah ) 106 . on the other hand endothelin ( et)-1 is a potent vasoconstrictor which stimulates contraction of mural cells via et - a and et - b receptors and release of no from ecs via endothelial et - b receptors 107 . several studies have suggested that tgf plays an important role in the regulation of vascular tone and reactivity , since tgf signaling is involved in regulating the expression of both vasodilators and vasoconstrictors . it was demonstrated that tgf/alk5/smad3 pathway induces ec expression of et-1 , resulting in decreased ec migration and proliferation 108 , 109 . in addition tgf1 induces pgi2 production by ecs 110 , and increases ec levels of cycloxygenase-1 111 and prostacyclin - stimulating factor 112 , which induce synthesis of the vasodilatory molecule prostacyclin . in addition , iloprost , a prostacyclin analog induces increased tgf1 expression by smcs 113 , suggesting a potential feedback mechanism . tgf regulates also enos activity , the enzyme responsible for baseline no signaling in ecs 114 , 115 . in contrast , addition of a synthetic no donor diethylenetriamine nitric oxide adduct ( deta)/no to cultured human coronary smcs was shown to induce tgf mrna and protein expression in human coronary smooth muscle cells 116 . another study has demonstrated that aortas from no synthase deficient mice displayed enhanced basal tgf1 activity ; ecs from these animals showed increased smad phosphorylation and transcriptional activity . in the same study it was shown that no reduced the half - life of ectopically expressed smad2 by enhancing its ubiquitination . thus , no pathway in ecs interferes with tgf/smad2 signaling by directing the proteasomal degradation of activated smads and may act as a feedback mechanism to limit activation of no by tgf 117 . these results suggest that , since tgf signaling influences both vasodilators and vasoconstrictors , misregulation of tgf signaling may result in perturbation of the vessel tone . consistent with this notion it was shown that intravascular infusion of tgf in mice results in decreased vascular resistance and vasolidation . in addition , it was suggested that chronically increased levels of tgf1 in the brain of alzheimer leads to impaired constriction of cerebral vessels due to impaired et-1 mediated contractions 118 . overexpression of soleng in mice resulted in impaired peripheral microvessel blood flow response and collapsed retinal microvessels 72 , 119 . in addition , soleng was found to inhibit tgf-mediated activation of enos , thereby affecting vascular tone 52 . similarly , mice heterozygous for endoglin have a severely vasoactive response as a result of decreased enos expression due to enos decreased half - life 115 . in addition eng+/ mice exhibit reduced matrigel - dependent capillary tube formation and delayed reperfusion in a hindlimb ischemia model 121 . moreover , it was shown that acetylcholine - induced vasorelaxation it was significantly increased in adult eng+/- vs. control vessels . these vasodilatory responses were inhibited by the nitric oxide synthase inhibitor n ( g)-nitro - l- arginine methyl ester ( l - name ) , suggesting enos dependence . in addition enos uncoupling was observed in lung tissues of adult , but not newborn , heterozygous mice and was associated with increased production of reactive o2 species ( ros ) in adult eng+/- vs. control lungs . these data suggest that enos - dependent h2o2 generation in eng+/- lungs accounts for the heightened pulmonary vasorelaxation 122 . in a recent study thalidomide induced vessel maturation by mural cell recruitment in eng - heterozygous mice and hereditary hemorrhagic telangiectasia ( hht ) patients , partly by inducing pdgf - b expression in ecs , further supporting the important role of endoglin in ec - smc interactions 123 . bmps were also shown to play important role in vessel tone regulation and vascular function . addition of exogenous bmp2 induces expression and activity of enos in intrapulmonary artery and pulmonary ec preparations 124 . in the same study it was shown that bmp2 heterozygous mice develop severe hypoxic pulmonary hypertension while bmp4 heterozygous mice are protected . this was shown to be due to decreased endothelial nitric oxide synthase ( enos ) expression and activity in the pulmonary vasculature of hypoxic bmp2+/- but not bmp4+/- mice . it was also shown that activation of bmprii promotes pulmonary artery ec ( paec ) survival , proliferation , and migration by activating enos 125 . interestingly functional deletion of the extracellular bmp modulator bmper results in reduced enos expression 126 . bmp4 infusion stimulated aortic nadph oxidase activity which led to endothelial dysfunction and subsequent impaired vasorelaxation and increased systolic blood pressure in mice 127 . addition of bmp4 in cultured human coronary arterial ecs exerts prooxidant , prohypertensive effects 128 . bmp4 treatment of mouse aortaes led to impaired endothelium - dependent relaxations , exaggerated endothelium - dependent contractions via cyclooxygenase-2 ( cox-2 ) upregulation in a reactive oxygen species ( ros ) dependent manner 129 . it has been also reported that increased pulmonary vascular remodelling in alk1+/ mice leads to signs of pah and it has been shown that this is associated with enos - dependent ros production and it can be averted by anti - oxidant treatment 130 . interestingly prostacyclin analogues were shown to enhance bmp induced smad1/5 phosphorylation by suppressing smad6 expression 131 . furthermore , prostacyclin analogues rescued the defects in smad1/5 phosphorylation in pasmcs from pah patients and inhibited smc proliferation and prevented pah progression in the monocrotaline rat model of pah 131 . the critical role of tgf signaling in vascular function was further verified by studies in human . genetic analysis revealed that mutations in genes of the tgf family or genes which affect tgf signaling function lead to vascular dysfunction and disease ( table 1 ) . mutations in the alk1 and endoglin genes lead to an autosomal dominant vascular dysplasia known as hht . hht type 1 is caused by mutations in endoglin 28 , 132 , whereas type 2 is due to mutations in alk1 23 , 24 . recently , it was reported that a subset of patients with juvenile polyposis ( jp ) , carrying mutations in the smad4 gene , can also develop hht 38 . interestingly targeted deletions of acvrl1 ( alk1 ) , tr1 ( alk5 ) , tr2 and eng in mice result in vascular abnormalities highly reminiscent of those described in patients with hht . the vascular abnormalities and fragile leaky vessels are due to abnormal ec proliferation and smc recruitment 3 . thalidomide treatment of hht patients was shown to enhance blood vessel stabilization and reduce nosebleed frequency 123 . moreover , it has been shown that vegf levels are elevated in skin telangiectatic lesions of hht patients and that anti - angiogenic drugs , such as thalidomide and bevacizumab ( anti - vegf antibody ) , are effective in treating gastrointestinal bleedings and liver avms , respectively 133 - 135 . endoglin can be shed from the cell surface by mmp14 and this result in a soluble form of endoglin 51 . excessive production of soleng by trophoblasts in placenta has been reported in patients affected by pre - eclampsia 52 , whether this is caused by excessive mmp14 activity remains to be shown . pre - eclampsia is characterized by ec dysfunction resulting in vasoconstriction , endo - organ ischemia and increased vascular permeability which leads to a raise in blood pressure , is a major cause of maternal , fetal , and neonatal mortality 52 . recent studies have shown that soleng can bind to bmp9 and bmp10 and inhibit their signaling 50 . although the exact mechanism is not known these results suggest that sol - endoglin may be used in anti - angiogenic therapeutic interventions . marfan syndrome ( mfs ) is a genetic disorder characterized by defects in the cardiovascular system and in aorta and heart valves due to increased tgf signaling activity . mfs is caused by mutations in the fibrillin gene 136 , 137 and affects the skeletal system , ocular system , cardiovascular system , pulmonary system as well as other organs . interestingly it was shown recently that mutations in trii associated with mfs2 and lds have differential effects on tgf-induced smad and erk signaling . it was suggested that the balance between tgf-induced smad and erk might be an important determinant of phenotypic severity in disorders related to different mutations in trii 139 . in a mouse model of mfs inhibition of tgf signaling by a tgf neutralizing antibody or the angiotensin ii type 1 receptor inhibitor losartan resulted in decreased aortic root dilatation , elastic fiber degeneration , smad2 activation , and reversal of skeletal myopathy 140 - 142 . these results further confirmed the contribution of excess tgf signaling to the vascular pathology of mfs . although the mechanism by which losartan protects against aortic aneurysm and reduces smad2 activation is not fully understood clinical studies have been initiated and losartan is now used in clinical trials with mfs 143 . recent observations demonstrated an essential role for the tgf non - smad signaling in aneurysm pathogenesis in mfs mice . it was demonstrated that tgf-induced erk1/2 activation contributes to aortic aneurysm progression and that antagonism of this pathway may be therapeutically useful 144 . the loeys - dietz ( lds ) syndrome is an aortic - aneurysm syndrome characterized by cardiovascular manifestations caused by inactivating mutations in the tgf type i or ii receptors 145 , 146 . although the molecular mechanism of lds is still poorly understood , lds is most likely related to tgf signaling defects . interestingly the aortae of patients with lds , who carry heterozygous inactivating mutations in either trii or tri display increased tgf-smad signaling and fibrosis 146 . recently , mutations in smad3 have been linked with a syndrome characterised by aneurysms , dissections and tortuosity throughout the arterial tree in association with mild craniofacial features and skeletal and cutaneous anomalies 35 . these findings implicate the tgf signaling pathway as a putative pharmacological target for the development of new treatment strategies for arterial wall anomalies and osteoarthritis . as discussed earlier tgf signaling is critical for vascular tone regulation and its perturbation leads to impaired blood flow . hypertension is a cardiovascular risk factor which contributes to the development of organ damage such as renal sclerosis , stroke , and coronary heart disease . recent studies have suggested that there is a link between increased levels of circulating tgf and hypertension 147 , 148 . it has been shown that emilin , an extracellular matrix protein , regulates tgf availability and that it is involved in the regulation of arterial diameter . genetic studies have shown that disruption of emilin-1 expression results in increased conversion of pro - tgf to the mature form and as a result increased tgf signaling 149 . this leads to a reduction in arterial lumen diameter with a resultant increase in vascular resistance and hypertension . it has been postulated that this is due to increased tgf signaling which leads to cytostasis of vsmcs and decreased arterial wall expansion . on the other hand , reduced arterial diameter and hypertension may be a secondary consequence of vascular remodelling in mice . this phenotype could be rescued genetically by disruption of one the tgf alleles and reduction of tgf levels 149 . interestingly , missregulation and reduction of tgf signaling in ecs , due to increased levels of soleng , results also in increased blood pressure in pre - eclampsia 52 . ipah is a disease characterized by constriction of the pre - capillary pulmonary arteries , which results in sustained pulmonary arterial pressure . pah is characterized by excessive proliferation of ecs and vsmcs 152 , 153 , increased vascular inflammation , increased vasoconstriction and reduced dilation . genetic studies in human revealed that loss of function mutations in bmp type ii receptor ( bmprii ) were linked with heritable pulmonary arterial hypertension ( hpah ) 26 , 154 - 157 . bmprii expression by ecs and vsmcs is significantly reduced not only in hpah cases with a bmprii mutation , but also in patients with secondary pah 158 - 160 . mice expressing a bmpr - ii mutant lacking c - terminal tail in the pulmonary smcs develop pulmonary vascular lesions similar to pah . interestingly it was shown that disruption of bmprii in pasmcs leads to reduced bmp2 and bmp4 signaling , while signaling by bmp6 and bmp7 is enhanced 161 . furthermore , it was shown that bmp4 is upregulated in hypoxia - induced pah and that heterozygous bmp4/lacz mice are protected from the development of hypoxia - induced pah , vsmc proliferation , and vascular remodelling . hypoxia induced pmvec to secrete bmp4 and promoted proliferation and migration of vsmcs in a bmp4-dependent manner 95 . in addition reduced bmp / smad signaling resulted in activation of the p38 mapk pathway which led in aberrant pasmc proliferation 99 , 162 , 163 . recently mutations in the downstream mediator smad8 , which is highly expressed in the pulmonary vasculature , were identified in patients with hpah 164 . this is surprising , since loss of smad8 function in canonical bmp signaling is largely compensated by smad1/5 . in the same study it was shown that smad8 mutations lead to decreased mir-21 levels and that mir-21 overexpression results in anti - proliferative effects in paec and pasmc . loss of smad8 function in mice results in abnormal vascular remodeling , increased vascular inflammation and pulmonary tumors 46 . interestingly , certain hht2 patients develop pah - like syndromes , suggesting that alk1 mutations can also be involved in pah 166 , 167 . abnormal tgf- signaling has been observed in the pulmonary vasculature of pah patients 96 , 168 . although tgf induced both smad2 and smad1/5 activation , only smad2 was shown to be important in tgf-induced apoptosis of pmvec . thus , imbalance of alk1 and alk5 may contribute to the development of pulmonary artery hypertension 169 . other studies provided evidence that increased tgf signaling participates in the pathogenesis of experimental pah . the development of monocrotaline - induced - pah was associated with increased vascular cell apoptosis and increased tgf signaling as documented by phosphorylated smad2 expression . inhibition of tgf signaling significantly attenuated the development of the pah and reduced pulmonary vascular remodeling 170 . although it is recognized that pah is characterized by increased number of smcs , this may not be only due to increased proliferation of smcs . it has been suggested that ecs may transdifferentiate into mesenchymal - like cells expressing sma , resulting thus in increased numbers of smc - like cells in vascular pathologies . this hypothesis is supported by the demonstration that transdifferentiation of pulmonary arteriolar ecs into smc - like cells occurs during hypoxia - induced pulmonary vascular remodeling and is regulated by myocardin 171 . this process during which ecs lose endothelial features , and acquire mesenchymal - like characteristics has been termed endothelial - to - mesenchymal transition ( endomt ) . for example , it was shown that endomt plays an important role in aortic and pulmonary artery development 172 , 173 . future studies will shed more light into the role of endomt in the pathology of pah and other vascular diseases . atherosclerosis is a chronic inflammatory disease of the arterial wall leading to regional ischemia and ischemic complications such as myocardial infarction and stroke , which greatly contribute to morbidity and mortality . atherosclerosis is characterised by immunological mechanisms leading to vascular inflammation and plaque formation 174 ( fig . tgf ligands , its receptors as well as smad proteins have been found to be expressed in fibro - fatty lesions and fibrous plaques 175 - 177 . consistent with such an expression profile , tgf has been found to affect the properties and function of all the cell types that are known to be present in atherosclerotic lesions such as ecs , smcs , monocytes / macrophages , platelets , t cells and myofibroblasts . it has been suggested originally that tgf exerts anti - atherogenic effects 178 thus having a protective role either by regulating vascular cell proliferation , plaque stability , immune system function or by its anti - inflammatory function 179 , 180 . interestingly , it was shown that there is an inverse relationship between serum tgf1 levels and the development of atherosclerosis and that concentration of active tgf1 is severely depressed in advanced atherosclerosis 181 - 183 . in addition , mutations of trii have been associated with atherosclerotic and restenotic vascular cells 184 . in contrast to the atheroprotective effects of tgf several studies have shown that atherogenic stimuli such as shear stress 185 , oxidized cholesterol 186 , angiotensin ii 187 or growth factors such as pdgf result in increased tgf expression 188 . at the same time increased tgf1 expression was found in atherosclerotic clinical specimens and increased levels of tgf1 were correlated with advanced atherosclerosis 189 , 190 ( fig . endoglin expression was shown to be upregulated in vsmc in human atherosclerotic plaques further supporting a role for endoglin in the maintenance of vascular integrity and in the response of the vessel wall to injury 89 , 191 . in addition it was shown that soleng levels are significantly higher in diabetic patients and in patients with hypertension and diabetes 192 . higher soleng levels were found in patients with diabetes who had retinopathy , in patients with diabetes who had a high probability of 10-year cardiovascular risk , and in patients with diabetes and hypertension who had three or more damaged target organs than in those with no organs affected 192 . changes in soleng plasma levels have been reported in other pathologies such as atherosclerosis and coronary artery disease 193 . also , changes of soleng plasma levels after an acute myocardial infarct are accurate predictors of acute mortality in these patients 194 . several studies suggested that bmps play also an important role in atherosclerosis by modulating ec inflammation and cell differentiation . it was shown that bmp4 is increased in response to the high fat diet and bmp2 and -4 have been implicated in proinflammatory effects in the endothelium 195 - 197 . furthermore , interference with the bmp pathway leads to reduced , while enhanced bmp activity results in increased atherosclerotic lesion formation in apolipoprotein ( apo)e knockout mice 195 - 197 . it was suggested that this might be due to the effects of bmp signaling on ec function . bmp2 and bmp4 have been implicated as mediators of endothelial inflammation in response to shear stress , oxidative stress and pro - inflammatory cytokines 198 , 199 . whether these effects are due to effects on mononuclear cell function is not known ( fig . although initially it was considered that vascular calcification is a passive process of accumulation of dying cells , later studies suggested that vascular calcification is a highly regulated process . several studies have provided evidence that vascular calcification is the result of an osteogenic process , which leads to mineralisation of the ecm and cell differentiation towards osteoblast - like lineages . it has recently become appreciated that under certain pathologic contexts and culture conditions , vsmcs can differentiate to acquire an osteogenic phenotype , and express lineage markers consistent with bone - forming cells as well as calcification - regulating proteins commonly found in bone 200 - 202 . considerable evidence suggests that tgf family members , particularly the bmps , are important modulators of vascular calcification 203 , 204 , probably by inducing ecm production that is amenable to mineralization and by favoring differentiation toward osteoblast - like lineages ( fig . is expressed in calcified human atherosclerotic plaques it was shown that bmps can direct osteogenic programming of vascular mesenchymal progenitors of the pericyte lineage 205 . later it was shown that bmp signaling as well as a variety of inflammatory stimuli can promote expression of osteoblast lineage markers such as alkaline phosphatase in cultured vsmcs 200 , 201 , 206 - 209 . treatment of smc with bmp2 enhanced vascular calcification via increased phosphate uptake and induction of osteogenic phenotype modulation in smc 210 . moreover , bmps may induce vsmc to express proteins , such as osteoprotegerin which may confer protection from vascular calcific and atheromatous disease 211 . recently , it was shown that inhibition of bmp signaling in apoe/ mice by transgene expression of mgp resulted in diminished smad1/5/8 signaling , as well as reduced inflammation , lesion formation , and calcification after fat feeding 197 . on the other hand mgp deficient apoe/ mice displayed enhanced bmp2 was shown to be expressed in adventitial myofibroblasts in the aorta of fat - fed diabetic low - density lipoprotein receptor ( ldlr)/ mice and to induce an osteogenic phenotype in a subset of the myofibroblasts by inducing msx2 expression , which is associated with bone formation 212 . in diabetic rats interestingly , transgenic expression of mgp resulted in reduced bmp activity and diminished calcification in the entire media 213 . mgp , noggin and chordin are downregulated in dedifferentiated vsmcs , which would make them more susceptible to bmp action 195 . thus , the balance between endothelial bmps and their inhibitors may determine the health of the endothelium , and an efficient limitation of endothelial bmp activity may confer protection against atherosclerosis . tgf is present in calcified aortic valves and regulates vascular calcification and osteoblastic differentiation of vsmcs 214 , 215 . it has been proposed that during lesion formation enhanced bmp signaling may promote developmental conditions and trigger the differentiation of multipotential susceptible cells into the osteogenic lineage . it has been suggested that several cell populations might enter into an osteocondrogenic lineage reprogramming , however , the relative contribution of the respective cell population to local osteogenesis and vascular calcification in different arteriosclerotic disease states remains to be established . myofibroblasts present in the adventitia of diabetic and hyperlipidemic ldlr/ mice 212 may also acquire an osteogenic phenotype . pericytes have recently emerged as multipotent adult mesenchymal stem cells and it was shown that they have osteogenic potential 217 . bmps can direct osteogenic programming of vascular mesenchymal progenitors of the pericyte lineage ; however , the relative contribution of pericytes to osteogenic vascular calcification has yet to be clarified . it was proposed that vsmc can differentiate and acquire an osteogenic phenotype and this may directly contribute to medial calcific vascular disease . vsmc transdifferentiation was also shown to contribute to the vascular endochondral ossification mechanisms in mice 218 . it has been suggested recently that vascular ecs might enter into an osteocondrogenic lineage reprogramming 219 . several studies have suggested that ec plasticity allows ec to transdifferentiate into messenchymal cells during embryonic development 220 or as a response to chronic inflammation 221 , 222 . it was demonstrated that expression of a constitutively active form of the alk2 type i receptor in mice causes endomt ; ec cells acquire a stem cell - like phenotype which enables them to undergo osteogenic differentiation 219 . patients with fibrodysplasia ossificans progressiva are characterized by heterotopic calcification , caused by mutations in the alk2 gene . in addition , treatment of huvecs and human cutaneous microvascular ecs ( hcmecs ) with either tgf2 or bmp - resulted in induction of endomt , whereas bmp7 and vegf blocked it 219 . tgf has also been reported to induce endomt in ecs in vitro 223 - 226 . tgf treatment resulted in decreased expression of endothelial markers and while inducing mesenchymal marker expression as well as cytoskeletal rearrangements , which promoted cells to acquire a contractile phenotype 225 . it is well appreciated that members of the tgf family play a crucial role in the regulation of both normal and abnormal vascular function . tgf ligands regulate angiogenesis through their actions either on ecs and/or on mural cells , affecting e.g. , proliferation , migration and most importantly cell differentiation , underlying the important role of tgf signals in angiogenesis . tgf signaling regulates also ec / smc interactions and by this way further control vascular function . missregulation of tgf signaling , both in human and mice , leads to vascular dysfunction and vascular pathologies . recently , it has become evident that tgf signaling acts as a rheostat rather than an on - off switch to regulate vascular function . ligand concentration , cell density , cell type , media and culture conditions may determine specific cellular responses to tgf family members . in addition the effects of tgf signals depend on the interplay between different members of the tgf family . due to its role in cardiovascular disease pathophysiology , tgf signaling components have been considered as potent therapeutic targets in a wide spectrum of cardiovascular disorders . several classes of inhibitors targeting ligand - receptor interactions have been developed to interfere with the tgf pathway . such inhibitors include tgf neutralizing antibodies , anti - receptor antibodies , receptor ectodomain proteins that act as ligand traps . in addition small chemical inhibitors have been developed which interfere with the kinase activity of type i tgf receptor . in a mouse model of mfs it was shown that systemic delivery of pan - tgf neutralizing antibodies prevents aortic aneurysm , developmental emphysema and skeletal myopathy 138 , 141 , 227 . administration of losartan had similar effects by inhibiting tgf ligand expression , receptors , and activators such as thrombospondin and mmps 227 . however , the same anti - tgf neutralizing antibody treatment induced lethal cardiovascular pathologies in mouse models of aneurysm and atherosclerosis 228 , 229 . in addition , preclinical observations suggested that angiotensin ii type i - receptor blockade results in decreased tgf signaling . this was translated into clinical studies and the effects of angiotensin ii type i inhibitors are being studied in mfs patients 140 , 143 . based on the observations that the pathology of pah results from a misbalance in bmp and tgf signaling pathways , targeting of these pathways has been considered as a potential therapeutic intervention for the treatment of pah . inhibition of tgf signaling as a potential treatment of pah is under investigation 230 , 231 . interestingly , administration of iloprost also restores deficiency in smad1/smad5 phosphorylation and id1 levels in bmprii deficient pasmcs , while prostacyclin analogues are currently used for treatment of pah in clinic since they restore id1 expression in pasmcs 131 . both tgf and bmp signaling several therapeutic approaches to interfere with bmp signaling such as recombinant ligands , antagonists , soluble receptors and neutralizing antibodies have been exploited 232 , 233 . recently dorsomorphin , a bmp receptor antagonist 233 , has been succesfully used for inhibition of bmp signaling . in addition , identification of novel molecules , such as ldn-193189 with enhanced specificity to antagonize bmp receptor activity 234 offer new therapeutic possibilities . in conclusion , due to its multifunctional and context dependent function interference with tgf-dependent processes must be carefully designed and the effects of tgf signaling or its inhibition on the different cell types involved must be carefully studied . thus , development of new methods through high throughput screening techniques will result in the identification of new generation molecules targeting tgf and bmp signaling . in this regard , targeting the downstream regulators smads might be a more selective diminution of the tgf signaling .

transforming growth factor ( tgf ) family members are involved in a wide range of diverse functions and play key roles in embryogenesis , development and tissue homeostasis . perturbation of tgf signaling may lead to vascular and other diseases . in vitro studies have provided evidence that tgf family members have a wide range of diverse effects on vascular cells , which are highly dependent on cellular context . consistent with these observations genetic studies in mice and humans showed that tgf family members have ambiguous effects on the function of the cardiovascular system . in this review we discuss the recent advances on tgf signaling in ( cardio)vascular diseases , and describe the value of tgf signaling as both a disease marker and therapeutic target for ( cardio)vascular diseases .

overactive bladder ( oab ) is a symptom complex comprising lower urinary tract symptoms of urinary urgency , with or without urgency urinary incontinence , usually with frequency and nocturia , and no proven infection or other obvious pathology.1 symptoms of oab are experienced by 11% of the worldwide population over the age of 20 years . this number is expected to increase , in line with population aging , by 20% to 546 million by 2018.2 the prevalence of oab increases with advancing age . the epic study found an increase in the prevalence of oab from 8% in men and 9% in women under 30 years to 15% and 16% , respectively , in those aged 6569 years , and a further increase to 21% and 22% , respectively , in older community - dwelling adults aged 70 years and over.3 available evidence from longitudinal studies suggests that oab symptoms appear to progress in terms of both prevalence and severity in men and women . in men interviewed in 1992 and again in 2003 , the proportion with oab increased from 15.6% to 44.4% and the proportion with urgency incontinence increased from 1.9% to 7.4%.4 a similar pattern was observed in women over a 16-year period , with a marked overall increase in the prevalence of urge incontinence from 17% to 26%.5 oab has a proven negative impact on health - related quality of life , and affected people score significantly worse than their age - matched counterparts without oab in the domains of physical and social functioning.6,7 in addition to having an effect on occupational , physical , psychological , and social well - being , the economic cost attributable to managing the condition is significant . oab sufferers in the us visit their physicians more often and have more urinary tract infections than those without the condition , and in 2007 , the average annual costs of oab were estimated at $ 1,925 per person ( $ 1,433 in direct medical costs , $ 66 in direct nonmedical costs , and $ 426 in indirect costs ) . applying these costs to the 34 million people with reported oab in the us , results in total national costs of $ 65.9 billion , and by 2020 this is predicted to rise to $ 82.6 billion.8,9 however , not everyone with symptoms will , according to the results of a uk study , want to seek health care for their symptoms , and these cost projections may reflect the worst case scenario.10 lifestyle changes and conservative therapies ( fluids , caffeine , obesity , bladder retraining , pelvic floor muscle exercises ) are the starting point for treatment of oab . should these measures prove ineffective , antimuscarinics currently remain the mainstay of pharmacological treatment for oab.11 however , antimuscarinic therapy is not always effective , and adherence in the community is low , with many people stopping their medication because of either adverse events or inefficacy . the agents available also vary in their propensity to be associated with adverse events , and this in turn has an impact upon perceived efficacy.1214 challenges in the treatment of oab comprise medication - related factors such as dosing frequency , dose flexibility , mode of administration , adverse events , and inadequate symptom control , which may require switching of medications to best suit the individual . in addition , there are patient - related factors , including unrealistic expectations of treatment , comorbidity , and issues related to taking regular medication . these factors result in poor adherence and intolerance.15,16 likewise , older people affected by oab constitute , if not a poorly managed group , then a difficult - to - treat sector of patients who often receive less optimal management than their younger counterparts . finding a well - tolerated treatment in these patients , who often have concomitant comorbidity and take multiple medications , is difficult . finally , local factors such as regulations concerning reimbursement and formulary inclusion may contribute to what might be perceived as poor management when regulatory opinion differs from that of clinical experts . this review considers the evidence for use of fesoterodine in patients who have responded suboptimally to other antimuscarinics , the evidence in favor of a switch from tolterodine to fesoterodine , and the evidence regarding the clinical efficacy and safety of fesoterodine in older people . it is a pro - drug , and is rapidly hydrolyzed by nonspecific esterases to 5-hydroxymethyltolterodine , the main active metabolite of tolterodine . this results in a more predictable pharmacokinetic profile then when tolterodine is given.17 there have been a number of phase iii trials comparing the clinical efficacy of fesoterodine and placebo.1821 two trials have compared fesoterodine with both tolterodine and placebo.20,21 in these studies , only chapple et al20 reported on previously treated patients ; here , an average of 41% of patients across the trial had previously been exposed to antimuscarinic agents for oab . there is no report on the proportion of patients who had discontinued their medication because of either inefficacy or dissatisfaction with treatment . the effect on disease - related variables in these trials is shown in table 1 . in each study , increasing the dose of fesoterodine from 4 mg to 8 mg was associated with a statistically significant increase in mean voided volume and an increase in number of continent days per week versus placebo . this response has not been consistently shown for other antimuscarinic agents at variable doses , including tolterodine , solifenacin , and darifenacin.2224 fesoterodine dosing , and its rapid conversion by gut esterases to 5-hydroxymethyltolterodine , appears to be associated with a more predictable dose - response relationship . in clinical practice , this should facilitate determination of the most effective dose for the patient , whilst attempting to curtail side effects . the dearth of direct comparisons between the newer antimuscarinics continues to hamper our ability to make informed decisions about the suitability of individual drugs . fesoterodine 8 mg was compared with tolterodine 4 mg in studies attempting to show the added benefit of a higher dose of medication , and in some , after a suboptimal response to tolterodine 4 mg.25,26 compared with tolterodine 4 mg extended - release , 8 mg fesoterodine showed significantly greater efficacy in reducing episodes of urgency urinary incontinence ( primary endpoint ) , and in increasing bladder capacity , as measured by mean volume voided . in addition , the diary dry rate at week 12 was also significantly greater in patients receiving fesoterodine 8 mg than in those receiving tolterodine 4 mg extended - release . in self - reported patient assessments of bladder - related problems , ie , urgency , symptom bother , and health - related quality of life , the statistically significant superiority of fesoterodine 8 mg over tolterodine 4 mg extended - release was observed as early as 3 weeks after escalation from fesoterodine 4 mg . this may encourage adherence with treatment , because those with perceived benefit from treatment are more likely to adhere to therapy , particularly older patients.12 the effects of flexible - dose fesoterodine in subjects with oab who were dissatisfied with previous tolterodine treatment may give some limited insight into its use in poorly managed patients . a 12-week , open - label , flexible - dose study was conducted in 516 adults with oab ( eight or more micturitions and three or more urgency episodes per 24 hours ) who had been treated with tolterodine ( immediate - release or extended - release ) within 2 years of screening and reported being somewhat dissatisfied or very dissatisfied with tolterodine on the treatment satisfaction question , a single item from the validated overactive bladder satisfaction questionnaire ( oab - q).27 eleven of the subjects included in this study did not report being dissatisfied with prior tolterodine treatment at the beginning of the study , but inclusion of their data did not alter the conclusions drawn from the study . in addition to prior tolterodine treatment , 216 subjects ( 42% ) had received at least one other antimuscarinic agent prior to enrollment in the study . fifty percent of patients opted to increase their dose of fesoterodine from 4 mg to 8 mg at week 4 . treatment with fesoterodine was associated with statistically significant within - group differences from baseline to week 12 with regard to micturitions ( 12.79.7 ) , urgency urinary incontinence episodes , ( 2.30.6 ) , urgency episodes per 24 hours ( 10.05.0 ) , and severe urgency episodes per 24 hours ( 5.01.5 ) . there were also statistically significant improvements in treatment satisfaction , with 1.0% of patients reporting that they were very satisfied or somewhat satisfied with their previous treatment and 80.2% reporting this level of satisfaction with fesoterodine . in terms of patient perception of bladder condition score following 12 weeks of treatment with fesoterodine , 63.4% reported that they had minor problems or fewer problems compared with pretreatment , and no subjects reported fewer than moderate problems . the proportion of subjects who reported that they were usually not able to hold their urine was reduced from 25% at baseline to 6% after 12 weeks , and the proportion who reported being able to finish what they were doing before going to the toilet was increased from 6.8% at baseline to 41% after 12 weeks . the lack of blinding and absence of a placebo arm limit the conclusions that can be drawn from this study ; however , unlike objective variables , the study may more closely reflect real clinical practice where outcomes are judged on patient perception and symptom bother.28 a further perspective on difficult - to - treat patients may be gleaned from the after29 ( fesoterodine after tolterodine er ) study , a pfizer - sponsored trial designed to determine the efficacy and safety of fesoterodine 8 mg versus placebo in patients with oab and urgency urinary incontinence who have responded suboptimally to tolterodine 4 mg extended - release . this was a 12-week , randomized , double - blind , placebo - controlled , multicenter study for which patients with a suboptimal response to tolterodine were identified by a 2-week , open - label treatment period consisting of daily treatment with tolterodine 4 mg extended - release . at the end of this 2-week period , those with a 50% reduction in mean urgency urinary incontinence episodes per 24 hours were randomized 1:1 to fesoterodine ( 4 mg for 1 week , 8 mg for weeks 212 ) or placebo once daily . the primary efficacy endpoint was the mean change from baseline to week 12 in urgency urinary incontinence episodes per 24 hours . missing data were imputed using the last observation carried forward technique , and there was a preplanned stepwise analysis whereby a one - sided paired t - test was used to assess the within - group mean reduction from baseline to week 12 in urgency urinary incontinence episodes for fesoterodine 8 mg , and if this was significant , a one - sided test of the between - group ( placebo versus fesoterodine ) change in mean number of urgency urinary incontinence episodes per 24 hours was then conducted using an analysis of covariance model . there were statistically significant changes ( p<0.0001 for each ) from baseline for both placebo ( 1.9 ) and fesoterodine ( 2.4 ) , allowing an analysis of between - group change . those treated with fesoterodine achieved a significantly greater reduction in urgency urinary incontinence episodes per 24 hours than the placebo - treated group ( p=0.0079 ) . fesoterodine was also associated with a statistically significant improvement over placebo in urgency episodes per 24 hours ( placebo 2.8 versus fesoterodine 3.5 , p=0.0438 ) . there was no statistically significant reduction in micturitions per 24 hours ( placebo 1.6 versus fesoterodine 2.0 , p=0.0931 ) . in terms of symptom relief , 69% of fesoterodine - treated patients versus 57% of placebo - treated patients ( p=0.0027 ) achieved a 50% reduction in urgency urinary incontinence ; 44.1% of placebo - treated patients and 58.9% of fesoterodine - treated patients achieved a 70% reduction in this symptom ( p=0.001 ) . complete resolution of incontinence was achieved in 32.3% of placebo - treated patients versus 39.0% of fesoterodine - treated patients ; this difference was not statistically significant . however , significant improvement was seen on the patient perception of bladder condition questionnaire ( p<0.0001 ) and urgency perception scale ( p=0.0095 ) for fesoterodine over placebo , for all domains of the oab - q30 ( p<0.05 ) , and for symptom bother ( placebo 18.1 versus fesoterodine 25.5 , p<0.001 ) . as might be expected , the most commonly reported adverse events were dry mouth ( placebo 4.0% versus fesoterodine 16.6% ) and constipation ( placebo 1.3% versus fesoterodine 3.9% ) . one of the main criticisms of this study concerns the limited exposure to tolterodine before ascribing a suboptimal response . although improvements are noted early on with antimuscarinic treatment , in most studies the plateau effect is not achieved for approximately 12 weeks . to what extent the transfer to fesoterodine was truly indicated in this group is unknown . in a similar vein , but with a markedly different trial design , a post hoc analysis of a retrospective , cross - sectional , observational study ( impacta [ assessment of reasons for overactive bladder treatment change])31 reported on factors contributing to treatment change in oab patients and the degree of satisfaction resulting from that change . this study used the validated treatment benefit scale32 and the clinical global impression of improvement . of the 2,038 patients evaluated , 1,407 had been treated previously with antimuscarinic therapy . in this study , 842 patients had previously received tolterodine , 748 of whom had data available for analysis . older patients were defined as those aged 65 years or older , and comprised 44.1% of the patient population . in the entire group , 69% of those younger than 65 years and the majority of those switching from tolterodine to fesoterodine did so because of lack of effectiveness ( 66.8% ) , although the details of this are not specified , and the duration of tolterodine treatment prior to the switch is not reported . adherence was generally low over the 6068 days of treatment ; 33.5% with fesoterodine 8 mg versus 24.9% with fesoterodine 4 mg . overall , 94.4% of patients improved , according to the judgment of the physician , which was in close agreement with the 94.2% of patients who showed improvement on the treatment benefit scale . a significantly higher proportion of patients were either assessed as having improved ( clinical global impression of improvement ) or reported improvement on the treatment benefit scale following treatment with fesoterodine 8 mg compared with the 4 mg dose , as might be expected . however , the adverse event rate was not reported for either dose of fesoterodine , with only adverse events precipitating a switch to fesoterodine being reported . the low adherence rate may give some guide to this , but would be confounded by other factors affecting adherence . there was no age differential in terms of the proportion of those who either reported improvement or were assessed as having improved . the effect of a switch from tolterodine to fesoterodine on patient - reported outcomes has recently been reported in a pooled analysis of two 12-week , double - blind , placebo - controlled , clinical trials.33 at week 12 , the analysis showed statistically significant improvements associated with fesoterodine 8 mg over tolterodine 4 mg and placebo for changes in the patient perception of bladder condition , urgency perception scale , all domains of the oab - q , and all bladder diary variables , except for nocturnal micturition frequency in women . in men , statistically significant improvements over tolterodine were limited to severe urgency episodes and to the symptom bother domain of the oab - q . the proportion of men and women who had previously been treated with antimuscarinic agents for oab was not reported in this study . the efficacy and safety of flexible - dose fesoterodine in older adults with oab has been reported in both the randomized , placebo - controlled and open - label follow - up portions of sofia ( the study of fesoterodine in an aging population).34,35 in this study , 67% of the fesoterodine - treated group ( n=392 ) had received antimuscarinic treatment for oab prior to entry into the trial ; there was no report of reasons for discontinuation , and it is likely that a proportion of patients would have stopped their previous treatment for the sole purpose of trial entry . however , both portions of the trial reported benefit in fesoterodine - treated patients , that reached statistical significance for the primary outcome , ie , urinary urgency episodes per 24 hours , in the placebo - controlled portion of the trial and the majority of secondary outcomes . there was no statistically significant difference between placebo and fesoterodine with regard to improved resolution of urgency urinary incontinence rates , which was probably due to a low level of urgency urinary incontinence at trial entry . in sofia , 47% of the patients were men , allowing a glimpse into the effect of fesoterodine in oab with benign prostatic enlargement , from which 41% of the men suffered and for which 28% had pre - existing treatment . there were low discontinuation rates due to urinary retention ; in the double - blind phase , six patients reported urinary retention ( three men and two women receiving fesoterodine , and one man receiving placebo ) . four of the six participants reporting urinary retention required catheterization ( three men receiving fesoterodine and one man receiving placebo ) . five of the six participants reporting urinary retention discontinued , including all four participants requiring catheterization . in the extension portion of the study , three men in the double - blind placebo / open - label fesoterodine group discontinued due to urinary retention . adverse effects of dry mouth and constipation in sofia were comparable with those reported for other antimuscarinics , although there was a higher discontinuation rate in the fesoterodine - treated and placebo - treated arms of the trial in the over 75-year age group compared with younger groups in other trials with fesoterodine . taking things a little further , into the realms of the physically frailer elderly , the recently reported vulnerable elders study36 utilized the vulnerable elders scale-13 to select older people scoring > 3 on this scale who had a high level of comorbidity , defining an increased risk of decline and death as well as oab , who were then randomized to a 12-week , controlled trial of fesoterodine versus placebo . patients had an option to increase their dose from 4 mg to 8 mg at 4 weeks with no opportunity to decrease the dose after that point . the mean number of comorbid conditions was approximately 8.5 ( range 127 ) , and 27% of patients had more than eleven concomitant medications at baseline . here , the primary outcome was urgency incontinence episodes and included patients who had to have a mean of more than two but fewer than 15 urgency urinary incontinence episodes on a 3-day bladder diary at baseline to be eligible for the study . reduction in urgency urinary incontinence episodes per 24 hours from baseline to week 12 was significantly greater with fesoterodine ( 2.84 ) than with placebo ( 2.2 , p=0.002 ) . likewise , there were statistically significant improvements in diary dry rate , nocturnal urgency , micturitions per 24 hours , and pad use associated with fesoterodine . patient - reported outcomes also improved , with statistically significant improvements over placebo - reported outcomes with regard to patient perception of bladder control score and the oab - q symptom bother , health - related quality of life , and coping and concern scores at week 12 . twenty - six ( 9.3% ) patients in the fesoterodine group and 14 ( 5.0% ) in the placebo group discontinued treatment because of adverse events . the total discontinuation rate was 19.6% in the fesoterodine group and 21.7% in the placebo group . there was no significant change in blood pressure or resting heart rate associated with fesoterodine . the use of drugs with anticholinergic properties in elderly patients has been associated with a decline in cognition and , in some , an increased risk of incident dementia.37 of the antimuscarinics used for oab in trials , only oxybutynin has been shown to be associated with a negative impact on cognition.38 concerns regarding this potential adverse impact have led pharmaceutical manufacturers to undertake studies specifically to examine cognitive function in older people exposed to such agents . in the case of fesoterodine , its effect on cognition has been examined in a randomized crossover study that ensured its subjects were exposed to chronic dosing of the drug at both 4 mg and 8 mg and used alprazolam as an active control . the trial reported on 18 cognitively intact older adults ( mean age 72 years ) and found no detectable impairment of cognition when assessing executive function , verbal and visual learning , or memory and reaction time with either fesoterodine 4 mg or 8 mg as compared with placebo . in contrast , alprazolam caused a significant reduction in performance on each test.39 although a short - term study , and conducted in cognitively intact elderly , some reassurance regarding lack of a significant cognitive effect can be taken from the low level of cognitive side effects reported in sofia ( three fesoterodine - treated patients , two of whom were thought to be unrelated to treatment ) and in the vulnerable elders study ( two fesoterodine - treated patients ) , with no overall change in mini - mental state examination40 scores over the duration of the study . whilst there is limited direct evidence of the efficacy of fesoterodine in patients with a well - defined suboptimal response to other antimuscarinic agents , fesoterodine appears to fulfill a role in the treatment of those who may not reach optimal efficacy with tolterodine . there are no stratified analyses comparing the efficacy of fesoterodine in patients who have previously received antimuscarinic treatment , and thus we are unable to draw any specific conclusions in this group of patients . in terms of groups that may be at risk of poorly managed oab , such as the elderly and medically complex patients , fesoterodine has an impressive portfolio of prospectively planned trials supporting its efficacy and safety . additionally , treatment with fesoterodine in these circumstances

overactive bladder ( oab ) , a clinically defined symptom complex comprising urinary urgency , usually accompanied by urinary frequency and nocturia , with or without urgency incontinence , is common and has a markedly negative impact on the sufferer s quality of life . following conservative and lifestyle management , the current pharmacological mainstay of treatment is antimuscarinic therapy . this review explores the role of fesoterodine , a relatively recently introduced antimuscarinic agent , in the treatment of patients who may have had a suboptimal response to initial therapy , who have switched treatment from tolterodine , or may be at risk of receiving poor treatment because of either multimorbidity or complex polypharmacy .

dental trauma is the most frequent cause of pulp necrosis in immature anterior permanent teeth . deep proximal fractures , especially if left untreated , can jeopardize vitality of the tooth . the walls of the root canals are frequently divergent , and the apices immature , making debridement and obturation difficult . calcium hydroxide is the material most commonly used for induction of the apical barrier formation in non - vital teeth.[24 ] the long term usage of calcium hydroxide could alter the collagen matrix of dentin due to its high alkalinity.[59 ] this would leave the dentin structure with reduced organic support , which may adversely influence the mechanical properties of dentin(i.e . , flexural strength ) , thus making teeth more susceptible to fracture from secondary injuries.[810 ] the newer adhesive materials can significantly increase the resistance to fracture of these weakened roots to the levels of intact teeth.[1020 ] this study compares the reinforcing effects of resin modified glassionomer cement ( rmgic ) , flowable compomer , and flowable composite on the root canal walls of simulated immature central incisor teeth treated with calcium hydroxide for varying periods of time . human permanent maxillary central incisor teeth extracted due to periodontal disease without signs of caries , obvious defects , or attrition were selected and stored in distilled water until further use . the mesiodistal ( md ) and buccolingual ( bl ) diameters of the specimens were determined at the crown limit of the root using a caliper . the mean values obtained were 6.02 mm for md and 6.64 mm for bl dimensions . samples presenting a difference of 20% from the mean were discarded , leaving a total of 160 central incisor teeth . the crowns of these specimens were removed to obtain a standard root length of 13 mm [ figure 2 ] . root canals were enlarged to a 120 file size at a working length of 12 mm . a thick mixture of pure calcium hydroxide powder mixed with distilled water was condensed into the root canals using plastic amalgam condenser and pluggers till the coronal limit of the root , and then the canals were sealed with restorative glassionomer cement ( gic ) at the coronal end . the apical ends of the specimen were sealed with intermediate restorative material ( irm ) . these specimens were thermo cycled using a regime of 250 cycles from 6c to 60c with a dwell time of 30 seconds and stored in a humidator at 37c . forty specimens were randomly selected from the lot at the end of 15 days and calcium hydroxide was cleaned off the root canals of the specimens using gentle instrumentation and saline irrigation , and these root specimens were obturated with gutta percha using lateral condensation technique with a zoe sealer and stored in a humidator at 37c until further use . at the end of 7 days , gutta - percha was removed using a no.6 peeso reamer creating 10 mm of post space , leaving 2 mm of apical gutta - percha fill . further grouping of the specimen was done as follows : group i-10 : non - reinforced root specimens served as control.group ii-10 : root specimens reinforced with rmgic [ figure 1 ] . the root canals of the specimens were gently dried using an absorbent point and treated with the primer provided by the manufacturer for 30 seconds , air dried for 15 seconds , and light cured for 20 seconds . vitremer ( 3 m ) powder and vitremer liquid was dispensed according to the manufacturer 's instructions and mixed on a paper pad for 45 seconds . the material was back loaded into delivery tip and the restorative was syringed into the root canal.group iii- 10 : root specimens reinforced with flowable compomer ( prima flow ) preparation of group iii specimensthe root canals of the specimens were gently dried using an absorbent point and treated with the contax primer and bond according to manufacturer 's instructions and light cured for 20 seconds . the canals were filled with flowable compomer ( prima flow ) [ figure 1 ] , which was applied directly from syringe in layers of maximum 2 mm.group iv-10 : root specimens reinforced with flowable composite ( filtek z350 ) [ figure 2 ] . the 10 root specimens were etched with 37% phosphoric acid ( scotch bond multipurpose ) for 1520 seconds , rinsed with sterile water for 20 seconds , and then blot dried . two coats of adper single bond 2 ( 3 m ) bonding agent was applied , gently air dried , and light cured for 20 seconds . the specimens were then filled with flowable composite ( filtek flow z350 ( 3 m ) directly from the syringes . the root canals of the specimens were gently dried using an absorbent point and treated with the primer provided by the manufacturer for 30 seconds , air dried for 15 seconds , and light cured for 20 seconds . vitremer ( 3 m ) powder and vitremer liquid was dispensed according to the manufacturer 's instructions and mixed on a paper pad for 45 seconds . the material was back loaded into delivery tip and the restorative was syringed into the root canal . group iii- 10 : root specimens reinforced with flowable compomer ( prima flow ) preparation of group iii specimens the root canals of the specimens were gently dried using an absorbent point and treated with the contax primer and bond according to manufacturer 's instructions and light cured for 20 seconds . the canals were filled with flowable compomer ( prima flow ) [ figure 1 ] , which was applied directly from syringe in layers of maximum 2 mm . group iv-10 : root specimens reinforced with flowable composite ( filtek z350 ) [ figure 2 ] . the 10 root specimens were etched with 37% phosphoric acid ( scotch bond multipurpose ) for 1520 seconds , rinsed with sterile water for 20 seconds , and then blot dried . two coats of adper single bond 2 ( 3 m ) bonding agent was applied , gently air dried , and light cured for 20 seconds . the specimens were then filled with flowable composite ( filtek flow z350 ( 3 m ) directly from the syringes . restorative materials ; a - rmgic , b - flowable compomer - flowable composite sectioning of tooth at the cervical level to cure the restorative materials , a transilluminating post ( luminex ) was used making sure it reaches the cervical limit of the apical filling [ figure 3 ] . after placing the post inside the canal and removing the excess , the material was light cured according to the manufacturer 's instructions . the root specimens were then re - subjected to thermo cycling regime for 250 cycles at 6c60c , with a dwell time of 30 seconds . process of reinforcement and light curing through the post the roots were embedded in color - coded acrylic blocks using a flat ended rod held vertically by a ney surveyor to position the roots during mounting so that they were parallel to the acrylic base with an exposure of 4 mm of the cervical portion of root above the acrylic . the acrylic was contained in a inch diameter pvc pipe 2 cm in length to standardize the size of the base . a stainless steel cone measuring 1.5 cm at the base and 3 cm in length was placed against the canal so that it was in line with the acrylic block . each specimen was tested for its failure using hounsfield universal testing machine with a cross head speed of 1 mm / min [ figures 45 ] . the same procedure was repeated at the end of 1 month ( phase ii ) , 3 months ( phase iii ) , and 6 months ( phase iv ) , on the remaining prepared specimens and the results tabulated . the recorded values were statistically analyzed using anova and newman - keul 's studentized range test . forty specimens were randomly selected from the lot at the end of 15 days and calcium hydroxide was cleaned off the root canals of the specimens using gentle instrumentation and saline irrigation , and these root specimens were obturated with gutta percha using lateral condensation technique with a zoe sealer and stored in a humidator at 37c until further use . at the end of 7 days , gutta - percha was removed using a no.6 peeso reamer creating 10 mm of post space , leaving 2 mm of apical gutta - percha fill . further grouping of the specimen was done as follows : group i-10 : non - reinforced root specimens served as control.group ii-10 : root specimens reinforced with rmgic [ figure 1 ] . the root canals of the specimens were gently dried using an absorbent point and treated with the primer provided by the manufacturer for 30 seconds , air dried for 15 seconds , and light cured for 20 seconds . vitremer ( 3 m ) powder and vitremer liquid was dispensed according to the manufacturer 's instructions and mixed on a paper pad for 45 seconds . the material was back loaded into delivery tip and the restorative was syringed into the root canal.group iii- 10 : root specimens reinforced with flowable compomer ( prima flow ) preparation of group iii specimensthe root canals of the specimens were gently dried using an absorbent point and treated with the contax primer and bond according to manufacturer 's instructions and light cured for 20 seconds . the canals were filled with flowable compomer ( prima flow ) [ figure 1 ] , which was applied directly from syringe in layers of maximum 2 mm.group iv-10 : root specimens reinforced with flowable composite ( filtek z350 ) [ figure 2 ] . the 10 root specimens were etched with 37% phosphoric acid ( scotch bond multipurpose ) for 1520 seconds , rinsed with sterile water for 20 seconds , and then blot dried . two coats of adper single bond 2 ( 3 m ) bonding agent was applied , gently air dried , and light cured for 20 seconds . the specimens were then filled with flowable composite ( filtek flow z350 ( 3 m ) directly from the syringes . the root canals of the specimens were gently dried using an absorbent point and treated with the primer provided by the manufacturer for 30 seconds , air dried for 15 seconds , and light cured for 20 seconds . vitremer ( 3 m ) powder and vitremer liquid was dispensed according to the manufacturer 's instructions and mixed on a paper pad for 45 seconds . the material was back loaded into delivery tip and the restorative was syringed into the root canal . group iii- 10 : root specimens reinforced with flowable compomer ( prima flow ) preparation of group iii specimens the root canals of the specimens were gently dried using an absorbent point and treated with the contax primer and bond according to manufacturer 's instructions and light cured for 20 seconds . the canals were filled with flowable compomer ( prima flow ) [ figure 1 ] , which was applied directly from syringe in layers of maximum 2 mm . group iv-10 : root specimens reinforced with flowable composite ( filtek z350 ) [ figure 2 ] . the 10 root specimens were etched with 37% phosphoric acid ( scotch bond multipurpose ) for 1520 seconds , rinsed with sterile water for 20 seconds , and then blot dried . two coats of adper single bond 2 ( 3 m ) bonding agent was applied , gently air dried , and light cured for 20 seconds . the specimens were then filled with flowable composite ( filtek flow z350 ( 3 m ) directly from the syringes . restorative materials ; a - rmgic , b - flowable compomer - flowable composite sectioning of tooth at the cervical level to cure the restorative materials , a transilluminating post ( luminex ) was used making sure it reaches the cervical limit of the apical filling [ figure 3 ] . after placing the post inside the canal and removing the excess , the root specimens were then re - subjected to thermo cycling regime for 250 cycles at 6c60c , with a dwell time of 30 seconds . the roots were embedded in color - coded acrylic blocks using a flat ended rod held vertically by a ney surveyor to position the roots during mounting so that they were parallel to the acrylic base with an exposure of 4 mm of the cervical portion of root above the acrylic . the acrylic was contained in a inch diameter pvc pipe 2 cm in length to standardize the size of the base . a stainless steel cone measuring 1.5 cm at the base and 3 cm in length was placed against the canal so that it was in line with the acrylic block . each specimen was tested for its failure using hounsfield universal testing machine with a cross head speed of 1 mm / min [ figures 45 ] . the same procedure was repeated at the end of 1 month ( phase ii ) , 3 months ( phase iii ) , and 6 months ( phase iv ) , on the remaining prepared specimens and the results tabulated . the recorded values were statistically analyzed using anova and newman - keul 's studentized range test . the results show that there were no significant differences in the reinforcement provided by the group i , group ii , group iii , and group iv specimens at phase ii and iii when compared to phase i [ tables 13 ] . however , at phase iv , specimens of group i , group ii , and group iii showed statistically significant differences when compared to phase i and group iv , but did not show statistical significant difference from phase i [ figure 6 and table 4 ] . load failures of various materials at determined time intervals intercomparison of failure load between various reinforcing materials at phase i intercomparison of failure load between various reinforcing materials at phase ii intercomparison of failure load between various reinforcing material at phase - iii inter - comparison of failure load between various reinforcing materials at phase - iv when intergroup comparisons in the mean failure load values between various groups at four different phases were made , the experimental groups had significant differences when compared to control group at each phase . among the experimental groups , flowable composite group had a statistically significant difference when compared to rmgic and flowable compomer groups . in this study , experimental models that simulate immature teeth with total crown loss after apexification were used . permanent maxillary central incisors were chosen because these teeth are most commonly affected by dental impact injuries . they were selected according to their md and bl dimensions in order to reduce variables related to the different anatomical structures . in order to create a wide root canal with thin dentinal walls , the root canals were enlarged till 120 file size , 1 mm short of their apex . in this study , it was decided to evaluate the failure load of the root specimens at the end of 15 , 30 , 90 , and 180 days . the rationale behind this choice of carefully spaced periods was done keeping in mind the number of days of calcium hydroxide therapy when used as an interappointment intracanal medicament , as a remedy for weeping canals and as an agent for apexification . a review by goodacre and spolnik recommended post length equal to of root canal length , if possible or at least equal to the length of the crown and 8 mm was the minimum length required for a post . in our study , a post length of 10 mm was left behind for reinforcement of the canals . the inherent problem with the light cured dentin bonded composites is the inability of the light to penetrate to a depth greater than 5 mm or 6 mm . since it has been impossible so far to transmit light down the length of the canal , this often presented a problem in the intraradicular space . the introduction of a light transmitting post enables the clinician to complete polymerization of a light - cured or dual - cured composite resin along the length and circumference of a prepared post channel . the testing force was directed along the long axis of the specimens , resulting in primarily a splitting stress applied above the cervical limit of the root . although previous studies applied the load 45130 to the long axis of the tooth , we decided to use a wedge effect on the root because of the absence of the crown and also since this is also the force the post induces once installed . a consistent finding in our study was that all the experimental groups at the end of each phase , significantly reinforced the calcium hydroxide - treated simulated immature incisor teeth compared to the control group . this finding confirmed the results of previous studies by ketabzadeh n , dalton c and trope m , goldberg f , kaplan a. roitman m , manfre s , picca m , and pene jr , nicholls ji and harrington gw who showed that restorative materials which bond chemically or micromechanically to teeth increased the resistance to fracture of roots with canals having increased diameters such as immature teeth . in a study conducted by andreason jo , farik b , munksgaard ec , they found that in teeth treated with calcium hydroxide for a period of one year , fracture resistance reduced by 45%50% . in our study , the failure load of the control group was significantly reduced by almost 40% at the end of 6 months when compared to the control specimens at 15 days . this shows that the long term therapies like apexification , which involved exposure of teeth to calcium hydroxide for time periods of six months or even greater resulted in significant weakening of the remaining tooth structure . at the end of 180 days , it was evident that the reinforcement provided by flowable composites was significantly greater than those provided by the flowable compomers and rmgic . the results of our study were comparable to the results of meyer jn , cattni - lorente ma and dupuis v , and that of i h el , kalla f and garcia - godoy , who evaluated the mechanical properties of rmgic , compomer , and composite and concluded that among the evaluated materials , the weakest was rmgic followed by compomer with the strongest being composite . the flowable composite group at the end of 180 days showed no significant reduction in the reinforcement values compared to that at the end of 15 days indicating that they were more effective in reinforcing the root specimens , despite the drastic drop in the failure load of control specimens at the end of 6 months . previous studies have postulated that one of the apparent short comings of composites used for reinforcement of root canals is the propogation of microcracks resulting from inherent porosities and flexure in the material , leading to subsequent failure of the restoration . this study was an unpretentious attempt to evaluate the time bound effects of exposing simulated immature root specimens to calcium hydroxide for varying periods of time and evaluating the reinforcing capabilities of three commonly used adhesive restorative materials . since the benefits of calcium hydroxide in dentistry are well documented , this material will continue to receive the patronage of dental practitioners for a variety of applications despite its inherent shortcomings . our study has described a restorative method to strengthen internally , immature traumatized incisors using luminex post system . hence , it would be prudent that the practitioners use flowable composites in conjunction with a light transmitting post when reinforcing calcium hydroxide - treated immature teeth . all the materials evaluated substantially increased resistance of the root specimens to fracture when compared with the non - reinforced specimens.flowable composites provided maximum reinforcement effect at the end of 180 days , followed by flowable compomer , and rmgic , which exhibited reduced reinforcement effects.within the limitations of this study and among the materials tested , flowable composites were found to be the best reinforcing material to strengthen the roots of teeth with weakened dentinal walls . all the materials evaluated substantially increased resistance of the root specimens to fracture when compared with the non - reinforced specimens . flowable composites provided maximum reinforcement effect at the end of 180 days , followed by flowable compomer , and rmgic , which exhibited reduced reinforcement effects . within the limitations of this study and among the materials tested , flowable composites were found to be the best reinforcing material to strengthen the roots of teeth with weakened dentinal walls .

one hundred and sixty human permanent central incisors were enlarged to a 120 file size after crown removal procedure to simulate immature teeth . the root canals were filled with calcium hydroxide and stored for 15 days ( phase i ) , 30 days ( phase ii ) , 90 days ( phase iii ) , and 180 days ( phase iv ) . at the end of these selected time periods , calcium hydroxide was cleaned off the root canals of forty teeth that were randomly selected and obturated with gutta - percha points in the apical 2 mm of the root canals with a sealer . the specimens were further equally divided into four groups . unrestored group i served as control and the root canals of teeth in the other three group specimens were reinforced with resin modified glassionomer cement ( rmgic ) ( group ii ) , flowable compomer ( group iii ) , and flowable composite ( group iv ) , respectively , using a translucent curing post . all specimens were subjected to compressive force using an instron testing machine , until fracture occurred . all the materials evaluated substantially reinforced the root specimens compared to the control . at the end of 180 days , flowable composites showed maximum reinforcement compared to the other groups ; however , no significant differences were found between the reinforcement capabilities of flowable compomer and rmgic .

cancer cachexia is characterized by an ongoing loss of skeletal muscle mass , with or without a loss of fat mass , leading to progressive functional impairment.1 prevalence varies according to cancer type , with the highest rates found for cancers of the lung and upper gastrointestinal tract , where over half of all patients are affected at diagnosis.1,2 variation in the development of cancer cachexia also occurs on an individual level , with genetic polymorphisms influencing hallmarks of the syndrome , for example inflammation , loss of lean or fat mass .. 3 the pathophysiology of cancer cachexia is complex but involves a combination of abnormal metabolism and a negative energy balance.4 functional consequences of cachexia include limiting symptoms,5 reduced muscle function,6,7 reduced exercise capacity,8 and physical inactivity,9 all of which impact adversely on a patient 's level of independence ( figure 1 ) . functional impairments and consequences of cancer cachexia . a proactive management approach is recommended , instigated before cachexia becomes refractory , with the aim to maintain or slow down the decline in physical function.1,4 multimodal intervention is also considered necessary , as it is unlikely any single intervention will address the metabolic , nutritional , and functional deficits caused by cachexia concurrently.4 three main component interventions are being developed : nutritional therapies , pharmacological agents , and exercise therapies.10 exercise is defined as a planned , structured and repetitive bodily movement done to maintain or improve one or more components of physical fitness.11 exercise programmes vary according to the frequency , intensity , volume , and type of training used , as well as contextual factors such as the setting and level of supervision . for example , both a 6 week hospital - based programme of twice weekly , high - intensity , treadmill walking , and a home - based programme of low - intensity , balance training throughout chemotherapy would be defined as exercise.11 exercise may attenuate the effects of cancer cachexia via modulation of muscle metabolism , insulin sensitivity , and levels of inflammation.12,13 exercise may help patients with or at risk of cancer cachexia to maintain their independence for longer , but empirical evidence is lacking . despite a growing evidence base for nutritional and drug interventions for cancer cachexia,10 studies of exercise in advanced cancer are few . this paper provides an executive summary of a recent cochrane collaboration systematic review,14 which synthesizes evidence for the use of exercise for cancer cachexia . the primary aim was to determine the effectiveness of exercise , compared with usual care or no treatment on lean muscle mass in adults with cancer . secondary aim were to examine the acceptability and safety of exercise in this setting , and if possible , to compare effectiveness according to the characteristics of the exercise intervention or patient population . criteria for review entry were randomized controlled trials or quasi - randomized studies in any setting involving adults ( 18 years of age ) with a histological or clinical diagnosis of cancer , offered any type of exercising programme as a sole intervention or in combination with another intervention . we adopted an international criteria for cancer cachexia,1 and patients could be at any stage in the proposed classification : pre - cachexia ; weight loss 5% with anorexia and metabolic changes cachexia ; weight loss > 5% in the past six months or body mass index ( bmi ) < 20 kg / m and ongoing weight loss > 2% or sarcopenia , anorexia or systemic inflammation refractory cachexia ; active catabolism , ongoing weight loss , not responsive to treatment , and life expectancy of <3 months . to include studies in populations relevant to the review , for example advanced cancer , but not explicitly performed to address cancer cachexia , we permitted studies where 50% of participants fell within the cachexia definitions . where baseline demographic data were insufficient to assess participants against these criteria , we contacted study authors to seek additional data for this purpose . the primary endpoint was lean body mass assessed at the first study timepoint following a programme of exercise . secondary outcomes included adherence to prescribed programmes , occurrence of adverse events and , subject to availability of data , muscle strength and endurance , maximal and submaximal exercise capacity , fatigue , and health - related quality of life . we developed a comprehensive search strategy using a combination of terms based on the target population and intervention . the following electronic databases were searched from their inception until june 2014 , central , medline ( ovid ) , embase ( ovid ) , dare and hta - health technology assessments ( on the cochrane library ) , isi web of science ( sci - expanded and cpci ) , lilacs ( latin american and caribbean health sciences ) , pedro ( the physiotherapy evidence database ) , sciverse scopus ; biosis previews premedline , open grey ( system for information on grey literature).we identified ongoing studies using : clinical trials.gov ; metaregister of controlled trials ( mrct ) ; international clinical trials registry platform ( ictrp ) ; pan african clinical trials ; and the eu clinical trials register . in addition , we hand - searched the following sources : proceedings from the society on sarcopenia , cachexia and wasting disorders ( scwd ) ; the american cancer society ; the british association for cancer research ; and the european clinical guidelines . in addition , we contacted corresponding authors , experts , and organizations in the field to seek potentially relevant research , including unpublished and ongoing studies . two review authors ( a.j.g . , v.s . ) independently assess titles and abstracts of articles for relevance.14 we extracted relevant data relating to the study source , methods , participants , and intervention , disagreements were discussed and resolved by consensus . we planned for two authors to independently assess each eligible study for risk of bias using the cochrane collaboration tool.15 criteria for review entry were randomized controlled trials or quasi - randomized studies in any setting involving adults ( 18 years of age ) with a histological or clinical diagnosis of cancer , offered any type of exercising programme as a sole intervention or in combination with another intervention . we adopted an international criteria for cancer cachexia,1 and patients could be at any stage in the proposed classification : pre - cachexia ; weight loss 5% with anorexia and metabolic changes cachexia ; weight loss > 5% in the past six months or body mass index ( bmi ) < 20 kg / m and ongoing weight loss > 2% or sarcopenia , anorexia or systemic inflammation refractory cachexia ; active catabolism , ongoing weight loss , not responsive to treatment , and life expectancy of <3 months . to include studies in populations relevant to the review , for example advanced cancer , but not explicitly performed to address cancer cachexia , we permitted studies where 50% of participants fell within the cachexia definitions . where baseline demographic data were insufficient to assess participants against these criteria , we contacted study authors to seek additional data for this purpose . the primary endpoint was lean body mass assessed at the first study timepoint following a programme of exercise . secondary outcomes included adherence to prescribed programmes , occurrence of adverse events and , subject to availability of data , muscle strength and endurance , maximal and submaximal exercise capacity , fatigue , and health - related quality of life . we developed a comprehensive search strategy using a combination of terms based on the target population and intervention . the following electronic databases were searched from their inception until june 2014 , central , medline ( ovid ) , embase ( ovid ) , dare and hta - health technology assessments ( on the cochrane library ) , isi web of science ( sci - expanded and cpci ) , lilacs ( latin american and caribbean health sciences ) , pedro ( the physiotherapy evidence database ) , sciverse scopus ; biosis previews premedline , open grey ( system for information on grey literature).we identified ongoing studies using : clinical trials.gov ; metaregister of controlled trials ( mrct ) ; international clinical trials registry platform ( ictrp ) ; pan african clinical trials ; and the eu clinical trials register . in addition , we hand - searched the following sources : proceedings from the society on sarcopenia , cachexia and wasting disorders ( scwd ) ; the american cancer society ; the british association for cancer research ; and the european clinical guidelines . in addition , we contacted corresponding authors , experts , and organizations in the field to seek potentially relevant research , including unpublished and ongoing studies . two review authors ( a.j.g . , v.s . ) independently assess titles and abstracts of articles for relevance.14 we extracted relevant data relating to the study source , methods , participants , and intervention , disagreements were discussed and resolved by consensus . we planned for two authors to independently assess each eligible study for risk of bias using the cochrane collaboration tool.15 after duplicates were removed , 3138 were deemed ineligible after title and abstract screening ( figure 2 ) . full texts of the remaining 16 potentially relevant titles were retrieved.14 we attempted to contact corresponding authors via electronic mail to determine the proportion of the sample meeting pre - cachexia or cachexia criteria . most authors did not explore this concept , and others did not respond.14 we did not identify any suitable studies for inclusion . in the absence of any suitable trials , we were unable to perform a meta - analysis to determine any effects from exercise intervention . using a comprehensive and systematic search strategy , we identified no randomized controlled trials of exercise for patients with cancer cachexia . in studies where exercise interventions were delivered to patients with cancer and at risk of cachexia , domains for the syndrome including weight loss , nutritional stores and intake , and muscle mass or function were seldom reported . to this end , it was not possible to determine the proportion of patients meeting pre - cachexia or cachexia criteria . the lack of studies in this field is at odds with the strong rationale for examining exercise as a therapeutic intervention . reviews have outlined the potential for exercise to impact positively on muscle mass and strength,16 inflammatory markers,13 and physical function,12 all of which are features of cancer cachexia which impairment the patient . it is plausible that the previous lack of a consensus definition has hindered study in the field . without agreed diagnostic or staging criteria , the prevalence and impact of cancer cachexia have been difficult to assess . moreover , interventions may have been inadvertently trialled in cachectic groups without formal recognition , as may be the case for exercise studies in advanced cancer . in this regard , the classification system proposed by fearon et al.1 is an important step forward , and recent international validation studies will add further momentum.1720 two candidate studies may have included patients with cancer cachexia , although this can not be confirmed . the first21 tested a multidisciplinary intervention with cognitive , emotional , social , and spiritual supportive care delivered over eight 90 min sessions . in 115 patients with advanced cancer undergoing radiation therapy , self - assessed physical well - being was improved in the intervention group , although no benefit in objectively measured physical function was observed . the second22 randomized 231 patients with incurable , metastatic cancer to physical exercise or usual care over 8 weeks . the exercise was delivered in two 60 min session each week , and led to improvements in handgrip strength and sit - to - stand performance . we also identified ongoing studies targeting cancer cachexia , most notably a randomized feasibility trial of a 6 week multimodal intervention comprising nutritional supplementation , home - based exercise , and anti - inflammatory treatment for patients with advanced non - small cell lung or pancreatic cancer ( nct01419145 ) . further ongoing studies are examining combined physical activity / exercise and nutrition interventions , over 12 weeks for patients with head and neck cancer during or following treatment ( nct01681654 ) and over 20 weeks for cachectic patients with lung cancer ( actrn12611000870954 ) . findings from these trials are eagerly awaited and will pave the way for future exercise interventions for cachexia . there is insufficient evidence to determine the safety and effectiveness of exercise for patients with cancer cachexia . despite a strong rationale for the use of exercise , there are no present data from rcts to elucidate the specific effects in this population . international criteria to diagnose and classify cancer cachexia provide a strong basis to define study populations within trials of interventions for this condition . an assessment of these domains must be included in cachexia trials and is strongly encouraged in populations where cachexia may be present . this would enhance the interpretation and generalization of findings from relevant study populations to the cachexia setting . antonio jose grande , valter silva , and matthew maddocks declare that they have no conflict of interest . the authors certify that they comply with the ethical guidelines for authorship and publishing of the journal of cachexia , sarcopenia and muscle ( von haehling s , morley ethical guidelines for authorship and publishing in the journal of cachexia , sarcopenia and muscle .

backgroundcancer cachexia is a complex syndrome characterized by an ongoing loss of skeletal muscle mass and progressive functional impairment . a proactive management approach is recommended , including physical exercise to maintain function via modulation of muscle metabolism , insulin sensitivity and levels of inflammation . the review aimed to determine the safety , acceptability and effectiveness of exercise in adults with cancer cachexia . secondary aims , subject to the data availability , were to compare effectiveness according to the characteristics of the study intervention or population.methodswe sought randomised controlled trials ( rcts ) in adults meeting international criteria for cancer cachexia , comparing a programme of exercise as a sole or adjunct intervention to usual care or an active control . central , medline , embase , dare and hta , isi web of science , lilacs , pedro , sciverse scopus , biosis previews premedline and open grey databases were searched up to june 2014 . two authors independently assessed studies for eligibility.resultswe screened 3154 separate titles and abstracts , and reviewed 16 full - texts . corresponding authors were contacted to determine if samples met cachexia staging criteria . most authors did not explore this concept . no trial met review eligibility criteria . we were unable to perform a meta - analysis to determine any effects from exercise intervention.conclusiondespite a strong rationale for the use of exercise , there is insufficient evidence to determine safety and effectiveness in patients with cancer cachexia . findings from ongoing studies are awaited . assessment of cachexia domains , ideally against international criteria , is required for future trials of exercise and supportive care interventions .

this work was supported by the following grants : pict 20133280 and pict 20111301 to glb , pict 20122493 and pip 205 - 2011 - 2013 to mgt . mgt and glb are investigators from the consejo nacional de investigaciones cientficas y tecnolgicas ( conicet ) , argentina .

abstractseveral cellular responses depend on translational regulation and in most cases , this involves the formation of cytoplasmic granules that contain repressed mrnas . in neurons , numerous mrnas travel along dendrites to be locally regulated upon synapse activity and we have recently shown that the exoribonuclease xrn1 forms dynamic aggregates at the post synapse that respond to specific stimuli.1 these foci were termed sx - bodies and are distinct from stress granules ( sgs ) , processing bodies ( pbs ) and other rna granules previously described . together with smaug1-foci and fmrp - granules , the sx - bodies contribute to dynamically shape the transcriptome available for translation at the post - synapse .

evidence of a link between degenerative intervertebral disc ( ivd ) and low back pain ( lbp ) is mounting . currently , the treatment of diseases related to ivd often involves surgical intervention or long - term rehabilitation therapy . the goals of biological therapy are to prevent or delay ivd degeneration and to alleviate its symptoms by promoting tissue repair . to better prevent and treat lbp , the comprehensive mechanisms and related biological and mechanical pathways of ivd degeneration remain poorly understood even though lbp is a common clinical condition . biomechanics and ivd degeneration are closely related , and many studies have shown that mechanical loading is one of the major factors leading to ivd . an epidemiological survey has also shown that mechanical loading is a risk factor for lbp . however , weight - bearing mechanical loading is the primary function of ivds , and it occurs in the natural environment . mechanical loading also stimulates ivd cells ; this stimulation is likely the underlying factor for the maintenance of cartilage cell activity . to better understand the mechanisms of ivd degeneration , an in - depth understanding of its cause and effect relationship with biomechanics is required . moreover , the establishment of an ideal experimental model might facilitate future - related studies . however , investigating the complex metabolism and signaling cascades that occur in ivds is difficult using in vivo models because of the lack of close control and difficulties with monitoring . in contrast , ex vivo culturing systems are more appealing because they allow for better control of biochemical and biomechanical factors . therefore , ex vivo organ models represent an experimental basis to observe the responses and changes of ivd tissues to specific external stimuli , such as mechanical loads or biologics . thus , an ivd motion segment was established and maintained in an organ culture , and it included the whole ivd organ and adjacent vertebral bodies ( vbs ) . first , it enables the ex vivo organ model to be more reflective of the true conditions . second , the adjoining vbs ( as rigid fixtures ) facilitate the application and control of complex mechanical loads . this study compared rabbit ivd motion segments cultured in custom - made apparatuses under static load between control tissues and fresh tissues . the tissue integrity , matrix synthesis , and matrix gene expression profile were assessed to determine the influence of static load on the ivds and to increase understanding of these conditions so that the effects of biomechanics on the ivds can be better controlled . all of the experimental procedures were conducted in accordance with the institutional guidelines for the care and use of laboratory animals of the china academy of chinese medical sciences , beijing , china . the experimental animals included 14 18-week - old ( 3.0 kg ) male new zealand white rabbits . these animals were used in accordance with protocols that were approved by the animal ethics committee of the institute of basic theory for chinese medicine , china academy of chinese medical sciences ( approval no . five minutes before death , 25,000 iu heparin was administered through the ear vein , and the lumbar spine was harvested under sterile conditions immediately after euthanasia via co2 asphyxiation . soft tissues and posterior elements of the spine were removed , and the motion segment models were dissected from consecutive levels ( n = 4/animal ) consisting of the whole disc organ , which included the vertebral end plates ( eps ) , annulus fibrous ( af ) , and nucleus pulposus ( np ) with adjacent vbs . the lengths of the ivd motion segments ranged from 280 to 310 mm , and the diameters of the ivds ranged from 110 to 140 mm . any debris on the cut surfaces was washed with phosphate - buffered saline using a syringe with an 18-gauge needle ; all of the ivds were rinsed for 2 min in hanks balanced salt solution containing heparin and 10% penicillin - streptomycin . a total of 28 ivd motion segments were maintained in 6-well plates without loading ( controls ) , and the other 28 ivd motion segments were kept in custom - made apparatuses under a constant compressive load ( 3 kg , 0.5 mpa ) . the 6-well plates and apparatuses were maintained in an incubator under standard culture conditions ( 37c , 5% co2 ; [ supplementary figure 1 ] ) . ( a ) the specimens were maintained in no - loading 6-well plates for the control conditions . ( b ) the specimens were maintained in load apparatuses under a constant compressive load . ( c ) the 6-well plates and apparatuses were situated inside of a 37c incubator with 5% co2 and 100% humidity . the specimens were maintained in dulbecco 's modified eagle 's medium ( dmem ) supplemented with 20% fetal bovine serum ( invitrogen life technologies , carlsbad , ca , usa ) , 50 mg / ml l - ascorbate , 100 u / ml penicillin , 100 mg / ml streptomycin , and 2.5 mg / ml fungizone . nacl was added to the dmem to raise the osmolarity to 410 mosm / kg . after 0 , 3 , 7 , and 14 days , seven ivds were assessed by hematoxylin and eosin ( he ) staining , alcian blue / periodic acid - schiff reaction ( ab / pas ) , and collagen type ii alpha 1 immunohistochemistry ( ihc ) , and real - time polymerase chain reaction ( pcr ) was performed to examine the matrix gene expression profile . loading and culturing apparatuses for the intervertebral disc motion segments the apparatuses were designed for the ex vivo culturing of ivd motion segments while simultaneously providing static axial compression . the loading frame provided a static load through the use of weights on a mobile loading plate that could be moved along the optical axis . because compression can cause the ivd heights to change slightly , the mobile weight ensured that the full load was being consistently applied to the specimen . a gland was fixed to the bottom of the loading plate , through which the weight was applied to the model . a gland also covered the top of the chamber to ensure that the culture environment was closed [ supplementary figure 2 ] . loading and organ culturing apparatuses for the rabbit ivd motion segments ( diagrammatic sketch ) . 1 : weights , 2 : loading plate , 3 : gland , 4 : optical axis , 5 : chamber , 6 : chamber fixed base , 7 : outlet , 8 : top pedestal , 9 : fluid level observation hole , 10 : ivd motion segments , 11 : base pedestal , and 12 : jackscrews . a disposable silicon tube was inserted at the bottom of culture chamber , and it functioned as an outlet for media changing ; the other end of the tube was plugged . a syringe was used for changing the media , and the manipulations were performed on an aseptic operation table . moreover , the tubing and needle were changed with each use to ensure for sterility of the culture [ supplementary figure 2 ] . based on the characteristics of the spinal motion segment , the device was arranged on the top and base pedestals . three jackscrews were inserted through the pedestal to fix one side of the vbs , allowing the motion segment to remain in an upright state so that the compression was always vertically loaded on the surfaces of the ivds [ supplementary figures 2 and 3 ] . ivd motion segments are prone to bending ( arrow ) under loading without fixation ( a ) . the specimen can be maintained in an upright state through fixation with jackscrews ( arrowhead ) though the pedestals ( b ) to ensure that full vertical loading is consistently applied on the surface of the model . all of the components could be disassembled from the device , and the materials were resistant to deformation and decomposition by disinfection treatments ( e.g. , high temperature and high pressure ) . moreover , the whole apparatus was sufficiently small ; thus , multiple loading stations could be placed in the incubator at the same time [ supplementary figure 1 ; patent number : zl 201420568511.9 ] . histological analysis the samples were fixed with a 10% buffered neutral formalin solution for up to 2 days , decalcified in ethylenediaminetetraacetic acid , and then embedded in paraffin . the sections were deparaffinized in xylene , rehydrated with graded ethanol , and stained with he . collagen type ii alpha 1 immunohistochemistry for detection of the np extracellular matrix ( ecm ) , a collagen type ii alpha 1 monoclonal antibody ( sigma , usa ) was used for ihc analyses . in brief , at each harvesting time point , 46 m paraffin sections of three samples from each group were dewaxed , rehydrated , and then blocked with hydrogen peroxide as an endogenous peroxidase . next , the sections were washed in dh2o and treated with 1 of 2 enzyme antigen retrieval reagents for 20 min at 37c . they were subsequently washed again , and nonspecific binding sites were blocked at room temperature for 45 min with 20% w / v goat serum . then , the sections were incubated with a mouse monoclonal primary antibody against collagen type ii alpha 1 ( sigma - aldrich , bale , switzerland ) diluted 1:20 overnight at 4c . negative controls were used for which the primary antibody was replaced with an equal concentration of mouse igg ( sigma ) . after the sections were again washed , those being assessed for collagen type ii alpha 1 were incubated with biotinylated rabbit anti - mouse antiserum ( sigma ) diluted 1:400 for 30 min at room temperature . the secondary antibody binding was visualized using the streptavidin - biotin complex technique with a 3,3-diaminobenzidine tetrahydrochloride solution as the colored reaction product . an nis - elements d2.30 image analysis system ( nikon , tokyo , japan ) was used for semiquantitative analysis of collagen type ii alpha 1 . real - time polymerase chain reaction analysis total rna from the np was analyzed for the expression of ecm genes . in brief , the af was cut with a blade , and the np tissue was removed using a micro curette and immediately frozen in liquid nitrogen at each harvesting time point . tissues from 3 ivds were pooled to ensure that a sufficient quantity of tissues was obtained for rna isolation , which was performed using trizol reagent ( invitrogen life technologies , carlsbad , ca , usa ) according to the manufacturer 's instructions . primers for the rabbitglyceraldehyde-3-phosphate dehydrogenase ( gapdh ) and ecm genes were custom designed using primer 5.0 software ( applied biosystems , foster city , ca , usa ) [ table 1 ] . details for the primers for the target and reference genes used in quantitative real - time pcr gapdh : glyceraldehyde-3-phosphate dehydrogenase ; col2a1 : collagen type ii alpha 1 ; agg : aggrecan ; f : forward ; r : reverse ; pcr : polymerase chain reaction . quantitative real - time polymerase chain reaction ( qrt - pcr ) was performed using an sybr green one - step qrt - pcr kit ( kapa biosystems , usa ) . the final qrt - pcr results were obtained using the comparative ct method with the following equation : fold change = 2 , where ct = ( cttarget ctreference ) control ( cttarget ctreference ) culture ( reference : mean ct of gapdh ; control : day 0 ; and culture : day 3 , 7 , 14 , or 21 ) . statistical analyses , including analysis of variance ( anova ) and post hoc pairwise comparisons , were performed using spss software ( version 16.0 , spss inc . , the between - group data were compared using the independent - samples t - test . all of the results are expressed as the mean standard deviation ( sd ) . all of the experimental procedures were conducted in accordance with the institutional guidelines for the care and use of laboratory animals of the china academy of chinese medical sciences , beijing , china . the experimental animals included 14 18-week - old ( 3.0 kg ) male new zealand white rabbits . these animals were used in accordance with protocols that were approved by the animal ethics committee of the institute of basic theory for chinese medicine , china academy of chinese medical sciences ( approval no . five minutes before death , 25,000 iu heparin was administered through the ear vein , and the lumbar spine was harvested under sterile conditions immediately after euthanasia via co2 asphyxiation . ivd motion segments were obtained from the 14 rabbits . soft tissues and posterior elements of the spine were removed , and the motion segment models were dissected from consecutive levels ( n = 4/animal ) consisting of the whole disc organ , which included the vertebral end plates ( eps ) , annulus fibrous ( af ) , and nucleus pulposus ( np ) with adjacent vbs . the lengths of the ivd motion segments ranged from 280 to 310 mm , and the diameters of the ivds ranged from 110 to 140 mm . any debris on the cut surfaces was washed with phosphate - buffered saline using a syringe with an 18-gauge needle ; all of the ivds were rinsed for 2 min in hanks balanced salt solution containing heparin and 10% penicillin - streptomycin . a total of 28 ivd motion segments were maintained in 6-well plates without loading ( controls ) , and the other 28 ivd motion segments were kept in custom - made apparatuses under a constant compressive load ( 3 kg , 0.5 mpa ) . the 6-well plates and apparatuses were maintained in an incubator under standard culture conditions ( 37c , 5% co2 ; [ supplementary figure 1 ] ) . ( a ) the specimens were maintained in no - loading 6-well plates for the control conditions . ( b ) the specimens were maintained in load apparatuses under a constant compressive load . ( c ) the 6-well plates and apparatuses were situated inside of a 37c incubator with 5% co2 and 100% humidity . the specimens were maintained in dulbecco 's modified eagle 's medium ( dmem ) supplemented with 20% fetal bovine serum ( invitrogen life technologies , carlsbad , ca , usa ) , 50 mg / ml l - ascorbate , 100 u / ml penicillin , 100 mg / ml streptomycin , and 2.5 mg / ml fungizone . nacl was added to the dmem to raise the osmolarity to 410 mosm / kg . after 0 , 3 , 7 , and 14 days , seven ivds were assessed by hematoxylin and eosin ( he ) staining , alcian blue / periodic acid - schiff reaction ( ab / pas ) , and collagen type ii alpha 1 immunohistochemistry ( ihc ) , and real - time polymerase chain reaction ( pcr ) was performed to examine the matrix gene expression profile . loading and culturing apparatuses for the intervertebral disc motion segments the apparatuses were designed for the ex vivo culturing of ivd motion segments while simultaneously providing static axial compression . the loading frame provided a static load through the use of weights on a mobile loading plate that could be moved along the optical axis . because compression can cause the ivd heights to change slightly , the mobile weight ensured that the full load was being consistently applied to the specimen . a gland was fixed to the bottom of the loading plate , through which the weight was applied to the model . a gland also covered the top of the chamber to ensure that the culture environment was closed [ supplementary figure 2 ] . loading and organ culturing apparatuses for the rabbit ivd motion segments ( diagrammatic sketch ) . 1 : weights , 2 : loading plate , 3 : gland , 4 : optical axis , 5 : chamber , 6 : chamber fixed base , 7 : outlet , 8 : top pedestal , 9 : fluid level observation hole , 10 : ivd motion segments , 11 : base pedestal , and 12 : jackscrews . a disposable silicon tube was inserted at the bottom of culture chamber , and it functioned as an outlet for media changing ; the other end of the tube was plugged . a syringe was used for changing the media , and the manipulations were performed on an aseptic operation table . moreover , the tubing and needle were changed with each use to ensure for sterility of the culture [ supplementary figure 2 ] . based on the characteristics of the spinal motion segment , the device was arranged on the top and base pedestals . three jackscrews were inserted through the pedestal to fix one side of the vbs , allowing the motion segment to remain in an upright state so that the compression was always vertically loaded on the surfaces of the ivds [ supplementary figures 2 and 3 ] . ivd motion segments are prone to bending ( arrow ) under loading without fixation ( a ) . the specimen can be maintained in an upright state through fixation with jackscrews ( arrowhead ) though the pedestals ( b ) to ensure that full vertical loading is consistently applied on the surface of the model . all of the components could be disassembled from the device , and the materials were resistant to deformation and decomposition by disinfection treatments ( e.g. , high temperature and high pressure ) . moreover , the whole apparatus was sufficiently small ; thus , multiple loading stations could be placed in the incubator at the same time [ supplementary figure 1 ; patent number : zl 201420568511.9 ] . histological analysis the samples were fixed with a 10% buffered neutral formalin solution for up to 2 days , decalcified in ethylenediaminetetraacetic acid , and then embedded in paraffin . the sections were deparaffinized in xylene , rehydrated with graded ethanol , and stained with he . collagen type ii alpha 1 immunohistochemistry for detection of the np extracellular matrix ( ecm ) , a collagen type ii alpha 1 monoclonal antibody ( sigma , usa ) was used for ihc analyses . in brief , at each harvesting time point , 46 m paraffin sections of three samples from each group were dewaxed , rehydrated , and then blocked with hydrogen peroxide as an endogenous peroxidase . next , the sections were washed in dh2o and treated with 1 of 2 enzyme antigen retrieval reagents for 20 min at 37c . they were subsequently washed again , and nonspecific binding sites were blocked at room temperature for 45 min with 20% w / v goat serum . then , the sections were incubated with a mouse monoclonal primary antibody against collagen type ii alpha 1 ( sigma - aldrich , bale , switzerland ) diluted 1:20 overnight at 4c . negative controls were used for which the primary antibody was replaced with an equal concentration of mouse igg ( sigma ) . after the sections were again washed , those being assessed for collagen type ii alpha 1 were incubated with biotinylated rabbit anti - mouse antiserum ( sigma ) diluted 1:400 for 30 min at room temperature . the secondary antibody binding was visualized using the streptavidin - biotin complex technique with a 3,3-diaminobenzidine tetrahydrochloride solution as the colored reaction product . an nis - elements d2.30 image analysis system ( nikon , tokyo , japan ) was used for semiquantitative analysis of collagen type ii alpha 1 . real - time polymerase chain reaction analysis total rna from the np was analyzed for the expression of ecm genes . in brief , the af was cut with a blade , and the np tissue was removed using a micro curette and immediately frozen in liquid nitrogen at each harvesting time point . tissues from 3 ivds were pooled to ensure that a sufficient quantity of tissues was obtained for rna isolation , which was performed using trizol reagent ( invitrogen life technologies , carlsbad , ca , usa ) according to the manufacturer 's instructions . primers for the rabbitglyceraldehyde-3-phosphate dehydrogenase ( gapdh ) and ecm genes were custom designed using primer 5.0 software ( applied biosystems , foster city , ca , usa ) [ table 1 ] . details for the primers for the target and reference genes used in quantitative real - time pcr gapdh : glyceraldehyde-3-phosphate dehydrogenase ; col2a1 : collagen type ii alpha 1 ; agg : aggrecan ; f : forward ; r : reverse ; pcr : polymerase chain reaction . quantitative real - time polymerase chain reaction ( qrt - pcr ) was performed using an sybr green one - step qrt - pcr kit ( kapa biosystems , usa ) . the final qrt - pcr results were obtained using the comparative ct method with the following equation : fold change = 2 , where ct = ( cttarget ctreference ) control ( cttarget ctreference ) culture ( reference : mean ct of gapdh ; control : day 0 ; and culture : day 3 , 7 , 14 , or 21 ) . statistical analyses , including analysis of variance ( anova ) and post hoc pairwise comparisons , were performed using spss software ( version 16.0 , spss inc . , chicago , il , usa ) . the between - group data were compared using the independent - samples t - test . all of the results are expressed as the mean standard deviation ( sd ) . before culturing , he staining showed that the central integrated np contained abundant cells and matrix material . in addition , the af was arranged in concentric circles with a clear hierarchy and clear boundaries between the np and the cartilage ep at the upper and lower ends . the section staining did not reveal any major structural change in the controls at 3 or 7 days compared with the fresh samples . the tissue structure remained intact for up to 14 days although a partial separation between the np and af was observed . culturing for 7 days led to a decrease in the number of np cells and their dispersal , as well as partial laceration of the af . culturing for 14 days led to loss of the tight concentric architecture of the af and separation of the np ; this change was more obvious under static load than under the control conditions [ figure 1 ] . hematoxylin and eosin staining of ivd midsagittal slices at each time point under control and under static load conditions ( original magnification , 10 ) . ( a ) fresh tissue ; ( b , d , and f ) tissue histological observations of organ controls on days 3 , 7 , and 14 , respectively ; ( c , e , and g ) histological observations of tissues under static load on days 3 , 7 , and 14 , respectively . in the controls , the tissue structure remained intact up ( a and d , arrows ) to 14 days although a partial separation between the np and af appeared ( f , arrows ) . under static load , culturing to 7 days led to a decrease in the number of np cells and their dispersal ( e , arrowheads ) . culturing for 14 days led to the loss of the tight concentric architecture of the af and separation of the np ( g , arrowheads ) , and this change was more obvious under the static load than that in the control condition . the specimens were stained with ab / pas to observe changes in the proteoglycan content . the staining within the matrix and in the immediate pericellular areas indicated the presence of proteoglycans . after 7 days , a marked loss of extracellular proteoglycans was observed . under static load , moreover , by 14 days , the number of cells in the np was obviously decreased , although no further decrease in staining was observed , suggesting a reduction in biosynthetic activity [ figure 2 ] . ab / pas staining of proteoglycans in the np at each time point ( original magnification , 200 ) . ( a ) fresh tissue ; ( b , d , and f ) ab / pas staining of organ control tissue on days 3 , 7 , and 14 , respectively ; ( c , e , and g ) ab / pas staining of tissues under static load on days 3 , 7 , and 14 , respectively . the staining within the matrix and in the immediate pericellular areas indicated the presence of proteoglycans ( a , arrows ) . after 7 days , a marked loss of extracellular proteoglycans was observed ( d , arrows ) . ( e ) under static load , by 7 days , the proteoglycan content decreased ( arrow ) and was unevenly distributed ( arrowheads ) . by 14 days , ab / pas : alcian blue and periodic acid - schiff ; np : nucleus pulposus . immunoreactive collagen type ii alpha 1 was observed within the matrix of the np in the controls and tissues cultured in the apparatuses at all times . the staining intensity remained consistent by 14 days in the controls , and no significant difference was observed between the control and fresh tissues ( p = 0.896 ) . the samples under static load showed a significant increase in ihc intensity during the initial 3 days , and significant differences were observed between the controls and fresh tissues ( p < 0.05 ) . however , the staining was obviously reduced after 3 days , and by 14 days , it was significantly decreased ( p < 0.05 ) and significantly different from that observed in the controls ( p < 0.001 ) [ figures 3 , 4 and table 2 ] . collagen type ii alpha 1 ihc staining of the np at each time point ( original magnification , 400 ) . ( a ) fresh tissue ; ( b , d , and f ) tissue collagen type ii alpha 1 staining of organ controls at days 3 , 7 , and 14 , respectively ; ( c , e , and g ) collagen type ii alpha 1 staining of tissues under static load at days 3 , 7 , and 14 , respectively . the intensity of staining remained constant by 14 days in the controls , and no significant difference was observed between the controls and fresh tissue ( a , arrow ) . the samples under static load showed a significant enhancement in ihc intensity for the initial 3 days ( c , arrowheads ) ; however , the staining was obviously reduced after 3 days , by 14 days ( g ) , the staining was significantly decreased ( arrow ) and significantly different from the controls . . collagen type ii alpha 1 ihc staining intensity of the np at each time point . no significant differences in collagen type ii alpha 1 staining intensity were observed between the fresh tissues and controls . the samples under static load showed a significant enhancement at 3 day , and significant differences were observed between the controls and fresh tissues . at 7 day , the staining was obviously decreased , and by 14 days , it was significantly decreased ( p < 0.05 ) and significantly different from that of the controls . the values represent the mean sd . * p < 0.05 versus day 0 of the same group ; p < 0.05 versus day 3 of the same group ; p < 0.05 versus controls . collagen type ii alpha 1 ihc staining intensity of the np during organ culture values are given as the mean sd . * the significance of differences between culture time point and fresh ( day 0 ) ; the significance of differences between culture time point and day 3 ; the significance of differences between static load and controls . ihc : immunohistochemistry ; np : nucleus pulposus ; sd : standard deviation , : not applicable . qrt - pcr revealed marked downregulation of aggrecan gene ( agg ) expression in the controls after 7 days compared with the fresh tissues whereas it was significantly downregulated under static load after day 0 compared with fresh tissues and controls [ figure 5 and table 3 ] . in contrast , collagen type ii alpha 1 ( col2a1 ) expression was upregulated under static load on day 3 , but tended to be downregulated under the control conditions ; this difference was statistically significant . however , col2a1 expression subsequently decreased rapidly to below detectable levels under static load until day 14 , but it was not significantly downregulated under the control conditions after 3 days ; further , statistically significant differences were observed on days 7 and 14 [ figure 5 and table 4 ] . an obvious downregulation of agg was observed in the controls after 3 days whereas a significant downregulation was observed under static load during culture compared with fresh tissue and controls . an upregulation of col2a1 was observed in response to static load by 3 days , and the expression of this gene subsequently decreased until day 14 to below detectable levels . col2a1 was downregulated in the controls by 3 days whereas no significant downregulation was observed after culturing , and significant differences were observed between the two groups after culture . * p < 0.05 versus day 0 of the same group ; p < 0.05 versus day 3 of the same group ; p < 0.05 versus controls . pcr : polymerase chain reaction ; agg : aggrecan ; col2a1 : collagen type ii alpha 1 ; sd : standard deviation . agg gene expression quantified by real - time pcr during organ culture values are given as the mean sd . * the significance of differences between culture time point and fresh ( day 0 ) ; the significance of differences between culture time point and day 3 ; the significance of differences between static load and controls . agg : aggrecan ; pcr : polymerase chain reaction ; sd : standard deviation , : not applicable . col2a1 gene expression quantified by real - time pcr during organ culture values are given as the mean sd . * the significance of differences between culture time point and fresh ( day 0 ) ; the significance of differences between culture time point and day 3 ; the significance of differences between static load and controls . col2a1 : collagen type ii alpha 1 ; pcr : polymerase chain reaction ; sd : standard deviation ; : not applicable . before culturing , he staining showed that the central integrated np contained abundant cells and matrix material . in addition , the af was arranged in concentric circles with a clear hierarchy and clear boundaries between the np and the cartilage ep at the upper and lower ends . the section staining did not reveal any major structural change in the controls at 3 or 7 days compared with the fresh samples . the tissue structure remained intact for up to 14 days although a partial separation between the np and af was observed . culturing for 7 days led to a decrease in the number of np cells and their dispersal , as well as partial laceration of the af . culturing for 14 days led to loss of the tight concentric architecture of the af and separation of the np ; this change was more obvious under static load than under the control conditions [ figure 1 ] . hematoxylin and eosin staining of ivd midsagittal slices at each time point under control and under static load conditions ( original magnification , 10 ) . ( a ) fresh tissue ; ( b , d , and f ) tissue histological observations of organ controls on days 3 , 7 , and 14 , respectively ; ( c , e , and g ) histological observations of tissues under static load on days 3 , 7 , and 14 , respectively . in the controls , the tissue structure remained intact up ( a and d , arrows ) to 14 days although a partial separation between the np and af appeared ( f , arrows ) . under static load , culturing to 7 days led to a decrease in the number of np cells and their dispersal ( e , arrowheads ) . culturing for 14 days led to the loss of the tight concentric architecture of the af and separation of the np ( g , arrowheads ) , and this change was more obvious under the static load than that in the control condition . the specimens were stained with ab / pas to observe changes in the proteoglycan content . the staining within the matrix and in the immediate pericellular areas indicated the presence of proteoglycans . after 7 days , a marked loss of extracellular proteoglycans was observed . under static load , moreover , by 14 days , the number of cells in the np was obviously decreased , although no further decrease in staining was observed , suggesting a reduction in biosynthetic activity [ figure 2 ] . ab / pas staining of proteoglycans in the np at each time point ( original magnification , 200 ) . ( a ) fresh tissue ; ( b , d , and f ) ab / pas staining of organ control tissue on days 3 , 7 , and 14 , respectively ; ( c , e , and g ) ab / pas staining of tissues under static load on days 3 , 7 , and 14 , respectively . the staining within the matrix and in the immediate pericellular areas indicated the presence of proteoglycans ( a , arrows ) . after 7 days , a marked loss of extracellular proteoglycans was observed ( d , arrows ) . ( e ) under static load , by 7 days , the proteoglycan content decreased ( arrow ) and was unevenly distributed ( arrowheads ) . by 14 days , ab / pas : alcian blue and periodic acid - schiff ; np : nucleus pulposus . immunoreactive collagen type ii alpha 1 was observed within the matrix of the np in the controls and tissues cultured in the apparatuses at all times . the staining intensity remained consistent by 14 days in the controls , and no significant difference was observed between the control and fresh tissues ( p = 0.896 ) . the samples under static load showed a significant increase in ihc intensity during the initial 3 days , and significant differences were observed between the controls and fresh tissues ( p < 0.05 ) . however , the staining was obviously reduced after 3 days , and by 14 days , it was significantly decreased ( p < 0.05 ) and significantly different from that observed in the controls ( p < 0.001 ) [ figures 3 , 4 and table 2 ] . collagen type ii alpha 1 ihc staining of the np at each time point ( original magnification , 400 ) . ( a ) fresh tissue ; ( b , d , and f ) tissue collagen type ii alpha 1 staining of organ controls at days 3 , 7 , and 14 , respectively ; ( c , e , and g ) collagen type ii alpha 1 staining of tissues under static load at days 3 , 7 , and 14 , respectively . the intensity of staining remained constant by 14 days in the controls , and no significant difference was observed between the controls and fresh tissue ( a , arrow ) . the samples under static load showed a significant enhancement in ihc intensity for the initial 3 days ( c , arrowheads ) ; however , the staining was obviously reduced after 3 days , by 14 days ( g ) , the staining was significantly decreased ( arrow ) and significantly different from the controls . collagen type ii alpha 1 ihc staining intensity of the np at each time point . no significant differences in collagen type ii alpha 1 staining intensity were observed between the fresh tissues and controls . the samples under static load showed a significant enhancement at 3 day , and significant differences were observed between the controls and fresh tissues . at 7 day , the staining was obviously decreased , and by 14 days , it was significantly decreased ( p < 0.05 ) and significantly different from that of the controls . * p < 0.05 versus day 0 of the same group ; p < 0.05 versus day 3 of the same group ; p < 0.05 versus controls . collagen type ii alpha 1 ihc staining intensity of the np during organ culture values are given as the mean sd . * the significance of differences between culture time point and fresh ( day 0 ) ; the significance of differences between culture time point and day 3 ; the significance of differences between static load and controls . ihc : immunohistochemistry ; np : nucleus pulposus ; sd : standard deviation , : not applicable . qrt - pcr revealed marked downregulation of aggrecan gene ( agg ) expression in the controls after 7 days compared with the fresh tissues whereas it was significantly downregulated under static load after day 0 compared with fresh tissues and controls [ figure 5 and table 3 ] . in contrast , collagen type ii alpha 1 ( col2a1 ) expression was upregulated under static load on day 3 , but tended to be downregulated under the control conditions ; this difference was statistically significant . however , col2a1 expression subsequently decreased rapidly to below detectable levels under static load until day 14 , but it was not significantly downregulated under the control conditions after 3 days ; further , statistically significant differences were observed on days 7 and 14 [ figure 5 and table 4 ] . an obvious downregulation of agg was observed in the controls after 3 days whereas a significant downregulation was observed under static load during culture compared with fresh tissue and controls . an upregulation of col2a1 was observed in response to static load by 3 days , and the expression of this gene subsequently decreased until day 14 to below detectable levels . col2a1 was downregulated in the controls by 3 days whereas no significant downregulation was observed after culturing , and significant differences were observed between the two groups after culture . the values represent the means sd normalized to fresh tissue . * p < 0.05 versus day 0 of the same group ; p < 0.05 versus day 3 of the same group ; p < 0.05 versus controls . pcr : polymerase chain reaction ; agg : aggrecan ; col2a1 : collagen type ii alpha 1 ; sd : standard deviation . agg gene expression quantified by real - time pcr during organ culture values are given as the mean sd . * the significance of differences between culture time point and fresh ( day 0 ) ; the significance of differences between culture time point and day 3 ; the significance of differences between static load and controls . agg : aggrecan ; pcr : polymerase chain reaction ; sd : standard deviation , : not applicable . col2a1 gene expression quantified by real - time pcr during organ culture values are given as the mean sd . * the significance of differences between culture time point and fresh ( day 0 ) ; the significance of differences between culture time point and day 3 ; the significance of differences between static load and controls . col2a1 : collagen type ii alpha 1 ; pcr : polymerase chain reaction ; sd : standard deviation ; : not applicable . the initial objective of this study was to investigate the effect of static load on the whole ivd motion segment in culture to establish conditions for the future study of the effect of complex dynamic loading on ivds . moreover , static loading can lead to alterations in the histological and biochemical properties of the ivds . however , the ivds are the key load - bearing components of the spine , and they are responsible for buffering shock , maintaining spinal stability , and distributing external force . therefore , it is essential to study the biology and mechanobiology of the ivds to improve the treatment of disc degeneration and lbp . all models have advantages and shortcomings depending on the specific hypotheses . because of the lack of close control and monitoring of the mechanical environment of in vivo models , it is difficult to examine specific aspects of cell signal transduction and matrix metabolism . the in vitro culturing of isolated disc cells is widely performed to analyze specific factors ; however , because they are separated from the original ecm , it is difficult to maintain the original cell properties and interactions between cells , leading to the loss of cell phenotypes . compared with the above - mentioned methods , culturing of the whole disc organ can enable the maintenance of cell - to - cell interactions , as well as interactions between cells and the ecm , which is conducive to studying the metabolism and degeneration of the discs under controlled conditions . moreover , this method is convenient for observing the responses of the ivd tissues to specific external stimuli . accordingly , lim et al . developed an in vitro organ culture model of the rat spinal motion segment and demonstrated the maintenance of viability and tissue integrity for 14 days . in previous studies , the loading was applied directly to the surface of the ivd organ , and thus , the pressure was applied through the cartilage ep or the af , leading to a specimen loading environment that significantly differed from the actual biological situation . in vivo loading is applied to the bone ep through the vbs , and then the cartilage ep is compressed , and the force is transferred to each part of the af to avoid uneven stress . the major difference between our study and the previous studies is that we tested the motion segment , which we believe enabled this ex vivo ivd organ model to more closely resemble the actual physiological state and , thus , provided a more accurate simulation of spinal loading conditions . in addition , the adjoining vbs ( as rigid fixtures ) might facilitate the application and control of complex mechanical loads and , thus , allow for the further study of ivd biomechanics . compared rabbit and rat ivd motion segments and found that glycosaminoglycans ( gags ) loss was minimal in rabbit ivds but that it was progressive and severe in rat ivds . further , the rat ivds showed increased expression of matrix metalloproteinase-3 and markedly decreased expression of collagen types i and ii compared to the rabbit ivds . accordingly , we used this motion segment as a model in the current mechanobiological study . although a decrease in col2a1 expression in the nucleus was observed in the control tissues cultured for 3 days compared with the fresh tissues , its expression was stable by 14 days , and the tissue structure was maintained during culturing . seol et al . did not detect any obvious change in the gag content in motion segments cultured for 14 days ; however , in our study , a significant decrease in proteoglycan gene expression was detected in the controls . regardless , we administered anticlotting agents before donor death and after tissue harvesting to prevent the blood clot formation as previously described . during prolonged culturing , vbs likely compromise the processing of nutrients through the ep channels , resulting in decreased biosynthetic activity . in addition , removal of the mechanical load significantly affected disc cell production by the matrix , and maintenance of the matrix content , as well as cell viability , was expected under mechanical load . numerous ex vivo studies have been performed using modified culture systems that have aimed to elucidate the effects of ivd biomechanics on the maintenance of tissue structure and function . for example , ohshima et al . developed a perfusion apparatus to use osmotic stress in lieu of mechanical stress to apply the load experienced by the discs in vivo ; in addition , lee et al . designed a chamber based on that of ohshima for long - term culturing to explore the possibility of the ex vivo culturing of large disc explants . however , the results revealed a marked decrease in cell viability after one week in the disc eps . this shortcoming might have been due to the sizes of the discs , given that the organ culturing of large animal ivds requires systemic anticoagulation . therefore , gantenbein et al . used anticoagulation treatments to avoid blood clotting in the ep capillary beds and established a bioreactor to apply diurnal loading to an ovine caudal ivd for 7 days ; jnger et al . improved this culture system by applying low - magnitude cyclic loading in addition to diurnal loading used a dynamic loading bioreactor to study the effect of more complex loading on disc degeneration . although the previous devices were applicable for analysis of the ivd organ , they were not suitable for the culturing or loading of the ivd motion segment because the middle disc is softer than both sides of the vbs ; moreover , the vertical axis was longer than the ivd organ . the segment was prone to bending deformation under loading , leading to difficult application of vertical compression and easily damaged disc tissues . therefore , we designed a loading and organ culturing apparatus for the ivd motion segment . as described previously , the use of jackscrews with pedestals enabled maintenance of the motion segment in an upright position , and the mobile loading plate ensured that full vertical loading was consistently applied on the surface of the model [ supplementary figure 3 ] . in this experiment , the osmolarity of the medium was adjusted to 410 mosm / kg to control disc swelling . a pilot experiment estimated that the weight can produce a disc compression force of 0.55 0.12 mpa . studies have shown that the ivds are subjected to pressure ranging from 0.1 to 1.1 mpa during the day and that compressive loading of over 0.8 mpa could induce early disc degeneration . because data on physiological loading magnitudes are incomplete for rabbit ivds , the loading magnitudes applied in this study were based on the physiological range of loading in humans . over the 14 days of culturing under constant static compression , the morphological integrity of the ivds gradually deteriorated after 3 days , and the proteoglycan content of the np was significantly decreased compared with that in the control tissues cultured in 6-well plates . however , the collagen type ii alpha 1 content was obviously increased at 3 days in the control tissues compared with the fresh tissues , after which it was markedly reduced . qrt - pcr analysis also confirmed that agg gene expression was significantly downregulated and significantly different than that in the controls ; however , col2a1 gene expression was upregulated at 3 days , and this trend was consistent with the results of histological and ihc analyses . these phenomena might be explained by wolff 's law , which states that an appropriate mechanical load can allow for the maintenance of tissue morphology and can stimulate bone metabolism to synthesize matrix components ; however , a persistent or excessive load inhibits these functions . ohshima et al . assessed the effect of static loading on intact bovine coccygeal discs and found that maximum tissue hydration occurred at a load of 510 kg and that lighter ( 0.5 kg ) or heavier loads ( 15 kg ) led to decreased incorporation . in the current experiment , in the presence of intradiscal pressure produced by a load of 3 kg , ihc and rt - pcr analyses revealed that the synthesis of collagen type ii alpha 1 was stimulated to resist compression within a brief period ( 3 days ) ; hence , the collagen type ii alpha 1 content was higher compared with those in the no - loading controls and fresh tissues . however , the constant static compression inhibited cell metabolic activity and led to a significant decrease in the collagen content after brief stimulation . during culture , however , the proteoglycan content did not increase . this finding might have been observed because the static load significantly inhibited the production of proteoglycans . cultured bovine coccygeal discs under a 5 kg static load and found that although np cell viability was maintained after 1 week , an approximately 50% decrease in proteoglycan synthesis occurred within 2 days of culturing . cultured models under cyclic diurnal loading ( 0.2 mpa for 6 h and 0.8 mpa for 16 h ) and demonstrated that gag synthesis was maintained after 7 days , showing that frequent loading can stimulate and maintain proteoglycan synthesis . under general conditions , the changes in the levels of col2a1 and agg are consistent ; however , neidlinger - wilke et al . found that ivds under low hydrostatic pressure ( 0.25 mpa , 30 min , 0.1 hz ) tend to exhibit increased agg expression in cell nuclei and decreased col2a1 expression . whereas the high hydrostatic pressure tended to decrease the expression of both agg and col2a1 , the author suggested that hydrostatic pressure might regulate disc matrix turnover in a dose - dependent manner . our experiment further showed that the changes in the two main matrix molecules were inconsistent up to 3 days and that the proteoglycan content was significantly decreased after 3 days but that collagen type ii alpha 1 production was briefly stimulated . the differences between our results and those of the above - mentioned studies are mainly attributed to differences in loading styles ; a constant static load can significantly inhibit the production of proteoglycans . in addition , static loading might have stronger effects on proteoglycans than on collagen type ii alpha 1 . thus , a shorter observational period will be used in a future study to test this hypothesis . future studies should aim to improve the ex vivo culture system and apparatus for application of a consistent physiologic load and complex dynamic load to maintain the functions of the model over a longer duration . in addition , the ex vivo model should be used to further examine ivd biomechanics and to observe the means through which degenerative discs can be manipulated to provide a basis for the clinical treatment of degenerative disc diseases . in conclusion , a loading and organ culture system for ex vivo rabbit ivd motion segments was developed . although degeneration was observed in the fresh ivds under the no - loading conditions , the tissue architecture and some of the matrix synthesis activity were maintained in the motion segment culture . however , constant static compression led to significant ivd degeneration , and specifically to a change in the proteoglycan content , which was markedly decreased after culturing . nevertheless , we found that static compression stimulated the synthesis of collagen type ii alpha 1 within a brief time period . thus , the results of this study indicate that an appropriate mechanical load can stimulate matrix synthesis but that constant static compression leads to progressive disc degeneration . supplementary information is linked to the online version of the paper on the chinese medical journal website . this work was supported by the grants from the special subject of scientific research of construction of the national clinical research base of traditional chinese medicine ( tcm ) by state administration of tcm ( no . jdzx2015274 ) , tcm innovation team of prevention and control osteoarticular degenerative diseases ( no . ys1304 ) , and bone and joint degenerative diseases subject research of wangjing hospital ( no . this work was supported by the grants from the special subject of scientific research of construction of the national clinical research base of traditional chinese medicine ( tcm ) by state administration of tcm ( no . jdzx2015274 ) , tcm innovation team of prevention and control osteoarticular degenerative diseases ( no . ys1304 ) , and bone and joint degenerative diseases subject research of wangjing hospital ( no .

background : the development of mechanically active culture systems helps increase the understanding of the role of mechanical stress in intervertebral disc ( ivd ) degeneration . motion segment cultures allow for preservation of the native ivd structure , and adjacent vertebral bodies facilitate the application and control of mechanical loads . the purpose of this study was to establish loading and organ culture methods for rabbit ivd motion segments to study the effect of static load on the whole disc organ.methods:ivd motion segments were harvested from rabbit lumbar spines and cultured in no - loading 6-well plates ( control conditions ) or custom - made apparatuses under a constant , compressive load ( 3 kg , 0.5 mpa ) for up to 14 days . tissue integrity , matrix synthesis , and the matrix gene expression profile were assessed after 3 , 7 , and 14 days of culturing and compared with those of fresh tissues.results:the results showed that ex vivo culturing of motion segments preserved tissue integrity under no - loading conditions for 14 days whereas the static load gradually destroyed the morphology after 3 days . proteoglycan contents were decreased under both conditions , with a more obvious decrease under static load , and proteoglycan gene expression was also downregulated . however , under static load , immunohistochemical staining intensity and collagen type ii alpha 1 ( col2a1 ) gene expression were significantly enhanced ( 61.54 5.91 , p = 0.035 ) and upregulated ( 1.195 0.040 , p = 0.000 ) , respectively , compared with those in the controls ( p < 0.05 ) . in contrast , under constant compression , these trends were reversed . our initial results indicated that short - term static load stimulated the synthesis of collagen type ii alpha 1 ; however , sustained constant compression led to progressive degeneration and specifically to a decreased proteoglycan content.conclusions:a loading and organ culture system for ex vivo rabbit ivd motion segments was developed . using this system , we were able to study the effects of mechanical stimulation on the biology of ivds , as well as the pathomechanics of ivd degeneration .

to find the relationship of periodontal status and dental caries status with oral health knowledge , attitude , behavior , among professional students in india . in a cross sectional study , a total of 825 students ( males : 577 , females : 248 ) from six professions were surveyed using a self administered structured questionnaire including 41 multiple choice questions and the who oral health assessment form ( 1997 ) . the data was analyzed using the spss version 13.0 to perform the student 's t - test , anova test , scheffe 's test and chi - square test , linear regression analysis . the mean percentage scores of the students for knowledge were 53.25 15.05 ; for attitude 74.97 20.48 ; and for behavior 59.09 18.77 . the percentage of students with calculus score was found to be significantly high ( 43.8% ) . the percentage of professional students with dmft>4 was 14.1% and the percentage of students with decayed teeth was 46.2% . the regression analysis showed that the oral health behavior of the students was dependent on the attitude ( p < 0.001 ) , but showed no significant linear relation with the knowledge . also , that the mean dmft score was dependent on the oral health behavior ( p < 0.05 ) , but showed no significant relationship with the knowledge and attitude of the students . the periodontal status was independent on the knowledge , but showed a significant relationship with attitude and behavior of the students . a positive attitude and adherence to good oral hygiene behaviors is associated with better oral health .

aimto find the relationship of periodontal status and dental caries status with oral health knowledge , attitude , behavior , among professional students in india.methodologyin a cross sectional study , a total of 825 students ( males : 577 , females : 248 ) from six professions were surveyed using a self administered structured questionnaire including 41 multiple choice questions and the who oral health assessment form ( 1997 ) . the data was analyzed using the spss version 13.0 to perform the student 's t - test , anova test , scheffe 's test and chi - square test , linear regression analysis.resultsthe mean percentage scores of the students for knowledge were 53.25 15.05 ; for attitude 74.97 20.48 ; and for behavior 59.09 18.77 . the percentage of students with calculus score was found to be significantly high ( 43.8% ) . the percentage of professional students with dmft>4 was 14.1% and the percentage of students with decayed teeth was 46.2% . the regression analysis showed that the oral health behavior of the students was dependent on the attitude ( p < 0.001 ) , but showed no significant linear relation with the knowledge . also , that the mean dmft score was dependent on the oral health behavior ( p < 0.05 ) , but showed no significant relationship with the knowledge and attitude of the students . the periodontal status was independent on the knowledge , but showed a significant relationship with attitude and behavior of the students.conclusiona positive attitude and adherence to good oral hygiene behaviors is associated with better oral health .

various intra - abdominal complications caused by ventriculoperitoneal ( vp ) shunt have been reported . among them , pyogenic liver abscess is very rare , especially the occurrence of multiple abscesses . to the best of our knowledge , only nine such cases have been reported in the english literature since the first report by fisher et al . in 19834 ) . we present another unusual case of multiple liver abscesses , review the previously reported cases , and postulate the most likely pathogenetic mechanism of multiple liver abscess formation after vp shunt . a 50-year - old woman who had been in a vegetative state for two months due to a spontaneous intracerebral and intraventricular hemorrhage was transferred to our hospital . a brain computed tomography ( ct ) scan revealed severe hydrocephalus without intracerebral hemorrhage and intraventricular hemorrhage . fifteen days later , the patient had a temperature over 38 and a chest x - ray showed patchy consolidation with partial collapse in the right lung . a sputum culture isolated pseudomonas aeruginosa and staphylococcus aureus . the patient received an initial regimen of intravenous antibiotics ( piperacillin / tazobactam and fluconazole ) , which was shifted to ciprofloxane due to a sustained high fever until the fever subsided . liver function tests yielded an alanine aminotransferase level of 115 u / l and an alkaline phosphatase level of 702 iu / l . fortunately , a chest x - ray showed gradual improvement of the right lung compared to the previous study . at that time , a subsequent abdominal ct showed multiple irregular - walled and diverse - sized cysts , including two large ones , which were not enhanced ( fig . the long segment of the distal shunt tube was found going through the subcapsular portion of the right hepatic lobe and the tip of the tube was embedded in the lower large cyst ( fig . ultrasonography - guided needle aspiration of the upper lesion and percutaneous catheter drainage of the lower lesion resulted in continuous gushing of yellowish pus . culture of the abscess isolated staphylococcus capitis ( s. capitis ) , which was sensitive to vancomycin . the patient became afebrile with the administration of intravenous vancomycin at a dose of 500 mg every 12 hours for several days . a vp shunt revision was then performed with the distal shunt tube extracted uneventfully from the liver , which was enveloped in yellowish pus . follow - up examinations have been unremarkable , with normal shunt function without the recurrence of liver abscesses . a distal shunt catheter retained within the peritoneal cavity as a foreign body can migrate to any viscera , subsequently creating various complications . we found nine cases of liver abscess associated with a vp shunt in english medline literature ; they are summarized in table 1 . in our case , a long segment of the distal peritoneal shunt tube was found passing through the subcapsular portion of the liver , and an abdominal ct showed the end tip embedded in the lower cystic cavity . although we can not explain why or how the catheter migrated to the liver and became fixed there , we postulate the most likely pathogenetic mechanism for the development of liver abscesses . the sharp and hard tip of the tube initially anchored around the liver and may have exerted repeated pressure on liver capsule ( glisson 's capsule)9 ) . combined with the water hammer effect of csf pulsation , liver perforation eventually occurred5 ) . in addition , mechanical irritation with the local inflammatory process between the liver capsule and catheter tip might also have facilitated the penetration of the shunt tube into the liver parenchyma7 ) . the tip then advanced , thereby drawing in the entire distal shunt catheter into the liver . in the review , it did not take long time to develop a liver abscess associated with vp shunt ( table 1 ) . the time interval from the last vp shunt operation to the development of a liver abscess ranged from 19 days to 3 months . exceptionally , one case was so long as much approximately two years but vp shunt infection was diagnosed approximately two weeks before the diagnosis of liver abscess9 ) . the clinical condition usually started with fever and nonspecific abdominal symptoms and continued with the development of neurologic symptoms . in our case , the diagnosis was somewhat delayed because our patient could not complain anything because of vegetative state and had only intermittent high fever . certain pathogens isolated from liver abscesses in previous cases were corynebacterium , staphylococcus albus , staphylococcus epidermidis , enterococcus faecalis , escherichia coli , and methicillin - resistant staphylococcus aureus . s. capitis is usually a nonpathogenic coagulase - negative species that is rarely associated with liver abscess . most pyogenic liver abscesses are polymicrobial , and mixed enteric facultative and anaerobic species are the most common pathogens6 ) . therefore , we presumed the liver abscesses in our case formed as a result of translocation of skin flora from the infected vp shunt rather than seeding into the liver via the portal veins due to intra - abdominal sepsis . the compromised state of our patient would potentiate this rare occurrence . of the 10 cases of liver abscesses including ours , eight cases were a single abscess and two were multiple ones ( table 1 ) . multiple liver abscesses are associated with higher mortality than a single liver abscess , especially in compromised patients1 ) . adequate treatment methods in such cases are beyond the scope of this report . although we delayed in establishing the exact diagnosis by neglecting the potential hepatic complications of a vp shunt , our patient responded favorably to the intravenous antibiotic therapy , needle aspiration , and a catheter drainage procedure . the postulated pathogenetic mechanism of liver abscess formation is migration , penetration of the infected shunt tube into the liver , and subsequent abscess formation . the compromised state of the patient will potentiate this rare occurrence . awareness and vigilance of the possibility of a liver abscess complicating vp shunting will help establishing an early accurate diagnosis and appropriate therapy .

liver abscess following ventriculoperitoneal ( vp ) shunting occurs very rarely . we report an unusual case of multiple liver abscesses caused by staphylococcus capitis in a 50-year - old compromised woman due to a complicating vp shunt infection . we reviewed the nine cases of vp shunt complications reported in the english literature , and speculated that the most likely pathogenetic mechanism in our case is an infected peritoneal tip that migrated to and penetrated the liver , which subsequently caused the formation of multiple liver abscesses . the patient was successfully treated with percutaneous aspiration , drainage of the abscesses , intravenous antibiotics , and shunt revision . awareness and vigilance of the possibility of liver abscess formation caused by vp shunt infection will help establish an early accurate diagnosis and therapeutic strategy .

we measured the height of the lesion by using the lesion s arc ( ) obtained from the difference between the angle of flexion at the beginning of the lesion and the angle of flexion at the end of the lesion . these records must be taken the same way for each arthroscopy and for each investigator . the lower limb was placed in an arthroscopic leg holder with the thigh horizontal ; the standard 4.5-mm arthroscope ( 30 tip angulation , wide - angle lens ) was introduced through a portal inferolateral or inferomedial to the patella . for the reference position , the scope was placed on the homolateral anterior meniscal horn , pointed horizontally toward the damaged condylar , and remained solidly against the tibia during movements . the knee was completely extended and gently flexed to 120 , the reading being done on the equator of the screen . when the anterior edge of the lesion appeared , the flexion angle of the knee was recorded visually on the lateral side of the limb ( a ) , and then , when the posterior edge of the lesion was on the equator of the screen , the second angular position was recorded ( b ) ( fig . the lesion s arc was obtained by the difference ( ) between these two positions ( b a ) . the angles of flexion a and b give information regarding the anterior - posterior location of the condylar lesion . arthroscopic measurement of the angle of the lesion in the laboratory : on the equator of the screen , the anterior border of the lesion is at 30 ( a ) and the posterior border is at 70 ( b ) . we validated this principle in the anatomical laboratory at the university of rennes , france . in the first series , the donor ages were 51 , 62 , 64 , 72 , and 77 years , with no degenerative knees on radiograph . the thigh was securely fixed in a leg holder with the leg free of flexion / extension movement . 4 quadrangular artificial lesions , grade 3c on the icrs scale , were carried out with a curette , under scope view , by an investigator not involved in the work . two lesions were created on the medial and two on the lateral condyles thus 4 defects in 1 knee and 20 defects in the first series . each lesion had been randomly evaluated using the lesional arc checked visually by 2 trained surgeons in arthroscopy ( jcl : observer 1 , hr : observer 2 ) . each measurement was randomly repeated after an interval of 1 hour , thus evaluating the intra- and interinvestigator reliability . then we did a second series of 20 defects in 5 other knees ( 54 , 56 , 63 , 78 , 89 years old ) , 1 month later , and as before , all the specimens had a bmi below 35 . again the flexion angles of the knees were measured using a goniometer solidly fixed on the lateral side of the thigh and the leg , the center of the goniometer superimposed on the epicondylar ( fig . were then disarticulated , and the true lesion arc was checked with a goniometer to assess the validity of the arthroscopic measurements . the center of the goniometer was superimposed on top of the epicondylar , and the lesion arc was read between the condylo - trochlear line ( point 0 ) and the posterior condyle ( point 90 ) . each actual lesion s arc directly measured on the disarticulated knee was compared with the previous results obtained by the 2 arthroscopists . second series : lateral view of the knee with the goniometer fixed on the lateral side of the knee . the lesion s arc ( ) obtained experimentally has to be converted into height ( mm ) for a clinical application . for this , we determined , from 30 lateral x - rays in scale 1/1 , 5 standard profiles with an increase of 3 mm . for each lateral x - ray , we defined the condylar center according to the methodology of grood and suntay for the description of the kinematics of bones . then we measured the length of the arc of flexion from 0 ( condylo - trochlear point ) to 90 and the length of each arc of 10 by using the orthogon software ( agfa healthcare , greenville , sc ) ( fig . 3 ) . thus , we could obtain an abacus for the calculation of the height of the lesion ( mm ) for these 5 standard profiles ( a to e ) . on x - ray , the center of the circle inscribed in the profile of the medial condyle allows an arc to be defined from 0 to 90 , with each arc length of 10 measured with the orthogon software . thus , we could obtain an abacus for the calculation of the height of the lesion ( mm ) for 5 standard profiles ( a to e ; see also fig . . the intraobserver reliability of the measurements of these angles was conducted using the bland and altman test limit of agreement ( see figs . 4 and 5 ) . the principle of the bland and altman test is to compare the difference between a pair of measurements ( e.g. , between 2 observers or between an observation and a reference ) against their mean values . if the measurements are comparable , the differences should be small , be centered around 0 , and show no systematic variation with the mean of the measurement pairs . for the analysis of agreement between 2 investigators , we chose the kappa coefficient . analyses of reproducibility within the observers for the 2 techniques were tested with all angular measurements of each anterior and posterior limit of the lesions ( i.e. , 2 per lesion ) . these measurements were transformed into categorical data for kappa agreement by rounding up the value to intervals of 10 ranging from 0 to 90 ( 010 , 1120 , 2130 , etc . ) , which seemed to be an acceptable error margin within the experience of the investigators . as each lesion was widened by the instrument once during the experiment , only half of the lesions were available for reading after disarticulation of the knee . the kappa coefficient calculated between the measurements of the 2 investigators is .66 , indicating poor agreement . the kappa coefficient calculated between the measurements of the 2 investigators is .78 , indicating good agreement . we measured the height of the lesion by using the lesion s arc ( ) obtained from the difference between the angle of flexion at the beginning of the lesion and the angle of flexion at the end of the lesion . these records must be taken the same way for each arthroscopy and for each investigator . the lower limb was placed in an arthroscopic leg holder with the thigh horizontal ; the standard 4.5-mm arthroscope ( 30 tip angulation , wide - angle lens ) was introduced through a portal inferolateral or inferomedial to the patella . for the reference position , the scope was placed on the homolateral anterior meniscal horn , pointed horizontally toward the damaged condylar , and remained solidly against the tibia during movements . the knee was completely extended and gently flexed to 120 , the reading being done on the equator of the screen . when the anterior edge of the lesion appeared , the flexion angle of the knee was recorded visually on the lateral side of the limb ( a ) , and then , when the posterior edge of the lesion was on the equator of the screen , the second angular position was recorded ( b ) ( fig . the lesion s arc was obtained by the difference ( ) between these two positions ( b a ) . the angles of flexion a and b give information regarding the anterior - posterior location of the condylar lesion . arthroscopic measurement of the angle of the lesion in the laboratory : on the equator of the screen , the anterior border of the lesion is at 30 ( a ) and the posterior border is at 70 ( b ) . we validated this principle in the anatomical laboratory at the university of rennes , france . in the first series , the donor ages were 51 , 62 , 64 , 72 , and 77 years , with no degenerative knees on radiograph . all the specimens studied had a body mass index ( bmi ) below 35 . the thigh was securely fixed in a leg holder with the leg free of flexion / extension movement . 4 quadrangular artificial lesions , grade 3c on the icrs scale , were carried out with a curette , under scope view , by an investigator not involved in the work . two lesions were created on the medial and two on the lateral condyles thus 4 defects in 1 knee and 20 defects in the first series . each lesion had been randomly evaluated using the lesional arc checked visually by 2 trained surgeons in arthroscopy ( jcl : observer 1 , hr : observer 2 ) . each measurement was randomly repeated after an interval of 1 hour , thus evaluating the intra- and interinvestigator reliability . then we did a second series of 20 defects in 5 other knees ( 54 , 56 , 63 , 78 , 89 years old ) , 1 month later , and as before , all the specimens had a bmi below 35 . the flexion angles of the knees were measured using a goniometer solidly fixed on the lateral side of the thigh and the leg , the center of the goniometer superimposed on the epicondylar ( fig . all 10 knees ( 5 in each series ) were then disarticulated , and the true lesion arc was checked with a goniometer to assess the validity of the arthroscopic measurements . the center of the goniometer was superimposed on top of the epicondylar , and the lesion arc was read between the condylo - trochlear line ( point 0 ) and the posterior condyle ( point 90 ) . each actual lesion s arc directly measured on the disarticulated knee second series : lateral view of the knee with the goniometer fixed on the lateral side of the knee . the lesion s arc ( ) obtained experimentally has to be converted into height ( mm ) for a clinical application . for this , we determined , from 30 lateral x - rays in scale 1/1 , 5 standard profiles with an increase of 3 mm for each lateral x - ray , we defined the condylar center according to the methodology of grood and suntay for the description of the kinematics of bones . then we measured the length of the arc of flexion from 0 ( condylo - trochlear point ) to 90 and the length of each arc of 10 by using the orthogon software ( agfa healthcare , greenville , sc ) ( fig . thus , we could obtain an abacus for the calculation of the height of the lesion ( mm ) for these 5 standard profiles ( a to e ) . on x - ray , the center of the circle inscribed in the profile of the medial condyle allows an arc to be defined from 0 to 90 , with each arc length of 10 measured with the orthogon software . thus , we could obtain an abacus for the calculation of the height of the lesion ( mm ) for 5 standard profiles ( a to e ; see also fig . the intraobserver reliability of the measurements of these angles was conducted using the bland and altman test limit of agreement ( see figs . 4 and 5 ) . the principle of the bland and altman test is to compare the difference between a pair of measurements ( e.g. , between 2 observers or between an observation and a reference ) against their mean values . if the measurements are comparable , the differences should be small , be centered around 0 , and show no systematic variation with the mean of the measurement pairs . for the analysis of agreement between 2 investigators , we chose the kappa coefficient . analyses of reproducibility within the observers for the 2 techniques were tested with all angular measurements of each anterior and posterior limit of the lesions ( i.e. , 2 per lesion ) . these measurements were transformed into categorical data for kappa agreement by rounding up the value to intervals of 10 ranging from 0 to 90 ( 010 , 1120 , 2130 , etc . ) , which seemed to be an acceptable error margin within the experience of the investigators . as each lesion was widened by the instrument once during the experiment , only half of the lesions were available for reading after disarticulation of the knee . the kappa coefficient calculated between the measurements of the 2 investigators is .66 , indicating poor agreement . the kappa coefficient calculated between the measurements of the 2 investigators is .78 , indicating good agreement . for the first series of tests , 20 artificial lesions were evaluated without a goniometer ( table 1 ) : the range for the 95% limit of agreement for the intraobserver analysis showed confidence intervals from 21.82 to 22.32 for observer 1 and 19.36 to 16.36 for observer 2 . means of differences were respectively 0.25 and 1.5 , which shows little bias . in each case , agreement was good . study without the goniometer note : cm1 = first lesion in the medial condyle ; cm2 = second lesion in the medial condyle ; cl1 = first lesion in the lateral condyle ; cl2 = second lesion in the lateral condyle . observer 1 , 1st = first registration by observer 1 ; observer 1 , 2nd = second registration by observer 1 ; observer 2 , 1st = first registration by observer 2 ; observer 2 , 2nd = second registration by observer 2 . for the second series , 20 artificial lesions were evaluated with a goniometer ( table 2 ) . we found a confidence interval from 8.9 to 8.1 and 6.94 to 7.94 for observers 1 and 2 , respectively . as the confidence interval was narrower with series with the goniometer for the 2 observers , we can approximately say that intraobserver reproducibility error was smaller with the goniometer . study with the goniometer note : cm1 = first lesion in the medial condyle ; cm2 = second lesion in the medial condyle ; cl1 = first lesion in the lateral condyle ; cl2 = second lesion in the lateral condyle . observer 1 , 1st = first registration by observer 1 ; observer 1 , 2nd = second registration by observer 1 ; observer 2 , 1st = first registration by observer 2 ; observer 2 , 2nd = second registration by observer 2 . for the series without the goniometer , kappa coefficient was evaluated at 0.66 , which could be classified as poor concordance . calculation for the series with the goniometer showed the kappa coefficient at 0.78 , with concordance between observers for this series thus being good . comparison to anatomical lesions , available only for half of the measurements , showed for both observers , for the first series , without goniometer , a 95% confidence interval from 27.69 to 21.32 and the mean of these differences around 3.19 ( fig . agreement was good , and bias was small . with the goniometer , in the second series , the confidence interval of the mean limits of agreement was from 16.69 to 10.81 ( fig . compared to anatomical lesions , we can hypothesize that reproducibility errors were lower with than without the goniometer . measurement of the lesion arc , without using the goniometer , after disarticulating the knee . measurement of the lesion arc , when using the goniometer , after disarticulating the knee . these results show that intra- and interobserver reliability and concordance between observers of reading angular limits of chondral lesions were better with the goniometer . the use of the abacus is relatively easy from the lesional arc and a strict x - ray profile of the patient s knee . the height or longitudinal dimension in millimeters of the lesion is obtained by subtraction of angle b and angle a on the abacus . for example , in the case of a lesion extending from 20 to 60 on a condyle sized c , the length of the arc is 44 14.6 = 29.4 mm ( see fig . ( a ) abacus for calculating lesion height based on condyle size ( a to e ) and lesion arc . ( b ) lateral x - rays of the 5 knees sized from a to e. for a scale 1/1 , the d segment has to measure 12 mm long . the patient s x - ray ( scale 1/1 ) is superimposed on the drawing to determine its size relative to the standard 5 sizes ( a to e ) . the main result of this work is to propose a method for measuring the height of condylar chondral lesions under arthroscopy from a lesion s arc defined by its anterior and posterior limits . for wilson et al . , the arthroscopic estimate of the extent of a lesion is considered the gold standard , but for oakley et al . , it can contain considerable errors . several authors have suggested their own techniques but without experimental validation of their accuracy . the determination of the surface area in the case of a focal defect is important for the choice of treatment ( microfracture , osteochondral graft , chondrocyte implantation ) . in the case of knee osteoarthritis , it is more important to know the percentage of cartilage damage compared to the total surface of the knee cartilage than in a localized lesion . in this instance , the french arthroscopic society composite scoring system proposed by ayral et al . in 1993 it is based on the combination of the beguin and locker classification for the severity and the percent area estimates for the size ; each compartment ( medial or lateral femorotibial , femoropatellar ) has a score between 0 ( normal cartilage ) and 100 points ( major chondropathy ) . the intraobserver reliability of percent area estimates was evaluated by examination of arthroscopic video recordings and was found to be good , but interobserver reliability was poor , explaining why a single trained observer is used to reread the videos . hunt s score also uses an evaluation system in percentage terms of each functional lesioned zone , but even the author states that the reliability and accuracy are both insufficient . these 2 composite scores require recording percent area estimates for each grade of chondropathy . in a plastic knee model , oakley et al . found that the accuracy and reliability of arthroscopic percent area estimates on the femoral surfaces were generally poor , and there was a clear trend to poorer reliability with larger lesions . it appears to us to be difficult in daily practice , as well as inaccurate for femoral condyles , due to their convexity and their large surface area . the use of variable - angle elongated probes especially designed for measurement of lesion diameter is one possible approach that has not been evaluated to date , to our knowledge . we have no direct experience of the commercially available flexible intra - articular rulers ( arthrex , naples , fl ) , and although their use is a possibility , they are actually single - use instruments . however , there are no data regarding reliability and validity of arthroscopic methods of localization ( e.g. , comparison of arthroscopy and anatomic dissection ) . our experimental study led to the development of an abacus based on radiological findings and arthroscopic measurements of the lesion arc . in the operating room , the width or transverse dimension is measured by a long graduated hook ( 0 - 20 mm ) inserted through the homolateral portal . the depth is estimated with a short angulated probe , of known size , according to the icrs score in 4 grades , and the height is obtained from the lesion s arc with the table ( fig . the lesion surface , similar to a quadrangular dome , is obtained by the following formula : width radius /180 . this formula does not take into account the spiral in the center of the curvature ( evolved fick curve ) . in practice , we can simply multiply the width by the height to calculate the lesion surface area assimilated to a rectangle after having chosen the exact size of the patient femur by superimposing a printout of figure 8b on the lateral x - ray ( scale 1/1 ) . figure 8c shows the superimposition of patient x - ray and trace d which best fits this case . the recording of the lesion s arc can also be used to locate the lesion on the icrs map . the location of the condylar lesion between 0 and 90 can be plotted on the 3 areas : anterior , middle , and posterior . there are two limitations of the work : first , we did not measure the height of the lesion in millimeters after dislocating the knees to validate the abacus , only the lesion arc . this was because we did not have at our disposal at that time a flexible and curved ruler , for example , those manufactured by the orteq ( london , uk ) . the technique of measuring the lesion s arc is reproducible , with care taken to respect the conditions required when taking records and using a standard goniometer , solidly fixed on the lateral side of the knee . care must be taken not to move the stationary arms of the goniometer when taking readings . by using the x - ray lateral view of the knee with the abacus , we can obtain the height , and a simple formula allows the calculation of the size of the lesion ( width height ) . preoperatively , the three - dimensional mr mapping of cartilage surface will permit the accurate measurement of focal defects , but arthroscopy remains the standard for assessing small or superficial lesions and the physical properties of cartilage . we propose a simple , reliable method to measure the height of a condylar lesion with the lesion s arc during arthroscopy . this method provides the height of the lesion from a simple - to - use abacus .

purpose : to present a new method of arthroscopic measurement of the surface and location of condylar lesions.methods:we propose measuring the height of the condylar lesion by using the lesion s arc ( ) obtained from the difference between the angle of flexion at the beginning of the lesion and the angle of flexion at the end of the lesion . the first goal of the study was to determine the intra and inter reliability of the lesion s arc . experiment 1 : 20 deep lesions were evaluated using the lesional arc by two arthroscopists . experiment 2 : in a second series of 20 lesions , the flexion angles of the knees were recorded using a goniometer . all 10 knees ( 5 in each series ) were then disarticulated and the true lesion arc was checked with a goniometer to assess the validity of the scopic measurements . the second goal was to obtain the height of the lesion from the lesion s arc . the lesion arc of the condylar is converted into height ( millimeters ) on the basis of a table obtained from 5 standard profiles of the lateral x - ray of the knee.results:experiment 1 : the intra observer reliability was good but the inter observer reliability was poor . experiment 2 : the intra and inter observers reliability were good . on the anatomic control after disarticulating the knee , the confidence interval was narrower when using the goniometer.conclusions:we propose a simple , reliable method to measure the height of a condylar lesion with the lesion s arc during arthroscopy .

basic and instrumental activities of daily living are tasks required to function on a daily basis , and are often impacted by disease processes that reduce cognitive ability such as alzheimer 's disease ( ad ) . basic activities of daily living ( adls ) include core tasks of everyday life such as eating , dressing , grooming , and bathing while instrumental activities of daily living ( iadls ) include more complicated , higher - level , tasks such as preparing meals , managing finances , shopping , doing housework , and using the telephone . driving and medication management are other iadls that are significantly disturbed in patients with ad . while both adls and iadls are impacted by ad , iadls are the first to decline and the level of functional impairment is the core clinical distinction between ad and milder conditions such as mild cognitive impairment ( mci ) . classification of mci subtypes , suggest that mci - amnestic type and mci patients with multiple - domain impairments are more predictive of later conversion to dementia and are more impaired functionally compared to mci of nonamnestic and single domain subtype . patients with ad experience a gradual loss in the abilities to live independently due to impairments in cognitive and memory functioning [ 1 , 3 ] and , as the disease progresses , the ability to carry out these essential activities eventually disappears . although there is not a large body of research on the relationship between neuropsychological measures and functional activities , several studies suggest that neuropsychological test performance is predictive of complex adls and iadls in elderly neuropsychiatric patients and those with ad [ 1 , 3 , 6 , 7 ] . evans found that performance on neuropsychological evaluations predicted functional capacity beyond negative symptoms in elderly with schizophrenia . however , these authors were unable to identify specific cognitive domains that impacted functional impairment . to date , most studies have not utilized comprehensive and conceptually sound measures to identify specific cognitive domains to predict particular areas of daily functioning . the limited existing literature suggests a correlation between objective neuropsychological assessment and informant reported level of functioning . this relative dearth of literature may be attributable to the difficulty in accurately measuring everyday functioning . trained observer ratings of functional level may be the gold standard but are very time consuming and impractical in outpatient settings . loewenstein et al . found that family members ' report of functional impairment is extremely accurate when compared with objective functional performance and is a useful mechanism to assess functioning . the present study evaluated the link between specific neurocognitive measures and informant report of adls and iadls in patients diagnosed with mild ad . as recommended by beck et al . , we address challenges in the current literature by utilizing a comprehensive neuropsychological battery to predict daily functioning in this clinical population . to our knowledge no research has addressed the possibility of gender - related differences in the cognitive mechanisms required for select areas of daily functioning . for instance , women inherently expend more effort than men in the area of dressing and grooming , which implies increased cognitive effort . the majority of studies have dealt with possible gender differences by covarying for gender , which tends to obscure any meaningful relationships that may be gender specific . our study directly evaluates gender differences in the ability of specific neuropsychological tests and cognitive domains to predict functioning . the total sample consisted of 202 ( 91 male , 111 female ) participants who met criteria for ad enrolled in the longitudinal research cohort of the texas alzheimer 's research consortium ( tarc ) . to reduce the effect of stage of decline on these measures , only individuals with a clinical dementia rating scale global score of 1.0 were included in the sample . collateral information on adls and iadls ratings was obtained from immediate caregivers who predominately were family members ( spouse and or children ) using the lawton - brody rating scales . briefly , the tarc project is a longitudinal multisite study of a cohort of ad patients and normal controls where each participant undergoes an annual evaluation that includes a medical examination , interview , neuropsychological testing , and blood draw . ad patients met consensus - based diagnosis for probable ad based on nincds - adrda criteria . male participants were 56 to 92 years of age ( m = 74.36 , sd = 8.21 ) , and females participants were 54 to 92 ( m = 76.95 , sd = 7.74 ) . the majority of participants were caucasian ( 98% ) , black or african american ( 1.5% ) was the next largest group . the tarc project received institutional review board approval , and all participants and/or caregivers provided written informed consent . the tarc neuropsychological core battery consists of the following instruments : wechsler digit span , logical memory , and visual reproduction , trail making test a & b , clock drawing test ( cdt ) , boston naming test , the geriatric depression scale ( gds-30 ) , and the clinical rating scale ( cdr ) . verbal memory was assessed with the wechsler logical memory i ( lm i ) and wechsler logical memory ii ( lm ii ) , visual memory was assessed with the wechsler visual reproduction i ( vri ) and ii ( vrii ) , attention was evaluated by performance on trails a and total digit span , linguistic capacity was assessed with boston naming test ( bnt ) and verbal fluency ( fas , category naming ( cowat ) ) , measures of executive functioning in this battery included the cdt and trails b. cognitive evaluation was administered in a controlled setting according to standardized instructions . in order to equate scores from digit span and story memory scales , all raw scores were converted to scale scores based on previously published normative data . for the boston naming test , the current group recently conducted an independent study that demonstrated the psychometric properties of an estimated 60-item bnt score that can be calculated from 30-item versions . descriptive statistics and one - way anova comparison of male and female samples ( presented in table 1 ) were conducted using spps version 17.0 . stepwise regression modeling was used to evaluate the link between each test of cognitive function and adls and iadls . independent variables were caregiver ratings on the physical self - maintenance scale for adls and the personal self - maintenance scale for iadls . each item has five descriptors from total independence to total dependence or total loss of functional control . the adls assessed were toileting , feeding , dressing , grooming , ambulation , and bathing . the iadls assessed were telephone use , shopping , food preparation , housekeeping , transportation , laundry management of medications and finances . logical memory i ( lmi ) and performance on cdt were significant predictors of iadls ( see table 2 ) . both lmi ( t = 2.34 , p = .022 ) and cdt ( t = 3.78 , p < .0001 ) significantly predicted total iadl score ( lmi / cdt combined ; t = 14.38 , p < .0001 ; effect size ( r = 0.26 ) . evaluation of specific iadls suggested that executive functioning ( i.e. , cdt ) predicted independence with medication management ( t = 12.23 , p < .0001 ; r = .32 ) , transportation ( t = 9.03 , p < .0001 ; r = .22 ) , laundry ( t = 7.67 , p < .0001 ; r = .22 ) , and housekeeping memory and learning capacity ( lmi ) significantly predicted ability to independently manage finances ( t = 11.11 , p < .0001 ; r = .24 ) , shop ( t = 12.70 , p < .0001 ; r = .30 ) , and telephone use ( t = 12.63 , p < .0001 ; r = .32 ) . measures of lmi and cdt ( t = 11.36 , p < .0001 ; r = .45 ) individually predicted food preparation ability . apoe4 status was excluded from stepwise regression modeling and did not impact the level of iadl functioning in women and men with mild ad . total adl scores were predicted by score on the cdt ( t = 14.38 , p < .0001 ; r = .26 ) . likewise , cdt score predicted the ability to bath ( t = 10.34 , p < .0001 ; r = .24 ) , groom ( t = 10.34 , p < .0001 ; r = .20 ) , and feed / eat ( t = 18.63 , p < .0001 ; r = .28 ) . trails a predicted the patient 's ability for self - dress ( t = 11.18 , p < .0001 ; r = .20 ) as demonstrated by table 3 . apoe4 status was excluded from stepwise regression modeling and did not impact the level of adl functioning in women and men with mild ad and as a result was not included in gender analyses . significant predictors of the ability of female ad patients to perform iadls included lmi , lmii , cdt , and the bnt . total iadl score was predicted by performance on both lmi ( t = 3.73 , p < .0001 ) and cdt ( t = 2.15 , p = .035 ) with combined value of ( t = 12.48 , p < .0001 ; r = .49 ) . the cdt alone significantly predicted independent functioning in the area of medication management ( t = 10.76 , p < .0001 ; r = .38 ) and housekeeping ( t = 8.35 , p < .0001 ; r = .36 ) . performance on lmi significantly predicted financial management ( t = 10.10 , p < .0001 ; r = .41 ) and the ability to shop independently ( t = 11.50 , p lmi and lmii significantly predicted telephone use ( t = 11.25 , p < .0001 ; r = .52 ) and performance on the bnt alone predicted transportation or driving capacity ( t = 9.04 , p < .0001 ; r = .25 ) in women with ad ( see table 4 ) . levels of adl functioning in women was predicted by performance on the cdt and trails a. performance on cdt predicted total adl score ( t = 11.55 , p < .0001 ; r = .27 ) and bathing capacity ( t = 8.37 , p < .0001 ; r = .26 ) . ability to eat / feed ( t = 14.72 , p < .0001 ; r = .32 ) independently was predicted by performance on trails a. performance on measures of vri ( t = 2.74 , p = .010 ) , vrii ( t = 2.29 , p = .029 ) , trails b ( t = 2.74 , p = .010 ) , and cowat ( t = 2.56 , p = .015 ) predicted medication management capacity in men ( t = 4.67 , p < .0001 ; r = .27 ) . food preparation was predicted by performance on the bnt ( t = 5.18 , p < vri , trails a , bnt , trails b , and cdt were all significant predictors of level of adl functioning in men with ad ( see table 5 ) . = .35 ) was significantly predicted by vri and trails a. performance on vri predicted bathing ability ( t = 6.26 , p < .0001 ; r = .41 ) and when combined with trails b also predicted grooming capacity ( t = 5.66 , p < .0001 ; r = .51 ) . the ability to eat / feed independently was predicted by bnt and cdt ( t = 16.65 , p < .0001 ; r = .56 ) . previous research has shown that cognitive functioning , as assessed by neuropsychological tests , is the strongest predictor of functional impairment [ 6 , 7 ] . specific cognitive domains of executive functioning , praxis / visuospatial skills , and memory have been found to be useful for predicting adl and iadl in assisted - living elders . our findings are consistent with previous research and demonstrate a significant relationship between performance of daily living activities and neurocognitive performance . unlike other studies , we found that attention is an important predictor of adls in ad patients . prior studies have been conducted in assisted care facilities where caregiver assistance may be sufficient to overcome inattention . however , individuals with mild dementia seen as outpatients in our study were likely responsible for basic adls , and , thereby , attention was necessary to facilitate functioning . memory and learning ( lmi , lmii , vr i , vrii ) , executive functioning ( cdt , trails b ) , and language ( bnt , cowat ) were significant predictors of adls and iadls . among the measures administered , cdt , lmi , and trails a were predictive of both adl and iadl functioning in analysis of the total sample . whereas prior reports suggested that cognitive abilities are most predictive of complex tasks of everyday functioning [ 15 , 16 ] , our results suggest that cognitive test performance also predicts basic adls ( e.g. , bathing , grooming , dressing , and feeding ) . this suggests that even in patients with mild ad , basic adls likely also require complex cognitive processes . another intriguing finding is that the presence of apoe4 genotype was not predictive of level of iadl and adl functioning in the current sample . presence of particular apoe genotype has been associated with greater disability in prior research with patients with mci . the current data suggests that the presence of apoe was not significantly associated with level of functioning in patients who have converted to ad status . we anticipated gender differences because there are ( a ) differences in task performance and ( b ) differences in strategies used to perform adls and iadls . for instance , it has been documented that women tend to use landmarks when driving and given directions where men are more likely to use street names [ 9 , 18 ] . in our research we administered several measures within each cognitive domain to facilitate understanding of not only which domain is different for male and female but also which specific measure best predicts functioning . figure 1 demonstrates gender differences in adls and iadls with regard to specific cognitive domains assessed . language also predicted iadl functions in men ( food preparation ) and women ( driving ) . gender differences remained in the domain of learning and memory , suggesting that men rely on this process for both adl and iadl whereas it is only predictive of iadl functions in women . a notable gender difference is that cognitive functioning is generally a better predictor of adl and iadl functioning for women compared to men . while the administered assessment battery predicted practically all daily tasks for women , it only predicted a few specific ones for men . this may be due in part to the likelihood that men especially of the generation in our sample are less likely to be involved in cooking , shopping , housekeeping and laundry and hence have little variability . for men , the iadl of medicine management was the only area predicted by performance on several different cognitive measures . among adls , only bathing , grooming , and feeding capacity was significantly predicted by vri , trails a and trails b , bnt , and the cdt . however , in women , the cdt , verbal learning and memory ( lmi and lmii ) and language were good predictors of capacity to perform almost all adls and iadls . these findings suggest that men tended to depend on visual learning and visual memory and women on verbal learning and verbal memory . lmi and lmii were predictive of functioning for women , whereas vri and vrii were predictive for men . one could speculate that women tend to problem - solve verbally using self - talk whereas men tend to conceptualize visually . the cdt appears to be a good measure in predicting functioning for women , but not for men . the cdt is typically seen as a measure of executive functioning and of frontal lobe processes . the clinical utility of the clock drawing test has been documented for diagnosing patients with dementia , but its relationship to specific functional activities has not been reported . the generalizability of our findings suffers from the relatively small sample size and the nature of subject recruitment . the current study is one of the first to examine gender differences , and efforts to replicate these findings is warranted due to several sample limitations that include lack of racial diversity and differences in educational levels and age among men and women in this sample . though it is unlikely these factors negate current results , it would be best to stratify according to education and age in future studies . additional studies with larger more representative samples are needed to further assess the impact of gender on predicting functioning . although late - life depression could impact cognitive functioning , gender differences were not significant ( table 1 ) in our sample , and , therefore , depression score was not accounted for in the analyses . however , effects of depression are significant and warrant future efforts . although the current research has its limitations this study has several advantages over earlier studies in terms of understanding patients with ad . first , the sample was limited to individuals with mild ad which helps control for the affect of disease severity on functional activities . third , in addition to evaluating predictive value of specific domains it also evaluated specific measures within those domains in both gender - specific and mixed - gender analyses . the findings of this study underscore the importance of gender and the gender - specific relations of neurocognitive measures to everyday activities . acknowledging gender differences is important as it may facilitate more accurate interpretation of neurocognitive tasks and its relationship to particular daily living activities . these findings also have clinical values for making informed decisions and recommendations of capacity in patients with ad . there is generally consensus that executive functioning is an important predictor of capacity to perform complex tasks ( iadls ; 15 ) . while this may be accurate for women , current findings suggest that it may be an irrelevant predictor for men . understanding how to most accurately predict level of function will also enable patients to maintain daily functions longer , reducing caregiver fatigue , and also social and economic burden .

objectives . to investigate the link between neurocognitive measures and various aspects of daily living ( adl and iadl ) in women and men with mild alzheimer 's disease ( ad ) . methods . participants were 202 ad patients ( 91 male , 111 female ) with cdr global scores of 1 . adls and iadls ratings were obtained from caregivers . cognitive domains were assessed with neuropsychological testing . results . memory and executive functioning were related to iadl scores . executive functioning was linked to total adl . comparisons stratified on gender found attention predicted total adl score in both men and women . attention predicted bathing and eating ability in women only . language predicted iadl functions in men ( food preparation ) and women ( driving ) . conclusions . associations between adls / iadls and memory , learning , executive functioning , and language suggest that even in patients with mild ad , basic adls require complex cognitive processes . gender differences in the domains of learning and memory area were found .

the formation of the protein shell of viruses has , due to its relative simplicity and importance in many diseases , become one of the most well - studied examples of self - assembly . although viruses are typically assembled within the cells of their host , the process may also be triggered in a bulk solution of viral proteins by changing the ph . such experiments have stimulated the application of simple computational models to help understand assembly processes . while much modeling has focused on the formation of virus capsids in the bulk , in recent work investigating the growth of viral shells around their genome , the assembly of simple subunits attracted to a flexible polymer was simulated . interaction with the polymer was found to allow assembly for parameters for which it would otherwise not occur . encapsulation of spherical nanoparticles has also been considered both in experiment and in simulation . experimentally , it was demonstrated that shells resembling different types of viral particles could be assembled by varying the nanoparticle diameter . beyond interactions with an encapsulated genome , there is also much evidence that membranes play an important role in assembly for many viruses . in a recent publication , we presented results on the effect of fluctuating membranes on the equilibrium of a system of self - assembling patchy colloids , designed to assemble viral core - like structures , from monte carlo ( mc ) simulations . we found a nonmonotonic dependence of the promotion of assembly on membrane stiffness , as well as the formation of membrane buds . it is of course true that such effects would be observable in an analogous experimental system after sufficient time and to be expected that they will influence the products of dynamical assembly . however , on relevant time scales , self - assembly processes may not reach equilibrium and the products may be affected , for example , by kinetic traps . key dynamical features that we capture in our simulations are the viscosity of the membrane and hydrodynamic interactions , the inclusion of which may alter dynamics both quantitatively and qualitatively . two key factors in the present work are attractions to the fluctuating membrane and hydrodynamic interactions . previous computational studies have looked into the effects of each of these individually on the clusters formed by isotropic spherical colloids . hydrodynamic interactions were found to change both the size and shape of clusters , while attraction to a membrane was found to induce the formation of linear chains on the surface . further , attractions of particles to a membrane surface may cause the formation of buds or tube - like structures . here , as a simple model to gain insight into the effect of membranes on the dynamics of self - assembly , we consider primarily the same , patchy - particle , subunits , which may assemble 12-component cores , as in our previous work , and simulate their assembly using a dynamically realistic method . as previously , our subunits are coupled to a membrane modeled using particles bonded to form a triangulated surface . the target core structure has icosahedral symmetry , similar to many viruses , although in reality enveloped viruses are larger . we describe our simulation models and in section 3 we present results from mc simulations on the equilibrium of the system . we then move on to dynamical simulations , describing simulation methods in section 4 . we present results for the 12-component cores in section 5 and compare them to some results for other cores in section 6 . rather than only considering enough subunits to form just one target structure as in our previous work , we now simulate 180 , allowing a maximum of 15 complete cores to be assembled . while it is expected that in experimental and biological situations it is also likely that a larger number of subunits will be available than required for one complete structure , this choice was additionally made for computational efficiency , so that , on a feasible time scale , although assembly of all possible cores may not occur , some complete cores will form . the interactions between subunits , ss , and between subunits and membrane particles , ms , are identical to those used in our previous work but we describe the important features again here . as shown in eq 1 , the potential is split into attractive , uatt , and repulsive , urep , parts . the interaction of two particles , i and j , separated by rij ( i j ) , with orientations i and j , either both subunits or a subunit and a membrane particle , is given by1where the forms of uatt and urep are shown in figure 1b . area , att and orient are dimensionless factors that take different forms for ss and ms interactions . for ss - interactions , area = att = 1 and , as depicted in figure 1a , there are 5 patches on each subunit , which are arranged symmetrically around a single ms patch . orient is used to control the patch width , and it has the form of a product of three functions of the form shown in figure 1c , see also the supporting information . for the first two factors , the argument is the angle between the interacting patches and the center - to - center vector , rij . the parameters for determining patch width , see figure 1c , are set to 0 = 1 = 0.2 . in contrast , for the third factor , the argument is the angle between the projections of the membrane patches onto the plane perpendicular to rij and 0 = 1 = 0.4 . subunits , which are all identical , are rendered in yellow , with positions , but not extents , of patches for interactions with other subunits in red . the particles forming the surface edge are confined to a frame region , which is located at a distance rframe from the periodic boundaries . in simulations with hydrodynamics , a stochastic rotation dynamics ( srd ) solvent composed of point particles interactions between srd particles are effected by first dividing the entire system into a grid of cells of side l0 . ( b ) the radial part of the inter - subunit or subunit - membrane potential , u(r ) , with a well - depth is split into attractive ( green ) and repulsive parts ( red ) . ( c ) the attractive part is multiplied by factors of the form fang( ) , where is an angle that depends on the relative orientation of the interacting particles . ( d ) sketch of momentum transfer between srd particles in a cell : ( i ) only particles within one cell interact . ( ii ) velocities are subtracted from all particles such that the center of mass velocity is 0 . ( iii ) all velocities are rotated , as signified by the heavy arrow , around a random axis , by a given angle . ( iv ) the subtracted velocities are added back on so that total momentum is conserved . for ms - interactions , parameters for the one orientational function composing orient , see figure 1c , are 0 = /4 and 1 = 0.2 , and there is no penalty for subunits rotating around rij . since , typically , assembling proteins will only be able to access one side of a membrane , we choose to make only one side of the membrane in our simulations attractive to subunits . this is achieved by setting att = 1 if a subunit interacts with the upper side and att = 0 if it interacts with the lower the area factor is proportional to the area of surface that surrounds the interacting membrane particle . the length scale for ss - interactions is chosen as ss = 2.5l0 and the length scale for ms interactions is ms = 1.75l0 , where l0 is a simulation unit of length . for the exact functional forms used in the ss and ms interactions , see the supporting information . as depicted in figure 1a , the membrane is composed of particles bonded to form a triangulated surface . to include membrane fluidity , the typical separation between bonded membrane particles is l0 , maintained by a potential that has a flat central region but diverges at 0.67l0 and 1.33l0 , see supporting information . we perform simulations in a box of size 45l0 45l0 45l0 , giving a subunit number density within the range for which yield was found to weakly depend on concentration . as in our previous work , we consider a range of ss that , at equilibrium in the bulk , covers the crossover to complete assembly of all cores . although approximately centered around the same ss value , for the larger number of cores considered here , the crossover is broader and so a wider range of ss is used . the same range of ms as in ref ( 22 ) is considered , chosen to cover the crossover from freely diffusing to membrane - bound structures . the stiffness of our membrane is controlled by a parameter b , through a potential , ubend = b(1 ninj ) , applied to all pairs of neighboring triangles in the surface , where ni and nj are the unit normal vectors of the triangles . we simulate using the three middle values from our previous work , b = 3kbt , 23kbt and 43kbt : at equilibrium , this covers the crossover from cores being able to cause budding of the membrane to them not being able to . as discussed in our previous work , this range of bending stiffness is at the lower end of that expected for biological membranes . given that in viral budding intrinsic curvature is expected to be important , which is neglected in our model , the bending stiffness in our simulation is most relevant in terms of the cost of deformation . although our focus is on dynamical simulation , we first investigate the equilibrium of the system for comparison . for this purpose , we use mc simulations , employing a similar approach as in our previous work . on the other hand , for molecular dynamics ( md ) simulations , we include hydrodynamic interactions using a stochastic rotation dynamics ( srd ) solvent , a coarse - grained method in which the fluid is represented by point particles . srd particle interactions are effected by dividing the system into a grid of cells , of side l0 , at regular intervals and exchanging momentum by a rotation through a certain angle of velocities relative to the cell center of mass velocity . this procedure is shown schematically in figure 1d . to understand the influence of hydrodynamic interactions , we also simulate using a method that neglects them , langevin dynamics ( ld ) , in which the effect of the solvent is represented by uncorrelated random , as well as drag , forces . to simulate a tensionless membrane , rather than box rescaling , we use a new membrane boundary condition , recently introduced by us , which is compatible with srd . the edge of the membrane is attached to a square frame , with sides positioned at a distance rframe into the simulation box , as depicted in figure 1a . for those triangles in the surface that have a side that forms part of the membrane edge , a bending potential of the same form as that between neighboring triangles is applied , except that the unit normal of the triangle is compared to a unit normal to the frame - plane . the distance rframe may increase and decrease during the simulation . to allow for deformation , the number of membrane particles bonded to the frame may also vary , with corresponding changes to the number of bonds in the bulk of the surface , nb bulk . for more details of the membrane boundary condition , see the appendix of ref ( 35 ) , the functional form of the confining potential is also given in the supporting information . for consistency , this approach is also used in mc and ld simulations , in which , of course , the solvent is absent . we first present , in figure 2 , results from mc simulations on the yield of complete cores , defined to be a cluster of 12 bonded subunits , each unit making 5 bonds to other cluster members . two subunits are defined to be bonded if their interaction energy is < 0.25ss . interaction strengths for different simulations lie on a grid from 0.12 to kbt in spacings of 0.08kbt for ms and from 4.5 to 8.02kbt in spacings of 0.32kbt for ss . systems at different parameters were run in parallel using multicanonical parallel tempering . for each data point in figure 2 , approximately 4 10 attempted mc moves were performed , including about 4 10 hybrid mc moves , as well as aggregate volume bias moves . the largest error for a single data point was estimated to be about 0.6 . similarly to our results with one core , for b = 3kbt and b = 23kbt at higher ms , the assembly of the cores causes the membrane to form buds , although these now generally contain multiple cores . for b again as for single cores , for high ms , assembly occurs for lower values of ss : the membrane promotes assembly . here , membrane - dependent , low ss assembly does not occur to the same extent as for high ss because , due to steric repulsion , only a fraction of the cores may interact with the membrane at once , typically about 4 cores in the case where a bud is formed . whereas for one core , the range over which promotion occurred was clearly largest for b = 3kbt , here the results for b = 23kbt are very similar . this may be because multiple cores together effectively form a larger object deforming the membrane . . average yield of complete cores , yield , as a function of subunit - membrane interaction strength , ms , and inter - subunit interaction strength , ss , for different membrane stiffnesses , b : ( a ) b = 3kbt ; ( b ) b = 23kbt ; ( c ) b = 43kbt . we next give details of our dynamical simulation methods . the srd particles have mass m and number density per cell = 5 . collisions are performed every tcoll = 10t0 and we use an srd rotation angle of /2 , giving a fluid viscosity of f = 2.5m / l0t0 . we apply a srd - cell level thermostat that conserves momentum to maintain the temperature . membrane particles are coupled to the srd solvent by including them in the collision step . there will typically be about one membrane particle per srd cell and we set their mass to m , giving a short - time friction coefficient mem = 15.8(m / t0 ) . unlike membrane particles , subunits have rotational degrees of freedom and so are coupled to the srd solvent using bounce - back boundary conditions . for their interactions with the fluid , subunits are treated as solid spherical particles of radius a = l0 , having mass m = ( 4/3)am and moment of inertia i = ( 2/5)ma . every tbound = 10t0 , the srd particles are checked . if an overlap with a subunit is detected , then the srd particle with velocity u is first moved by ( 1/2)tboundu and then shifted radially to the edge of the subunit , r from the center , where |r| = a. this scheme is based on the fact that for srd particles the average crossing of the subunit boundary is halfway through a time step . a bounce - back collision is then performed : the radial , u , and tangential , u , components of u are updated according to2here , a = 2m/(m+m ) , b = 7m/2 m and the surface velocity v = v + r , where v is the center of mass velocity of the subunit and is its angular velocity around an axis that passes through the center of mass after all overlapping srd particles have been rebounded , corresponding changes to the subunit velocity and angular velocity , v = ( m / m)i(uiold uinew ) and = ( m / i)iri ( uiold uinew ) , where i indexes the different rebounded particles , are applied so that momentum and energy are conserved . if m m , srd particle velocities relative to the surface are completely reversed ; for our parameters , m 20 m . overlapping of embedded particles in an srd fluid may lead to a spurious depletion attraction . in fact , even if particles are prevented from overlapping , the bounce - back scheme may need to be iterated due to the possibility of a fluid particle interacting with more than one solute particle within tbound . we avoid these issues by choosing the excluded volume length for subunit interactions , ss = 2.5l0 , so that the typical closest approach of two subunit fluid surfaces 0.5l0 is much greater than the typical displacement of a fluid particle 10l0 . bounce - back interactions between srd particles and embedded colloids lead to spurious slips at the colloid surface . methods exist to ameliorate this by the introduction of virtual particles but , for mobile colloids , this was found to lead to deviations from expected thermal distributions . in our simulations , the concern is moot anyway , because of the discrepancy between the radii chosen for inter - subunit and subunit - fluid interactions . effectively , there is a slip - velocity at the subunit surface , as defined by its interactions with other subunits , which has contributions from these two different sources . given that the subunits are typically representing protein complexes , which are rough on length - scales up to many solvent molecules , rather than smooth colloids , this is reasonable . for bounce - back boundaries , the short - time friction coefficients for the subunits may be calculated using a modified enskog theory . for our parameter choice , this gives coefficients of v = 62.0(m / t0 ) and = 73.3(ml02/t0 ) , for linear and angular velocities respectively . comparing the corresponding correlation times , m/v and i/ , to typical thermal velocities , we obtain values of 0.07l0 and 0.04 for the typical length and angular displacements over which the subunit motion is correlated . these are smaller than the typical separation of subunits and patches , given by ss = 2.5l0 and 0.4 respectively , so that at the scale that assembly occurs on , subunit motion is diffusive . similarly , the length scale over which membrane particle motion is correlated is 0.1l0 . to obtain parameters for simulations without hydrodynamic interactions , we simulated single subunits and membrane particles in a box of the same size as that used for assembly with an srd solvent . the friction coefficients extracted were lower than the short - time values due to long - time hydrodynamic contributions . the hydrodynamic contribution to the self - diffusion coefficients is included but hydrodynamic interactions between different particles are neglected . an alternative approach to simulating without hydrodynamics is to use an srd fluid and randomize particle velocities at every step . for colloids , however , membrane fluidity is included by performing a certain number of attempts to flip bonds between neighboring pairs of membrane particles every 10t0 . the membrane viscosity is set by the level of attempted bond - flips and we consider three different rates : nb bulk , 10nb bulk and 10nb bulk attempted bond - flips per 10t0 , where nb bulk is the number of bonds in the bulk of the membrane and the resulting numbers are rounded to integers . by considering poiseuille flows in two - dimensional membranes , the corresponding membrane viscosity , m , may be estimated . for the highest rate of flips the value is estimated to be 35.1 0.1m / t0 , whereas for the lower rates we estimate 133.3 0.6m / t0 and 1190 60m / t0 respectively . for a lipid bilayer in water , the ratio of membrane to fluid viscosities , l , is typically around 110 m . in our simulations the solvent viscosity f = 2.5m / l0t0 so that , if our subunits represent capsomers with a size on the order of 10 nm , then the ratio of their hydrodynamic radius to l is around the expected range . interaction strengths for dynamical simulations were chosen to coincide with those for mc simulations , although fewer were considered due to higher computational costs . a closer spacing between the highest interaction strengths was chosen as it was expected that the most interesting results would be found here . all averages are taken over at least 5 independent runs and in some cases over 10 . we consider the same values of b , and also ms and ss in the same range , as for the equilibrium mc simulations . we simulate primarily using srd but , for b = 3kbt and 23kbt , we also simulate using ld for comparison , to gain insight into the importance of hydrodynamic interactions . ld simulations were essentially identical to the srd ones expect that , rather than having regular interactions with an explicit fluid , subunits and membrane particles were , at each md integration step , subject to random and friction forces . the system was initially simulated for either 8 10t0 with srd , or 2 10t0 with ld , without attractive interactions . subsequently , attractions were switched on and the system was simulated for a further 5 10t0 to gather results . in contrast to the mc results , for the stiffest membrane , b = 43kbt , with the highest membrane - subunit interaction strength only , ms = kbt , in some , though not all runs , budding occurred . were it possible to run the dynamical simulations indefinitely , it is expected that results would eventually converge to those found for the equilibrium simulations . however , as the simulation progresses , further assembly becomes increasing slow as the supply of free subunits is depleted and eventually relies on rearrangement of subunits between partially formed structures , possibly moving into or out of a membrane bud . it is thus necessary to choose a finite simulation time shorter than that required for complete assembly and inevitably the results obtained will depend on it . for our chosen simulation time , the highest yield observed in any simulation is 50% of the possible maximum . it is nonetheless sufficient for the effect of the membrane on assembly to be apparent . however , the finite time chosen should be borne in mind when considering the results . first , in figure 3 , we plot the averages of various quantities as a function of ms and ss at different simulation times , t , for b = 23kbt and m = 133.3m / t0 . considering figure 3a , at later times , the largest number of correctly assembled cores are obtained for the second highest ss , 7.38kbt . this is close to the optimal value obtained in previous work with a very similar model of about 7.14kbt . although for the highest value , ss = 8.02kbt , the total interaction energy between subunits relative to the interaction strength is somewhat lower , see figure 3b , this corresponds to many incomplete cores assembling , thus starving the system of free subunits . this kinetic trap is not related to the membrane and has often been observed previously . in contrast , for high ss , increasing attraction to the membrane hinders complete assembly due to the membrane enveloping , or partly surrounding , partial cores too quickly , thus preventing subunits or other partial cores from approaching them . the fast envelopment is apparent in figure 3 , where it may be seen that , for high ms and ss , the interaction energy of the subunits with the membrane approaches its final value much more quickly than the yield . similarly to the mc results , a promotion of the finite - time assembly for low ss at high ms occurs . plots as a function of subunit - membrane interaction strength , ms , and inter - subunit interaction strength , ss at different times , t , increasing from left to right in intervals of 1 10t0 , for membrane stiffness , b = 23kbt and membrane viscosity m = 133.3m / t0 , from srd simulations . ( a ) average yield of complete cores , yield. ( b ) average total interaction energy between subunits , relative to the interaction strength , uss/ss. ( c ) average total interaction energy between subunits and the membrane , relative to the interaction strength , ums/ms. comparing results for equilibrium , figure 2 , to those from dynamical simulations , there are several differences . clearly , for equilibrium results , kinetic traps do not play a role . furthermore , the interaction strength at which assembly starts is slightly lower at equilibrium than after a finite time in dynamical simulation . for the lowest ms , ms = 0.12kbt , where the membrane does not play a significant role and assembly occurs in the bulk , whereas at equilibrium there are complete cores at ss = 5.46kbt , albeit at a relatively small yield , no complete cores were formed within the allowed time in dynamical simulations . similarly , the range of parameters for which there is assembly promotion is larger for the equilibrium results . considering figure 3a , we note that a time lag before complete cores are assembled , seen in previous work , is apparent for many data points at t = 10t0 , including for ss = 7.38kbt and low ms , where the yield is highest at later times . however , for some data points , primarily with high ms , some complete cores are already present at t = 10t0 : as well as causing a higher yield for low ss once assembly has progressed significantly , attraction to the membrane may also speed up the formation of a single core . by confining subunits to a surface , the effective size of the space that they must search to find each other is reduced . the membrane may also mediate effective attractions , directing subunits and partial cores toward each other and , if deformation occurs , it may bring membrane - attached subunits closer together . conversely , deformation of the membrane may also tend to block assembly , preventing partial cores from being accessed by subunits or other partial cores , leading to an increase in assembly time . the effect of the membrane on single - core assembly times is also shown in figure 4 , where we plot the average time until the first complete core in the system is formed , which we denote , as a function of ms . we note that , since this quantity is based on a single assembly event , large fluctuations were seen for lower interaction strengths and for some parameters additional simulations were run . for both membrane stiffnesses shown , b = 23kbt and 43kbt , tends to be lower for high ms for ss = 6.42kbt , whereas for ss = 7.38kbt the curve is flatter . for subunit interaction strengths that are approximately optimal for bulk assembly , the process is sufficiently fast that the membrane does not affect , whereas for lower values it may cause a significant speed - up . however , we note that , for high ms with some parameters , shows an increase . average time until the first complete core is assembled , , as a function of membrane - subunit interaction strength , ms , for inter - subunit interaction strength , ss = 6.42kbt ( red ) and ss = 7.38kbt ( green ) , with different membrane viscosities : m = 35.1m / t0 ( ) ; m = 133.3m / t0 ( ) ; m = 1190m / t0 ( ) . we next consider the average yield of complete cores , yield , measured at the end of the simulation , at time t = 5 10t0 . we present results for all the different parameter sets we have simulated , except for the lowest ss , for which only a very small amount of assembly occurred at the highest ms . we split the results into 3 figures by membrane stiffness : b = 3kbt in figure 5 ; b = 23kbt in figure 6 ; b = 43kbt in figure 7 . within each figure , yield is plotted as a function of ms , with curves corresponding to different membrane viscosities and simulation methods indicated by different colors , symbols and line types . rather than describe in detail the specific features of each figure , we discuss the general trends that arise and pick out interesting features . plots of the average yield of complete cores , yield , as a function of membrane - subunit interaction strength , ms , for membrane stiffness , b = 3kbt at t = 5 10t0 . from simulations with srd ( solid lines , filled symbols ) or ld ( dashed lines , open symbols ) , for different membrane viscosities : m = 35.1m / t0 ( blue , / ) ; m = 133.3m / t0 ( green , / ) ; m = 1190m / t0 ( red , / ) ; and subunit interaction strengths , ss , as indicated in panels a d . plots of the average yield of complete cores , yield , as a function of membrane - subunit interaction strength , ms , for membrane stiffness , b = 23kbt at t = 5 10t0 . from simulations with srd ( solid lines , filled symbols ) or ld ( dashed lines , open symbols ) , for different membrane viscosities : m = 35.1m / t0 ( blue , / ) ; m = 133.3m / t0 ( green , / ) ; m = 1190m / t0 ( red , / ) ; and subunit interaction strengths , ss , as indicated in panels a d . plots of the average yield of complete cores , yield , as a function of membrane - subunit interaction strength , ms , for membrane stiffness , b = 43kbt at t = 5 10t0 . from simulations with srd , for different membrane viscosities : m = 35.1m / t0 ( blue , / ) ; m = 1190m / t0 ( red , / ) ; and subunit interaction strengths , ss , as indicated in panels a d . overall , as ss is increased , we identify three different trends for finite - time assembly with realistic dynamics . first , at ss = 5.46kbt and 6.42kbt , both lower than the bulk - assembly optimal value , increasing ms , which tends to increase the membrane deformation rate in all regimes , may promote finite - time assembly . with high ms , assembly also occurs for values of ss where there is no bulk assembly within our simulation time . it is influenced by various factors , for example the strength of the attraction of subunits to the membrane or membrane viscosity , and results in competing effects on assembly . increasing it via ms may at first aid assembly , as seen in the initial increase in yield with ms in parts a and b of figures 57 , but when it is too high , the yield may decrease again , as is seen particularly clearly in figure 6a and b. results depend on factors such as membrane viscosity and hydrodynamic interactions : decreasing membrane viscosity or including hydrodynamic interactions both increase the deformation rate . attraction to , deformation of , and encapsulation within , the membrane is expected to decrease the subunit entropy such that the difference in entropy between unassembled and assembled states is less . additionally , the deformation of the membrane may help to guide subunits attached to it toward each other . it is expected that these effects will all play a role in promoting assembly , although their relative importance may not be easily deduced . however , if the deformation occurs too quickly , before complete cores are formed , the membrane will hinder further subunits , or other partial cores , from approaching the partial structure , preventing its completion . interestingly , in this low ss regime , finite - time assembly is promoted even for b = 43kbt , figure 7a , b , although for this membrane stiffness budding only occurs for the highest ms . for this membrane stiffness , results do not depend on membrane viscosity , confirming that here budding does not play a role . despite a lack of envelopment , it is expected that attachment to the membrane will nonetheless reduce the entropic cost of forming a partial core . a second plausible mechanism is a local increase in subunit density near the membrane surface . in contrast , for the lower two b , figure 5a , b and figure 6a , b , the membrane deformation rate does play a role . comparing srd and ld results , simulations with hydrodynamic interactions show a larger promotion of finite - time assembly as ms is increased , at least initially . for some parameters the yield decreases again as ms is increased further and this drop off occurs earlier with hydrodynamic interactions . furthermore , particularly for b = 23kbt , membrane viscosity , m , is also important . especially for srd results , decreasing m shifts the point at which the finite - time yield begins to decrease to lower ms . interestingly , the effect of hydrodynamic interactions and membrane viscosity are much stronger for b = 23kbt than for b = 3kbt . in the second regime , for ss = 7.38kbt , at about the bulk - assembly optimal value , increasing ms tends to decrease the finite - time yield . here , there is no strong dependence on membrane viscosity or hydrodynamic interactions and , additionally , results are quite similar for all three membrane stiffnesses . this suggests membrane deformation is not crucial , rather the decrease in yield may occur because attraction to the membrane promotes the faster assembly of partial cores , bringing the system into the monomer starvation trap that is only seen in the bulk for higher ss . finally , for the highest ss , ss = 8.02kbt , where there is a monomer starvation kinetic trap for bulk assembly , there is no clear effect of the membrane on finite - time assembly . since assembly , at least of partial cores , occurs very quickly in the bulk , results here are likely dominated by nonmembrane - associated assembly . in figure 8 , we show snapshots of final configurations from simulations with b = 23kbt and m = 133.3m / t0 . for ms = kbt and ss = 7.38kbt , figure 8a , for which the average yield was reduced compared to the low ms value , three of the four cores encapsulated in a bud that are depicted are incomplete . in contrast , for ms = 0.6kbt and ss = 6.42kbt , figure 8b , for which the average yield was enhanced compared to low ms value , only one of the four cores encapsulated , or partially encapsulated , in a bud that are depicted is incomplete . although these snapshots only depict the situation in two individual runs , they illustrate how the membrane may block assembly completion when its deformation rate is too high by preventing partial cores from being accessed by subunits or other partial cores . snapshots from simulations with srd at t = 5 10t0 with membrane stiffness , b = 23kbt and membrane viscosity , m = 133.3m / t0 : ( a ) membrane - subunit interaction strength , ms = kbt and inter - subunit interaction strength , ss = 7.38kbt ; ( b ) ms = 0.6kbt , ss = 6.42kbt . completed cores are circled in blue , while partially assembled ones are circled in red . a useful quantity to indicate the amount of membrane deformation is rframe , the distance from the edge of the simulation box of the frame to which the edge of the membrane is bound , which increases as the membrane distorts its shape out of the plane . to show how changing membrane viscosity , and including hydrodynamic interactions , alters the rate and extent of membrane deformation , we plot , in figure 9 , rframe as a function of time with b = 23kbt and ss = 6.42kbt for different ms from 0.36kbt . apart from the lowest ms , the membrane deformation occurs faster and to a greater extent for simulations with hydrodynamics . since budding requires the whole of the membrane to move , correlations mediated by hydrodynamics promote it . furthermore , for srd simulations at the highest two ms , there are also significant differences between membrane viscosities with a trend as expected : rframe is largest for the smallest viscosity . plots of the average position of the frame , rframe , as a function of time , t , for membrane stiffness , b = 23kbt and inter - subunit interaction strength , ss = 6.42kbt . from simulations with srd ( solid lines , filled symbols ) or ld ( dashed lines , open symbols ) , for different membrane viscosities : m = 35.1m / t0 ( blue , / ) ; m = 133.3m / t0 ( green , / ) ; m = 1190m / t0 ( red , / ) ; and subunit - membrane interaction strengths : ( a ) ms = 0.36kbt ; ( b ) ms = 0.6kbt ; ( c ) ms = 0.84kbt ; ( d ) ms = kbt . we next consider the distributions of cluster size , nc , in our srd simulations , h(nc ) . in figure 10 , we plot distributions for b = 23kbt and m = 35.1m / t0 at three different times . for ss = 6.42kbt , the lowest ss for which , in dynamical simulations , there is bulk assembly , there are few clusters with intermediate sizes when ms is low . at all times considered , the majority of clusters are of size two ; at later times there is an additional peak at size 12 , corresponding to complete cores . in contrast , when ms is high , the attraction to the membrane stabilizes intermediate cluster sizes at early times . at later times , the clusters have grown but the peak near 12 is less sharp , with similar numbers of cores of size 10 and 11 , and also some larger ones . this shows the effect of the membrane blocking the completion of partial cores . it also seen for higher ss , ss = 7.38kbt , where the distribution for low ms is much flatter at early times with many clusters of intermediate sizes . histograms , h , of cluster size , nc , for membrane stiffness , b = 23kbt and membrane viscosity , m = 35.1m / t0 at different times , t : t = 1 10t0 ( red ) ; t = 3 10t0 ( green ) ; t = 5 10t0 ( blue ) ; with different parameters : ( a ) membrane - subunit interaction strength , ms = 0.12kbt and inter - subunit interaction strength , ss = 6.42kbt ; ( b ) ms = kbt , ss = 6.42kbt ; ( c ) ms = 0.12kbt , ss = 7.38kbt ; ( d ) ms = kbt , ss = 7.38kbt . in figure 11 , we show the effect of membrane viscosity on the cluster size distribution . we plot results from the end of the simulations , at time t = 5 10t0 , again for b = 23kbt . with ss = 6.42kbt , we see that , for the highest viscosity , increasing ms leads to a distribution that is more strongly peaked at 12 . in contrast , for the lowest viscosity , although increasing ms does lead to more larger clusters , it also gives a much broader distribution around 12 . a similar effect occurs for ss = 5.46kbt : for this ss too , when membrane viscosity is low , high ms causes the membrane to encapsulate the assembling capsids too quickly , blocking their completion . histograms , h , of cluster size , nc , for membrane stiffness , b = 23kbt at time , t = 5 10t0 for different membrane - subunit interaction strength values : ms = 0.12kbt ( red ) ; ms = 0.6kbt ( green ) ; ms = kbt ( blue ) ; with different parameters : ( a ) membrane viscosity , m = 1190m / t0 and inter - subunit interaction strength , ss = 6.42kbt ; ( b ) m = 35.1m / t0 , ss = 6.42kbt ; ( c ) m = 1190m / t0 , ss = 5.46kbt ; ( d ) m = 35.1m / t0 , ss = 5.46kbt . as in similar previous models , the concentration of our subunits is relatively high compared to experimental systems , and furthermore the number of subunits in a completed core is low . these choices are necessary for computational tractability but have the consequence that the assembly rates in our simulations are much higher than experimental ones . assuming that subunits correspond to capsomers of size on the order of 10 nm , and matching the drag coefficient of our subunits , we estimate that our simulation length is around 5 ms , whereas in vitro(24 ) and in vivo(52 ) experiments have observation times on the order of minutes . our results rather demonstrate how the rate of membrane deformation compared to the assembly rate may affect the success of the latter . furthermore , they show how properties such as membrane viscosity , which might be varied experimentally by changing lipid composition are expected to impact on the assembly process . the interactions between assembling viral capsomers are typically relatively weak as this aids the avoidance of kinetic traps and thus the first , low ss regime identified is most likely to be relevant to these systems . we have investigated the effect of a membrane on core assembly of icosahedral cores . it is expected that many of the qualitative features of the results , such as the interplay between the membrane promoting assembly by confining subunits and hindering it by blocking additional subunits or other partial cores from approaching partial structures , will be general to other target structures . to gain more insight into the transferability of the findings to other core shapes , we finally , in figure 12 , present results on the yield obtained in srd simulations when the target structure is changed from the icosahedral core . as for icosahedra , we define a cluster to be a complete structure when it contains the correct number of subunits for the target structure and each subunit patch on each subunit forms a bond with another member of the cluster . we choose subunits with patches such that their interactions are minimized for cubic and dodecahedral structures . otherwise , parameters such as patch width are unchanged , with the membrane patch still lying on the symmetry axis as defined by the subunit patches and pointing outward in a complete structure . in both cases , the subunits only have three patches for bonding with other subunits and thus form fewer bonds in a complete structure than the icosahedral subunits . correspondingly , the range of ss was shifted up by about 2kbt but the range of ms remained the same . plots of the average yield of complete structures , yield , as a function of membrane - subunit interaction strength , ms and inter - subunit interaction strength , ss at time t = 5 10t0 for membrane stiffness , b = 23kbt and membrane viscosity , m = 133.3m / t0 : ( a ) subunits with interactions to form a cube . ( b ) subunits with interactions to form a dodecahedron . note the different scales for yield and also the higher values of ss as compared to the results for icosahedral cores . for both cubes and dodecahedra , as for icosahedra , budding of the membrane occurred for high ms . in figure 12a , many of the features of the finite - time assembly of icosahedra are reproduced for cubes . for high ms , there is finite - time assembly at lower ss than for low ms . there is also an increase in finite - time yield with increasing ms for the lowest ss for which assembly occurs without significant attraction to the membrane . unlike for icosahedra , at least for these membrane parameters , the yield does not drop off again as ms is increased further . this may be because , since they are composed of less subunits , cubes assemble faster than icosahedra . for the highest ss , however , there is a reduction in finite - time yield with ms , similar to results for icosahedra . as observed in previous work , finite - time yields of dodecahedra , figure 12b , however , here again , there is evidence that attraction to the membrane may promote assembly for ss for which it would otherwise not occur . although it is not apparent in our results , as seen in previous work with a very similar model , it is expected that , if the subunit interaction strength were increased sufficiently , the same nonmembrane - related kinetic trap that is observed for icosahedra would also be seen for cubes and dodecahedra . to summarize , we have applied a simple patchy - particle model to investigate the effect of interactions with a fluctuating membrane on the dynamics of the assembly of core structures with the same symmetry as many viral cores . as well as we also considered the effect of hydrodynamic interactions by simulating both with srd and ld . as at equilibrium , for assembly with realistic dynamics , attraction to a membrane may promote finite - time assembly , also for subunit interaction strengths , ss , for which it does not occur in the bulk . furthermore , for ss less than the optimal bulk value , attraction to the membrane also decreases the single - core assembly time . membrane budding occurred in dynamically realistic simulations and its rate was strongly increased by hydrodynamic interactions , as well as by lowering the membrane viscosity . the rate of membrane deformation is important in determining the assembly yield after finite time . relatively high rates may promote assembly by increasing the envelopment of assembling cores and thus decreasing the entropic penalty and also by guiding subunits toward each other . however , if the rate is too high , the membrane may block partial cores from being completed . for ss less than the bulk optimum , finite - time yields depend intricately on a combination of all parameters and may both increase and decrease as attraction to the membrane is increased . for ss about equal to the bulk optimum , finite - time yields do not depend strongly on the membrane deformation rate and tend to decrease as attraction to the membrane is increased . for ss higher than the bulk optimum , assembly in the bulk is affected by a monomer starvation kinetic trap and the membrane has little influence . finally , results with qualitative similarities were also found for core structures with cubic and dodecahedral symmetries . in future work it would be interesting to investigate more different structures , in particular much larger cores .

a coarse - grained computational model is used to investigate the effect of a fluctuating fluid membrane on the dynamics of patchy - particle assembly into virus capsid - like cores . results from simulations for a broad range of parameters are presented , showing the effect of varying interaction strength , membrane stiffness , and membrane viscosity . furthermore , the effect of hydrodynamic interactions is investigated . attraction to a membrane may promote assembly , including for subunit interaction strengths for which it does not occur in the bulk , and may also decrease single - core assembly time . the membrane budding rate is strongly increased by hydrodynamic interactions . the membrane deformation rate is important in determining the finite - time yield . higher rates may decrease the entropic penalty for assembly and help guide subunits toward each other but may also block partial cores from being completed . for increasing subunit interaction strength , three regimes with different effects of the membrane are identified .

anomalies in the development of branchial clefts can lead to four unique but closely related lesions , cysts , external sinuses , internal sinuses , and complete fistulas . the branchial fistula arising from each arch can be identified from the position of the internal and external openings . branchial fistulae are formed due to the abnormal persistence of the embryonic second branchial cleft . branchial fistulae arising from second and third arches are common than from first and fourth arches . in majority of cases , we report a rare case of complete second arch branchial fistula in a 7-year - old child , which was successfully treated by excision . a 7-year - old boy presented with complaints of watery discharge from an opening in the left side of neck , on and off since 4 years of age . there was a history of few episodes of swelling at the site of the opening , associated with fever . on examination , there was pinhead - sized opening seen at the junction of middle and lower third of left sternocleidomastoid at its anterior border [ figure 1 ] . watery discharge could be seen coming from the opening on making the child to drink water [ video 1 ] . a clinical diagnosis of branchial fistula was made . a contrast study was done by injecting gastrografin into the external opening , which showed the internal opening at the lateral aspect of pharyngeal wall in the supratonsillar fossa . the patient was able to feel the taste of contrast material in and the esophagus too showed the swallowed contrast , confirming the diagnosis of complete branchial fistula [ figure 2 ] . the fistulous tract was traced along carotid sheath where it turned medially to pass between the internal and external carotid arteries . the embryonic appearance and differentiation of branchial apparatus occur between the 3 and 7 week in the human embryo . each ridge has a core of mesenchyme , which is covered externally and internally by ectoderm and endoderm , respectively . failure of the second arch tract to obliterate would result in the formation of a branchial sinus and fistula . patients commonly present in the first two decades of life with intermittent , mucopurulent discharging sinus in the neck . although bilateral branchial fistulas have been documented , unilateral right - sided lesions are commonly seen . external opening is often situated between the upper two - thirds and lower one - third of sternocleidomastoid . radiologically demonstrable complete branchial fistulae with complete patency from internal to external opening are extremely uncommon in clinical practice . the tract in second arch fistula extends deep to the platysma , along the carotid sheath , passing between the bifurcation of the carotid arteries after crossing over the hypoglossal and glossopharyngeal nerves and passes below the stylohyoid ligament . it opens internally in the lateral wall of the pharynx region of the tonsillar fossa . contrast fistulogram can trace the tract up to the internal opening and is a commonly done preoperative evaluation . contrast fistulogram can differentiate second and third arch fistulas by demonstrating the internal opening and can obviate the need for further imaging . rarely , squamous cell lining , mixed cell lining , and branchiogenic carcinoma have also been documented . stepladder approach with two incisions in the neck gives good exposure of the fistulous tract with less tissue dissection . the tract has to be traced till the internal opening and excised completely to prevent recurrence . recurrence rate of 3% has been reported with open approach alone , probably due to incomplete surgical excision fistula tract in the parapharyngeal space . in complete branchial fistula with a probe in situ surgery can be delayed in infants with uncomplicated branchial fistula up to 3 years of age . infective exacerbations should always be treated before surgery is planned . in conclusion , we report a rare case of radiologically demonstrable complete second arch branchial fistulae in a 7-year - old male child . the child presented with intermittently discharging sinus in the neck and the diagnosis was confirmed by fistulogram .

branchial fistulae are formed due to the abnormal persistence of the embryonic branchial clefts . complete branchial fistula with internal and external opening is extremely rare . we report a rare case of complete second arch branchial fistulae in a 7-year - old boy , which was confirmed by a fistulogram . the tract was completely excised and the patient was successfully treated .

music is used synchronously to accompany repetitive endurance tasks such as cycle ergometry , walking and running ( e.g. , karageorghis et al . , 2009 ; simpson & karageorghis , 2006 ) . the validity of several early studies was compromised by tasks that were difficult or impossible to standardise ( see karageorghis & terry , 1997 ) . the psychophysical benefits of synchronous music use are equivalent to those observed in the asynchronous use of music , while the ergogenic effects consistently exceed those found in its asynchronous application . schneider , askew , abel , and strder ( 2010 ) proposed that human movement and rhythmical perception are both bound to the same optimal frequency of 120 beats per min ( bpm ) . this argument was predicated on the fact that participants adopt a spontaneous tempo of 120 bpm when asked to finger tap ( e.g. , kay , kelso , saltzman , & schoner , 1987 ) or walk ( hirasaki , moore , raphan , & cohen , 1999 ) . moreover , an analysis of over 70,000 pieces of popular music until 1990 indicated that 120 bpm was the most prevalent tempo ( macdougall & moore , 2005 ) . however , following a treadmill - based experiment , schneider et al . reported that 2.72.8 hz may represent the optimally efficient frequency for running at various intensities in line with the predictions of gutmann , jacobi , butcher , and bertram ( 2006 ) . the music choices of participants to accompany the treadmill test appeared to fall in a similar range of 2.62.8 hz ( approximately 156168 bpm ) . in criticism , the notion of optimal frequency does overlook the fact that this variable is specific to each individual and their intended work rate ( cf . hayakawa , miki , takada , and tanaka ( 2000 ) assessed the effects of traditional japanese folk music ( used asynchronously ) and the synchronous use of aerobics dance music on several dimensions of mood during a 30-min bench - stepping exercise . both music conditions lessened feelings of fatigue when compared to the metronome control condition . furthermore , aerobic dance music was responsible for increases in feelings of vigour when compared to a metronome control condition , whereas the folk music was not . also evaluating the psychological effects of synchronous music , van der vlist , bartneck , and maeler ( 2011 ) developed an interactive music feedback system akin to the nike plus interface . the system , named mobeat , adjusts the musical tempi to synchronise with participants cycling pace and issues recorded instructions to increase or reduce speed as appropriate . whereas the system did enhance participants intrinsic motivation and attentional focus when compared to a heart rate ( hr ) monitor reference condition , no differences were evident in terms of perceived exertion and compliance to the prescribed movement tempo . although at the vanguard of technological development in this field , the results may have been influenced by an elementary drawback . the synthesised music used was unfamiliar to participants and consequently had no motivational qualities by virtue of association . this is highly likely to have limited its impact . a number of studies found that synchronous music improved performance in motoric tasks ( karageorghis et al . , 2009 ; simpson & karageorghis , 2006 ; terry , karageorghis , mecozzi saha , & d'auria , 2012 ) . in each case , the ergogenic effects of motivational music exceeded those of oudeterous ( neutral ) music . in the former study , music was used synchronously to ergogenic effect during a 400-m sprinting task . the runners strode in time with a piece of motivational music that was delivered during the task . this music elicited faster times than a no - music control condition . a second synchronous condition consisting of music that was lacking in motivational qualities also improved performance relative to the control , but not to the same degree . hence , both synchronisation and the motivational qualities of the music appear to be of benefit to high - intensity physical activity . terry et al . also used a running task : a submaximal treadmill run to the point of volitional exhaustion . a sample of elite triathletes was able to endure longer in the presence of two synchronous music conditions ( self - selected and an alternative that was neutral in motivational terms ) . furthermore , mood responses and feeling states were more positive under the motivational music compared to either neutral or no - music conditions . based on physiological indices , the authors contested that the synchronous music improved running economy by virtue of lower oxygen consumption and blood lactate levels . ( 2009 ) investigated the ergogenic effects of the synchronous application of music during treadmill walking . participants began the task at 75% of their maximum heart rate and continued walking until voluntary volitional exhaustion . in addition to the no - music control , two synchronous conditions were employed : motivational and oudeterous music . both music conditions promoted greater endurance than the control , with the motivational music proving superior to the oudeterous selection . indeed , the motivational music elicited a marked 15% increase in treadmill endurance when compared to the control , and a clear 6% increase relative to the oudeterous condition . the experimental conditions appeared to lower perceptions of exertion at the start of the test relative to the remaining conditions . nonetheless , as the intensity of exercise increased throughout the duration of the test , ratings of perceived exertion ( rpe ) ceased to differ by music condition . this trend marginally failed to achieve significance , but is in keeping with the prediction that the influence of music on attentional processes wanes at higher exercise intensities ( cf . ( 2009 ) study also showed that music conditions exerted a strong influence on feeling states , which were more positive under the motivational condition until the point of volitional exhaustion . this result contradicts existing theory ( rejeski , 1985 ; tenenbaum , 2001 ) given that the physiological strain associated with high - intensity exercise is expected to prevent participants from fully attending to musical stimuli . the findings supported the notion that , although music may not moderate what one feels during high - intensity exercise ( i.e. , rpe ) , it can moderate how one feels it ( see hardy & rejeski , 1989 ) , therefore intense exercise can be more pleasurable with appropriate musical accompaniment . this result was mirrored by shaulov and lufi ( 2009 ) who investigated the effects of dance music when used synchronously during stationary cycling . while music did not lead to greater energy expenditure ( calories burnt ) when compared to a no - music control , participants did report heightened states of pleasure during the music condition . styns , van noorden , moelants , and leman ( 2007 ) conducted multiple trials to investigate the precise contingencies of the synchrony between walking and various music tempi ( 50190 bpm ) . whereas the overwhelming majority of participants chose to synchronise their movement to the musical tempo without solicitation , also , the authors proposed that aspects of the music additional to tempo may influence the spatial aspects of movement . these aspects included the number of events per musical beat ( meter ) , the number of musicians performing , and the energy with which instruments appeared to be played . finally the authors proposed that any musical rhythm perpetuates an internalised model of movement which may be generalised ( not specific to listener ) and is likely to consist of dance elements . hypothesising that the synchronous effects of music were more likely to become evident when motoric tasks of greater complexity were used , karageorghis et al . ( 2010 ) assessed whether the performance of strength endurance exercises was affected by three synchronous conditions each delivered at a tempo of 120 bpm . motivational music , oudeterous music and an audible metronome ( control ) were used to accompany sit - ups , squats , jumping jacks , heel raisers , step - ups and press behind neck . each of these circuit - type exercises was performed until synchronous movement could no longer be maintained , and a gender comparison was a key feature of the study . the music conditions neither led to increases in the number of repetitions performed nor influenced feeling states when considering both genders together . however , a gender condition interaction emerged : both genders performed worse when listening to oudeterous as opposed to motivational music , but whereas women performed similarly whether listening to oudeterous music or the control , men performed as well under the control condition as they had when listening to motivational music ( see figure 1a ) . women also reported more positive feeling states than men in both music conditions ( see figure 1b ) . the men appeared to find it harder than the women to coordinate their movements with musical tempo and this is perhaps reflected in the popularity of choreographed exercise classes among women . motivated by music than males ( priest , karageorghis , & sharp , 2004 ) . age was an even stronger determinant of musical reactivity in the exercise setting , with older participants ( > 46 years ) starkly preferring less stimulative music , being less likely to report feeling motivated by music , and regarding music during exercise as less important than their younger counterparts ( < 26 years ) . ( reprinted from psychology of sport and exercise , 11 , karageorghis , c.i . , priest , d. l. , williams , l.s . , hirani , r.m . , , & bates , b.j . , ergogenic and psychological effects of synchronous music during circuit - type exercise , 551559 , 2011 , with permission from elsevier . ) music condition gender interaction for affect ( p < .05 ) . seath and thow ( 1995 ) used pop music and an auditory metronome control condition ( both 132 bpm ) to accompany a four - week course of biweekly exercise sessions . the activity consisted of 15 min of aerobic exercise taking the form of a simplified exercise - to - music dance routine . the music exerted large differences both on measures of rpe and positive feeling states , lowering the former and raising the latter . a minor criticism of this study is that rpe and affect measures were taken every 30 s , a procedure that may have proved quite distracting / irritating for participants . annesi ( 2001 ) reported that , over 14 weeks , members of a large private health club in the us experienced less dropout and trend towards greater attendance when exposed to an intervention that combined self - selected music with television ( 62 changeable channels ) as opposed to interventions that comprised only music , only television , and neither of the above . although clearly high in terms of ecological validity , the internal validity of this study is questionable on several grounds , chief among these being the small group sizes ( n = 1114 ) for independent sample analysis . notably , the music - only condition fell marginally short of significance on several of the measures despite apparent meaningful differences ( effect sizes were not reported ) . further corroboration of chronic effects that stem from listening to music during exercise can be found in the qualitative work based on semi - structured interviews ( priest & karageorghis , 2008 ) and nationwide surveys ( priest et al . , 2004 ) . in both studies respondents stated that music may positively ( and in some cases negatively ) influence their adherence to exercise and gym attendance . the post - task use of music is typically recuperative in nature in that it aids recovery from injury , competition or training ( terry & karageorghis , 2011 ) . at present , there has been very little empirical investigation into the capacity of music to aid recovery from training , competition or injury . however , music has been shown to offer pronounced recuperative effects in other areas such as education and health ( e.g. , denora , 2007 ; srkm et al . , 2008 ; sokhadze , stewart , & hollifield , 2007 ) . for example , knight and rickard ( 2001 ) reported that relaxing music prevents stress - induced increases in subjective anxiety , systolic blood pressure and heart rate . have demonstrated that various types of music accelerated recovery from exposure to a stressful visual stimulus ( images previously rated as similarly , hirokawa and ohira ( 2003 ) found that listening to music after a stressful task led to measureable improvements in immune function , neuroendocrine responses and emotional states in college students . finally , labb , schmidt , babin , and pharr ( 2007 ) observed that listening to self - selected or classical music , after exposure to a stressor , significantly reduced the incidence of negative emotional states and physiological arousal when compared to listening to heavy metal music or sitting in silence . jing and xudong ( 2008 ) have conducted what is possibly the inaugural study into the effects of post - task music in the exercise domain . a sedative instrumental piece ( acupuncture on the mind composed by the second author ) was used to aid the recovery of male college students for 15 min following an exhaustive cycle ergometer trial which began at 8085% blood lactate threshold . decreases in heart rate , urinary protein ( indicative of post - exercise kidney function ) and rpe were greater in the music group than the control . however , there was no difference in terms of the remaining measures jump height , blood glucose , blood lactic acid and simple reaction time . this result indicates that post - exercise music use may have beneficial consequences although further studies to investigate the contingencies of its use are clearly required . for example , a vertical jump is a questionable measure of fatigue while the 15-point borg scale was certainly not designed for post - exercise use . savitha , mallikarjuna , and chythra ( 2010 ) reported that slow music accelerated the haemodynamic ( blood pressure and hr ) recovery from 5 min of intense treadmill work when compared to fast music and a control . despite the fact that the study may have been flawed , for example no counterbalancing procedure appears to have been used , the results augment those of jing and xudong ( 2008 ) and may be used to support the proposition that sedative music hastens post - exercise recovery . at this juncture , there is clearly scope for considerable additional work in the area of post - task music use . seath and thow ( 1995 ) used pop music and an auditory metronome control condition ( both 132 bpm ) to accompany a four - week course of biweekly exercise sessions . the activity consisted of 15 min of aerobic exercise taking the form of a simplified exercise - to - music dance routine . the music exerted large differences both on measures of rpe and positive feeling states , lowering the former and raising the latter . a minor criticism of this study is that rpe and affect measures were taken every 30 s , a procedure that may have proved quite distracting / irritating for participants . annesi ( 2001 ) reported that , over 14 weeks , members of a large private health club in the us experienced less dropout and trend towards greater attendance when exposed to an intervention that combined self - selected music with television ( 62 changeable channels ) as opposed to interventions that comprised only music , only television , and neither of the above . although clearly high in terms of ecological validity , the internal validity of this study is questionable on several grounds , chief among these being the small group sizes ( n = 1114 ) for independent sample analysis . notably , the music - only condition fell marginally short of significance on several of the measures despite apparent meaningful differences ( effect sizes were not reported ) . further corroboration of chronic effects that stem from listening to music during exercise can be found in the qualitative work based on semi - structured interviews ( priest & karageorghis , 2008 ) and nationwide surveys ( priest et al . , 2004 ) . in both studies respondents stated that music may positively ( and in some cases negatively ) influence their adherence to exercise and gym attendance . the post - task use of music is typically recuperative in nature in that it aids recovery from injury , competition or training ( terry & karageorghis , 2011 ) . at present , there has been very little empirical investigation into the capacity of music to aid recovery from training , competition or injury . however , music has been shown to offer pronounced recuperative effects in other areas such as education and health ( e.g. , denora , 2007 ; srkm et al . , 2008 ; sokhadze , stewart , & hollifield , 2007 ) . for example , knight and rickard ( 2001 ) reported that relaxing music prevents stress - induced increases in subjective anxiety , systolic blood pressure and heart rate . sokhadze et al . have demonstrated that various types of music accelerated recovery from exposure to a stressful visual stimulus ( images previously rated as disgusting ) . similarly , hirokawa and ohira ( 2003 ) found that listening to music after a stressful task led to measureable improvements in immune function , neuroendocrine responses and emotional states in college students . finally , labb , schmidt , babin , and pharr ( 2007 ) observed that listening to self - selected or classical music , after exposure to a stressor , significantly reduced the incidence of negative emotional states and physiological arousal when compared to listening to heavy metal music or sitting in silence . jing and xudong ( 2008 ) have conducted what is possibly the inaugural study into the effects of post - task music in the exercise domain . a sedative instrumental piece ( acupuncture on the mind composed by the second author ) was used to aid the recovery of male college students for 15 min following an exhaustive cycle ergometer trial which began at 8085% blood lactate threshold . decreases in heart rate , urinary protein ( indicative of post - exercise kidney function ) and rpe were greater in the music group than the control . however , there was no difference in terms of the remaining measures jump height , blood glucose , blood lactic acid and simple reaction time . this result indicates that post - exercise music use may have beneficial consequences although further studies to investigate the contingencies of its use are clearly required . for example , a vertical jump is a questionable measure of fatigue while the 15-point borg scale was certainly not designed for post - exercise use . savitha , mallikarjuna , and chythra ( 2010 ) reported that slow music accelerated the haemodynamic ( blood pressure and hr ) recovery from 5 min of intense treadmill work when compared to fast music and a control . despite the fact that the study may have been flawed , for example no counterbalancing procedure appears to have been used , the results augment those of jing and xudong ( 2008 ) and may be used to support the proposition that sedative music hastens post - exercise recovery . at this juncture , there is clearly scope for considerable additional work in the area of post - task music use . the purpose of this two - part review paper was to critically evaluate and synthesise research into the effects of music in an exercise context , with the aim of identifying future directions for research and providing evidence - based guidelines for practitioners . the principal delineator of the studies reviewed was the time of music 's introduction relative to the task . it is evident that pre - task music can promote heightened arousal , both of the psychological and physiological variety . it is logical to infer that music can also act as a sedative when used pre - task , although there is currently insufficient empirical evidence in support of this ; very few people need to be sedated or calmed before engaging in exercise . those engaging in motoric activities that require elevated levels of psychomotor arousal are likely to prefer stimulative music ( north & hargreaves , 2008 ) . the efficacy of pre - task music use depends on how functional such changes in arousal / activation are for the listener and task in question . for example , an extravert would likely benefit from such an intervention prior to a bench press effort . qualitative research ( e.g. , priest & karageorghis , 2008 ) has begun to detail a more precise picture of the cognitions that accompany this psyching - up process and , consequently , the paths that practitioners may use to activate individual athletes . of the 32 eligible studies in this review , 24 report an ergogenic effect when music is used in - task 27 refer to the asynchronous use of music and 23 report an ergogenic effect . the majority of studies that include a performance measure ( e.g. , distance travelled , repetitions completed ) are focused on high - intensity exercise ( e.g. , tasks completed to the point of volitional exhaustion ) . nevertheless , all six studies that focus on a low - to - moderate - intensity task and include a performance measure report that music promotes an ergogenic effect . furthermore , during exercise of a low - to - moderate intensity , music is associated with a 10% reduction in rpe . this result is consistent across studies , even when the music is arbitrarily selected ( e.g. , classical music ; szmedra & bacharach , 1998 ) although studies that have manipulated music preference show that the largest benefits to rpe are associated with music that is preferred ( e.g. , dyrland & wininger , 2008 ) . there are also instances of participants performing greater work output with music while rpe remains stable or does not rise in the expected manner ( e.g. , edworthy & waring , 2006 ; elliott , carr , & orme , 2005 ) . in essence , the key role of music in recreational exercise is one of lowering perceptions of exertion and thereby increasing the amount of work performed without the shift towards negative feeling states typically associated with more intense exercise ( see ekkekakis , hall , & petruzzello , 2004 ; hutchinson & tenenbaum , 2006 ) . typically , the psycho - physiological changes promoted by music are masked by those which accompany exercise . for example , urakawa and yokoyama ( 2005 ) reported that listening to favoured musical selections during a 15-min submaximal cycling bout led to an increased ratio of low- to high - frequency heart rate variability , which is indicative of sympathetic nervous system activity and hence increased arousal . in total , 10 studies were concerned with the use of music during short - duration , high - intensity tasks ; in all but two , music promoted ergogenic effects . when in - task music was used over a longer duration , during bouts of work completed to exhaustion , similar results ensued in that ergogenic effects were reported in 12 of the 14 studies cited . although the internal feedback associated with exertion during high - intensity exercise dominates the perceptual field , music has the capacity to enhance positive feeling states during this type of exercise . indeed , this improvement in affect appears to alter the way judgements are made about the exercise experience . hence , the affective impact of music may be key to its positive influence on endurance during high - intensity exercise . this would not be illogical , as high - intensity exercise is associated with significant decrements in affect ( see , e.g. , ekkekakis et al . , 2004 ) . future research may be used to pinpoint whether the potency of music during higher - intensity work is mediated by lyrical affirmations and task - related imagery . music , especially when selected according to its motivational qualities , renders moods and feeling states more positive across a range of exercise modalities and tasks ( e.g. , karageorghis et al . , 2009 ; shaulov & lufi , 2009 ; terry et al . , 2012 ) . this positive effect appears to hold regardless of exercise intensity or the synchronicity of the music , and ranges from 319% depending on how well the music is selected ( i.e. , with participants preferences and task characteristics in mind ) . well - selected music is associated with increases in positive affect that are greater than 10% . the finding that music 's affective qualities impact upon the psychological state of the exerciser when working at high intensities ( > 70% max heart rate reserve [ hrr ] ) challenges the information processing model advanced by rejeski ( 1985 ) who proposed that the internal feedback associated with physical exertion would predominate . however , convincing evidence has been revealed herein that , while music can not alter what one feels when working at high intensities , it can still impact upon how one feels ( hardy & rejeski , 1989 ; hutchinson et al . a number of authors ( e.g. , annesi , 2001 ; karageorghis , terry , & lane , 1999 ; miller , swank , manire , robertson , & wheeler , 2010 ; schwartz , fernhall , & plowman , 1990 ) have proposed that the positive effects of music on feeling states can lead to increased adherence to exercise ( i.e. , attendance ) . this notion is also supported by interview - based evidence ( denora , 2000 , pp . 89103 ; priest & karageorghis , 2008 ) . to elaborate on the distinction that scherer ( 2004 ) drew between utilitarian and aesthetic emotions , it is probable that the aesthetic emotional response applies to low - to - moderate - intensity exercise whereas utilitarian emotions , which are adaptive and associated with pressing circumstances , are more relevant to high - intensity work . while music that is selected according to its motivational qualities appears to have the greatest impact , it is clear that music per se leads to measurable benefits , both in terms of lowered rpe and an ergogenic effect . ( 1999 ) prediction that only motivational music is associated with reductions in perceived exertion has not been borne out by the 12 years of research that have followed . when music is used asynchronously , it appears to be at its most effective when used in conjunction with self - paced tasks , which mirror the ad hoc behaviour of exercise participants in vivo . indeed , music use was associated with an ergogenic effect in each of the six studies where participants were permitted to select their own exercise intensity . finally , music appears to be of greater benefit to untrained or recreationally active individuals than it does to those who are highly trained ( e.g. , brownley , mcmurray , & hackney , 1995 ; mohammadzadeh , tartibiyan , & ahmadi , 2008 ) . this may be attributable to motivational differences between the two groups or the fact that , whereas trained individuals tend to work at high intensities , the observed ergogenic effect of music when used synchronously is concurrent with the documented predisposition of the human organism to respond physically to the rhythmical qualities of music ( see , e.g. , crust , 2008 ; karageorghis et al . , 1999 ; lucaccini & kreit , 1972 ) . various neuropsychological mechanisms have been advanced to explain this predisposition , including the presence of a neural pacemaker ( wilson , 1987 , pp . 131132 ) , a property of the nervous system used to control repetitive movement known as timeform printing ( clynes & walker , 1982 ) and the dual involvement of certain brain sectors in music response and movement ( kornysheva , von cramon , jacobsen , & schubotz , 2010 ) . there is a weakness in approach that has limited research into the synchronous use of music . very few studies have examined the relative effects of music used synchronously vs. asynchronously ( e.g. , anshel & marisi , 1978 ) . hence , a detailed comparison between the effectiveness of these types of music use is not possible at the present time . yet it is logical to infer that , when movements are synchronised to music , additional benefits are obtained both in terms of dissociation ( through having to focus on keeping time ) and energy efficiency in repetitive endurance activities and those with a marked rhythmic component ( e.g. , callisthenic - type exercises ) . while relatively little is known about the impact of music heard post - task and used in a recuperative capacity , it is logical to infer from studies outside the sport and exercise domain that music has the capacity to relieve stress and improve affective states , thus aiding recovery ( e.g. , srkm et al . , 2008 ; sokhadze et al . , 2007 ) . this area is worthy of further systematic investigation as there are currently only two studies that the authors could locate , neither of which were particularly strong in methodological terms . a prominent trend to emerge from the present review is that music appears to have a greater effect when external validity is relatively high , especially when either the task or music selection is self - regulated ( as is often the case during exercise ) . this draws on self - determination theory insofar as autonomy is one of the three primary needs that underlie intrinsic motivation ( ryan & deci , 2000 ) . in support of this point is the finding of dwyer ( 1995 ) who found that post - exercise intrinsic motivation was higher in a group who were exposed to music they believed they had chosen , as opposed to a group who listened to the same music but were not informed that it was selected as a result of their surveyed preferences . the effects of music with video in combination have received scant attention despite the fact that this type of delivery is rapidly usurping the former role of music , particularly in health and fitness facilities . in this sense , there is a dearth of work examining the motivational effects of moving images and whether the effects of music and video appose or interact in some manner . both pieces of work featuring a music - video condition reported beneficial effects which bettered those of music alone ( annesi , 2001 ; barwood , weston , thelwell , & page , 2009 ) . furthermore , loizou and karageorghis ( 2009 ) report that the use of primes incorporated into pre - task motivational video presentations led to performance increments in anaerobic work . go ) which appeared momentarily ( 40 ms ) on screen during the video presentations . finally , qualitative work has revealed many subtleties in musical response hitherto unreported in the experimental literature . for example , exercise participants have reported a strong anticipation effect whereby particular segments of musical pieces were awaited and used as a conscious trigger for increased work output ( priest & karageorghis , 2008 ) . this effect was expressed in terms of heightened arousal during the bars leading up to the preferred segment . such findings indicate a particularly nuanced and complex relationship between exerciser , task and music . experimental work by lim , atkinson , karageorghis , and eubank ( 2009 ) using cycle ergometry with differentiated music exposure has also pointed towards similar anticipation effects , therefore this is a phenomenon that warrants further investigation . the present review bears several implications for future research through the trends and gap teeth in the literature that it has covered . notably , the role of age , gender and other personal variables in musical reactivity within the exercise context is currently an underexplored area . for example , is it the case , as has been suggested in qualitative investigations ( e.g. , priest & karageorghis , 2008 ) , that music is a more powerful stimulus for younger exercise participants than it is for seniors ? perhaps the lack of research into age - congruent music has limited our understanding and so researchers should consider experimental protocols that entail age - congruent and age - incongruent music programmes . as noted by crust and clough ( 2006 ) , an important personal factor that has been largely overlooked in the literature an obvious entre into this line of research would be to examine extraverted vs. introverted exercise participants with the hypothesis that extraverts would be likely to respond more favourably to loud / stimulative musical selections than introverts ( cf . similarly , attentional style and musical response in the exercise domain has received scant attention ; it is thought that dissociators are likely to derive greater benefit from music use than associators , although an important mediating variable is task intensity . there is emerging evidence that cognitive strategies change in accordance with the intensity of an exercise task ( hutchinson & tenenbaum , 2006 , 2007 ) which means that low - intensity tasks leave greater attentional capacity for parallel processing tasks such as music listening ( cf . hence , examining responses to music across a range of exercise intensities while employing participants who exhibit different attentional styles would be an especially propitious endeavour . researchers should also consider the extent to which music moderates ratings of perceived exertion beyond the anaerobic threshold , when physiological cues tend to predominate attentional processes . when a multi - dimensional approach is taken in the measurement of perceived exertion ( e.g. , hutchinson & tenenbaum , 2006 ) it appears likely that carefully selected music ( e.g. , motivational music ) can influence the motivational - affective component of perceived exertion beyond the anaerobic threshold , even if it can not alter gestalt perceptions of exertion at such intensities . it is certainly the case that the degree to which the motivational qualities of music mediate reductions in perceived exertion warrants further empirical investigation . work to date has not systematically investigated the role of the lyrical content of music in determining psychological or psycho - physiological responses during exercise . it is plausible that the additional information processing capacity required to process lyrics can increase the dissociative influence of music , but there is a notable lack of exercise - related studies that compare responses to the same piece of music with and without lyrics . there is just one recent conference paper which showed that although lyrics contributed to the perceived motivational qualities of music , they had no bearing on psycho - physiological responses or work output in a submaximal cycle ergometer task ( sanchez , moss , twist , & karageorghis , 2011 ) . self - selected vs. experimenter - selected music has also been seldom tested ( e.g. , dyrlund & wininger , 2008 ) although researchers should be aware of the inherent difficulties pertaining to self - selection ; there is a greater likelihood for the emergence of hawthorne effects and experimenter effects ( see lucaccini & kreit , 1972 ) . with reference to the cubic trend identified in the exercise heart rate music tempo preference relationship ( karageorghis , jones , et al . , 2011 ) , future researchers should seek to investigate the link between music tempo preference and a range of affective and motivational outcomes . this approach will reveal the psychological consequences of congruity between exercise heart rate and preferred music tempo . study using other exercise modalities ( e.g. , cycling , motorised stepping , dance aerobics , etc . ) and longer exercise bouts would shed light on the stability and applicability of the relationship . with reference to stability , it is unknown whether the cubic trend would manifest itself among middle aged and senior exercise participants , as it emerged through an investigation of young adults . given that maximal heart rate gradually declines with age , one possibility is that the trend line will shift downwards and flatten when investigated among older groups of exercisers . research needs to be undertaken to evaluate the comparative effects of music used synchronously and asynchronously . in particular , do females derive greater benefit from the synchronous use of music when undertaking complex motor tasks such as callisthenic - type exercises or aerobic dance exercise ( cf . researchers may also consider the role of rhythmic ability in response to the synchronous use of music ( cf . accordingly , there is scope to examine how syncopated ( off beat ) rhythms are processed by high and low rhythmic ability groups . there is a strong likelihood that individuals with high rhythmic ability and/or musical training will process such complex rhythms more easily , thus responding with greater bodily precision than their less musical counterparts ( see , e.g. , vuust et al . , 2005 ) . research into post - task music use is in its infancy so there is considerable scope for work that ascertains how music facilitates recovery from injury and intense exercise . for this reason , it would be advantageous for researchers to develop and validate a tool , similar to the brunel music rating inventory ( bmri ) , for the assessment of the sedative qualities of music in an exercise context . in such investigations , an insightful approach would be the use of psychobiological measures , such as salivary cortisol , to examine the mechanisms that underlie the potential benefits of post - task music . the few studies that have examined training status as a moderator of exercisers response to music have shown that this is an important personal variable that should be considered for inclusion in future research ( brownley et al . , 1995 ; mohammadzadeh et al . , two tentative findings that require more detailed investigation are that untrained participants tend to derive greater benefit from music , while those who are highly trained prefer to listen to their bodies ( i.e. , use an associative attentional strategy ) . nonetheless , the evidence presented herein indicates that , under certain circumstances ( long - duration , low - intensity activities ) , highly trained individuals stand to benefit from the judicious use of music . work on the interactive effects of music and video is virgin territory for researchers , yet it represents a modality that is increasingly becoming ubiquitous in the gym environment ( see barwood et al . , 2009 ; loizou & karageorghis , 2009 ) . potential paths include the use of interview and observational techniques to gauge how exercisers respond to music and video across a range of exercise modalities . the ultimate potential avenue for research is an investigation of the neurophysiological correlates of the musical response before , during and after exercise the development of new technology , particularly fmri scanners that can be used during gross motor tasks , would facilitate such investigations . for example , this approach would be a possible means by which to explore the proposed attentional switching ( rejeski , 1985 ) between internal and external cues that may take place at anaerobic threshold . a further possible exploration would entail evaluating the role of associations and memory in musical response and the shared activation between music and other emotive stimuli . the evidence reviewed and synthesised in parts i and ii of this paper lead to a number of implications for exercise practitioners and participants . music selected as an accompaniment for exercise should be congruent with participants personal characteristics ( e.g. , age and socio - cultural upbringing ) , the exercise environment and desired outcomes . when working with a mixed group in terms of age and/or sociocultural upbringing , practitioners might consider democratising the music selection process so that all participants musical predilections are accounted for and reflected in the music programme ( dyrlund & wininger , 2008 ; nakamura et al . , 2010 ) . music tempo should be selected with the expected exercise intensity in mind , and be sequenced to contour in accordance with changes in heart rate . other than when warming up , warming down or recuperating , the appropriate band of tempi for exercise intensities in the range 4090% max hrr is 125140 bpm when the music is used asynchronously ( see karageorghis , jones , et al . , 2011 ) . it is also advantageous for the rhythm of the music to approximate the motor patterns entailed where possible ( crust , 2008 ; schneider et al . , 2010 ) . in group exercise classes when music is used synchronously , it appears that the rhythmic abilities of participants warrant careful consideration in the sequencing of music selections ( see , e.g. , denora , 2000 , pp . synchronous motor activity may occur in association with complex rhythmical segments that involve syncopation or span musical measures , not simply with the principal beat that marks musical tempo ( styns et al . , 2007 ) . music containing affirmations of exercise or inspirational references drawn from popular culture should be selected in order to promote motivational imagery and self - talk . positive affect is thought to be consequent to the harmonic and melodic features of the music and its lyrical content ( karageorghis et al . , 1999 ; lucaccini & kreit , 1972 ) . in order to have a stimulative effect , the music should be up - tempo ( > 120 bpm ) and possess prominent percussive and rhythmical features . in order to sedate , the inverse is true in terms of percussion and rhythmical features , and tempi < 80 bpm are recommended ( see terry & karageorghis , 2011 ) . a novel finding that has emanated from qualitative work is that music selection for exercise is concerned with the entire programme of music , not just individual pieces ( e.g. , denora , 2000 , pp . 89103 ; priest & karageorghis , 2008 ) , a perspective not countenanced in the experimental literature . for example , selectors should consider the congruence of musical pieces that appear in close proximity on a playlist and aim to achieve variety in terms of churn ( i.e. , varying selections from session to session ) . with specific reference to congruence , taking into account factors such as beat matching , style matching , artist matching , era matching , etc moreover , a change or absence of music can be an effective marker of the next exercise phase or unit ( denora ) , such as the progression from a cardiovascular segment to a warm - down phase . the findings reported in this review lead us to the conclusion that self - selected music is especially efficacious in an applied context ( e.g. , bharani , sahu , & mathew , 1984 ; priest & karageorghis , 2008 ) . it is exceedingly unlikely that a motivational music programme selected by a practitioner would yield stronger effects than a programme selected by the participant themselves , unless they make inept choices . self - selection also invokes a sense of autonomy , which is a central tenet of intrinsically motivated behaviour ( ryan & deci , 2000 ) . bearing in mind the individual variation in the desired psychological state during exercise and the subjective nature of music preference , there is a clear basis for encouraging the self - selection or democratisation of music selection where possible . the role of exercise practitioners should be limited to guiding and focusing the selection process by helping individuals to identify motivational music and develop track selections into coherent programmes that contour the mental and physical demands of their workout ( see , e.g. , karageorghis , jones , et al . , 2011 ) . the use of objective music selection procedures ( e.g. , bmri-2 ) would seem to be most apposite in the case of large groups of exercise participants , especially those that fluctuate over time such as the usership of a gym . in these cases , individual preferences and needs of the few this is especially so when such groups are mixed in terms of gender , age and socio - cultural background . special attention should be given to the distinction between pre- , in- , and post - task music selection . we contend that at present practitioners give insufficient consideration to pre- and post - task music . pre - task music can be used as an effective pre - exercise prime while post - task music can aid mental and physical revitalisation . while there should be a clear demarcation among the various applications , it is possible to maintain coherence in a music programme by using pieces from the same era , the same genre , or by the same artist when moving between the pre- , in- , and post - task phases . terry and karageorghis ( 2011 ) recently proposed guidelines to aid the selection of post - task music . it should possess sedative qualities , such as a tempo in the range 6070 bpm ( around resting heart rate ) , a simplistic rhythmical structure , regular pulsation and repetitive tonal patterns based on a limited number of pitch levels . in terms of emotional tone , the music should be neutral or relaxing , while the instrumentation might be comprised of soothing , warm instruments , such as strings , oboe or gentle piano . the inclusion of natural sounds , such as breaking waves , bird song or a babbling brook , may also be effective for this purpose . according to karageorghis , terry , lane , bishop , and priest ( 2011 ) , while music is a beneficial accompaniment to exercise in most circumstances , it is contraindicated under certain conditions : ( a ) when it may distract users from safety - relevant information ( e.g. , on public roads ) ; ( b ) when exercisers need to focus their full attention on learning a demanding motor skill ( e.g. , a power clean ) ; and ( c ) when exercising at high intensities that require an associative attentional style ( i.e. , listening to the body ) . it is probable that , as a stimulant , music should be used intermittently ( i.e. , not all of the time ) . this approach will prevent desensitisation to its stimulative effects and permit exercisers to habituate themselves for occasions when they may not be able to use it ( e.g. , long - distance running events in which music is not permitted ) . excessively loud music combined with high - intensity exercise is to be avoided owing to the potential threat to the structures of the inner ear . research shows that even moderate - intensity activity accompanied by very loud music ( > 100 db sound pressure level ) can cause temporary hearing loss ( lindgren & axelsson , 1988 ) . the present review bears several implications for future research through the trends and gap teeth in the literature that it has covered . notably , the role of age , gender and other personal variables in musical reactivity within the exercise context is currently an underexplored area . for example , is it the case , as has been suggested in qualitative investigations ( e.g. , priest & karageorghis , 2008 ) , that music is a more powerful stimulus for younger exercise participants than it is for seniors ? perhaps the lack of research into age - congruent music has limited our understanding and so researchers should consider experimental protocols that entail age - congruent and age - incongruent music programmes . as noted by crust and clough ( 2006 ) , an important personal factor that has been largely overlooked in the literature an obvious entre into this line of research would be to examine extraverted vs. introverted exercise participants with the hypothesis that extraverts would be likely to respond more favourably to loud / stimulative musical selections than introverts ( cf . similarly , attentional style and musical response in the exercise domain has received scant attention ; it is thought that dissociators are likely to derive greater benefit from music use than associators , although an important mediating variable is task intensity . there is emerging evidence that cognitive strategies change in accordance with the intensity of an exercise task ( hutchinson & tenenbaum , 2006 , 2007 ) which means that low - intensity tasks leave greater attentional capacity for parallel processing tasks such as music listening ( cf . hence , examining responses to music across a range of exercise intensities while employing participants who exhibit different attentional styles would be an especially propitious endeavour . researchers should also consider the extent to which music moderates ratings of perceived exertion beyond the anaerobic threshold , when physiological cues tend to predominate attentional processes . when a multi - dimensional approach is taken in the measurement of perceived exertion ( e.g. , hutchinson & tenenbaum , 2006 ) it appears likely that carefully selected music ( e.g. , motivational music ) can influence the motivational - affective component of perceived exertion beyond the anaerobic threshold , even if it can not alter gestalt perceptions of exertion at such intensities . it is certainly the case that the degree to which the motivational qualities of music mediate reductions in perceived exertion warrants further empirical investigation . work to date has not systematically investigated the role of the lyrical content of music in determining psychological or psycho - physiological responses during exercise . it is plausible that the additional information processing capacity required to process lyrics can increase the dissociative influence of music , but there is a notable lack of exercise - related studies that compare responses to the same piece of music with and without lyrics . there is just one recent conference paper which showed that although lyrics contributed to the perceived motivational qualities of music , they had no bearing on psycho - physiological responses or work output in a submaximal cycle ergometer task ( sanchez , moss , twist , & karageorghis , 2011 ) . self - selected vs. experimenter - selected music has also been seldom tested ( e.g. , dyrlund & wininger , 2008 ) although researchers should be aware of the inherent difficulties pertaining to self - selection ; there is a greater likelihood for the emergence of hawthorne effects and experimenter effects ( see lucaccini & kreit , 1972 ) . with reference to the cubic trend identified in the exercise heart rate music tempo preference relationship ( karageorghis , jones , et al . , 2011 ) , future researchers should seek to investigate the link between music tempo preference and a range of affective and motivational outcomes . this approach will reveal the psychological consequences of congruity between exercise heart rate and preferred music tempo . study using other exercise modalities ( e.g. , cycling , motorised stepping , dance aerobics , etc . ) and longer exercise bouts would shed light on the stability and applicability of the relationship . with reference to stability , it is unknown whether the cubic trend would manifest itself among middle aged and senior exercise participants , as it emerged through an investigation of young adults . given that maximal heart rate gradually declines with age , one possibility is that the trend line will shift downwards and flatten when investigated among older groups of exercisers . research needs to be undertaken to evaluate the comparative effects of music used synchronously and asynchronously . in particular , do females derive greater benefit from the synchronous use of music when undertaking complex motor tasks such as callisthenic - type exercises or aerobic dance exercise ( cf . researchers may also consider the role of rhythmic ability in response to the synchronous use of music ( cf . accordingly , there is scope to examine how syncopated ( off beat ) rhythms are processed by high and low rhythmic ability groups . there is a strong likelihood that individuals with high rhythmic ability and/or musical training will process such complex rhythms more easily , thus responding with greater bodily precision than their less musical counterparts ( see , e.g. , vuust et al . , 2005 ) . research into post - task music use is in its infancy so there is considerable scope for work that ascertains how music facilitates recovery from injury and intense exercise . for this reason , it would be advantageous for researchers to develop and validate a tool , similar to the brunel music rating inventory ( bmri ) , for the assessment of the sedative qualities of music in an exercise context . in such investigations , an insightful approach would be the use of psychobiological measures , such as salivary cortisol , to examine the mechanisms that underlie the potential benefits of post - task music . the few studies that have examined training status as a moderator of exercisers response to music have shown that this is an important personal variable that should be considered for inclusion in future research ( brownley et al . , 1995 ; two tentative findings that require more detailed investigation are that untrained participants tend to derive greater benefit from music , while those who are highly trained prefer to listen to their bodies ( i.e. , use an associative attentional strategy ) . nonetheless , the evidence presented herein indicates that , under certain circumstances ( long - duration , low - intensity activities ) , highly trained individuals stand to benefit from the judicious use of music . work on the interactive effects of music and video is virgin territory for researchers , yet it represents a modality that is increasingly becoming ubiquitous in the gym environment ( see barwood et al . , 2009 ; loizou & karageorghis , 2009 ) . potential paths include the use of interview and observational techniques to gauge how exercisers respond to music and video across a range of exercise modalities . the ultimate potential avenue for research is an investigation of the neurophysiological correlates of the musical response before , during and after exercise . the development of new technology , particularly fmri scanners that can be used during gross motor tasks , would facilitate such investigations . for example , this approach would be a possible means by which to explore the proposed attentional switching ( rejeski , 1985 ) between internal and external cues that may take place at anaerobic threshold . a further possible exploration would entail evaluating the role of associations and memory in musical response and the shared activation between music and other emotive stimuli . the evidence reviewed and synthesised in parts i and ii of this paper lead to a number of implications for exercise practitioners and participants . music selected as an accompaniment for exercise should be congruent with participants personal characteristics ( e.g. , age and socio - cultural upbringing ) , the exercise environment and desired outcomes . when working with a mixed group in terms of age and/or sociocultural upbringing , practitioners might consider democratising the music selection process so that all participants musical predilections are accounted for and reflected in the music programme ( dyrlund & wininger , 2008 ; nakamura et al . , 2010 ) . music tempo should be selected with the expected exercise intensity in mind , and be sequenced to contour in accordance with changes in heart rate . other than when warming up , warming down or recuperating , the appropriate band of tempi for exercise intensities in the range 4090% max hrr is 125140 bpm when the music is used asynchronously ( see karageorghis , jones , et al . , 2011 ) . it is also advantageous for the rhythm of the music to approximate the motor patterns entailed where possible ( crust , 2008 ; schneider et al . , 2010 ) . in group exercise classes when music is used synchronously , it appears that the rhythmic abilities of participants warrant careful consideration in the sequencing of music selections ( see , e.g. , denora , 2000 , pp . synchronous motor activity may occur in association with complex rhythmical segments that involve syncopation or span musical measures , not simply with the principal beat that marks musical tempo ( styns et al . , 2007 ) . music containing affirmations of exercise or inspirational references drawn from popular culture should be selected in order to promote motivational imagery and self - talk . positive affect is thought to be consequent to the harmonic and melodic features of the music and its lyrical content ( karageorghis et al . , 1999 ; lucaccini & kreit , 1972 ) . in order to have a stimulative effect , the music should be up - tempo ( > 120 bpm ) and possess prominent percussive and rhythmical features . in order to sedate , the inverse is true in terms of percussion and rhythmical features , and tempi < 80 bpm are recommended ( see terry & karageorghis , 2011 ) . a novel finding that has emanated from qualitative work is that music selection for exercise is concerned with the entire programme of music , not just individual pieces ( e.g. , denora , 2000 , pp . 89103 ; priest & karageorghis , 2008 ) , a perspective not countenanced in the experimental literature . for example , selectors should consider the congruence of musical pieces that appear in close proximity on a playlist and aim to achieve variety in terms of churn ( i.e. , varying selections from session to session ) . with specific reference to congruence , taking into account factors such as beat matching , style matching , artist matching , era matching , etc moreover , a change or absence of music can be an effective marker of the next exercise phase or unit ( denora ) , such as the progression from a cardiovascular segment to a warm - down phase . the findings reported in this review lead us to the conclusion that self - selected music is especially efficacious in an applied context ( e.g. , bharani , sahu , & mathew , 1984 ; priest & karageorghis , 2008 ) . it is exceedingly unlikely that a motivational music programme selected by a practitioner would yield stronger effects than a programme selected by the participant themselves , unless they make inept choices . self - selection also invokes a sense of autonomy , which is a central tenet of intrinsically motivated behaviour ( ryan & deci , 2000 ) . bearing in mind the individual variation in the desired psychological state during exercise and the subjective nature of music preference , there is a clear basis for encouraging the self - selection or democratisation of music selection where possible . the role of exercise practitioners should be limited to guiding and focusing the selection process by helping individuals to identify motivational music and develop track selections into coherent programmes that contour the mental and physical demands of their workout ( see , e.g. , karageorghis , jones , et al . , 2011 ) . the use of objective music selection procedures ( e.g. , bmri-2 ) would seem to be most apposite in the case of large groups of exercise participants , especially those that fluctuate over time such as the usership of a gym . in these cases , individual preferences and needs of the few are subsumed into a collective response ( of the many ) . this is especially so when such groups are mixed in terms of gender , age and socio - cultural background . special attention should be given to the distinction between pre- , in- , and post - task music selection . we contend that at present practitioners give insufficient consideration to pre- and post - task music . pre - task music can be used as an effective pre - exercise prime while post - task music can aid mental and physical revitalisation . while there should be a clear demarcation among the various applications , it is possible to maintain coherence in a music programme by using pieces from the same era , the same genre , or by the same artist when moving between the pre- , in- , and post - task phases . terry and karageorghis ( 2011 ) recently proposed guidelines to aid the selection of post - task music . it should possess sedative qualities , such as a tempo in the range 6070 bpm ( around resting heart rate ) , a simplistic rhythmical structure , regular pulsation and repetitive tonal patterns based on a limited number of pitch levels . in terms of emotional tone , the music should be neutral or relaxing , while the instrumentation might be comprised of soothing , the inclusion of natural sounds , such as breaking waves , bird song or a babbling brook , may also be effective for this purpose . according to karageorghis , terry , lane , bishop , and priest ( 2011 ) , while music is a beneficial accompaniment to exercise in most circumstances , it is contraindicated under certain conditions : ( a ) when it may distract users from safety - relevant information ( e.g. , on public roads ) ; ( b ) when exercisers need to focus their full attention on learning a demanding motor skill ( e.g. , a power clean ) ; and ( c ) when exercising at high intensities that require an associative attentional style ( i.e. , listening to the body ) . it is probable that , as a stimulant , music should be used intermittently ( i.e. , not all of the time ) . this approach will prevent desensitisation to its stimulative effects and permit exercisers to habituate themselves for occasions when they may not be able to use it ( e.g. , long - distance running events in which music is not permitted ) . excessively loud music combined with high - intensity exercise is to be avoided owing to the potential threat to the structures of the inner ear . research shows that even moderate - intensity activity accompanied by very loud music ( > 100 db sound pressure level ) can cause temporary hearing loss ( lindgren & axelsson , 1988 ) . the evidence in this field demonstrates that music has the capacity to exert beneficial psychological and ergogenic effects across a wide range of exercise tasks and through a number of applications , pre - task , in - task and post - task . the evidence is not unilateral and there are certain circumstances , tasks and participant groups that are more conducive to these benefits than others . nevertheless , with the exception of the psycho - physiological responses to music , the effects described herein are of a consistent nature . it is notable that the findings which support these conclusions are influenced heavily by the heightened methodological scrutiny and theoretical framework that emerged in the late 1990s . from this point , researchers have generally adopted a more judicious approach to music selection and become more sensitive to participants musical preferences and socio - cultural background . researchers are now more aware of the inherent subjectivity of the stimulus under investigation and the complexity of its effects . music is now rarely viewed in a manner akin to the vitamin model described by sloboda ( 2008 , p. 32 ) wherein one can ascribe immutable effects to a specific musical selection for all listeners and at all times . the beneficial consequences of music use stem from an interaction between elements of the musical stimulus itself and factors relating to the traits and experiences of the listener , and aspects of the exercise environment and task . in particular , the role of music is dependent on when it is introduced in relation to the task and the intensity of the exercise undertaken . in closing , the evidence presented in this review demonstrates that music has a consistent and measurable effect on the psychological state and behaviour of exercise participants .

since a 1997 review by karageorghis and terry , which highlighted the state of knowledge and methodological weaknesses , the number of studies investigating musical reactivity in relation to exercise has swelled considerably . in this two - part review paper , the development of conceptual approaches and mechanisms underlying the effects of music are explicated ( part i ) , followed by a critical review and synthesis of empirical work ( spread over parts i and ii ) . pre - task music has been shown to optimise arousal , facilitate task - relevant imagery and improve performance in simple motoric tasks . during repetitive , endurance - type activities , self - selected , motivational and stimulative music has been shown to enhance affect , reduce ratings of perceived exertion , improve energy efficiency and lead to increased work output . there is evidence to suggest that carefully selected music can promote ergogenic and psychological benefits during high - intensity exercise , although it appears to be ineffective in reducing perceptions of exertion beyond the anaerobic threshold . the effects of music appear to be at their most potent when it is used to accompany self - paced exercise or in externally valid conditions . when selected according to its motivational qualities , the positive impact of music on both psychological state and performance is magnified . guidelines are provided for future research and exercise practitioners .

abdominal surgery that was previously performed via a large incision is now more commonly performed laparoscopically . the laparoscopic surgeries involve structures such as the gall bladder , colon , small intestine , stomach , liver , and pancreas . in laparoscopy , intraoperative pulmonary changes are due to decreased pulmonary compliance secondary to upward movement of the diaphragm during insufflation and to changes in carbon dioxide ( co2 ) homeostasis secondary to absorption of insufflated co2 from peritoneum . general anesthesia and surgery related pain may lead to changes in the ventilation pattern resulting in the patient taking shallow breaths which reduce the ability to clear sputum from the chest [ 46 ] . studies have reported altered pulmonary function after both conventional and laparoscopic abdominal surgeries [ 712 ] . postoperative pulmonary dysfunction in laparoscopic surgery is approximately 20% to 25% depending upon the type of surgery [ 79 ] . pulmonary dysfunction leads to pulmonary complications which includes atelectasis , pneumonia , tracheobronchial infection , and respiratory failure . reduction of pulmonary function , forced vital capacity ( fvc ) , and forced expiratory vital capacity ( fev1 ) have been reported on the basis of functional alterations . chest physiotherapy has been employed as an alternative intervention to reduce occurrence of pulmonary function loss and its complications . it includes breathing exercises , percussion , vibration , splinted huffing / coughing , positioning , and mobilization . diaphragmatic breathing exercises are used in order to augment diaphragmatic descent while inhalation and diaphragmatic ascent while expiration . the beneficial effects of diaphragmatic breathing are as follows : inflation of the alveoli , reversing postoperative hypoxemia , improvement of ventilation and oxygenation , decreasing the work of breathing , and increasing the degree of excursion of the diaphragm [ 16 , 17 ] . mechanical breathing device such as the incentive spirometry ( is ) has been introduced into clinical practice . incentive spirometry encourages the patient to take long , slow deep breath mimicking natural sighing and also provides a visual positive feedback . incentive spirometers are available either by volume of inspiration ( volume - oriented ) or flow rate ( flow - oriented ) [ 46 , 1820 ] . the flow - oriented incentive spirometer ( triflow device ) consists of three chambers in series , each of which contains a ball . when the patient 's effort generates a subatmosphere pressure above the ball , it rises in the chamber . an inspiratory flow of 600 ml / s is required to raise the first ball , an inspiratory flow of 900 ml / s is required to elevate the first and second balls , and a flow of 1200 ml / s is required to elevate all three balls . the volume - oriented incentive spirometer is a compact device of 4000 ml capacity and has a one - way valve to prevent exhalation into the unit . a sliding pointer indicates the prescribed inspiratory volume and an inspiratory flow guide coaches the subject to inhale slowly [ 1820 ] . studies suggest a physiologically significant difference in the effect of the flow- and volume - oriented incentive spirometer . flow - oriented devices ( triflow device ) enforce more work of breathing and increase muscular activity of the upper chest . volume - oriented devices ( coach 2 device ) enforce less work of breathing and improve diaphragmatic activity [ 6 , 1821 ] . earlier studies show that the volumetric incentive spirometer is better in case of cardiac and thoracic surgeries because it provides the appropriate feedback for a slow sustained inspiration and volume . studies show that slow sustained inspirations are much more effective to promote lung expansion rather than fast inspirations . studies also show that diaphragmatic breathing exercise encourages more diaphragmatic movement [ 17 , 18 ] . studied thirty - six subjects , in order to assess the effect of respiratory kinesiotherapy on respiratory muscle strength and pulmonary function following laparoscopic cholecystectomy . three breathing exercises were performed by seventeen subjects while other nineteen served as a control group . all the subjects were assessed for maximal inspiratory pressure ( mip ) and maximal expiratory pressure ( mep ) , pef , and spirometry ( fvc , fev1 , and fev1/fvc ) . both groups registered a decrease in all variables on the first day after surgery . on the second postoperative day , the exercise group showed decreased values for all variables . however , values of all variables for the control group begin to normalize only on the fifth postoperative day . . carried out a study on two experimental groups of patients in order to evaluate the effects of aerobic exercise training and incentive spirometry in controlling pulmonary complications following laparoscopic cholecystectomy . one group was given aerobic walking raining and incentive spirometry as well as traditional physical therapy ( group a ) ; the other ( group b ) was given traditional physical therapy . results indicated a significant reduction in heart rate , sao2 , and inspiratory capacity for both groups . the researchers concluded that aerobic exercise and incentive spirometry were beneficial in reducing the postoperative pulmonary complications after laparoscopic cholecystectomy . kundra et al . carried out a comparative study on the effect of preoperative and postoperative incentive spirometry on the pulmonary function of fifty patients who had undergone laparoscopic cholecystectomy . the study group had to carry out incentive spirometry fifteen times before surgery , every four hours , for one week . pulmonary function was recorded before surgery and 6 , 24 , and 48 hours postoperatively and at the time of discharge . the authors concluded that pulmonary function is well - preserved with preoperative than postoperative incentive spirometry . reviewed forty - four studies in order to evaluate the effects of chest physiotherapy interventions in laparoscopic and open abdominal surgery . but the results showed that breathing exercises were efficacious in preventing postoperative pulmonary complications in patients undergoing open surgery . the review also showed that laparoscopic procedures impair respiratory function to a considerably lower degree than open surgery . one study in the review showed that routine treatment is not called for in upper gastrointestinal features such as , for instance , fundoplication and vertical banded gastroplasty . studied forty - one morbidly obese to assess use of incentive spirometry preoperatively which could help patients to preserve their pulmonary function ( inspiratory capacity ) better in the postoperative period following laparoscopic bariatric surgery . subjects were randomly sorted into two groups ( the exercise and the control group ) . the exercise group used the incentive spirometer for ten breaths , five times per day . the control group used incentive spirometer three breaths , once per day . pulmonary function ( inspiratory capacity ) the author concluded that preoperative use of the incentive spirometer does not lead to significant improvement of pulmonary function ( inspiratory capacity ) . various chest physiotherapy techniques are used clinically as part of the routine prophylactic and therapeutic regimen in postoperative respiratory care . however , the efficacy of flow and volume incentive spirometry and diaphragmatic breathing exercise is still controversial [ 6 , 17 ] . there are no retrievable studies that have been done on the clinical efficacy of diaphragmatic breathing exercise and flow and volume incentive spirometry after laparoscopic abdominal surgery . with this background the present study aim is to compare the effect of diaphragmatic breathing exercise , flow and volume incentive spirometry , on pulmonary function and diaphragm excursion , following laparoscopic surgery . inclusion criteria involved subjects of either gender in the age group of 18 to 80 years who were posted for laparoscopic abdominal surgery . exclusion criteria were as follows : patients who had undergone open abdominal surgery and laparoscopic obstetrics and gynecological surgery.patients with unstable hemodynamic parameters ( arterial pressure < 100 mmhg systolic and < 60 mmhg for diastolic and mean arterial pressure ( map ) < 80 mmhg).patients with postoperative complications requiring mechanical ventilation.uncooperative patients or patients unable to understand or to use the device properly.patients with inadequate inspiration characterized by vital capacity < 10 ml / kg . patients with unstable hemodynamic parameters ( arterial pressure < 100 mmhg systolic and < 60 mmhg for diastolic and mean arterial pressure ( map ) < 80 mmhg ) . patients with postoperative complications requiring mechanical ventilation . equipment used was as follows : ultrasonography machine ( voluson730).pulmonary function test machine ( easyone plus portable diagnostic spirometer machine , ndd medical technologies , inc . massachusetts , usa).flow - oriented incentive spirometry machine ( triflow device , igna medical devices , mumbai).volume - oriented incentive spirometry machine ( coach 2 device , smiths medical international ltd . pulmonary function test machine ( easyone plus portable diagnostic spirometer machine , ndd medical technologies , inc . flow - oriented incentive spirometry machine ( triflow device , igna medical devices , mumbai ) . volume - oriented incentive spirometry machine ( coach 2 device , smiths medical international ltd . the study was carried out in kasturba medical college hospitals mangalore over a period of four years starting from january 2011 to december 2014 . the purpose of study was made clear to each patient and a written informed consent was obtained prior to involving them in the study . the patients were divided into four groups : flow - oriented incentive spirometry group ( triflow device ) . the entire sample was divided into 13 blocks with 20 patients in each , 5 belonging to each group . group information was concealed in a sealed opaque envelope and revealed to the patients only after they were recruited into the treatment group or the control group done by primary investigator . following the allocation to groups , the patients in the treatment group were visited one day prior to the surgery ; preoperative information was offered and , based upon his / her group , flow - oriented incentive spirometry , volume - oriented incentive spirometry , or diaphragmatic breathing exercise was taught to each patient . other therapies like airway clearance techniques , thoracic expansion exercise , and mobilization were also taught to every patient in all treatment groups ( see steps 15 ) . the first step is diaphragmatic breathing exercise , flow or volume incentive spirometry ( 3 sets , 5 repetitions of deep breaths ) . the first step is diaphragmatic breathing exercise , flow or volume incentive spirometry ( 3 sets , 5 repetitions of deep breaths ) . the third step is circulation ( foot and ankle pumping , hip and knee bending 10 times each hour ) . the third step is circulation ( foot and ankle pumping , hip and knee bending 10 times each hour ) . the fourth step is thoracic expansion exercise ( position patient in long sitting in bed / high sitting over the side of the bed ) . the fourth step is thoracic expansion exercise ( position patient in long sitting in bed / high sitting over the side of the bed ) . the fifth step is mobilization : sitting out of the bed in a chair ( one hour twice daily).walking ( three times per day).stair climbing done before the patient was discharged from the hospital . the fifth step is mobilization : sitting out of the bed in a chair ( one hour twice daily).walking ( three times per day).stair climbing done before the patient was discharged from the hospital . sitting out of the bed in a chair ( one hour twice daily ) . walking ( three times per day ) . an experienced radiologist carried out ultrasonography for diaphragm excursion on the preoperative as well as the 1st and 2nd postoperative day , for all groups . pulmonary function tests ( pft ) measured the following variables : forced vital capacity ( fvc ) , forced expiratory volume in the first second ( fev1 ) , peak expiratory flow rate ( pefr ) . these were taken on the preoperative day and 1st and the 2nd postoperative day , for all groups . the patient lays in the supine position and diaphragm movements were recorded in the b - mode . the probe was positioned between the midclavicular and anterior axillary lines , in the subcostal area , so that the ultrasound beam entered the posterior third of the right hemidiaphragm perpendicularly . the procedure began at the end of normal expiration with the subjects being instructed to inhale as deeply as possible . a fixed point at the edge of the image on the screen and the diaphragm margin at maximal inspiration and again at maximal expiration served as reference points between which measurements were made , with the average of three values being taken for both maximal inspiration and maximal expiration [ 26 , 27 ] . pulmonary function test procedures ( easyone plus portable diagnostic spirometer machine ) were carried out according to the american thoracic society / european respiratory society guidelines . the following variables have been recorded : forced vital capacity ( fvc ) , forced expiratory volume in the first second ( fev1 ) , and peak expiratory flow rate ( pefr ) the best value of 3 acceptable tests . the patient was placed in a semirecumbent position ( 45 ) , with a pillow under the knees . the patient was instructed to inhale with a slow and deep sustained breath , holding it for a minimum of 5 seconds and then to exhale passively in order to avoid any forceful expiration . first , the patient was given demonstration and then asked to perform in order to ensure that she / he understood the process [ 15 , 17 ] . the patient was instructed to hold the spirometer upright and to perform flow - oriented incentive spirometry by inhaling slowly and thereby raising the ball , followed by volume incentive spirometry in order to raise the piston or plate in the chamber to the set target [ 19 , 20 ] . the therapist administered the exercise four times a day and the patient was instructed to perform the same for the rest of the day . the patient was asked to keep a record of the exercise performed by entering in a log book which was provided beforehand . the patient assumed a semi - fowler 's position ( back and head are fully supported and abdominal wall is relaxed ) and performed diaphragmatic breathing . the therapist placed his hands just below the anterior costal margin , on the rectus abdominis , while the patient was instructed to inhale slowly and deeply through the nose , from functional residual capacity to total lung capacity with a three - second inspiratory hold . the patient was then instructed to relax the shoulders , keep the upper chest quiet in order that the abdomen be raised a little . the patient was then instructed to exhale slowly through the mouth [ 16 , 28 ] . the patient was made to experience a slight rise and subsequent fall of the abdomen during inspiration and expiration , by placing his or her own hand below the anterior costal margin . the patient was instructed to perform 3 sets of 5 deep breaths with the therapist administering them four times a day and the patient being instructed to perform the same once every waking hour for the rest of the day . in between the repetitions of the diaphragmatic breathing exercise , the patient was asked to keep a record of the exercise performed by entering in a log book which was provided beforehand . the sample size was calculated based on the values obtained from pulmonary function test in a pilot study ( 20 subjects , 5 in each group ) [ 30 , 31 ] . the following formula was used for calculating the same : ( 1)n=2z+zd / s2,where n is the number of subjects in each group and z and z are constants and they are substituted . selected power for the study was 90% and d is effect size which is the absolute value of the difference in means and represents what is considered a clinically meaningful or practically important difference in means . d is taken from the pilot study which used the same variable , which compared pulmonary function test in subjects , and s is the standard deviation of the means . the sample size is 65 in each group ( total 260 subjects ) . anova and post hoc analysis ( bonferroni 's t - test ) were carried out to verify the within - groups differences . between - groups we selected 274 patients posted for laparoscopic abdominal surgery , of which 260 were included in the study . baseline demographic characteristics of the participants such as age , height , weight , bmi , risk factors , and duration of surgery are presented in table 1 . data about patients who underwent different types of laparoscopic abdominal surgery are summarized in table 1 . of the 260 patients included , 140 patients underwent cholecystectomy , 53 hernioplasty , 43 appendectomy , 11 umbilical hernia repair , 8 laparoscopic diagnostic , 3 bariatric surgery , and 2 hemicolectomy . forced vital capacity ( fvc ) was compared within the intervention groups and the control group before and after operation , and the same is summarized in table 2 . there was a statistically significant decrease in forced vital capacity ( fvc ) in the 1st and 2nd post - op day when compared with the preoperative period in all groups . forced expiratory volume in one second ( fev1 ) was compared within the intervention groups and the control group before and after the operation and is summarized in table 3 . there was a statistically significant decrease in forced expiratory volume at the end of the first second ( fev1 ) on the 1st and 2nd postoperative day when compared with the preoperative period in all groups . peak expiratory flow rates ( pefr ) were compared with the intervention groups and control group before and after operation and are summarized in table 4 . in all groups there was a statistically significant decrease in peak expiratory flow rate ( pefr ) on the 1st and 2nd postoperative day compared to the preoperative period . diaphragm excursions were compared within intervention groups and the control group before and after operation and are summarized in table 5 . there was a statistically significant decrease in diaphragm excursion in the 1st and 2nd postoperative period when compared with the preoperative period in all groups except in diaphragmatic breathing exercise group and volume incentive spirometry group which almost came back to normal . forced vital capacity ( fvc ) , forced expiratory volume in one second ( fev1 ) , peak expiratory flow rate , and diaphragm excursion were compared between the intervention groups and the control group during the preoperative and 2nd postoperative day and are summarized in table 6 . there was a statistically significant difference between intervention groups ( diaphragmatic breathing exercise group and volume incentive spirometry group ) and control group in terms of forced vital capacity ( fvc ) and diaphragm excursion ( p < 0.001 ) , the said variables being significantly lower in the control group than in the diaphragmatic breathing exercise group and volume incentive spirometry group . the main purpose of this study was to compare diaphragmatic breathing exercise , flow- and volume incentive spirometry , on pulmonary function and diaphragmatic excursion in patients undergoing laparoscopic abdominal surgery . to the best of our knowledge , this study is the first to compare the effects of diaphragmatic breathing exercise with two different kinds of incentive spirometry and also against a control group . there were 65 patients included in each group and the four groups were homogenous in terms of all demographic parameters . in our study we found that diaphragmatic breathing exercise and volume incentive spirometry improve lung function and diaphragm excursion in patients undergoing laparoscopic abdominal surgery . in our study pulmonary function ( fvc , fev1 , and pefr ) and diaphragm excursion showed a decrease on the 1st postoperative day when compared to the preoperative values in all four groups with an average decrease of 27% in forced vital capacity , 28% in forced expiratory volume in one second , 37% in peak expiratory flow rate , and 28% in diaphragm excursion . the present study finding of reduction in pulmonary function during postoperative day is similar to those reported in a previous study [ 3 , 2124 ] . our results are in accordance with schauer et al . who found 30% to 38% reduction in postoperative pulmonary function ( fvc , fev1 , and fef25%75% ) in laparoscopic cholecystectomy . ramos et al . found 20% to 30% reduction in postoperative pulmonary function ( fvc and fev1 ) in laparoscopic cholecystectomy . found 21% to 31% reduction in postoperative day pulmonary function variables ( fvc , fev1 , and fef25%75% ) in laparoscopic cholecystectomy . possible reasons for decrease in pulmonary function and diaphragm excursion during the postoperative period in laparoscopic abdominal surgery are as follows . during the postoperative period , patients exhibit shallow breathing without the intermittent sigh or breaths which are inspired approximately ten times an hour . patients will breathe shallowly which leads to a decrease in ventilation to dependent lung regions [ 7 , 32 , 33 ] . in the present study , reduced pulmonary function ( fvc , fev1 , and pefr ) and diaphragm excursion in postoperative laparoscopic abdominal surgery subjects might be due to postoperative pain , location of surgical ports , along with anaesthetic , analgesic usage [ 7 , 34 ] . the effects of general anaesthesia on distribution of ventilation and chest wall and lung mechanics lead to ventilation - perfusion mismatch , increased dead space , shunt , and hypoxemia [ 9 , 35 , 36 ] . narcotic / opioid analgesics and other drugs affect the central regulation of breathing , changing the neural drive of the upper airway and chest wall muscles , which lead to hypoventilation , a diminished sensitivity of the respiratory center to carbon dioxide stimulation , an increase of obstructive breathlessness , the suppression of the cough reflex , and irregular mucus production . the location of surgical ports involves trauma near the diaphragm and chest wall / ribs , leading to postoperative incisional pain and reflex inhibition of the phrenic nerve and diaphragmatic reflex paresis resulting in functional disruption of respiratory muscle movement . in addition , when patients remain lying down for long periods during the postoperative period their abdominal content limits diaphragmatic movement . several studies found that diaphragmatic dysfunction is due to gas insufflation in the abdominal cavity which might also be responsible for the increase of resistance and reduced diaphragmatic excursion , leading to reduced lung volume . all these factors lead to a change in postoperative lung function usually resulting in development of a restrictive pattern and decreased diaphragm excursion in laparoscopic abdominal surgery . our results are in accordance with ford et al . , who showed that reduction in inspiratory muscle activity , mainly the diaphragm , was the main determinant of impaired pulmonary function . several studies suggested that laparoscopic abdominal surgery causes reflex inhibition of the phrenic nerve which might lead to shallow breaths and reduced pulmonary ventilation . showed a decrease of 27% in the respiratory muscular activity of patients who underwent laparoscopy abdominal surgery . possible reasons for improved pulmonary function and diaphragm excursion in the diaphragmatic breathing exercise group are as follows . the present study showed that the diaphragmatic breathing exercise group was able to improve pulmonary mechanics thus leading to a beneficial effect on pulmonary function ( fvc ) and diaphragm excursion . diaphragmatic breathing exercise improves diaphragmatic descent and diaphragmatic ascent during inspiration and expiration , respectively . slower deep inspiration ensures more even distribution of air throughout the lung , particularly to the dependent lung . the physiological effects of diaphragmatic breathing exercise are that breathing through full vital capacity and holding for 35 seconds ensure full inflation of the lungs thus opening up alveoli which have low volume and stimulating the production of surfactant . diaphragmatic breathing exercise will also decrease activity of accessory muscles , ensure that breathing patterns are as close to normal as possible , and also reduce the work of breathing [ 16 , 31 ] . our results are in accordance with the findings of tahir et al . who showed that diaphragmatic breathing exercise will improve basal ventilation . weber and prayar and menkes and britt found that diaphragmatic breathing exercise will improve tidal volume and also facilitate secretion removal [ 43 , 44 ] . blaney and sawyer observed that tactile stimulation over the subject 's lower costal margin as well as verbal instruction served to significantly increase diaphragmatic movement during diaphragmatic breathing exercises . found that diaphragmatic breathing exercise was able to improve pulmonary mechanics and lead to beneficial effect on forced vital capacity ( fvc ) . grams et al . evaluated the efficacy of diaphragmatic breathing exercise for the prevention of postoperative pulmonary complications and for the recovery of pulmonary mechanics and found that diaphragmatic breathing exercise appeared to be more effective . possible reasons for improved pulmonary function and diaphragmatic excursion in the volume incentive spirometry group are as follows . the present study showed that the volume incentive spirometry group also had improved pulmonary mechanics that led to a beneficial effect on pulmonary function ( fvc ) and diaphragm excursion . after laparoscopic abdominal surgery , it may be hard to take a deep breath and if patients do not breathe deeply it may lead to postoperative pulmonary complications . the volume incentive spirometer is a mechanical device used to take slow , deep long breaths that encourage patients to breathe to total lung capacity , to sustain that inflation and open up collapsed alveoli . the volume incentive spirometer will be more physiological because the training volume is constant until it reaches the maximum inspiratory capacity ( level preset by physiotherapist ) . it provides a low level of resistance training while minimizing the potential fatigue to the diaphragm . our study results are in accordance with paisani et al . who showed that when volume incentive spirometry was performed with low inspiratory flow it promoted diaphragmatic excursion and improved the expansion of the basal area of chest wall . observed that patients treated with incentive spirometry would have early recovery of the pulmonary volume . found that the use of incentive spirometry in the preoperative period leads to greater improvement in the lung functions than if given in the postoperative period . so use of the volume incentive spirometer will result in active recruitment of the diaphragm and other inspiration muscles which may lead to improved pulmonary function and diaphragm excursion . there was no blinding in the study procedure ; the same investigator who randomized the patients into the experimental groups and the control group measured the outcome variables ( pulmonary function test ) and the same investigator taught the exercises to all experimental groups . diaphragm excursion measurement was not done by the same radiologist throughout the study and the finding would have been confounded by the expertise of professional . type of anaesthesia , analgesia , and postoperative pain was not recorded which could affect the findings . there was no follow - up in the study as all patients were discharged on the 2nd postoperative day . patient adherence to the intervention programs was recorded by providing a log book to each subject , in which they had to make an entry the very time they did the prescribed technique but there is no way to verify the authenticity of these entries . future research could be directed at long - term follow - up to see which group sustains improvement for a long duration and the functional aspect of recovery . future studies can be carried out to compare the effect of the techniques on patients who have undergone upper and lower abdominal laparoscopic surgeries , using a larger sample size . effect of combining therapy like incentive spirometer and diaphragmatic breathing exercise can be studied on laparoscopic abdominal surgery patients . future research can be done by assessing and using respiratory muscle strength and patient comfort with different technique as an outcome in laparoscopic abdominal surgery . similar studies can be conducted on patients following open abdominal surgeries and cardiac and thoracic surgeries . based on the results of the study we strongly recommend the following : volume - oriented incentive spirometry and diaphragmatic breathing exercise can be recommended for all patients preoperatively and postoperatively over flow - oriented incentive spirometry as an intervention for the generation and sustenance of pulmonary function and diaphragm excursion in the management of laparoscopic abdominal surgery . volume - oriented incentive spirometry and diaphragmatic breathing exercise can be recommended for all patients preoperatively and postoperatively over flow - oriented incentive spirometry as an intervention for the generation and sustenance of pulmonary function and diaphragm excursion in the management of laparoscopic abdominal surgery . ( i)from our study we conclude that in laparoscopic abdominal surgery patients there is a significant decrease in pulmonary function ( fvc , fev1 , and pefr ) and diaphragm excursion in all four groups on the 1st postoperative day when compared with the preoperative day.(ii)a greater improvement in pulmonary function and diaphragm excursion between the first and second postoperative day was seen in all experimental groups when compared to the control group.(iii)from our study we conclude that pulmonary function and diaphragm excursion was better preserved in the diaphragmatic breathing exercise group and volume incentive spirometry group when compared with the flow incentive spirometry group and the control group . from our study we conclude that in laparoscopic abdominal surgery patients there is a significant decrease in pulmonary function ( fvc , fev1 , and pefr ) and diaphragm excursion in all four groups on the 1st postoperative day when compared with the preoperative day . a greater improvement in pulmonary function and diaphragm excursion between the first and second postoperative day was seen in all experimental groups when compared to the control group . from our study we conclude that pulmonary function and diaphragm excursion was better preserved in the diaphragmatic breathing exercise group and volume incentive spirometry group when compared with the flow incentive spirometry group and the control group .

objective . to evaluate the effects of diaphragmatic breathing exercises and flow and volume - oriented incentive spirometry on pulmonary function and diaphragm excursion in patients undergoing laparoscopic abdominal surgery . methodology . we selected 260 patients posted for laparoscopic abdominal surgery and they were block randomization as follows : 65 patients performed diaphragmatic breathing exercises , 65 patients performed flow incentive spirometry , 65 patients performed volume incentive spirometry , and 65 patients participated as a control group . all of them underwent evaluation of pulmonary function with measurement of forced vital capacity ( fvc ) , forced expiratory volume in the first second ( fev1 ) , peak expiratory flow rate ( pefr ) , and diaphragm excursion measurement by ultrasonography before the operation and on the first and second postoperative days . with the level of significance set at p < 0.05 . results . pulmonary function and diaphragm excursion showed a significant decrease on the first postoperative day in all four groups ( p < 0.001 ) but was evident more in the control group than in the experimental groups . on the second postoperative day pulmonary function ( forced vital capacity ) and diaphragm excursion were found to be better preserved in volume incentive spirometry and diaphragmatic breathing exercise group than in the flow incentive spirometry group and the control group . pulmonary function ( forced vital capacity ) and diaphragm excursion showed statistically significant differences between volume incentive spirometry and diaphragmatic breathing exercise group ( p < 0.05 ) as compared to that flow incentive spirometry group and the control group . conclusion . volume incentive spirometry and diaphragmatic breathing exercise can be recommended as an intervention for all patients pre- and postoperatively , over flow - oriented incentive spirometry for the generation and sustenance of pulmonary function and diaphragm excursion in the management of laparoscopic abdominal surgery .

the hands in apert syndrome often demonstrate so - called spoon hand or mitten hand deformity , including syndactyly , symphalangism and soft - tissue anomalies . consideration of blood flow to the digits of each patient , not only arterial supply but also venous drainage , is very important in the division surgery . an apert syndrome infant with upton 's type ii hands underwent a series of surgeries . at the age of 14 months , separation between the middle and ring fingers was done . under general anaesthesia , just prior to the operation , infrared venography was done with genial - viewer ( genial light co. ltd . , hamamatsu , japan ) [ figure 1 ] and recorded in a laptop computer . full thickness skin graft was taken from the lower abdomen and replanted to cover the defect . separation of index - middle and ring - little fingers was performed 5 months after [ figure 2 ] . genial - viewer is consisted of measuring module ( front ) , power supply unit ( back right ) and a laptop computer for data processing and storage infrared venography through finger splitting surgeries for an apert patient . volar venous arch placed distally to the metacarpophalangeal joint , where incision had to be performed to divide fingers sufficiently . ( a , b ) snap shots of palmar side venogram , immediately before the separation surgery . ( c , d ) designs for the first separation surgery , ( e and f ) immediately after the first separation surgery . ( g , h ) snap shots of venogram , immediately before the second separation surgery . ( i , j ) fifteen months after the second separation surgery infrared venography was able to visualise superficial veins , which could not be seen with bare eyes . real - time venogram was able to be seen through display of a laptop computer . the images were captured and recorded . furthermore , video could be recorded . at the time before the separation procedures , vein of digits the arch placed distally to the metacarpophalangeal joint , where incision has to be done to divide fingers sufficiently . longitudinal vein of palm was observed very thin . on the dorsal aspect , for his left hand , veins could be observed on the radial margin of index , radial side of middle and ulnar margin of little finger . no proper dorsal vein of the ring finger could be identified at that moment . on the dorsum of his right hand , a vein runs on the index finger supplying branches to the middle finger . the other runs slightly radial side of ring - little inter - digital space , which seemed likely to be damaged by separation procedure [ figure 3 ] . at the time of immediately before secondary splitting surgery , palmar venous arch revealed to be severed by the first surgery [ figure 2 ] . venogram of the apert patient 's hands , based on infrared video and snap shots . there were palmar venous arches on both hands , which placed distally to the metacarpophalangeal joint infrared venography was able to visualise superficial veins , which could not be seen with bare eyes . real - time venogram was able to be seen through display of a laptop computer . the images were captured and recorded . furthermore , video could be recorded . at the time before the separation procedures , vein of digits the arch placed distally to the metacarpophalangeal joint , where incision has to be done to divide fingers sufficiently . longitudinal vein of palm was observed very thin . on the dorsal aspect , for his left hand , veins could be observed on the radial margin of index , radial side of middle and ulnar margin of little finger . no proper dorsal vein of the ring finger could be identified at that moment . on the dorsum of his right hand , a vein runs on the index finger supplying branches to the middle finger . the other runs slightly radial side of ring - little inter - digital space , which seemed likely to be damaged by separation procedure [ figure 3 ] . at the time of immediately before secondary splitting surgery , palmar venous arch revealed to be severed by the first surgery [ figure 2 ] . venogram of the apert patient 's hands , based on infrared video and snap shots . there were palmar venous arches on both hands , which placed distally to the metacarpophalangeal joint upton reported , based on his operative records , that a dominant deep palmar arch was present usually abnormally far into webspaces before digital bifurcation was demonstrated . with three - dimensional computed tomography ( ct ) and magnetic resonance imaging , holten et al . reported that common digital arteries tended to bifurcate after the metacarpophalyngeal joint . only writing could be found was a report with digital video angiography , stating normal venous return . this is the first report of venous abnormality in hands of an apert syndrome patient . light transmission through tissues is greatest within the red and near infrared wavelength . at near infra - red band , haemoglobin has lower absorbance than at visible band , though it is relatively high in comparison with other protein in the tissue . therefore , absorbance contrast between vessels and surrounding tissue can be used to visualise vascular pattern . infrared venogram of an author 's hand nowadays , infrared venography of finger or palm has been widely used for personal identification in automated teller machines . based on the same theory , genial - viewer was designed to visualise vasculature beneath skin . it is consisted of measuring module , power supply unit and lap - top computer for data processing and storage . measuring module is composed of infrared submission table and camera , connected with an arm . it is light and handy . it can be used in the operative field only have to be wrapped with an ordinal sterile plastic bag . as infrared ray has strong diffusional scattering character through tissue , it can not penetrate thick part of the body . it is suitable to observe superficial veins . in finger replantation surgery , it is useful to find vessels to anastomose . x - ray angiography , including three - dimensional ct , offers extensive information , but requires complicated sequence and time . there are a few infra - red imaging systems , utilising reflected infrared light while genial - viewer employs transmitted light . for visualising blood vessels of hand , early surgical intervention is recommended for hand in apert syndrome , to minimise progressive growth deformity and influence on patients psychological development . though , splitting all fingers by one stage , performing surgery on both sides of the same digit should be avoided . by means of ultra - red venography , abnormal venous distribution was detected . though , it was confirmed that it is safer to perform staged separation for apert hand , not only because of the arterial abnormality , but also the abnormal venous distribution .

as well as craniofacial synostosis , complex syndactyly of hands is a distinctive feature of apert syndrome . consideration of blood flow to the digits is very important in separation surgery . several reports offer information about arterial distribution in apert 's hands . though , venous pattern has not been well discussed . infrared venography offers a real - time image with minimal invasion . an apert syndrome patient underwent a series of finger splitting surgeries . infrared venography was carried out to assess veins . there was a palmar venous arch , placing distally to the metacarpophalangeal joint . the arch had to be cut to divide fused fingers sufficiently . as well as arterial abnormality , venous uniqueness should be noted in apert syndactyly surgeries . infrared venography , which can be carried out easily , offers good information that surgeon require .

the onset of seizures is usually before the age of 12 months in developmentally normal infants . the first seizure type is usually clonic , generalized or unilateral and triggered by fever . the diagnosis of ds is based on the clinical phenotype ; however , mutations in the scn1a gene can be found in 7585% of patients with ds 2 , 3 . the estimated incidence of ds ranges from 1 in 15 700 to 1 in 40 000 live births 2 , 4 . the current treatment strategy for patients with ds involves the use of multiple antiepileptic drugs ( aeds ) , including combinations of valproate , topiramate , clobazam , stiripentol and others 5 , 6 . in a recent survey of european patients with ds , about 40% of patients were treated with three aeds and approximately 40% were treated with four aeds 6 . despite these multidrug regimens , 45% continue to experience 4 tonic clonic seizures / month 6 . to reduce frequent and disabling seizures , fenfluramine ( 3trifluoromethylnethylamphetamine ) , originally approved as an appetite suppressant 7 , has also been reported to exhibit activity in refractory epilepsy 8 . due to the retrospective character of previous studies 9 , 10 , we wanted to confirm our data in a welldesigned prospective trial in a new group of patients with ds . here we describe the effectiveness , tolerability and safety of fenfluramine in a new cohort of patients with ds who began treatment after 2010 . patients of between 6 months and 50 years of age with a ds diagnosis based on the core phenotype 1 with or without scn1a mutation and whose epilepsy was uncontrolled despite adequate therapy with standard aeds were eligible . the major exclusion criteria included cardiovascular pathology , hypertension treated with medication , glaucoma , or hypersensitivity to fenfluramine or any other amphetaminelike drug . all patients who enrolled between january 2011 and december 2015 and who had at least 3 months of observation after starting fenfluramine were included in this analysis . this prospective openlabel study began with a 3month baseline period during which patients continued treatment with their current aeds . a daily diary was used to record the date , time , type and duration of seizures . a 24h electroencephalographic study was conducted during the baseline period as was an echocardiographic examination to evaluate cardiac valvular structure and function . after the 3month baseline period , fenfluramine was added at a dose of 0.251.0 mg / kg / day . the daily dose could be adjusted during the study based on efficacy or tolerability issues , with a maximum of 20 mg / day . concomitant aeds were kept stable during the first 3 months , thereafter adjustments could be made if necessary ( figure s1 ) . echocardiographic examinations were conducted every 3 months during the first year of treatment , every 6 months during the second year of treatment and annually thereafter . beginning in 2014 all echocardiograms were evaluated by a pediatric ( f.m . ) and an adult ( b.p.p . ) all data from inclusion until the last study visit ( before april 2016 ) were analyzed . fenfluramine was obtained from zogenix international limited , emeryville , ca , usa and its purity was assessed and confirmed by the belgian pharmaceutical association . fenfluramine was compounded into capsules by the pharmacy of antwerp university hospital at doses of 2.5 , 5 , 10 and 20 mg / capsule . most patients in this study swallowed the capsules but , for a few , the contents were sprinkled prior to consumption . the key objective of this study was to assess the overall change in frequency of all major motor seizures during the fenfluramine treatment duration compared with the 3month baseline period . in addition , the change in major motor seizure frequency was assessed at specific time points ( 3 , 6 , 9 and 12 months postinitiation of fenfluramine treatment ) . clonic , tonic , clonic , atonic and myoclonic seizures lasting > 30 s. seizure frequency ( seizures / month ) during the baseline period was calculated as the total number of seizures divided by the number of days in the baseline period multiplied by 30 . during treatment with fenfluramine , seizure frequency taking into account the small sample size and the nongaussian distribution , a wilcoxon signedrank test for paired data was used to analyze the treatment effect . statistical analysis was performed with spss software ( ibm corp spss statistics for windows , version 23.0 , armonk , ny , usa ) . global impression of change , quality of life ( qol ) and sleep quality were assessed at each study visit since june 2015 . the qol and sleep quality of parents and patients were assessed using a visual analog scale ( 0 , extremely bad to 10 , very good ) . global impression of change was evaluated with a fivepoint scale ( 2 , very much improved ; 1 , much improved ; 0 , no change ; 1 , much worse ; and 2 , very much worse ) . following the withdrawal of fenfluramine in 1997 , its use as an aed for the treatment of intractable epilepsy was allowed in belgium following the issuance of a royal decree ( kb 2002/22215 ) . the study protocol was reviewed and approved by the ethics committee at antwerp university hospital prior to the initiation of this study ( registration no . the parents or guardians of each patient were fully informed about the study and provided written informed consent prior to participation in the study . patients of between 6 months and 50 years of age with a ds diagnosis based on the core phenotype 1 with or without scn1a mutation and whose epilepsy was uncontrolled despite adequate therapy with standard aeds were eligible . the major exclusion criteria included cardiovascular pathology , hypertension treated with medication , glaucoma , or hypersensitivity to fenfluramine or any other amphetaminelike drug . all patients who enrolled between january 2011 and december 2015 and who had at least 3 months of observation after starting fenfluramine were included in this analysis . this prospective openlabel study began with a 3month baseline period during which patients continued treatment with their current aeds . a daily diary was used to record the date , time , type and duration of seizures . a 24h electroencephalographic study was conducted during the baseline period as was an echocardiographic examination to evaluate cardiac valvular structure and function . after the 3month baseline period , fenfluramine was added at a dose of 0.251.0 mg / kg / day . the daily dose could be adjusted during the study based on efficacy or tolerability issues , with a maximum of 20 mg / day . concomitant aeds were kept stable during the first 3 months , thereafter adjustments could be made if necessary ( figure s1 ) . echocardiographic examinations were conducted every 3 months during the first year of treatment , every 6 months during the second year of treatment and annually thereafter . beginning in 2014 all echocardiograms were evaluated by a pediatric ( f.m . ) and an adult ( b.p.p . ) all data from inclusion until the last study visit ( before april 2016 ) were analyzed . fenfluramine was obtained from zogenix international limited , emeryville , ca , usa and its purity was assessed and confirmed by the belgian pharmaceutical association . fenfluramine was compounded into capsules by the pharmacy of antwerp university hospital at doses of 2.5 , 5 , 10 and 20 mg / capsule . most patients in this study swallowed the capsules but , for a few , the contents were sprinkled prior to consumption . the key objective of this study was to assess the overall change in frequency of all major motor seizures during the fenfluramine treatment duration compared with the 3month baseline period . in addition , the change in major motor seizure frequency was assessed at specific time points ( 3 , 6 , 9 and 12 months postinitiation of fenfluramine treatment ) . clonic , tonic , clonic , atonic and myoclonic seizures lasting > 30 s. seizure frequency ( seizures / month ) during the baseline period was calculated as the total number of seizures divided by the number of days in the baseline period multiplied by 30 . during treatment with fenfluramine , seizure frequency taking into account the small sample size and the nongaussian distribution , a wilcoxon signedrank test for paired data was used to analyze the treatment effect . statistical analysis was performed with spss software ( ibm corp spss statistics for windows , version 23.0 , armonk , ny , usa ) . adverse events were monitored at each visit during the study . global impression of change , quality of life ( qol ) and sleep quality were assessed at each study visit since june 2015 . the qol and sleep quality of parents and patients were assessed using a visual analog scale ( 0 , extremely bad to 10 , very good ) . global impression of change was evaluated with a fivepoint scale ( 2 , very much improved ; 1 , much improved ; 0 , no change ; 1 , much worse ; and 2 , very much worse ) . following the withdrawal of fenfluramine in 1997 , its use as an aed for the treatment of intractable epilepsy was allowed in belgium following the issuance of a royal decree ( kb 2002/22215 ) . the study protocol was reviewed and approved by the ethics committee at antwerp university hospital prior to the initiation of this study ( registration no . were fully informed about the study and provided written informed consent prior to participation in the study . nine patients with ds between the ages of 1.2 and 29.8 years enrolled in the study between january 2011 and december 2015 , and had been treated with fenfluramine for a median duration of 1.5 ( range , 0.35.06 ) years at the time of the last assessment . the initial dose of fenfluramine in each patient was 5 or 10 mg / day , divided and administered as two separate doses , with a mean weightadjusted daily dose of 0.24 ( range , 0.160.50 ) mg / kg / day . fenfluramine doses were increased in six patients in order to optimize effectiveness during their treatment and , at the most recent study visit , the mean dose was 0.35 ( range , 0.160.69 ) mg / kg / day . individual patient demographic and clinical information clb , clobazam ; ffa , fenfluramine ; lev , levetiracetam ; stp , stiripentol ; tpm , topiramate ; vns , vagal nerve stimulation ; vpa , valproic acid clonic , tonic , clonic , atonic and myoclonic seizures lasting > 30 s. monthly seizure frequency was calculated as the total number of seizures during the treatment period divided by the total number of treatment days multiplied by 30 days / month . percent reduction refers to the entire treatment period compared with the seizure frequency per month in the baseline period . tonic clonic seizures were the only major motor seizures observed in these patients both before and during treatment with fenfluramine . during the 3month baseline period , the frequency of major motor seizures ranged from 0.4 to 39.7/month ( median , 15.0/month ) . only tonic clonic seizures were observed in patients 16 , whereas additional types of major motor seizures were observed in patients 79 . all patients experienced a decrease in the frequency of major motor seizures after treatment with fenfluramine was initiated ( table 2 ) . compared with the 3month baseline period , a reduction in the median frequency of major motor seizures was observed from baseline ( 15.0/month ) to 3 months ( 2.0/month ) , 6 months ( 1.1/month ) , 9 months ( 1.1/month ) and 12 months ( 1.6/month ) ( fig . 1 ) . a wilcoxon signedrank test showed a significant difference between the 3month baseline period compared with the first 3 months of treatment with fenfluramine ( z = 2.67 , p = 0.008 ) . over the entire treatment period the median percentage reduction in major motor seizures was 75% ( range , 28100% ) . the effect of addon fenfluramine on the frequency of major motor seizures in patients with dravet syndrome . * significant differences in wilcoxon nonparametric test ( p < 0.05 ) compared with baseline period . six of the nine ( 67% ) patients demonstrated 50% reduction ( defined as a responder ) in major motor seizures by month 3 and remained responders throughout the entire observation period ( table 2 , fig . one became a responder at month 6 and remained a responder for the rest of the observation period . thus , as of the last clinic visit , 78% ( seven of nine patients ) of patients were 50% responders . the aeds were kept stable during the first 3 months except in one patient ( patient 8) . the parents of this boy suddenly stopped his ethyl loflazepate 1 week after starting fenfluramine ; 2 weeks after this he experienced an increase in seizures with development of a nonconvulsive status epilepticus for which he was hospitalized . he recovered after the addition of clobazam . due to the long followup period and good seizure control in some patients , we tried to optimize the aed treatment regime . in three patients , one of the other aeds ( clobazam , bromide or stiripentol ) was successfully discontinued without any increase in seizures . several patients experienced clinical benefits in addition to reductions in seizure frequency . at the most recent visit , mean sleep quality for patients and parents was 8.1/10 and 7.9/10 , respectively ( 10 , very good ) , mean qol scores were 7.4/10 and 7.6/10 , respectively , and five parents indicated a much to very much improved global impression . patient 3 , a 5.9yearold boy , had a low frequency of major motor seizures during the baseline observation period . prior to starting fenfluramine he regularly experienced atypical absences with increasing somnolence and the impression of decreasing cognitive function . after starting fenfluramine , all seizure activity ceased , including the absence seizures and his school performance improved . patient 4 , an 11.9yearold boy , typically had motor seizures occurring in clusters and , during treatment with fenfluramine , the duration of the clusters was diminished with a faster recovery between clusters . patient 7 , a 20.3yearold male , demonstrated the smallest decrease in motor seizure frequency in this cohort ( 28% ) ; however , his seizures were described as less severe and his level of alertness throughout the day as being increased . all patients experienced one or more treatmentemergent adverse events ( teaes ) during treatment with fenfluramine . the most commonly reported teaes were somnolence ( n = 5 ) and anorexia ( n = 4 ) . in addition , fatigue ( n = 3 ) , sleep difficulties ( n = 2 ) and nonconvulsive status epilepticus ( n = 3 ) occurred in two or more patients during treatment with fenfluramine . except for episodes of nonconvulsive status epilepticus , all teaes were deemed to be of mild to moderate severity . one patient ( patient 2 ) , who was hospitalized 11 times for status epilepticus before the addition of fenfluramine , experienced four separate episodes of status epilepticus with fever due to respiratory infection during the 4.7 years of treatment with fenfluramine . between two and nine echocardiographic examinations were performed in each patient ( including the baseline examination ) . no evidence of change in cardiac valve structure or function was observed in any patient during the entire treatment observation period and nor were there any echocardiographic findings suggestive of pulmonary arterial hypertension . in this new cohort we prospectively evaluated nine patients with ds treated with fenfluramine for a median duration of 1.5 ( range , 0.35.06 ) years . all patients demonstrated a reduction in major motor seizure frequency during treatment , with a median reduction of 75% . in addition , a tendency towards a positive effect on the sleep quality and qol of both patients and parents was seen in combination with an improved global impression . importantly , no echocardiographic evidence of cardiac valvulopathy or pulmonary hypertension was observed with repeated echocardiography . thus , this study confirms the previously published original cohort of patients with ds who were successfully treated with fenfluramine 10 , 11 . 10 have followed patients with ds treated with fenfluramine for periods of up to 27 years . in their study of the original cohort of 12 patients with ds , seven of the 10 patients with ds who were still being treated as of the end of 2010 had been seizure free for 1 year at their last study visit . a recent 5year followup report on the 10 patients from this original cohort demonstrated continued longterm efficacy and good tolerability 11 . three patients were seizure free for the entire 5 years and four patients experienced seizurefree intervals of at least 2 years during the new study period . two patients had mild ( stable ) valve thickening on the last echocardiography , which was deemed clinically insignificant . the lack of effective medical treatment has led to investigation of new agents for treatment of ds 12 , 13 . devinsky and colleagues have recently reported the results of an openlabel trial of cannabidiol addon therapy in 214 patients with childhoodonset intractable epilepsy , including 32 patients with ds 14 . the patients with ds had a 49.8% median reduction in monthly motor seizures and 16 patients ( 50% ) had a reduction of 50% . multiple logistic regression analysis revealed that only treatment with clobazam independently predicted being a responder ( defined by a 50% decrease in seizure frequency ) . somnolence was the most common adverse event , reported by 25% of patients , followed by decreased appetite ( 19% ) , diarrhea ( 19% ) and fatigue ( 13% ) . in the present study , acknowledging the small sample size , we did not observe any association between the degree of seizure frequency reduction with fenfluramine in patients who were and were not being treated with clobazam or any other anticonvulsant ( tables 1 and 2 ) . it remains to be fully assessed whether the association of fenfluramine with cardiac valvulopathy 7 , 15 that was seen at higher doses ( > 60 mg / day ) , often in combination with phentermine , in the treatment of adult obesity translates to its use in low doses in children with ds . it was removed from the market in 1997 following a report of 113 cases of valvulopathy described by the united states centers for disease control and prevention . in those cases , only 2% involved fenfluramine monotherapy and 79% occurred with both fenfluramine and phentermine 16 . although no echocardiographic evidence of changes in cardiac valve structure or function during fenfluramine treatment was observed in the present study , the relatively short period of exposure in this new cohort of patients may be insufficient for valvulopathy to become evident . in the original cohort of patients with ds treated with fenfluramine , eight of 10 patients had normal echocardiograms at their most recent study visit ( average treatment duration of 16.6 years ) , whereas two patients had slight , but stable , cardiac valve thickening without any clinical manifestations 11 . it is important to note that cardiac valvulopathy has been reported to be dose related . showed that , in adult obese patients , severe valvulopathy was more common with 60 mg / day compared with patients treated with < 40 mg / day 17 . in the present study , lower doses of fenfluramine were employed ; the maximum dose in these patients was 20 mg / day and the median dose was 10 mg / day . major limitations of the current study include the small sample size , use of a study diary , openlabel design , absence of systematic electroencephalographic monitoring and lack of a control group . sleep quality , qol and global impression were not assessed at baseline for the majority of the patients . it is , however , encouraging that the findings in this group of patients are consistent with those reported in the original cohort 10 . the ongoing phase 3 randomized , placebocontrolled studies on the use of fenfluramine in ds will be required to confirm these initial observations and aid in further defining the benefit risk profile in patients with ds . these results on the use of lowdose fenfluramine in this new cohort of patients with ds further support the observations reported in the original cohort . patients with ds experienced sustained periods of clinically meaningful improved seizure control during fenfluramine treatment with a favorable tolerability and no echocardiographic or clinical evidence of cardiac valvulopathy or pulmonary hypertension . a.s.s . is a phd student at the university of antwerp and received an educational grant from zogenix . the seizure frequency is indicated by a red line ( , left yaxis ) .

background and purposedravet syndrome ( ds ) is a severe , drugresistant epilepsy . fenfluramine has been reported to have a longterm clinically meaningful anticonvulsive effect in patients with ds.methodsthis prospective , openlabel study assessed the safety and effectiveness of lowdose fenfluramine in a new cohort of patients with ds . following a 3month baseline period , fenfluramine was added to each patient 's current antiepileptic drug regimen at a dose of 0.251.0 mg / kg / day ( max . 20 mg / day ) . the incidence of major motor seizures ( tonic , clonic , tonic clonic , atonic and myoclonic seizures lasting > 30 s ) in both the baseline and treatment periods was assessed via a seizure diary . periodic echocardiographic examinations during the treatment period were used to assess cardiovascular safety.resultsnine patients ( aged 1.229.8 years ) enrolled in the study and were treated with fenfluramine for a median duration of 1.5 ( range , 0.35.1 ) years . median frequency of major motor seizures was 15.0/month in the baseline period . all patients demonstrated a reduction in seizure frequency during the treatment period with a median reduction of 75% ( range , 28100% ) . seven patients ( 78% ) experienced a 50% reduction in major motor seizure frequency . the most common adverse events were somnolence ( n = 5 ) and anorexia ( n = 4 ) . no evidence of cardiac valvulopathy or pulmonary hypertension was observed.conclusionsthe effectiveness and safety of lowdose fenfluramine as an addon therapy for ds in this new prospective cohort supports previous findings .

japanese encephalitis virus ( jev ) is a mosquito - borne microorganism that causes encephalitis in humans and reproductive failure in pregnant sows . jev has been recently recognized as a reemerging pathogen as it has expanded into new territories such as the torres strait and australia [ 5,10,18 - 20 ] . based on a sequence analysis of the c / prm and e genes , jev has been classified into five genotypes ; their relationships have been characterized . jev genotype 1 , which was distributed in limited regions such as thailand and cambodia before the 1990s , has expanded into northeastern asian countries including vietnam , china , japan , and korea . although the exact mechanisms for the expansion of this genotype are unknown , several factors such as bird migration , wind - blown mosquitoes , and the transport of animals infected with jev have been suggested . additionally , climate changes caused by recent global warming and extreme weather patterns may have significantly impacted the transmission of vector - borne diseases such as jev , west nile virus ( wnv ) , and tick - borne encephalitis virus ( tbev ) . this is because rapid weather and climate changes can directly or indirectly affect migratory birds and mosquitoes . sero - epidemiological studies are critical for predicting potentially important viral disease outbreaks and preventing the introduction of new jev genotypes into korea . recently , saito et al . suggested that wild ducks captured in hokkaido , japan can transmit vector - borne viruses into new territories . migratory birds may serve as viral reservoirs or amplifying hosts , but they do not develop clinical symptoms . although migrating wild birds may be a major jev vector , no epidemiological survey of jev , which could provide valuable information for establishing control measures to prevent jev outbreaks in swine and humans , has been properly conducted for wild birds in korea . thus , we performed a serological survey to determine the prevalence of antibodies against jev in wild birds captured on the korean peninsula . a total of 1,316 blood samples were collected from wild birds in 16 locations of six provinces ( gyeonggi - do ; 3 site , gyeongsangnam - do ; 1 site , jeollanam - do ; 4 sites , jeollabuk - do ; 3 sites , chungcheongnam - do ; 3 sites , chungcheongbuk - do ; 2 sites ) of korea between april 2007 and december 2009 for our seroprevalence study . all wild birds were lured by rice seed and captured using cannon or mist net . blood sample from wing vein of each bird using sterile syringe of 3 ml was taken . the nine species of wild birds tested in this study were oceanodroma castro ( four birds ) , anas formosa ( seven ) , anas penelope ( 20 ) , fulica atra ( 30 ) , anas acuta ( 89 ) , anas crecca ( 154 ) , anas platyrhynchos ( 214 ) , aix galericulata ( 310 ) , and anas poecilorhyncha ( 488 ) . clotted blood samples were separated by 3,000 g , and the sera were stored at -20 until use . before performing the hi test , the kv1899 ( genotype 1 strain ) strain of jev was used as the positive antigen for the hi test . this strain was isolated from korean pig blood in 1999 and has been passed nine times in vero cells after isolation . to estimate the jev antibody prevalence in the wild bird sera , an hi test was performed in 96-well microplates ( corning , usa ) using slightly modified standard methods . a sucrose - acetone extraction method was used to prepare viral antigens from the brains of suckling mice infected with the korean isolate of jev strain kv1899 . briefly , 10 l of the serum samples collected from wild birds and 50 l of 4% bovine albumin were mixed with 40 l of 25% kaolin ( sigma , usa ) and incubated for 30 min to remove non - specific inhibitors at room temperature . after mixing , the clear supernatant was then mixed with 5 l of packed goose erythrocytes to remove any natural agglutinins . after incubating at 37 for 1 h , the treated sera were separated from the goose erythrocytes by 3,000 g at 4. the sera ( 25 l ) were diluted two - fold from 1 : 20 to 1 : 10,240 in round - bottom 96-well microplates ( corning , usa ) and incubated with 8 hemagglutinating antigen ( ha ) units of jev . after incubating at 37 for 1 h , 50 l of 0.33% goose erythrocytes were added and the microplates were incubated for 30 min at 37. to confirm the test reliability , porcine control sera positive and negative for jev were used in all hi tests . hi titer was expressed as the reciprocal of the highest dilution of serum showing complete hemagglutination inhibition . jev prevalence was estimated as the number of positive samples compared to the number of samples tested and expressed as a percentage . a 95% confidence interval ( ci ) additionally , each titer was classified according to bird species , time ( year ) , and place ( capture location ) . a total of 1,316 blood samples were collected from wild birds in 16 locations of six provinces ( gyeonggi - do ; 3 site , gyeongsangnam - do ; 1 site , jeollanam - do ; 4 sites , jeollabuk - do ; 3 sites , chungcheongnam - do ; 3 sites , chungcheongbuk - do ; 2 sites ) of korea between april 2007 and december 2009 for our seroprevalence study . all wild birds were lured by rice seed and captured using cannon or mist net . blood sample from wing vein of each bird using sterile syringe of 3 ml was taken . the nine species of wild birds tested in this study were oceanodroma castro ( four birds ) , anas formosa ( seven ) , anas penelope ( 20 ) , fulica atra ( 30 ) , anas acuta ( 89 ) , anas crecca ( 154 ) , anas platyrhynchos ( 214 ) , aix galericulata ( 310 ) , and anas poecilorhyncha ( 488 ) . clotted blood samples were separated by 3,000 g , and the sera were stored at -20 until use . before performing the hi test , the sera were inactivated by incubating at 56 for 30 min . the kv1899 ( genotype 1 strain ) strain of jev was used as the positive antigen for the hi test . this strain was isolated from korean pig blood in 1999 and has been passed nine times in vero cells after isolation . to estimate the jev antibody prevalence in the wild bird sera , an hi test was performed in 96-well microplates ( corning , usa ) using slightly modified standard methods . a sucrose - acetone extraction method was used to prepare viral antigens from the brains of suckling mice infected with the korean isolate of jev strain kv1899 . briefly , 10 l of the serum samples collected from wild birds and 50 l of 4% bovine albumin were mixed with 40 l of 25% kaolin ( sigma , usa ) and incubated for 30 min to remove non - specific inhibitors at room temperature . after mixing , the clear supernatant was then mixed with 5 l of packed goose erythrocytes to remove any natural agglutinins . after incubating at 37 for 1 h , the treated sera were separated from the goose erythrocytes by 3,000 g at 4. the sera ( 25 l ) were diluted two - fold from 1 : 20 to 1 : 10,240 in round - bottom 96-well microplates ( corning , usa ) and incubated with 8 hemagglutinating antigen ( ha ) units of jev . after incubating at 37 for 1 h , 50 l of 0.33% goose erythrocytes were added and the microplates were incubated for 30 min at 37. to confirm the test reliability , porcine control sera positive and negative for jev were used in all hi tests . hi titer was expressed as the reciprocal of the highest dilution of serum showing complete hemagglutination inhibition . jev prevalence was estimated as the number of positive samples compared to the number of samples tested and expressed as a percentage . a 95% confidence interval ( ci ) additionally , each titer was classified according to bird species , time ( year ) , and place ( capture location ) . jev seroprevalence as determined by our study is shown in tables 1~3 , and figs . 1 and 2 . the overall prevalence of antibodies against jev was 86.7% ( 95% ci , 84.7~88.5% ) for the 1,316 serum samples consisting of nine wild bird species captured in the korean peninsula . prevalence according to year was 68.2% ( 189 positives/277 tested ; 95% ci , 62.4~73.7% ) for 2007 , 86.4% ( 367/425 ; 95% ci , 82.7~89.5% ) for 2008 , and 95.3% ( 585/614 ; 95% ci , 93.3~96.8% ) for 2009 ( fig . the regional seroprevalence of positive hi titers 1 : 20 ranged from 82.9 to 100% . compared to other provinces , the gyeongsangnam - do showed the highest prevalence ( 100% ) ; however , no significant difference in regional prevalence was found . the regional seroprevalences were 100% ( 80/80 ) in gyeongsangnam - do , 93.0% ( 80/86 ; 95% ci , 85.4~97.4% ) in jeollanam - do , 91.1% ( 216/237 ; 95% ci , 86.8~94.4% ) in jeollabuk - do , 84.7% ( 210/248 ; 95% ci , 79.6~88.9% ) in gyeonggi - do , 84.6% ( 176/208 ; 95% ci , 79.0~89.2% ) in chungcheongbuk - do , and 82.9% ( 379/457 ; 95% ci , 79.2~86.3% ) in chungcheongnam - do . seroprevalence according to wild bird species were 100% ( 4/4 ; 7/7 ; 89/89 ) in oceanodroma castro , anas formosa , and anas acuta ( winter visitor ) ; 96.7% ( 29/30 ; 95% ci , 82.8~99.9% ) in fulica atra , 90.8% ( 443/488 ; 95% ci , 87.9~93.2% ) in anas poecilorhyncha ( migratory breeder ) , 87.9% ( 188/214 ; 95% ci , 82.7~91.9% ) in anas platyrhynchos , 80% ( 16/20 ; 95% ci , 56.3~94.3% ) in anas penelope ( winter visitor ) , 78.7% in aix galericulata ( migratory breeder ) , and 78.6% ( 121/154 ; 95% ci , 71.2~84.8% ) in anas crecca . the prevalence of wild birds with an hi antibody titer above 1 : 20 or 1 : 1,280 were 86.7% and 21.2% , respectively . wild birds captured in 2009 had a higher jev seropositive rate than those captured in 2007 ( fig . 2 ) . among the wild birds captured in 2009 that had an hi antibody titer of 1 : 20 or more , the most frequent hi titer was 1 : 640 ( 16.2% ) . climate change , global warming , environmental destruction , and rapid urbanization can alter wild bird migratory paths and increase the incidence of arboviral disease outbreaks . under these conditions , vector - borne diseases caused by jev , wnv , and tbev may be easily introduced into disease - free countries by migratory wild birds . since jev was first isolated in korea in 1946 , several korean jev isolates have been reported and characterized based on a c / prm and e gene nucleotide analysis . phylogenetic analyses to determine the genetic relationships between jev isolates found that korean jev isolates changed from genotype 3 to genotype 1 around 1993 . however , few studies have addressed the possibility of jev introduction into korea via wild birds . thus , we conducted a nationwide survey of jev antibodies in wild birds , including migratory birds . in this study , the sera of wild birds that were captured in 16 locations of the korean peninsula were tested for the presence of jev antibodies . this hi test result was surprisingly high and suggested that most of the wild birds we captured in korea had been exposed to jev . saito et al . reported that 85.9% and 5.4% of wild ducks captured in hokkaido , japan have antibodies against jev according to the focus - reduction neutralization test ( frnt50 and frnt90 ) , which is more specific than the hi test . additionally , 7.2% , 20.4% , and 28.1% of wild birds in 2007 , 2008 , and 2009 , respectively , had a titer > 1 : 1,280 . sugiura and shimada reported that horses experimentally inoculated with jev had an antibody titer of 1 : 1,280 within 2 or 3 weeks after inoculation . therefore , wild birds with titers of 1 : 1,280 or higher may have been infected with wild jev within 3 weeks . although we conducted our serosurveillance study with an hi test , which is known to be less specific than the frnt , our survey results correspond well with those of a previous study in wild ducks in japan . however , cross - reaction between jev and wnv in serodiagnoses is a well - known occurrence in flavivirus - infected animals , including birds . therefore , it is necessary to perform a more specific method , such as a plaque - reduction neutralization test ( prnt ) , for jev serosurveillance in birds . several positive jev serosurveillance results have been reported in different animals including swine , goats , cattle , horses , and wild boars in japan and korea . it has been suggested that goats may serve as good sentinel animals for serological monitoring of jev infection because they are currently not vaccinated against jev in korea . unlike domestic animals , wild birds can not be inoculated with a jev vaccine , but they are easily exposed to several kinds of arthropods such as mosquitoes , ticks , and midges . the high number of positive jev titers in this study does not indicate viral intrusion into korea since jev viremia ends when jev - specific antibodies are detected in ducks . however , wild birds with viremia may carry jev to several countries . according to data obtained from the korean center for disease control and prevention , 45 jev cases in human more than half of these cases occurred in 2010 , suggesting that climate change or a high jev infection rate among wild birds may , in part , explain the increasing jev infection rate in the human population . gyeonggi - do had a high incidence rate ( 42.3% ) of jev infection in humans , but no significant relationship was found between the seropositive rates in the capture areas and the jev incidence rate in humans . nevertheless , new jev genotypes may be introduced into korea via wild or migratory birds . it is therefore necessary to monitor jev antibodies using the prnt in wild birds . taken together , these findings lead us to predict that jev infections may appear in domestic animals such as pigs . in conclusion , the results of the present study suggested that wild birds with a high incidence rate ( 86.7% ) of jev antibodies may be introducing new jev genotypes into korea . further studies are needed to isolate all jev genotypes from blood obtained from migratory birds . various research projects examining disease distribution and transmission associated with climate change in the korean peninsula should also be carried out in the near future .

climate change induced by recent global warming may have a significant impact on vector - borne and zoonotic diseases . for example , the distribution of japanese encephalitis virus ( jev ) has expanded into new regions . we surveyed the levels of hemagglutination - inhibition ( hi ) antibodies against jev ( family flaviviridae , genus flavivirus ) in wild birds captured in korea . blood samples were collected from 1,316 wild birds including the following migratory birds : oceanodroma castro ( n = 4 ) , anas formosa ( n = 7 ) , anas penelope ( n = 20 ) , fulica atra ( n = 30 ) , anas acuta ( n = 89 ) , anas crecca ( n = 154 ) , anas platyrhynchos ( n = 214 ) , aix galericulata ( n = 310 ) , and anas poecilorhyncha ( n = 488 ) . all were captured in 16 locations in several korea provinces between april 2007 and december 2009 . out of the 1,316 serum samples tested , 1,141 ( 86.7% ) were positive for jev . wild birds captured in 2009 had a higher seroprevalence of ant - jev antibodies than those captured in 2007 . wild birds with an hi antibody titer of 1 : 1,280 or higher accounted for 21.2% ( 280/1,316 ) of the animals tested . these findings indicated that wild birds from the region examined in our study have been exposed to jev and may pose a high risk for introducing a new jev genotype into korea .

incident hiv cases ( i.e. , recent infection ) and new aids diagnoses are disproportionately among african americans and latinos in los angeles county1 . the los angeles aids epidemic primarily involves men who have sex with men ( msm ) and men who have sex with men and women ( msm / w ) , groups who account for 76.1% of all aids cases . increasing numbers of women with aids , particularly african americans and latinas , factors that might promote hiv moving from a concentrated epidemic involving msm and msm / w to a more generalized epidemic involving women remain poorly specified . one factor that may contribute is high - risk sexual behavior of men with both male and female sexual partners driven by internalized homonegativity23 . internalized homonegativity ( or homophobia ) reflects a lack of positive beliefs about being gay , about valuation of the larger gay community , and about the morality of being gay2 . in msm and msm / w , internalized homophobia correlates with low self - esteem and high - risk sex4 . by contrast , levels of internalized homophobia inversely correspond with sexual satisfaction ratings among white msm5 . some have speculated that msm with high levels of internalized homophobia are not exposed to cultural norms and corresponding hiv - risk reduction messages that reinforce safer sexual behaviors6 . recent data show that msm / w , however , restrict unprotected sex solely to regular / main male or female partners7 . the men presumably are more likely to know the hiv status of these regular / main partners and consequently limit unprotected sex with other men or women in response to perceived messages regarding hiv risks associated with unprotected sex with status unknown partners7 . higher scores of internalized homophobia have been associated with higher expectations that substance use during sex would enhance sexual experiences , both factors that associate with practice of unprotected anal intercourse ( uai)8 . internalized homonegativity and sexual risk behaviors are factors that may be strongly correlated in communities of msm and msm / w of color ; experiences with internalized homophobia are thought to be particularly profound and involve incorporation of disapproval of msm from both minority and dominant cultures9 . some msm and msm / w of color may resist or even reject adoption of a sexual identity that recognizes their experiences with other men and instead nurture a heterosexual identity10 . long - term consequences to maintaining a heterosexual identity while engaging in same sex behaviors include high - risk sex , substance abuse , psychological distress , and negative physical health effects , including hiv11 . in turn use of stimulants and other drugs cover feelings of shame and internalized homonegativity to facilitate sexual behaviors for msm and msm / w that are not possible when not under the influence1213 . prior studies show that msm , including men of color , use substances to facilitate sex14 , though african american msm use significantly less substances than white msm for this purpose15 . this study evaluates associations between reported demographic factors , drug use behaviors , sexual risk behaviors , hiv status , and levels of internalized homonegativity among msm and msm / w who are poor , urban , and of ethnic descent in los angeles . participants participants were msm or msm / w who participated in one of the two waves of data collection ( 20052006 ; 20062008 ) at the los angeles site for nida s sexual acquisition and transmission of hiv cooperative agreement program ( sath - cap ) . participants were : ( 1 ) over 18 years of age ; ( 2 ) msm or msm / w who engaged in the past 6 months in any anal intercourse ( ai ) and/or a male or female drug user ( i.e. , self - reported use of powder cocaine , crack cocaine , heroin , methamphetamine , or any injection drug use)or a sexual partner of a sath - cap participant . only msm and msm / w are included in this analysis as only they completed the homonegativity questions . study sample the sample was compiled using respondent - driven sampling16 . for each of two waves of data collection , seeds ( individuals willing to participate in the study ) were passively recruited via flyers that described the research project , subsequently made an appointment , arrived at the clinic , completed questionnaires and provided biological samples for which they were compensated $ 50 . upon completion , participants were provided with coupons to recruit people to the study who they knew to be msm and/or drug users or who were their sexual partners , with the potential of earning an additional $ 140 in vouchers for the referrals ( see iguchi et al . measures an audio computer - assisted self - interview ( acasi ) collected detailed information regarding : ( 1 ) drug use past 6 months use of powder cocaine , crack cocaine , methamphetamine , heroin , and speedballs ( heroin plus cocaine ) ; ( 2 ) sexual risk behaviors numbers of male and female sexual partners in the prior 6 months , numbers of specific behaviors engaged in over the past 6 months while having sex with male and female partners ( particularly those under the influence of drugs ) , numbers of specific sexual and drug use behaviors with each of the last three sexual and drug - using partners over the past 6 months ; ( 3 ) the internalized homonegativity inventory2a 23-item questionnaire that yielded a total score and three subscales ( personal homonegativity , gay affirmation , morality of homosexuality ) . personal homonegativity included 11 items on feelings of shame , anxiety , and resentment over being homosexual . the total and subscales are compiled using sums ; ( 4 ) self - reported variables measuring i.e. , the number of men you know who have sex with other men , the number of friends / family who know you have sex with other men ; ( 5 ) self - reported sexual identification , defined as ( a ) gay or homosexual , ( b ) bisexual , ( c ) straight , ( d ) down low , same gender loving , messing around on the other team , ( e ) male to female transgender , ( f ) no label ; ( 6 ) biological specimens oral hiv rapid test with confirmatory elisa for positive results , and urine drug screens tested for metabolites of cocaine , methamphetamine , and heroin ( opioids ) . men in the sample who reported having had sex with other men , but not within the previous 6 months , were categorized as having had no sex . data analysis prior to addressing study questions , the pattern of missing data was explored . for cases missing three or fewer items on the ihni , this allows retention of a significant number of cases that would otherwise be lost in the analyses . psychometric properties of the ihni total and the subscales were assessed using cronbach s alphas for internal consistency and a principal components factor analysis for latent structure of the data . ihni scores were correlated with self - reported measures of outness to examine content validity.chi-square analyses compared the distributions of percentages of msm and msm / w among demographic , drug and sex variables . participants reporting no sex in the previous 6 months who identified as msm or msm / w were included separately . anovas tested univariate differences between ihni scores and interval level demographic , drug and sex variables . hypotheses for condom use in the last three reported sexual partners were fit with a random intercept model in proc glimmix in sas version 9.1 ( sas institute , cary , nc , usa ) and were used to evaluate whether internalized homonegativity scores predicted condom use when controlling for ethnicity / race and sexual identification . linear regression was used to evaluate the effect of ihni and subscale scores on the number of reported sexual partners and the number of reported ai partners . when evaluating the effects of ihni score and subscale scores on sexual risk behaviors , ethnicity / race and self sexual identification were used as covariates . logistic regression was used to determine the association of demographic , drug use , and sexual risk behaviors with confirmed hiv status.all scientific and research procedures were overseen by the ucla human subjects protection committee and the rand institutional review board . the final sample of 722 msm and msm / w included data from 55 participants who had three or fewer ihni items missing and were imputed . participants with three or fewer missing items differed significantly from those with complete data only along biological and self - report of hiv serostatus ( both p < 0.001 ) . participants with imputed ihni items were more likely to be hiv seronegative than those who completed the survey . table 1demographic factors , drug use , sexual risk behaviors , and hiv status for male participants who reported having sex with men ( msm ) and with men and women ( msm / w ) in the past 6 monthsnno sex ( % ) msm ( % ) msm / w ( % ) chi - sq(df ) , p valuedemographic variablesage(8 ) = 22 , 0.0041<3084116227303918113602840492991553325059122302545 > = 6020164044race(6 ) = 47 , < 0.0001white152135730black312154144hispanic198146917other44184834education level(4 ) = 23 , 0.0001less than high school156224730high school233154540more than high school316116128employment status(4 ) = 15 , 0.0045unemployed581154935part - time5696823full - time69136819income in past month ( legal)(4 ) = 23 , 0.0001$0$500424144639$501$1,000171185923>$1,000102106625homeless in past year(2 ) = 17 , 0.0002no351156026yes355144640marital status(6 ) = 37 , < 0.0001single461165827married / cohabitating70145927formerly married160113652other15204040drug use variablesinject drugs past 30 days(2 ) = 11 , 0.0051no572135631yes130184042inject drugs ever(2 ) = 5 , 0.0688no494155530yes212154739urine drug screen , cocaine(2 ) = 10 , 0.0069negative580145631positive125184042urine drug screen , methamphetamine(2 ) = 16 , 0.0004negative639145235positive66246412urine drug screen , heroin(2 ) = 7 , 0.0359negative663145333positive42265519self - report , methamphetamine , last 6 months(2 ) = 0.1 , 0.9567no379145333yes326155233self report , cocaine / crack , last 6 months(2 ) = 30 , < 0.0001no265176320yes440134740self - report , heroin , last 6 months(2 ) = 26 , < 0.0001no576145729yes128183349self - report , speedball , last 6 months(2 ) = 27 , < 0.0001no597155629yes108143253self - report , any drug , last 6 months(2 ) = 13 , 0.0016no144116623yes560164935sexual behaviorsnumber of sexual partners past 6 months(6 ) = 742 , < 0.0001none103100001106085152127053473 or more37005842condom use last 6 months(2 ) = 154 , < 0.0001no unprotected sex313334720unprotected sex37205644sexual identification(10 ) = 314 , < 0.0001gay or homosexual32117803bisexual19372766straight60301060dl , same gender , mess around57123058male > female3020773no label32132563sexual behavior(8 ) = 459 , < 0.0001only with men35717821mostly with men10854946equal with men and women5181280mostly with women1519981only with women2665431hiv statusconfirmed hiv status(2 ) = 161 , < 0.0001negative370133354positive335167410self - report hiv status(6 ) = 178 , < 0.0001test , do nt know65155529negative283113158positive29815768no test59253737the sample was msm and msm / w , predominantly african american ( 44% ) or latino ( 28% ) men , likely to report drug use in the past 6 months ( 79.5% ) and many who are hiv positive ( 47.5% ) . biological testing for hiv showed latino men had the highest hiv prevalence ( 64.6% ) followed by white ( 43.4% ) and black ( 39.2% ) men . among the smaller number of men with other ethnicities , 47.7% tested hiv positive . in the smaller group of men who did not know their hiv status , 26% ( of 66 ) who reportedly had tested previously but did not know their results and 26% ( of 62 ) men who reportedly never tested table 1 shows significant associations between msm and msm / w by demographic , drug use , sexual behavior , and hiv status characteristics . while 52% of msm / w informed their female partners about having sex with men , only 18% of msm / w reported informing their male partners that they also have had sex with women . demographic factors , drug use , sexual risk behaviors , and hiv status for male participants who reported having sex with men ( msm ) and with men and women ( msm / w ) in the past 6 months psychometric properties ihni scores in this sample were substantially higher for both the total scores and for all three subscale scores in comparison to the original sample used in test construction ( higher scores indicate higher levels of internalized homonegativity ) . cronbach s alphas for the ihni total ( = 0.91 ) and the personal homonegativity ( = 0.90 ) , gay affirmation ( = 0.83 ) , and morality of homosexuality ( = 0.76 ) scales were in the excellent - to - good range . the factor analysis yielded two factors that confirmed the first two factors ( personal homonegativity , gay affirmation)2 ; the items that did not load on the two large factors were on the morality of homosexuality subscale . as there were no statistically significant differences between waves along ihni or subscale scores , the data from both samples were combined for analyses.table 2 shows means and standard deviations for the ihni total and the personal homonegativity , gay affirmation , and morality of homosexuality scores by demographic , drug use , sexual behavior , and hiv status characteristics . univariate analyses showed statistically significant differences by age on the ihni total , gay affirmation , and morality subscales , with scores increasing with age . scores for the ihni total , gay affirmation , and morality scores were significantly higher for african american men than for those of other ethnicities . higher scores were observed for the ihni total , personal homonegativity , gay affirmation , and morality of homosexuality scores for participants who attained high school or less educational levels . significant differences also were found by employment status and income levels for the past month with ihni total , gay affirmation , and morality of homosexuality scores being lower among those who reported full - time employment and higher monthly income levels . homeless men scored significantly higher on the ihni total score than those who denied homelessness . ihni total , gay affirmation , and morality of homosexuality subscales differed significantly by marital status , with higher levels observed for participants who were formerly married . table 2mean internalized homonegativity index scores by demographic , drug use , sexual behaviors , and hiv statusnihni totalpersonalgay affirmationmoralitymeansdmeansdmeansdmeansddemographicsagef(4,717 ) = 2.5 , p = 0.04f(4,717 ) = 0.27 , p = 0.89f(4,717 ) = 5.1 , p = 0.0005f(4,717 ) = 2.5 , p = 0.04<308659.524.826.513.822.09.311.05.830391357.622.825.713.020.88.710.95.7404936061.322.526.412.922.99.312.06.2505912764.922.427.013.125.29.112.75.8 > = 602067.422.028.514.225.88.913.27.2ethnicity / racef(3,718 ) = 9.2 , p < 0.0001f(3,718 ) = 2.5 , p = 0.05f(3,718 ) = 12.9 , p < 0.0001f(3,718 ) = 10.5 , p < 0.0001white15356.924.624.814.122.19.210.15.9black31965.822.127.813.225.09.013.06.1hispanic20656.520.625.311.420.28.811.05.3other4460.326.527.614.920.79.212.06.8educationf(2,717 ) = 12.3 , p < 0.0001f(2,717 ) = 6.6 , p = 0.002f(2,717 ) = 7.5 , p = 0.0006f(2,717 ) = 13.0 , p < 0.0001less than high school15963.020.126.811.723.79.412.55.7high school23865.723.128.613.824.18.812.96.2more than high school32356.423.324.613.021.39.310.55.8employment statusf(2,719 ) = 5.1 , p = 0.007f(2,719 ) = 2.1 , p = 0.12f(2,719 ) = 5.7 , p = 0.003f(2,719 ) = 3.4 , p = 0.036unemployed59362.022.926.813.223.39.212.06.1part - time5959.523.026.512.721.79.311.35.6full - time7053.022.023.412.019.59.010.15.5income in past month ( legal)f(2,708 ) = 9.2 , p = 0.0001f(2,708 ) = 2.5 , p = 0.08f(2,708 ) = 15.4 , p < 0.0001f(2,708 ) = 5.8 , p = 0.003$0$50043263.822.827.313.324.29.112.46.1$501$1,00017457.520.724.911.521.59.311.15.5>$1,00010555.025.125.414.219.18.510.56.3homeless in past yearf(1,720 ) = 3.9 , p = 0.05f(1,720 ) = 1.9 , p = 0.17f(1,720 ) = 3.5 , p = 0.06f(1,720 ) = 2.6 , p = 0.107no36159.322.725.812.822.19.311.46.0yes36162.623.127.113.323.49.112.16.0marital statusf(3,717 ) = 7.2 , p < 0.0001f(3,717 ) = 2.2 , p = 0.09f(3,717 ) = 11.9 , p < 0.0001f(3,717 ) = 2.9 , p = 0.033single47559.823.426.013.222.29.311.66.0married / cohabitating7055.222.624.812.319.88.410.75.8formerly married16167.720.828.613.126.28.512.96.1other1553.517.924.611.718.56.610.45.1drug use variablesinject drugs past 30 daysf(1,716 ) = 9.7 , p = 0.002f(1,716 ) = 3.1 , p = 0.08f(1,716 ) = 13.3 , p = 0.0003f(1,716 ) = 6.0 , p = 0.015no58759.622.526.012.822.19.211.55.9yes13166.423.928.213.925.49.212.96.1inject drugs lifetimef(1,720 ) = 8.8 , p = 0.003f(1,720 ) = 4.7 , p = 0.03f(1,720 ) = 9.28 , p = 0.003f(1,720 ) = 3.6 , p = 0.059no50759.322.525.712.722.19.311.56.0yes21564.823.628.013.824.39.012.46.1urine drug screen , cocainef(1,719 ) = 12.0 , p = 0.0006f(1,719 ) = 2.5 , p = 0.11f(1,719 ) = 14.6 , p = 0.0001f(1,719 ) = 15.0 , p = 0.0001negative58959.523.226.013.022.19.311.45.9positive13267.120.728.013.625.58.713.66.4urine drug screen , methamphetaminef(1,719 ) = 13.8 , 0 = 0.0002f(1,719 ) = 8.7 , p = 0.003f(1,719 ) = 9.6 , p = 0.002f(1,719 ) = 8.7 , p = 0.003negative65561.922.926.913.223.19.212.06.0positive6651.020.921.911.319.49.19.75.4urine drug screen , heroinf(1,719 ) = 0.8 , p = 0.38f(1,719 ) = 0.6 , p = 0.46f(1,719 ) = 0.08 , p = 0.77f(1,719 ) = 1.8 , p = 0.19negative67960.722.926.313.122.79.211.76.0positive4263.923.827.913.823.19.513.06.2self - report , methamphetamine , last 6 monthsf(1,719 ) = 0.8 , p = 0.36f(1,719 ) = 0.01 , p = 0.93f(1,719 ) = 2.1 , p = 0.15f(1,719 ) = 1.2 , p = 0.28no38861.621.326.512.823.29.212.06.0yes33360.124.726.413.522.29.211.56.0self - report , cocaine / crack , last 6 monthsf(1,719 ) = 8.0 , p = 0.005f(1,719 ) = 1.9 , p = 0.17f(1,719 ) = 14.3 , p = 0.0002f(1,719 ) = 3.9 , p = 0.048no27457.922.725.613.221.19.511.26.0yes44762.822.926.913.123.88.912.16.0self - report , heroin , last 6 monthsf(1,718 ) = 26.4 , p < 0.0001f(1,718 ) = 13.8 , p = 0.0002f(1,718 ) = 26.2 p < 0.0001f(1,718 ) = 12.3 , p = 0.0005no59058.922.325.612.721.99.211.45.9yes13070.123.430.214.026.48.413.46.5self - report , speedball , last 6 monthsf(1,719 ) = 25.1 , p < 0.0001f(1,719 ) = 14.0 , p = 0.0002f(1,719 ) = 22.2 , p < 0.0001f(1,719 ) = 15.7 , p < 0.0001no61059.122.325.712.722.19.211.45.8yes11171.023.830.714.326.58.713.86.6self - report , any drug , last 6 monthsf(1,718 ) = 4.6 , p = 0.03f(1,718 ) = 0 .9 , p = 0.35f(1,718 ) = 9.2 , p = 0.003f(1,718 ) = 2.1 , p = 0.144no14957.321.025.512.620.79.111.16.0yes57161.823.326.613.223.39.211.96.0sexual behaviorsnumber of sexual partners past 6 monthsf(3,702 ) = 0.5 , p = 0.66f(3,702 ) = 0.5 , p = 0.69f(3,702 ) = 3.6 , p = 0.014f(3,702 ) = 0.8 , p = 0.49none10361.021.725.212.623.510.412.46.2110660.725.126.114.322.49.912.26.5212763.221.326.813.024.88.411.65.93 or more37060.223.326.813.021.98.911.55.9condom use last 6 monthsf(1,683 ) = 1.0 , p = 0.31f(1,683 ) = 3.3 , p = 0.07f(1,683 ) = 0.02 , p = 0.88f(1,683 ) = 0.02 , p = 0.87no unprotected sex31360.022.825.513.022.79.611.86.0unprotected sex37261.823.027.313.122.68.911.86.0sex of sex partnersf(2,703 ) = 55.5 , p < 0.0001f(2,703 ) = 26.8 , p < 0.0001f(2,703 ) = 54.8 , p < 0.0001f(2,703 ) = 29.7 , p < 0.0001men , but no sex last 6 months10361.021.725.212.623.510.412.46.2men only37256.721.423.712.119.78.710.35.4men and women23172.621.331.413.627.27.513.96.1female partners know have sex with menf(1,128 ) = 6.0 , p = 0.015f(1,128 ) = 1.8 , p = 0.18f(1,128 ) = 6.3 , p = 0.013f(1,128 ) = 5.0 , p = 0.028yes6367.620.929.513.625.47.612.76.0no6776.017.932.612.828.46.014.95.5male partners know have sex with womenf(1,128 ) = 0.00 , p = 0.97f(1,128 ) = 0.1 , p = 0.71f(1,128 ) = 0.04 , p = 0.84f(1,128 ) = 0.2 , p = 0.63yes10771.919.531.312.926.96.813.75.6no2371.821.530.214.927.27.914.47.1sexual identificationf(5,703 ) = 32.8 , p < 0.0001f(5,703 ) = 11.9 , p < 0.0001f(5,703 ) = 39.8 , p < 0.0001f(5,703 ) = 19.6 , p < 0.0001gay or homosexual33051.620.423.011.718.98.69.75.1bisexual19769.120.829.912.525.87.413.46.0straight6077.322.730.616.231.27.415.57.1dl , same gender , mess around5772.218.830.113.028.28.113.95.9male > female3149.916.520.98.618.39.310.73.8no label3465.224.329.214.623.68.912.56.5sexual behaviorf(4,704 ) = 41.8 , p < 0.0001f(4,704 ) = 16.3 , p < 0.0001f(4,704 ) = 52.8 , p < 0.0001f(4,704 ) = 23.6 , p < 0.0001only with men36651.820.122.911.518.98.710.05.2mostly with men11063.521.528.612.623.28.211.75.9equal with men and women5372.421.932.013.826.17.514.46.3mostly with women15474.851.631.214.329.06.714.66.3only with women2671.711.925.712.131.18.415.06.1hiv statusconfirmed hiv statusf(1,719 ) = 55.4 , p < 0.0001f(1,719 ) = 24.4 , p < 0.0001f(1,719 ) = 52.6 , p < 0.0001f(1,719 ) = 38.1 , p < 0.0001negative37666.822.828.713.525.09.113.16.4positive34554.521.323.912.220.28.710.45.3self - report hiv statusf(3,717 ) = 22.6 , p < 0.0001f(3,717 ) = 10.6 , p < 0.0001f(3,717 ) = 20.1 , p < 0.0001f(3,717 ) = 15.9 , p < 0.0001test , do nt know6666.024.329.115.324.28.812.77.0negative28765.822.828.313.524.89.212.76.2positive30653.221.023.411.619.86.910.15.2never tested6270.920.030.212.726.28.414.65.5only answered by those who answered that they have had sex with both men and women in the past 6 months , n = 275ihni scores varied significantly by drug use ; injection drug users ( lifetime and in the past 30 days ) scored significantly higher on the ihni total and gay affirmation scores than those who denied injection drug use . the morality subscale was significantly higher for recent injectors ( past 30 days ) , though not for injectors lifetime . urine drug screening results for heroin were not associated with ihni or subscale scores , though self - report of use of heroin and of speedballs ( heroin plus cocaine ) was significantly associated with increased ihni total and the personal , gay affirmation , and morality subscales . participants who tested positive for cocaine screens were significantly higher on the ihni total , gay affirmation , and morality subscale scores than those who tested negative . methamphetamine drug screen results showed opposing patterns with participants who tested positive for methamphetamine being significantly lower on the ihni total , personal , gay affirmation , and morality subscales than those who tested methamphetamine negative . self - report of methamphetamine use was not associated with ihni scores.ihni total and subscale scores were highest for msm / w , lowest for msm , and intermediate for the men who reported having no sex ( all p < 0.0001 ) . an hiv seropositive test strongly and significantly correlated with lower ihni total and subscale scores . a similar pattern was observed for participants self - report of hiv status , with lower scores for those who reported being hiv positive . as expected , ihni scores were highest for men who self - identified as straight or as any label indicating bisexuality . lowest ihni scores were observed among men who identified as gay or homosexual.table 3 shows that when the effects of ethnicity / race and sexual identification were controlled for , the gay affirmation subscale significantly and inversely predicted the number of sexual partners over the past 6 months . these analyses adjusted for ethnicity / race and sexual identification but not the factor of msm / msm / w ( sexual orientation in the past 6 months ) due to collinearity . only the gay affirmation subscale correlated significantly with number of sexual partners in the previous 6 months when controlling for race and self sexual identification , with higher gay affirmation scores associating with fewer partners . evaluation of the coefficients in this model showed this effect significantly pronounced for the men who identified as straight ( 0.42 , se = 0.19 ; t = 2.18 ; p = 0.0295 ) , with very high gay affirmation scores and few sexual partners . table 3multivariate analysis of ihni total and subscale scores by sexual risk behavior variablesnumber of sexual partners last 6 months ( natural log)number of unprotected anal sex partners reported ( natural log)condom use last 3 sexual partnersestsdf / tdfp valueestsdf / tdfp valueestsdf / tdfp valueihni scorerace0.43(3,683)0.73001.70(3,663)0.16530.56(3,571)0.6410sex id3.41(5,683)0.00472.37(5,663)0.03821.03(5,571)0.4016ihni score0.00060.00160.390.69330.00110.00170.650.51690.00290.00370.780.4344personal scorerace0.54(3,683)0.65501.81(3,663)0.14440.59(3,572)0.1253sex id3.34(5,683)0.00552.62(5,663)0.02360.99(5,572)0.4240personal score0.00390.00271.450.14720.00120.00290.420.67700.00950.00621.540.1253gay affirmation scorerace0.51(3,683)0.67801.83(3,663)0.13960.47(3,571)0.7016sex id3.43(5,683)0.00462.08(5,663)0.06661.13(5,571)0.3440gay affirmation score0.00930.00412.250.02470.00660.00441.510.13220.00180.00960.190.8502morality scorerace0.33(3,683)0.80541.55(3,663)0.19990.44(3,572)0.7955sex id3.37(5,683)0.00512.30(5,663)0.04361.14(5,572)0.3381morality score0.00870.00601.450.14810.00730.00651.130.25790.00370.01410.260.7955this was run using the last three partner data for every participant . this is a fixed effects ( repeated measure logistic regression ) model of condom use ( unprotected sex = 1/no unprotected sex = 0 ) for up to three possible partners . only those with at least 20 items and with at least one sexual partner data were included in the analyses mean internalized homonegativity index scores by demographic , drug use , sexual behaviors , and hiv status only answered by those who answered that they have had sex with both men and women in the past 6 months , n = 275 multivariate analysis of ihni total and subscale scores by sexual risk behavior variables this was run using the last three partner data for every participant . this is a fixed effects ( repeated measure logistic regression ) model of condom use ( unprotected sex = 1/no unprotected sex = 0 ) for up to three possible partners . only those with at least 20 items and with at least one sexual partner data were included in the analyses findings showed the ihni scale to have adequate psychometric properties and similar factor structure to the scale s original sample2 . ihni scores averaged ten points or more higher than the original sample , which suggests aspects of internalized homonegativity are more pronounced in poor , urban msm and msm / w of color than white middle - class , midwest gay men . the ihni total score and the three subscales captured distinct aspects of internalized homonegativity and described meaningful differences along meaningful participant characteristics , including social indicators of the participant s outness . levels of internalized homonegativity increased with age , with lower educational levels , with african american ethnicity , with experiences of poverty and homelessness , with recent use of cocaine ( lower levels of homonegativity with methamphetamine use ) , with experiences of being incarcerated , with being a man who is behaviorally bisexual , and with being hiv seronegative . the sampling frame involving rds did not promote immediate convergence of ihni scores for participants enrolled in the first versus the latter halves of each wave of data collection . in both waves of data collection , enrollment of african americans ( and concomitant higher ihni scores ) increased as the linked referrals proceeded . this application of rds did not yield a sample that could be considered representative of the general population of msm or msm / w in los angeles county , particularly along the factors of ethnicity / race , poverty and hiv . hence , findings are understood to reflect a unique sample of very poor msm and msm / w of color in los angeles county . drug - specific behaviors interacted with the ihni scores and ethnicity / race such that african american msm / w were more likely to have positive urine cocaine screens and higher ihni scores , while white and hispanic msm were more likely to provide positive urine methamphetamine screens and lower ihni scores . although substance use is an efficient method to cover over feelings of internalized homophobia17 , its functions appear to be divergent for cocaine and for methamphetamine using men . another distinction is the finding that african american men reported similar levels of drug use as white and latino men , which contrasts with work showing lower levels of substance use in african american msm / w15 . high ihni scores for african american msm / w validates the work of many and indicates that the sociocultural milieu of most african american men prohibit expressions of non - heterosexual behaviors and identities101718 . african american msm / w may face potential rejection of cultural affiliation when openly acknowledging either male male sexual behaviors or gay or bisexual identities719 . consistent with prior work15 , the highest homonegativity scores were reported by msm / w who reportedly had no prior tests for hiv ; hiv prevalence in this group was high . as such , homonegativity may function within this group of men as a barrier to hiv testing . still , this sample of men with high homonegativity scores completed their rapid tests and learned their results . design of prevention strategies with the goal of increasing hiv testing among men who have never tested may benefit from rapid testing procedures and/or monetary incentives . ihni scores generally did not predict hiv - related sexual risk behaviors after controlling for race / ethnicity and self sexual identification . one exception is that high scores on the gay affirmation subscale significantly predicted low numbers of sexual partners , particularly for men who self - identified as straight . that only one model showed significant associations between ihni scores and behavioral outcomes after holding race / ethnicity and self sexual identification constant indicates that there is no homogenous experience of sexual behaviors and internalized homonegativity for msm and msm / w of differing racial / ethnic groups who adhere to differing sexual identification labels . these include collecting all data from a single convenience sample in los angeles county and reliance primarily on subject reports . yet , participants were scattered throughout the los angeles basin and comprised a coherent sample of predominantly low - income msm and msm / w of color . the size of the sample allowed sufficient design effect for findings to be considered significant , even if some participants misrepresented self - reports . as well , acasi was used to increase privacy and findings comparing urine data with self - report of drug use indicating participants approached the questionnaire straightforwardly . finally , there is a limitation to the concept of internalized homonegativity that involves emphasis on individual pathology rather than on institutional / societal oppression3 . other limitations to these findings deserve mention that are related to the rds method . in our use of dual cores of drug users and/or msm in the rds procedure , we compiled a sample that showed high levels of similarity between participants and the recruits they referred into the study ( i.e. , homophily ) for most of the variables measured . these included hiv status , race / ethnicity , drug use , and levels of income , even though the sample was overwhelmingly poor . representative sample of drug users and/or msm in both this and another rds study in los angeles20 , which also recruited a very poor sample with high hiv prevalence . this suggests findings should be constrained to similar urban groups of older msm and msm / w of color with high hiv prevalence and who are drug users . despite these limitations , findings still show internalized homonegativity to correlate significantly and strongly with a variety of demographic factors , drug use , sexual behaviors , and hiv status in this sample of very poor , largely minority msm and msm / w , which provide a rare glimpse into associations between internalized homonegativity , sexual behaviors , and drug use for the men in this understudied group . findings also emphasize the value of using rapid testing procedures with those who do not know their hiv status and imply that optimally effective prevention interventions that address homonegativity and sexual risk may be constructed differently for msm methamphetamine users than for msm / w cocaine users .

this study evaluates associations between internalized homonegativity and demographic factors , drug use behaviors , sexual risk behaviors , and hiv status among men who have sex with men ( msm ) and with men and women ( msm / w ) . participants were recruited in los angeles county using respondent - driven sampling ( rds ) and completed the internalized homonegativity inventory ( ihni ) and questionnaires on demographic and behavioral factors . biological samples were tested for hiv and for recent cocaine , methamphetamine , and heroin use . the 722 msm and msm / w participants were predominantly african american ( 44% ) and hispanic ( 28% ) , unemployed ( 82% ) , homeless ( 50% ) , and hiv positive ( 48% ) who used drugs in the past 6 months ( 79.5% ) . total and personal homonegativity , gay affirmation , and morality of homosexuality ihni scores were significantly higher for african american men than for other ethnicities , for msm / w than for msm , for recent cocaine users than for recent methamphetamine users , and for hiv - seronegative men than for hiv - seropositive men . linear regression showed the gay affirmation scale significantly and inversely correlated with the number of sexual partners when controlling for effects of ethnicity / race and sexual identification , particularly for men who self - identified as straight . highest ihni scores were observed in a small group of msm / w ( n = 62 ) who never tested for hiv . of these , 26% tested hiv positive . findings describe ways in which internalized homophobia is a barrier to hiv testing and associated hiv infection and signal distinctions among participants in this sample that can inform targeted hiv prevention efforts aimed at increasing hiv testing .

current biometry techniques are precise in the correction of spherical refractive errors ( myopia and hyperopia ) . however , a failure to correct refractive errors associated with astigmatism during cataract surgery may compromise the patient 's ability to be free of glasses . recent studies on different ethnic groups have confirmed that among cataract patients ; approximately 60% present a prevalence of corneal astigmatism lower than 1.5 diopters ( d ) and greater than 0.75 d [ 1 , 2 ] . among the treatment options for astigmatism , the toric intraocular lens ( iol ) implantation is considered to be the most effective . despite the excellent refractive results for toric lenses , which provide a visual acuity greater than 20/40 in more than 80% of cases and freedom from glasses in approximately 70% of cases , residual refractive astigmatism and complaints regarding vision still persist after surgery . one important instrument is the 25-item visual functioning questionnaire ( vfq-25 ) produced by the national eye institute ( nei ) of the united states . the questionnaire measures the influence of low vision and visual symptoms on specific areas of overall health , such as the emotional state and social functioning . the objective of this study was to evaluate the refractive and the quality of vision outcomes of toric iol implantation in patients with low astigmatism . it included patients with cataracts who repeatedly visited a public hospital and underwent phacoemulsification , preoperative testing , and postoperative testing to measure improvements in distance best corrected ( bcva ) and uncorrected ( ucva ) visual acuity and contrast sensitivity ( cs ) . the cataract classification , demographic and epidemiological data , and the visual quality questionnaire were performed . the inclusion criteria were cataract diagnosis confirmed by biomicroscopy , corneal astigmatism range from 0.75 d to 1.5 d , and no previous ocular surgeries or ocular diseases that could compromise vision . the exclusion criteria were incomplete postoperative follow - up and intraoperative and postoperative complications like posterior capsule rupture and iol misalignment . the following data were used in the study : ucva and bcva.cs.refraction and spherical equivalent ( se).score on the visual quality questionnaire . this study was planned to identify a difference between questionnaire domains of at least 20 points , a level of significance of 5% , and 80% power . the standard deviation of a previous study was found to be 20 points , and this was therefore considered the minimal difference for the present study . the sample size was calculated after considering quantitative differences in the group before and after the intervention ; accordingly , a minimum of 21 group participants were determined . the same surgeon ( e.s.b . ) performed all surgeries . the surgery performed was phacoemulsification with a toric iol implantation while the patient was under topical anesthesia . the main incision was a 2.5 mm clear corneal incision and it was made at 120. rayner t flex iols were used in this study . this is an aspheric toric iol , the iol edges are squared , and its spherical aberration is neutral . rayner t flex has an optic body diameter of 5.75 or 6.25 mm and an overall length of 12 or 12.5 mm . the estimated a - constant is 118.9 and the spherical power ranges from + 6 to + 30 d and the cylinder power ranges from + 1 to + 6 d. the data obtained by the iol master 500 were inserted into the supplier 's website ( raynercalculator ) , and the intraocular lens that came closest to emmetropia was chosen . the surgically induced astigmatism was set at 0.3 d on the axis where the main incision was made . the ophthalmologic evaluation was performed by a specialist ( l.f.p . ) and included the patient 's clinical history and the following exams : bcva , ucva , cs , tonometry , corneal topography , and retinal evaluation . biomicroscopy was performed to characterize cataracts by type ( cortical , nuclear , or posterior subcapsular ) and by grade ( 1 to 6 ) according to the international lens opacity classification system ( locs iii ) . the participants were evaluated on the first and seventh postoperative days , as well as 6 weeks after surgery . the rotational stability of the iol was evaluated 6 weeks postoperatively at the slit - lamp . visual acuity at 6 meters was measured using the snellen chart ; values were obtained up to the decimal notation . this was measured using the pelli robson chart . as recommended in the chart , the test was performed in a room with uniform lighting , and the table presented a mean luminance of 85 cd / m . this situation was considered photopic , with an acceptable range of 60 to 120 cd / m . the patient was seated 1 meter from the table , thus allowing legibility of the optotypes , with a spatial frequency of approximately 1 cycle per degree ( cpd ) at this testing distance . the cs value considered was that which corresponded to the last group of three letters in which the patient was able to read at least two correctly . corneal astigmatism was evaluated using the iol master 500 device and 6 central points that were 2.5 mm in diameter . autorefraction was performed using the huvitz hrk-7000 device with three measurements from which the mean value was calculated . the se was calculated as the sum of the value of the spherical degree and half of the cylindrical degree . the questionnaire is composed of 25 questions that evaluate various domains of quality of life and visual functioning , as outlined in table 1 . the patients themselves completed the questionnaires ; they were aided by the specialist when questions arose . each question corresponds to a numerical value established so that a score of 100 represents the best conditions and a score of 0 reflects the worst conditions . the means were calculated according to the following equation : the mean is defined as the sum of the numerical values of each question within a given domain divided by the number of items evaluated in that domain . noncontinuous variables were expressed as frequency , and the continuous variables were expressed as means and standard deviations . the variables were statistically analyzed using the paired t - test when the values were continuous and presented normal distribution and using the wilcoxon signed - rank test when they did not present normal distribution . the pearson correlation coefficient ( for normal distribution variables ) and the spearman correlation coefficient ( for variables that did not present normal distribution ) were used to compare vfq with visual acuity , cs , and refractive values . the mean age was 68.9 10 ( sd ) years and age ranged from 50 to 87 years . of these participants , fifteen participants ( 71.4% ) had their right eye operated and 6 participants ( 28.6% ) had their left eye operated . the results of cataract type as classified by the locs iii were shown in figures 1 and 2 . as shown in table 2 , the mean preoperative ucva was 0.2 0.17 ( sd ) and average postoperative ucva was 0.80 0.19 ( sd ) ; the range of means was 0.60 , and the p value was less than 0.001 between preoperative and postoperative values in the paired t - test . with regard to postoperative ucva , 47% of patients exhibited visual acuity better than or equal to 20/25 , and 86% presented visual acuity better than or equal to 20/30 ( figure 3 ) . the mean preoperative and postoperative bcva were 0.28 0.15 ( sd ) and 0.97 0.15 ( sd ) respectively , range of means was 0.69 , and the p value was less than 0.001 between preoperative and postoperative values in the wilcoxon signed - rank test . with regard to postoperative bcva , 100% of patients presented visual acuity that was better than or equal to 20/25 . when the paired t - test was applied , no significant difference in bcva was found between the eye included in the study and the contralateral eye ( p = 0.1053 ) . the mean preoperative and postoperative photopic cs were 1.22 0.32 ( sd ) and 1.74 0.19 ( sd ) , respectively , range of means was 0.52 , and the p value was less than 0.001 between preoperative and postoperative values in the paired t - test . the mean preoperative corneal cylinder ( iol master ) was 1.06 0.27 d , ranged from 0.75 to 1.46 d. of these , 9.5% were oblique astigmatisms , 28.5% were with - the - rule astigmatisms , and 62% were against - the - rule astigmatisms . the mean topography astigmatism was 1.04 0.3 d , ranged from 0.46 to 1.48 d. the mean preoperative and postoperative refractive astigmatism were 1.23 0.53 d and 0.34 0.39 d , respectively ( figure 4 ) . among the participants , 86% presented a postoperative refractive cylinder lower than or equal to 0.5 d , and 52% of participants exhibited a refractive cylinder lower than or equal to 0.25 d ( figure 5 ) . a significant linear correlation was observed between the variation in ucva , bcva , and cs and variation in vfq results ( table 5 ) , as shown in the scatterplots ( figures 6 , 7 , and 8) . it is important to note that an astigmatism greater than 0.5 d is found in approximately 80% of the cataract patient population [ 1 , 2 ] ; its treatment is therefore fundamental . according to visser et al . , toric iol implantation is currently considered the most effective method of treatment . despite the success of modern cataract surgery techniques and their adequate refractive results , patients report many complaints regarding their eyesight , particularly in terms of visual quality ( such as halos and blurriness ) , which lead to decreased quality of life among these patients . thus , the current study sought not only to present visual and refractive results on a toric iol implantation in patients with low astigmatism but also to correlate these results with the quality of vision according to a visual functioning questionnaire . the study sample was composed of 61.9% females and 38.1% males , and the mean patient age was 68.9 years . this sample was similar to those in other studies that have evaluated cataracts in the elderly [ 1 , 2 ] . no statistically significant difference was observed between ucva of the studied eye and that of the contralateral eye ; thus , the impact of cataract surgery on the quality of life was not affected by disproportionate visual acuity . with regard to cataract classification , most patients exhibited nuclear cataracts ( 71.4% ) , followed by posterior subcapsular cataracts ( 23.8% ) and cortical cataracts ( 4.8% ) . according to prokofyeva et al . , the risk factors for nuclear cataracts are an unbalanced diet and poor socioeconomic conditions . this finding explains the higher prevalence of nuclear cataracts among the participants in the present study , which was performed in a developing country . the cataract grades exhibited by the sample were the following : 76.2% of patients were of grades 4 , 5 , and 6 . presented a study in which 16.4% of patients had cataracts of grade 4 or higher ; this difference may be explained by the socioeconomic conditions and limited access to healthcare among developing country populations . ucva is an important parameter when evaluating the refractive effectiveness of cataract surgery . in the present study , the mean postoperative ucva was 0.8 , with 47% of patients experiencing vision that was better than or equal to 0.8 and 96% experiencing vision that was better than or equal to 0.5 . recent reviews of the literature on the surgical outcomes of toric iols have presented results in which ucva greater than or equal to 0.8 was observed in 23% to 100% of cases and in which ucva greater than or equal to 0.5 was observed in 68% to 100% of cases [ 3 , 10 ] . statistically significant variation between preoperative and postoperative values ( p < 0.001 ) was also found by agresta et al . . in this study , the mean postoperative bcva was 0.97 , with 100% of patients exhibiting bcva greater than 0.8 . preoperative and postoperative variations were found to be statistically significant ( p < 0.001 ) . in addition , a statistically significant variation was obtained ( p < 0.001 ) in the evaluation of cs , with a mean value of 1.74 db . similar mean values were observed under similar photopic conditions with low spatial frequency [ 10 , 12 ] . an important result of the present study was the sample 's mean corneal and refractive astigmatisms of 1.06 0.27 d and 1.23 0.53 d , respectively , in the preoperative period and a mean postoperative refractive astigmatism of 0.34 0.39 d. leon et al . studied a population with low astigmatism , and the preoperative and postoperative refractive astigmatism values were 1.59 0.52 d and 0.40 0.20 d , respectively . their results were therefore similar to those of the present study . in their study , the postoperative refractive cylinder was lower than or equal to 0.5 d in 86% of patients and lower than or equal to 0.25 previous studies have reported 25% to 100% patients with a postoperative refractive cylinder lower than or equal to 0.5 d . obtained a preoperative and postoperative refractive astigmatism of 1.75 0.71 d and 0.62 0.46 d , respectively . the mean spherical equivalent values increased without statistical significance ( from 0.17 2.33 d to 0.52 0.35 d ) ; however , an important decrease in the standard deviation reflected greater homogeneity of the postintervention results . the trend toward negative spherical equivalent values occurred due to the total spherical degree , the goal of which is emmetropia with a residual negative cylinder . presented values similar to those of the present study , with a spherical equivalent ranging from 0.13 2.31 d to 0.26 0.62 d . a search of the literature on pubmed in august 2016 ( using the keywords vfq 25 and toric iol ) did not reveal articles in which the vfq 25 was applied to patients who had undergone cataract surgery with toric iol implants . however , greater satisfaction was reported among patients who had received the toric iol implantation as opposed to those who had received the spheric iol implantation . these results were consistent with those of the present study , which also found a significant improvement in all of the items evaluated by the questionnaire . it is important to consider the gain in vision quality and quality of life as a result of the simple visual recovery provided by cataract surgery , as demonstrated by other studies in which the questionnaire was applied to patients who had undergone cataract surgery [ 15 , 16 ] . to et al . reported a variation from 65.19 16.80 points to 88.02 14.51 points in the nei vfq 25 of cataract patients who had received surgery for one eye . these results were similar to those of the present study ( 43.20 15.76 to 79.70 10.11 ) , both with a significant p value . lin et al . presented a mean variation between preoperative and postoperative aspheric and spheric iols of 13.95 and 13.02 , respectively ; the present study found a mean positive variation of 36.49 . more important than refractive , visual , and quality of life data is the interpretation of these findings based on their correlations . the current study revealed statistically significant correlations between patient satisfaction and the visual function items . when the variation in the nei vfq 25 and cs was analyzed , pearson 's coefficient was 0.409 ( p = 0.033 ) , indicating that the improvement in cs provided by cataract surgery is directly correlated with an increase in visual quality and quality of life . when variations in bcva were correlated with the results of the questionnaire , spearman 's correlation coefficient was 0.508 ( p = 0.009 ) . in addition , when ucva was correlated with the results of the questionnaire , pearson 's coefficient was 0.548 ( p = 0.005 ) . these findings reflect the importance of refractive results in cataract surgery , particularly in terms of freedom from glasses . the lack of a statistically significant difference between ucva of the eye included in the study and the contralateral eye decreased the influence of visual acuity of the contralateral eye as a confounding factor when the benefit of the surgery was analyzed in terms of the results of the quality of life questionnaire . thus , according to the results presented here , cataract surgery with a toric iol implant to achieve better visual and refractive results is associated with a greater patient perception of visual quality and a greater quality of life . patients with low astigmatism who underwent phacoemulsification with a toric iol implant experienced significant decrease in refractive astigmatism and improvement in their quality of life ; this finding positively correlated with variations in cs , ucva , and bcva .

purpose . to evaluate the refractive and the quality of vision outcomes of toric iol implantation in patients with low astigmatism . design . prospective study of single - arm . methods . patients with corneal astigmatism range from 0,75 d to 1,5 d and cataract that underwent cataract surgery with toric iol . the measurements were performed preoperatively and 6 weeks after the surgery . patients were evaluated for visual acuity with and without correction , contrast sensitivity , static and dynamic refraction , and quality of life questionnaire . pre- and postoperative values were compared and their variations were evaluated for linear correlation . results . 21 eyes of 21 patients . postoperative mean uncorrected visual acuity was 0.80 0.19 , and the best corrected visual acuity was 0.97 0.15 . p < 0.001 compared to preoperative values . the average postoperative refractive cylinder was 0.34 0.39 . the questionnaire 's total value before and after surgery was , respectively , 43.20 15.76 and 79.70 10.11 ( p < 0.001 ) . the correlation coefficients between the values of the questionnaire variation and the ucva , bcva , and cs variation were , respectively , 0.548 ( p = 0.005 ) , 0.508 ( p = 0.009 ) , and 0.409 ( p = 0.033 ) . conclusion . patients with low astigmatism who underwent phacoemulsification with toric iol implantation experienced significant decrease in refractive astigmatism and improvement in their quality of life .

tissue - specific autoimmunity may be defined as a progressive inflammatory immune response to specific proteins originating from cells in a tissue or organ resulting in diminished organ function or organ failure . the identification of immune cells responsible for initiation of autoimmunity remained elusive until a new class of immune cells , dendritic cells ( dc ) , was discovered in the early 1970s by nobel laureate ralph steinman . until this time , cells representing the innate and adaptive arms of the immune system were considered to be separate entities . however , steinman 's discovery that dendritic cells were the missing link responsible for coordinating innate and adaptive immune responses has revolutionized the relationship between these two segments of the immune system . in addition , this discovery allowed the proliferation of many novel therapeutic strategies for prevention and treatment of tissue - specific autoimmunity . the first clues to interactions between innate immunity and the adaptive immune response became apparent in 1973 when steinman and cohn detected the presence of an unusual immune cell type while observing cells from mouse spleen by phase contrast microscopy [ 1 , 2 ] . the authors identified a specific population of immune cells that did not display the typical morphology of macrophages and which possessed long dendrite - like cytoplasmic processes capable of dynamic extension or retraction . from the time of this observation , it took nearly 5 years for steinman 's laboratory to develop a method for obtaining a pure population of dendritic cells . this accomplishment led to important discoveries that dendritic cells expressed high levels of major histocompatibility complex ( mhc ) proteins and that they were the most potent antigen presenting cells in the immune system for inciting a mixed leukocyte reaction ( mlr ) . prove to be a critical accessory cell for the generation of many immune responses . since steinman 's early discovery , dendritic cells identified in human blood were shown to uniquely capture and present antigens to t cells of the adaptive immune system [ 5 , 6 ] . in the early 1990s , steinman and his colleagues developed a method for production of human langerhans dendritic cells from cd34 progenitors with granulocyte macrophage colony stimulating factor ( gm - csf ) and tnf- . although this method increased the availability of dcs for future study , the amplification of dcs remained limited due to the scarcity of cd34 progenitors in adult blood . this obstacle was overcome by the finding that large numbers of dendritic cells could be generated from peripheral blood mono nuclear cells ( pbmcs ) following treatment with gm - csf and interleukin 4 ( il-4 ) . this new monocyte - derived dc amplification system energized the study of dendritic cells by permitting the study of human dc functions in both healthy and diseased states . a detailed history of the discovery of dendritic cells is beyond the scope of this review and are covered in the cited reviews [ 3 , 911 ] . dendritic cells are the primary line of immune cell defense against pathogens and toxins that invade the body . representing the innate portion of the immune system , dendritic cells recognize and destroy invading bacterial , viral , protozoan , and fungal pathogens and other foreign molecules that escape the body 's passive defenses . in innate immunity , monocytes recruited into inflammatory sites differentiate into dendritic cells under the influence of th1 cytokines . the dendritic cell response comprises secretion of tnf- and no to aid in the clearing of pathogens . the inflammatory cytokine tnf- can bind to receptors on gram - negative bacteria facilitating phagocytosis by macrophages . dendritic cells may also activate nk cells through both contact - dependent and contact - independent mechanisms . when the pathogen load becomes excessive , dendritic cells act primarily as antigen presenting cells by migrating to the spleen or peripheral lymph nodes and delivering portions of the invading pathogens to lymphocytes of the adaptive immune system to amplify the immune response . an increased awareness of the intricate relationship between dendritic cells and other cells of the adaptive immune system will be essential for understanding how immunological homeostasis is achieved and maintained . further improvements in understanding how dendritic cells generate peripheral and central tolerance will be needed before more effective and safer therapeutic strategies can be constructed to prevent or reverse the process of autoimmunity . in central tolerance , dendritic cells residing in the thymus present antigens on mhcii and cross present self - antigens on mhci receptors to bone marrow - derived t cells to stimulate apoptosis of potentially autoreactive t cells ( negative selection ) [ 16 , 17 ] . further , peripheral dendritic cells were shown to migrate to the thymus to induce clonal t cell deletion or development of t regulatory ( treg ) cells [ 18 , 19 ] . although not yet demonstrated for all endogenous self - antigens , dendritic cells can induce peripheral tolerance through presentation of immunodominant antigens expressed at high levels . further , dendritic cells are thought to make a major contribution to peripheral tolerance by facilitating induction and/or maintenance of peripheral treg cells . additional experimental evidence supports the role of dendritic cells in the induction of autoimmunity , reinforcing the notion that the activation state of dendritic cells is largely responsible for the induction of autoimmunity or tolerance . ablation of dendritic cells in mice with an autoimmunity - prone background was shown to ameliorate disease onset . together , this data shows that dendritic cells can stimulate the development of autoreactive t cells as well as generate immunological tolerance . later in this review , we discuss the origin of dendritic cell subsets and their respective functions in the initiation of inflammation as well as immune tolerance . further , we review the involvement of dendritic cells in the induction of tissue - specific autoimmunity with comparisons to systemic autoimmunity to demonstrate how development of dendritic cell - based therapies for treatment of tissue - specific autoimmunity may become broadly applicable . in order to efficiently encounter , process , and transport foreign antigens to lymph nodes for presentation to t cells , a variety of dendritic cell subsets exist that are specialized in morphology , function , and location . dendritic cells , like most cells of the immune system , comprise a number of groups or subsets that are distinct either in origin , function , or both . although classification of dc subsets may vary , this review focuses on the four major categories of dendritic cells currently recognized : ( 1 ) conventional dendritic cells ( cdcs ) , ( 2 ) langerhans cells , ( 3 ) plasmacytoid dendritic cells ( pdcs ) , and ( 4 ) monocyte - derived dendritic cells ( modcs ) . due to differences in phenotype , an alternative subset of dendritic cells , the myeloid dcs ( mdcs ) , dendritic cell hematopoiesis was originally established in mice and the corresponding dc subsets were identified in humans . the origin of dendritic cells begins in the bone marrow with cd34 flt3 common myeloid progenitor ( cmp ) cells . the cmps differentiate into restricted macrophage dendritic cell progenitors ( mdp ) . the mdps branch to create two separate dendritic cells lineages : the monocyte and the common dendritic cell progenitor ( cdp ) . monocytes circulate in the periphery and during inflammation , differentiate into cd11c , cd11b , and mhcii modcs . the cdps differentiate in the bone marrow into plasmacytoid dcs and into pre - dcs which give rise to conventional dcs in peripheral tissues . the only dendritic cells that do not originate in the bone marrow are the langerhans cells . the langerhans subset is derived from a local ly6c myelomonocytic precursor in the skin that originates from macrophages during early embryonic development . conventional dendritic cells are a highly specialized dc subset that is efficient in antigen processing and presentation . this major group of dendritic cells can be categorized as either migratory or lymphoid tissue resident dcs . migratory dendritic cells develop from precursors in both lymphoid and nonlymphoid tissues but are not found in the spleen . after taking up and processing antigens from resident tissues , cdcs migrate to the peripheral draining lymph nodes via afferent lymph vessels to present sequestered antigens to nave t cells . the migration of cdcs is markedly amplified during inflammatory conditions . in contrast , lymphoid tissue resident dendritic cells are located in the spleen , thymus , and lymph nodes . in comparison with the migratory dc subset , lymphoid tissue resident dendritic cells do not migrate but rather develop from dendritic cell precursors already residing in lymphoid tissues . during immunological homeostasis , cdcs remain immature , have high endocytic capacity , and synthesize low levels of mhc relative to other dendritic cell subsets . due to their predominant location in the spleen , lymphoid tissue resident dendritic cells are well suited to sample antigens transported by the blood . they reside in the skin and can be identified by the expression of the monocyte / macrophage and endothelial cell differentiation antigen ly6c . their function is to capture , identify , and present antigens from the external environment to nave t cells . both cdcs and langerhans cells are necessary for the maintenance of immunological homeostasis , with their major functions occurring during the steady state . plasmacytoid dendritic cells arise from lymphoid progenitors and are broadly distributed throughout the body . in humans , this dc subset can be identified through expression of immunoglobulin - like transcript 7 ( ilt7 ) and cd45r . plasmacytoid dendritic cells are generally quiescent , but when stimulated , they secrete large amounts of type 1 interferons which induce antiviral responses in other immune cells . these toll - like receptors bind pathogen nucleic acids , especially motifs rich in cpg , and play a major role in pdcs response to viral infections . because pdcs have low antigen presentation capabilities , their role in promoting adaptive immunity remains relatively unclear . however , pdcs are known to play a major role during inflammation as opposed to cdcs which are more involved in the maintenance of immunological homeostasis . monocyte - derived dendritic cells arise from myeloid progenitors and are crucial for immune responses because they provide a pool of antigen presenting cells that can effectively initiate an adaptive immune response following the onset of infection . during inflammation , it is well known that circulating monocytes express receptors for gm - csf , m - csf , il-4 , and other differentiation and chemoattractant molecules that recruit them to the site of inflammation and differentiate them into modcs . monocyte - derived dendritic cells are highly potent in antigen processing , presentation , and cross presentation . they are cd11c positive and express high levels of mhc ii in comparison with other dc subsets . the function of modcs is similar to cdcs because they both process foreign antigens from tissues and migrate to the nearest draining lymph nodes where they present the antigens to nave t cells . upon activation , the lifespan of all dc subsets is relatively similar , as opposed to the length of time dcs reside in tissues during their immature state . the major responsibility of modcs and pdcs is to incite an adaptive immune response through activation of t cells that can resolve inflammation and return the immune system to immunological homeostasis . the interaction of modcs and pdcs with autoreactive t cells can either induce autoimmune dysfunction or initiate tolerance . however , it is their ability to link the innate and adaptive immune system that confers them a role in autoimmunity . in adaptive immunity , most importantly , dcs transfer innate signals to the adaptive immune system by priming nave t cells , by stimulating th1 , th2 , and th17 responses , by cross presentation of antigens to cd8 t cells , and by regulating t cell differentiation [ 3942 ] . the breakdown of dendritic cell functions is considered to be the driving force behind the onset of tissue specific autoimmunity . dendritic cell subsets differ in their specialized functions , including the location of activity , cytokine profiles , types of antigens detected , migratory , or tissue resident status and presence during immunological homeostasis or during inflammation . however , the common function of these dendritic cell subsets is the communication of inflammatory or immunosuppressive signals among the innate and adaptive cells of the immune system . the second step is the presentation of antigens along with the appropriate secondary signals required to induce an adaptive t cell response . dendritic cells identify antigens via pathogen - associated molecular patterns ( pamps ) or damage - associated molecular patterns ( damps ) . comprised of molecules common to a variety of organisms but absent in the host , pamps are an exogenous signal to dendritic cells for infection . for example , viral envelope proteins and ssdna from viruses , lipopolysaccharide and flagellin from bacteria , zymosan from fungus , and profilin from toxoplasma gondii are pamps which are recognized by specific receptors on dendritic cells . in comparison release of atp , dna , or uric acid can be a warning sign to dendritic cells for stress , microbial invasion , or necrotic cell death such as in the case of cancer . dendritic cells detect pamps and damps through several classes of surface and intracellular receptors called pattern recognition receptors ( prrs ) . these classes include the receptor for advanced glycation end products ( rage ) , rig - i - like receptors ( rlrs ) , nod - like receptors ( nlrs ) , and toll - like receptors ( tlrs ) . other damps , such as atp and uric acid activate nlrs and induce the formation of inflammasomes which trigger the downstream secretion of inflammatory cytokines il-1 and il-18 . all immunogenic nucleotides bind rlrs which need subsequent recognition to induce a signaling response within the dendritic cells . lastly , tlrs , the most common of the prrs , recognize a variety of pamps and damps and are largely implicated in autoimmune disease . surface tlr4 , tlr5 , and heterodimers of tlr1/2 and tlr2/6 all recognize bacterial membrane components while tlrs 3 , 7 , 8 , and 9 are located within endosomes and recognize immunogenic nucleotides . the high expression of tlr- 2 and tlr-4 on mdcs and tlrs 7 and 9 on pdcs may have a role to play in tissue - specific autoimmunity where molecular mimicry or autoantibodies to nucleic acids is a potential underlying mechanism . once a dendritic cell has encountered an antigen , it processes and transfers this information to t cells . mature dcs interact with t cells through ( 1 ) dendritic cell mhc - ii / antigen complex interactions with the t cell receptor , ( 2 ) dendritic cell cd86/cd80 costimulation of t cell cd28 , and ( 3 ) dendritic cell cytokine signaling to t cells . because the extent of dc activation determines whether interaction with a t cell will induce tolerance or immunity , antigen presentation in the presence of inflammatory signals results in immunity while ag presentation during the steady state after results in tolerance . during inflammation , dendritic cells can induce nave t cell ( th0 ) differentiation or activate memory t cells . upon dendritic cell maturation , nave cd4 t cells can differentiate into memory and th1 , th2 , and th17 effector cells associated with autoimmunity . nave cd8 t cells differentiate into cytotoxic t lymphocytes and cd8 memory cells . during immunological homeostasis , dc interaction with t cells generates and maintains a population of tregs . under conditions of autoimmunity , autoantigens which should induce tolerance during homeostasis generate an inflammatory response . in general , the role of dcs during the development of autoimmunity is to induce autoreactive cd4 and cd8 proinflammatory t cell differentiation rather than immunosuppressive treg development or autoreactive t cell anergy . dendritic cells play a critical role in both the prevention and onset of autoimmune responses . in the nondiseased state , the induction of t cell tolerance is dependent on whether a dc is tolerogenic or immunogenic during autoantigen presentation , and also on the contribution of autoreactive t cells that may escape from the thymus . although the continuously developing t cell repertoire is rigorously screened in the thymus to develop central tolerance , autoreactive t cells which may escape apoptosis in the thymus are poised to induce autoimmunity unless regulated by dendritic cells in the periphery . normally , presentation of a self - antigen to an autoreactive t cell results in t cell anergy , deletion , activation , or induction of treg cells . there are several factors that influence whether a dc will induce tolerance when presenting self - antigens including but not limited to activation state and method of antigen capture / antigen source . the activation state of a dc is crucial to the fate of the t cells with which it interacts . typically , tolerogenic dcs express low levels of costimulatory molecules ( cd80 , cd86 ) , generate increased secretion of il-10 and tgf- , and reduce secretion of proinflammatory il-12 , il-1 , il-6 , and tnf cytokines . in addition to these hallmarks of tolerogenic dcs , the secretion of il-2 and a variety of enzymes such as retinaldehyde dehydrogenase-2 and indolamine 2,3 dioxygenase are involved in the suppression of th1-mediated autoimmunity in addition to the induction of treg cells [ 4648 ] . the dcs ability to capture process and tam receptor kinases ( tyro3 , axl , mer ) present on apoptotic cell membranes inhibit tlr and cytokine - induced signaling cascades , therefore preventing immunogenicity of the autoantigens presented . in addition , tgf- is associated with treg induction and is secreted from dcs upon uptake of apoptotic cells [ 50 , 51 ] . dendritic cells can capture highly glycosylated proteins through c - type lectin receptors ( clrs ) present in the plasma membrane . because clrs are involved in the clearance of multiple soluble self - antigens , alternatively , acetylation of proteins can produce neoantigens to which the immune system has not been tolerized . all of these factors are in place to ensure a tolerogenic phenotype in dcs during presentation of self - antigens . however , these mechanisms to prevent the human immune system from recognizing self - antigens may occasionally malfunction . in general , this malfunction can involve self - antigen presentation in the presence of danger signals , therefore breaking tolerance . for each autoimmune disease , different mechanisms for dc initiation of autoimmunity are suggested ; nonetheless , they still remain unclear . for example , in the case of type 1 diabetes , genetic abnormalities in dc subsets and viral infection have been linked as initiators of autoimmunity . nod mice have increased cd11b cdcs which have been demonstrated to be responsible for the presentation of type 1 diabetes autoantigens to autoreactive t cells [ 55 , 56 ] . in the diseased state , these cdcs cross - present islet antigens to autoreactive cd8 t cells and secrete increased il-12 , tnf , and il-1 . the breach of tolerance induced by cdcs is speculated to be regulated by pdcs . in some cases , certain viral tropisms induce the secretion of type 1 interferons by binding tlrs within pdcs . a localized type 1 interferon response within the pancreas can activate autoreactive t cells and act as a danger / maturation signal to resident cdcs , therefore promoting presentation of steady state antigen but in a proinflammatory context . this immunogenic presentation of islet autoantigens to cd8 t cells may allow for the homing of cytotoxic t cells to the islets , destruction of the target cells , and the perpetuation of autoimmunity . systemic lupus erythematosus is an example of a systemic autoimmune disease for which there are models describing the chain of events linking dc activation to an adaptive immune response . in genetically susceptible individuals , immune complexes consisting of nucleic acid - associated autoantigens and autoantibodies which are internalized by pdcs via fcriia receptors this tlr activation , just like a response to viral infection , induces the secretion of type 1 interferon . type 1 interferons induce maturation of cdcs , increasing the expression of mhc class i and ii , costimulatory molecules , and chemokines and chemokine receptors . this scenario allows for the uninhibited presentation of autoantigens by proinflammatory dcs and thus the expansion of autoreactive t cells . for other autoimmune diseases such as multiple sclerosis , the chain of events linking dc activation by pamps or damps to induction of autoreactive t cells is relatively unknown and is an important area for future research . in summary , the induction of autoimmunity by dcs requires excessive production of pamps or damps to switch dc presentation of autoantigens from tolerogenic to immunogenic , usually on a genetically susceptible background . the switch of the dc to a proinflammatory state disrupts tolerance by activating and inducing differentiation of autoreactive t cells via tcr ligation and cytokines such as il-2 , il-12 , il-6 , type 1 interferons , and tgf-. in the following segments , we will review current evidence for dendritic cell - induced autoimmunity in tissue - specific autoimmune diseases . we will also identify potential dendritic cell targeted therapies and discuss their mechanisms of action . tissue - specific autoimmunity requires the release of specific autoantigens characteristic of a given tissue or organ that can be recognized by dendritic cell prrs . prototypical tissue - specific autoimmune disease examples presented in this review are type 1 diabetes ( t1d ) and multiple sclerosis ( ms ) , and for comparison examples of systemic autoimmune diseases discussed are rheumatoid arthritis ( ra ) and systemic lupus erythematosus ( sle ) . the pancreatic -cell is one of the most specialized cells in the body and is central to type 1 diabetes onset . in addition to the production , storage , and secretion of the peptide hormone insulin , the cell is capable of sensing and responding to changes in blood glucose levels . during the earliest stages of t1d research , specific islet autoantigens signaling diabetes onset were discovered through their recognition by islet cell autoantibodies ( icas ) . since identification of icas in 1976 , their -cell protein targets were only slowly revealed [ 61 , 62 ] . with the exception of insulin as an obvious t1d candidate autoantigen , it was not until 1990 that the 64 kda glutamic acid decarboxylase ( gad ) protein was discovered to be an important secondary antigen for t1d development [ 61 , 63 ] . multiple sclerosis ( ms ) is a devastating inflammatory disease of the brain and spinal cord resulting from autoimmune attack against antigens in the central nervous system . proinflammatory t cell responses to the major myelin proteins , myelin basic protein ( mbp ) , and proteolipid protein ( plp ) are considered to be important for the development of ms . however , dc - mediated autoimmune responses to other minor myelin antigens such as myelin - associated glycoprotein ( mag ) and myelin oligodendrocyte glycoprotein ( mog ) may also play a significant role in disease initiation or progression . unlike organ - specific autoimmune diseases , a major challenge exists in finding individual representative antigens for initiating the onset of systemic autoimmunity ( figure 1 ) . this difficulty is based on observations by many investigators that the onset of systemic autoimmunity may be initiated at different times and locations in the connective tissues of rheumatoid arthritis ( ra ) and systemic lupus erythematosus ( sle ) patients . further , the problem of progressive antigen spreading can further complicate this issue . in confirmation that tissue specificity underlies systemic autoimmunity , it was recently shown that hyperactivation of myd88-adapter - dependent signaling in dcs is sufficient to drive pathogenesis of lupus - like autoimmunity . systemic lupus erythematosus ( sle ) is an autoimmune disease that progressively invades many tissues throughout the body and is frequently characterized by the formation of anti - nuclear and anti - chromatin antibodies . these autoantibodies are generated in response to aberrant apoptosis and decreased clearance of apoptotic cells which increases the abundance of apoptotic cell blebs containing chromatin . interestingly , mdcs can take up , process , and present chromatin found in these apoptotic blebs to t cells . dendritic cell presentation of this modified chromatin stimulates activation of autoreactive t helper cells , leading subsequently to the formation of autoantibodies by autoreactive b cells . the deposition of immune complexes formed by anti - chromatin autoantibodies and modified chromatin on cell basal membranes stimulates a local inflammatory response . further , autoreactive t cells that bind nuclear antigens such as dna and histones or small ribonucleoproteins including smith ( sm ) antigens and u1 and heterogeneous ribonucleoprotein ( hnrnp ) a2 to their t cell receptors were shown to be associated with sle development . taken together , this data suggests that chromatin , smith antigens , and ribonucleoproteins are potential autoantigen candidates for development of sle . rheumatoid arthritis ( ra ) is another well - known systemic autoimmune disease that affects connective tissues throughout the body . ra - related autoantibodies demonstrate reactivity to citrullinated proteins and peptides designated as anti - citrullinated protein antibodies ( acpa ) . the process of citrullination removes positive charges from the antigen via replacement of arginine with citrulline . the alteration in charge can modify secondary and tertiary protein structure , thereby increasing the binding affinity to mhc receptors . the citrullinated proteins , now altered in structure from normal body proteins , may be recognized by the immune system as foreign and thus , potential autoantigens integral to the development of ra . in agreement with this concept , autoreactivity towards a variety of self - proteins has been associated with the onset and progression of ra . several of the antigens described are joint - derived proteins , such as type ii collagen and human cartilage - derived glycoprotein hcgp39 . these experimental findings suggest the possibility of treating both tissue - specific and systemic autoimmune diseases by targeting several specific autoantigens characteristic of disease development . type 1 diabetes is a juvenile onset form of diabetes resulting from autoimmune destruction of insulin - producing cells in the pancreatic islets of langerhans . type 1 diabetes can be treated in a palliative fashion with exogenous insulin injection . however , the increasing prevalence of this disease , its progressive complications , and the lack of effective curative and preventive strategies demand a significant research effort to identify promising therapies capable of restoration of immunological tolerance . at present , no effective , safe , and economical treatment exists to control the onset and progression this life - long debilitating disease . for this reason , the nonobese diabetic ( nod ) mouse , a widely used animal model presenting the dominant symptoms of human t1d autoimmunity , is frequently used for studying the mechanisms underlying t1d onset and progression . based on the presence of specific autoantigens known to elicit diabetes onset , t1d has been identified as a model prototypic tissue - specific autoimmune disease . hyperglycemia develops in t1d when insulin presenting dcs encounter naive insulin reactive t cells in the periphery . during this interaction , the dcs guide autoreactive t cell differentiation into inflammatory effector cells that arrest insulin production by inducing -cell apoptosis little is known concerning the kinetics and phenotype of dcs in the nod mouse pancreas during t1d development . while peri - islet accumulation of cdcs can be observed in nod mice as early as 4 weeks of age , pdcs were shown to accumulate around the islets of langerhans beginning later at 10 weeks of age . peri - islet dendritic cell accumulation was found to be concomitant with the influx of lymphocytes . ablation of total dcs in nod mice led to loss of cd4 t cell activation , insulitis , antibody production , and pancreatic infiltration with proinflammatory th1/th17 cells . the authors further demonstrated that reintroduction of mdcs to the dc ablated mice induced insulitis and diabetes . together , these results demonstrate that dcs are key players in the onset of tissue specific autoimmunity . the state of dendritic cell activation is critical for determination of their function as tolerogenic or inflammatory dcs . an important feature of tolerogenic dcs is their ability to secrete the immunoregulatory cytokine tgf- as well as the anti - inflammatory cytokine il-10 which can suppress t cell responses by inhibiting t cell secretion of il-2 and ifn- . presence of the anti - inflammatory cytokine il-10 was shown to control a number of different immune cell types implicated in the inflammatory response , including dcs . in addition , il-10 was shown to upregulate the expression of tolerogenic molecules like ilt3 and ilt4 , allowing them to minimize immune responses and induce treg morphogenesis [ 79 , 80 ] . during the immunological steady - state also referred to as homeostasis , dcs were shown to secrete high levels of il-10 that can modulate activation of neighboring myeloid dc and promote de novo induction of tolerogenic dcs . in view of their pivotal role in regulating t cell immunity , dendritic cells could be expected to alter the balance between pathogenic t cells and tregs in type 1 diabetes . studies in the nod mouse have shown that mdcs can exhibit a hyper - inflammatory phenotype [ 81 , 82 ] . specifically , nod mouse mdcs have been shown to generate an elevated capacity for stimulation of t cells and secretion of proinflammatory cytokines such as il-12 . further , in vitro studies have shown that maturation of modcs is inhibited in the presence of exogenous il-10 . multiple sclerosis is a chronic inflammatory disease of the central nervous system ( cns ) associated with an autoimmune response against components of myelin , including myelin basic protein . experimental autoimmune encephalomyelitis ( eae ) , the animal equivalent of ms , is the prominent tool by which researchers studying ms have investigated the disease process . various versions of the model are under investigation to study the mechanisms of immunopathogenesis and new treatments for ms . new treatment strategies are frequently tested in a chronic eae mouse model , and when successful , the treatment can be considered for human therapy . over the last two decades , the knowledge of immunopathogenesis in ms has grown to define ms as a multifocal demyelinating disease mediated by an autoimmune response to several self - antigens . although neurological deficits in ms may be the result of a combined cellular and humoral autoimmune attack on the myelin sheath , ms has long been considered a predominantly t cell - mediated autoimmune disease . however , it is clear that t cells are not the only immune cell type involved in ms and eae disease pathogenesis . the characterization of dcs during the course of eae development indicates that different dc subsets serve distinct functions . for example , conventional dcs are involved in disease development , while plasmacytoiddcs that produce interferons ( ifn ) are important in the development of treg cells and disease resolution . in ms , recent experimental findings highlight the upregulation of tlr7 mediated by interferon- ( ifn- ) in pdcs . upregulation of tlr7 in pdcs and consequently increased activation of pdcs by tlr7 ligands could be considered novel immunoregulatory mechanisms for ifn- . a critical role for ifn- was demonstrated in a recent finding in which the proinflammatory cytokine induces expression of the il7r receptor . expression of il7r is unequivocally associated with susceptibility to ms and was shown to be increased in mdcs in a haplotype - dependent manner in response to increases in ifn. active participation of dcs in the pathology of ms is supported by their presence and activation in the cns of ms patients . myeloid and plasmacytoid dendritic cells are present in cerebrospinal fluid in noninflammatory neurological diseases and elevated in multiple sclerosis and in acute monosymptomatic optic neuritis . particular emphasis has been given to the study of pdcs involvement based on their importance in stimulating or inhibiting effector t cells in ms . in addition to the presence of dcs in cerebrospinal fluid ( csf ) and cns lesions in ms patients , both phenotypic and functional impairments have also been observed to be dependent on dc subsets and ms subtypes . circulating cdcs expressing upregulated levels of costimulatory molecules and proinflammatory cytokines stimulate proinflammatory cytokine secretion by effector t cells . their infiltration into the inflamed brain can be attributed to upregulation of c - c chemokine receptor 5 ( ccr5 ) . it was suggested that pdcs promote priming of autoimmune th17 lymphocytes in eae , whereas depletion of pdcs prior to induction of the disease decreases its severity . abundant expression of tlr9 in pdcs appears to be important in the pathogenesis of eae . activation of apcs through tlr9 can overcome tolerance and precipitate eae while tlr9 knockout mice show a decreased susceptibility to eae [ 93 , 94 ] . antigen or epitope spreading is a process in which immunoreactive segments of a protein ( epitopes ) distinct from and non - cross - reactive with initial disease - inducing antigen epitopes become targets of a progressive inflammatory immune response . the phenomenon of epitope spreading has been defined in experimental and naturally occurring inflammatory responses as a consequence of acute or persistent infection characteristic of the chronic tissue destruction that occurs during autoimmune disease progression . epitope spreading has been described in different models of autoimmunity , including t1d and eae . epitope spreading may occur as increasing numbers of cells of the affected tissue or organ undergo necrosis or necroptosis following the initial acute immune response . the progress of inflammatory cell death may allow for the capture , processing , and presentation by dcs of an increasing number of cellular antigens . thus , the number of autoantigens presented in this progressive inflammatory state would increase and tolerance to these autoantigens would be lost . in type 1 diabetes , pancreatic resident dcs are generally the first cells of the immune system to process -cell autoantigens , and by promoting autoreactivity , they play a major role in the onset of pancreatic inflammation ( insulitis ) . protection from the onset of autoimmune disease may be induced by the introduction of candidate autoantigens ( in the case of t1d : proinsulin , insulin , heat shock protein 60 , or glutamic acid decarboxylase ) considered to be the initial major offending autoantigens . results of mechanistic studies have confirmed that downregulation of the immune response specific to the disease autoantigen can rapidly extend to other candidate autoantigens [ 97 , 98 ] . these experimental findings suggest that therapeutics may not need to be targeted toward an ever increasing number of autoantigens found both in tissue - specific and systemic autoimmunity but may be effective through targeting one of the major autoantigens found during the acute phase of disease onset . because the specific role of dcs in epitope spreading in type 1 diabetes has yet to be elucidated , epitope spreading remains an obstacle in autoantigen targeted therapy . dendritic cells have been shown to be particularly critical in epitope spreading in the experimental autoimmune encephalomyelitis ( eae ) model . in eae , reactivity to myelin epitopes generated during the initial clinical episode of relapsing eae ( r - eae ) , for example , epitope spreading , plays a major role in the mediation of further clinical relapses [ 99102 ] . determination of the capacity for antigen delivery by antigen - presenting cell ( apc ) populations purified from the central nervous system ( cns ) of mice with established r - eae shows that peripherally derived cd11bcd11ccd45 myeloid dendritic cells ( mdcs ) are efficient in presentation of endogenous myelin antigens that stimulate the differentiation of both preactivated effector myelin - specific t cells and nave t cells . the mdcs , which drive epitope spreading , preferentially polarize pathogenic th17 responses that correlate with their enhanced expression of cytokines tgf - beta1 , il-6 , and il-23 . in the same r - eae model , it was shown that dcs , macrophages ( f4/80cd45 ) , and microglia ( f4/80cd45 ) activate a plp139 - 151-specific t helper cell line . the data from this study and others suggest that dcs presenting cns antigens migrate from cns tissue to prime encephalitogenic myelin - reactive t cells in lymphoid organs , thereby inducing antigen spreading and recruitment of t cells into the inflamed cns . in rheumatoid arthritis ( ra ) , autoantibodies targeting several innate immune cell ligands including citrullinated histones , fibrinogen , and biglycan have provided insights into the earliest autoantigen targets and potential mechanisms responsible for the onset and development of ra autoimmunity . in addition , expansion of anti - citrullinated protein antibodies ( acpa ) has strongly predicted increases in many inflammatory cytokines in ra including tnf- , il-6 , il-12p70 , and ifn-. thus , it was observed that the preclinical phase of ra can be characterized by accumulation of multiple autoantibody specificities that reflect the process of antigen spreading . autoantibodies targeted against nuclear components are a characteristic feature of sle . due to disturbed apoptosis and/or an insufficient clearance of apoptotic debris recent studies have identified apoptosis - induced acetylation of histone h2bk12 as a target for autoantibodies in sle . since anti - h2bk12ac reactivity was found mainly in prediseased lupus mice , this epitope seems to be important in the early phase of antichromatin autoimmune responses leading to subsequent epitope spreading to unmodified histone h2b . plasmacytoid dcs can internalize immune complexes formed by anti - chromatin antibodies and present autoantigen to b cells . this presentation , under proinflammatory conditions , could induce b cell differentiation to plasma cells or induce the secretion of more autoreactive abs which could form more immune complexes and ultimately induce a feed - forward proinflammatory loop that might contribute to antigen spreading in sle . understanding antigen spreading in autoimmunity will be crucial for the design of effective dc - interfering therapeutics ; therefore , additional research will be needed to further define the role of dcs in autoimmunity - mediated antigen spreading . a variety of molecular and cellular strategies for suppression of tissue - specific autoimmunity are currently under development ( figure 2 ) . these strategies include agonist or antagonist mediated interactions , pharmaceuticals , cytokine targeted antibody therapies , and immunosuppressive vaccines . mechanisms by which dcs may be able to mediate the suppression of tissue - specific autoimmunity are discussed below . how dendritic cells become activated and the nature of the activation state remains a question for further investigation . however , it has been shown that toll - like receptor 2 ( tlr2 ) can recognize molecular motifs in atypical lps , peptidoglycan , lipoteichoic acid , lipoproteins , and lipopeptides . more recently , it was shown that chronic administration of the tlr2 agonist pam3csk4 could prevent diabetes onset in nod mice by inducing dc - mediated tolerance . further corroborating these experimental findings , additional research has shown that tlr2 signaling can modulate immune regulation and alter the progression of autoimmunity in the nod mouse . these experimental results suggest a role for tlr2 in enhancement of cd4cd25 treg proliferation both in a nave t cell context and during viral infection to provide increased protection against development of autoimmune diabetes . treatment of prediabetic mice with a synthetic tlr2 agonist diminished the onset of t1d and increased the number and function of cd4cd25 tregs , thereby conferring tolerogenic properties to dcs . the ligation of dendritic cell tlr2 was also shown to increase their capacity for autoimmune disease prevention and to promote the proliferation of tregs . cyclosporine and tacrolimus , complex nonantibiotic macrolide compounds isolated from soil fungi and bacteria , are widely used as immunosuppressive agents following solid organ transplantation . these immunosuppressant drugs were shown to dampen the inflammatory activity of the immune system by interfering with the activity and growth of t cells . cyclosporine and tacrolimus cause immune suppression by binding cytoplasmic cyclophilin and fk - binding proteins , respectively . generation of these protein complexes induces binding to calcineurin , therefore blocking calcineurin 's activation of the t cell transcription factor nfatc . consequently , t - cell proliferation is suppressed as evidenced by the ability of cyclosporine treated myeloid dcs to suppress the proliferation of allogenic peripheral blood mononuclear cells ( pbmcs ) . similarly , stimulation of memory cd8 t cells by dcs was impaired by cyclosporine pretreatment . from this study , it was concluded that cyclosporine differentially alters the function and phenotype of mdcs leading to a partially impaired capacity to stimulate the activation of allogenic and autologous t cells . in another study , it was found that cyclosporine a ( csa ) impaired the migration of mouse bone marrow - derived dcs toward macrophage inflammatory protein-3beta ( mip-3beta ) and induced them to retain responsiveness to mip-1 after lipopolysaccharide ( lps ) stimulated dc maturation in vitro . administration of csa in vivo was shown to inhibit the migration of dcs out of skin and into the secondary lymphoid organs . further , it was also shown that cyclosporine suppresses -cell autoimmunity and rescues islet -cell function . however , this study indicates that the therapeutic effect of csa is sustained only with continuous cyclosporine administration , which unfortunately is associated with significant adverse effects . cyclosporine was shown to downregulate dc synthesized inflammatory cytokines il-2 and il-12 , suggesting this class of inhibitory molecules may have an important role in the regulation of dc - mediated inflammatory immune responses . sirolimus is a complex organic molecule isolated from bacteria that exerts a suppressive effect on the immune system but acts differently from the calcineurin inhibitors cyclosporine and tacrolimus . sirolimus was shown to inhibit responses to the inflammatory cytokine il-2 , thereby blocking t and b cell activation . in contrast to cyclosporine and tacrolimus , sirolimus inhibits il-2 secretion by binding to the cytosolic protein fk - binding protein 12 ( fkbp12 ) . the sirolimus - fkbp12 complex blocks the mammalian mtor pathway through direct binding to the mtor complex1 ( mtorc1 ) . resistance to maturation and tolerogenic properties of dcs were shown to be supported and preserved by conditioning with sirolimus . the ability of sirolimus to suppress dc activation suggests that sirolimus / rapamycin - based therapeutic strategies may be effective for the inhibition of tissue - specific autoimmunity . the notch signaling pathway is a highly conserved cell signaling system present in most multicellular organisms . the notch family of proteins is transmembrane proteins with extracellular epidermal - like growth factor ( egf ) domains . ligand proteins binding to the egf domains induce proteolytic cleavage and release of the intracellular domain , which enters the cell nucleus to modify gene expression . interaction between notch receptors and their ligands represents an evolutionarily conserved signaling pathway important for cell fate commitment in hematopoiesis and thymus development [ 120122 ] . a new addition to the delta family of notch ligands , named delta - like ligand 4 ( dll4 ) , dii4 is characterized by an extracellular region containing several egf - like domains and a delta / serrate / lag-2 ( dsl ) domain required for receptor binding . studies have shown that dll4 is an essential and nonredundant notch1 receptor ligand and its specific inactivation on thymic epithelial cells ( tecs ) leads to a block in t cell development accompanied by ectopic appearance of an alternative b cell lineage within the thymus [ 120 , 124 ] . in a recent finding , an anti - delta - like ligand 4 ( dll4)-notch signaling treatment was shown to fully prevent t1d in nod mice via a treg cell - mediated mechanism . further treatment with anti - dll4 was shown to convert cd4cd8c - kitcd44cd25 ( dn1 ) t cell progenitors to immature dcs that induce ex vivo differentiation of naive cd4 t cells into treg cells . these results identify dll4-notch as a novel pathway that may be important for regulating dc - mediated treg cell homeostasis and autoimmunity . glucocorticoids are a class of steroid hormones synthesized in the adrenal cortex that act in the regulation of glucose levels in the blood but have feedback properties that inhibit inflammation through modulation of gene transcription . prednisone , combined with azathioprine , was shown to improve cell function in new - onset t1d patients [ 126128 ] . as glucocorticoids have been shown to downregulate dendritic cell function both in vitro and in vivo , they may continue to occupy a significant role in the suppression of tissue - specific autoimmunity . targeted antibody immunotherapies hold great promise for the treatment and cure of tissue - specific autoimmune diseases . antibodies that bind dc costimulatory factors cd83 , cd86 , and cd80 were shown to arrest dc maturation by blocking dc costimulatory factor interaction with cd28 receptors on autoreactive t cells , thereby reducing or inhibiting dc stimulation of effector t cell development [ 131 , 132 ] . psoriasis , a form of skin autoimmunity , is characterized by dc induction of autoreactive th1 and th17 effector cell differentiation . administration of antibodies specific for the p40 subunit of il-12 and il-23 ( anti - il-12p40 ) reduced mrna expression of proinflammatory cytokines and chemokines in psoriatic skin lesions following a single administration of anti - il-12p40 . these studies demonstrate the efficacy of anti - il-12 antibody immunotherapy for suppression of chronic inflammatory skin disorders . further studies have shown that anti - il-17 antibodies are effective in suppression of experimental uveoretinitis and rheumatoid arthritis [ 134 , 135 ] . in t1d , anti - il-17 antibodies were shown to inhibit diabetes during the effector phase of disease progression in nod mice ( at 10 weeks of age ) , but not during the initiation of disease ( in mice less than 5 weeks of age ) . this data suggests that dc stimulation of il-17 secreting th17 cells does not occur until t1d disease progression . anti - cd3-specific antibodies demonstrate a unique capacity to restore self - tolerance in established autoimmunity . they induce long - term remission of overt diabetes both in nonobese diabetic ( nod ) mice and in human t1d . the potency of anti - cd3-specific monoclonal antibody therapy in mice and humans results from its ability to reestablish immune homeostasis in treated individuals , likely through a concerted dendritic cell and regulatory t - cell - mediated mechanism . anti - cd3 binds the t - cell receptor- ( tcr- ) cd3 complex ( also termed antigenic modulation ) and induces apoptosis of activated autoreactive t cells . this t cell clearance allows for homeostatic remission , survival , and expansion of treg cell populations which effectively control pathogenic effectors including dcs . there is compelling evidence that regulatory t cells exert their control over pathogenic t cells through suppression of dc activation rather than from direct t cell - t cell interactions . the immunoregulatory cytokine tgf- , which in this model is not only produced by regulatory t cells but also potentially by dcs and other stromal cell types , is an ideal candidate cytokine for the maintenance of a broad anti - inflammatory environment through its action on effector t cells , regulatory t cells , and dcs . suppression of tissue - specific autoimmunity may be accomplished based on an innovative therapeutic strategy in which susceptible subjects are treated with their own mdcs . monocytes isolated from the patient can be differentiated in vitro to obtain large numbers of modcs that can be transfected with genes encoding immunosuppressive cytokines such as il-10 , tgf- , or il-4 . in addition to suppression of inflammatory cytokine synthesis , the activated dcs would provide an element of safety because they have a limited lifetime of approximately 5 days and therefore would have only a transient effect on the immune response . additional studies have demonstrated prevention and reversal of type 1 diabetes in nod mice using costimulation impaired , immunosuppressive bone marrow - derived dcs generated ex vivo with a mixture of antisense oligonucleotides targeting the primary transcripts of dc costimulatory factors cd40 , cd80 , and cd86 . parenteral vaccination is generally considered to be the most effective form of protection against infectious diseases . more recently , however , vaccination at mucosal surfaces and combinatorial vaccination strategies that link immunostimulatory molecules ( adjuvants ) to antigens have been developed to further enhance vaccine efficacy . prominent among immunological enhancement strategies are the group of bacterial and plant ab toxins , which include shiga toxin , anthrax toxin , ricin toxin , the heat sensitive enterotoxin from e. coli , and the cholera toxin cta and ctb subunits . in contrast to the toxic cta subunit , the nontoxic ctb subunit displays both carrier and mild immunostimulatory properties . when linked to pathogen antigens , ctb can impart immunostimulatory properties that convey increased levels of immune system stimulation in response to the linked antigen . these vaccination strategies have been broadened further to include ctb linkage to self proteins , which paradoxically often result in enhanced immunological suppression of autoimmunity . linkage of ctb to an autoantigen was shown to provide up to a 10,000-fold reduction in the amount of autoantigen required for generating immune - tolerance [ 142 , 144 , 145 ] . in t1d , for example , self - proteins become more strongly immunosuppressive when linked to ctb . in addition to its known capacity to induce a proinflammatory response , oral administration of ctb subunit coupled with insulin or gad35 autoantigen was shown to induce immunological tolerance in nod mice [ 142 , 144 , 146 ] . morphological changes in dcs incubated with ctb coupled to an autoantigen included cell enlargement , elongation of dendrites , and increased migration of dcs to draining lymph nodes , as well as increased expression of dendritic cell b7 - 2/cd86 costimulatory molecules [ 142 , 147 ] . in a recent finding in our laboratory , incubation of human immature modcs with ctb - ins autoantigen fusion protein showed an increase in surface expression of tlr2 with no significant upregulation in tlr4 expression . in contrast , inoculation of immature dendritic cells ( idcs ) with ctb stimulated the biosynthesis of both cd86 and cd83 costimulatory factors demonstrating an immunostimulatory role for ctb in both dc activation and maturation . in comparison , incubation of idcs with proinsulin partially suppressed dc activation , while incubation of idcs with ctb - ins fusion protein suppressed idc biosynthesis of both cd86 and cd83 costimulatory factors . inoculation of idcs with ctb - ins fusion protein was shown to dramatically increase secretion of the immunosuppressive cytokine il-10 while suppressing synthesis of the proinflammatory cytokine il12/23p40 subunit . this result suggests that linkage of ctb to proinsulin ( ins ) could play an important role in mediating dc guidance of th0 cell development into treg cells . taken together , the experimental data suggests that tlr2 may play a dominant role in ctb - ins - mediated prevention of human t1d onset . further , fusion of ctb to proinsulin was found to be essential for enhancement of immune suppression as codelivery of ctb and insulin did not significantly inhibit dendritic cell cd86 biosynthesis . thus , the experimental data supports the hypothesis that ctb - autoantigen - mediated suppression of islet cell inflammation and hyperglycemia development is dependent on ctb stimulation of dendritic cell tlr2 receptor activation and coprocessing of both ctb and the autoantigen in the same dc . our laboratory also found that linkage of ctb to a 5 kda c - terminal protein fragment of the major diabetes autoantigen gad35 can block dc functions including biosynthesis of costimulatory factor proteins cd86 , cd83 , cd80 , and cd40 and secretion of the inflammatory cytokine il-12 . inoculation of idcs with ctb - gad35 protein dramatically suppressed levels of cd86 , cd83 , cd80 , and cd40 costimulatory factor protein biosynthesis in comparison with idcs inoculated with gad35 alone . surprisingly , incubation of idcs in the presence of the ctb - autoantigen and the strong immunostimulatory molecules pma and ionomycin revealed that ctb - gad35 was capable of arresting pma + ionomycin induced dc maturation and activation . consistent with this finding , ctb - gad35-mediated suppression of dc maturation was accompanied by a dramatic decrease in the secretion of the proinflammatory cytokines il-12/23p40 and il-6 and a significant increase in secretion of the anti - inflammatory cytokine il-10 . taken together , the experimental data suggests that linkage of the weak adjuvant ctb to the dominant type 1 diabetes autoantigens ins and gad inhibits dc maturation through downregulation of major dc costimulatory factors and inflammatory cytokine biosynthesis . these experimental results also emphasize the possibility that ctb - autoantigen fusion proteins enhance dc priming of th0 cell differentiation into treg cells . the above described immunological phenomena establish a basis for improvement of adjuvant augmented multicomponent subunit vaccine strategies capable of complete suppression of organ - specific autoimmune diseases in vivo . the first enzyme in the tryptophan degradation pathway , indoleamine 2,3 dioxygenase , ( ido ) may be an important contributor to dc - mediated suppression of autoimmunity . ido is the rate - limiting catabolic enzyme encoded by the ido1 gene responsible for the degradation of l - tryptophan ( l - trp ) to n - formyl kynurenine and its further degradation products . ido was shown to inhibit dc maturation through tryptophan starvation via a generalized reduction in cellular energetics and through the generation of secreted kynurenines shown to be effective in stimulating t cell apoptosis and treg proliferation [ 125128 ] . further , it was recently found that the tryptophan metabolite 3-hydroxyanthranilic acid ( 3-haa ) directly inhibits dc activation and is responsible for suppression of inflammatory th1 cell functions . treatment with 3-haa was shown to significantly reduce production of the proinflammatory cytokines il-12 , il-6 , and tnf- in bone marrow - derived dendritic cells stimulated with lps . the role of ido in dendritic cell function may differ among dc subsets because the ability of dcs to produce ido does not seem to be equally distributed among the various dc subsets . the cd8 positive dcs in mice were shown to express higher amounts of ido in comparison with cd8-negative dcs . in response to ifn , cd8-positive dcs were shown to rapidly express ido and establish immunological tolerance [ 152154 ] . the immunosuppressive activity of ido was first speculated to be solely a function of the physical depletion of tryptophan from the intracellular environment , thus starving dcs , t cells , and other effector cells of the immune system . tryptophan starvation is sensed in eukaryotic cells through activation of the general control nonrepressed 2 ( gcn2 ) kinase , which directly binds uncharged trnas [ 152 , 155 ] . tryptophan depletion was shown to result in the induction of the gcn2 pathway , the downregulation of cd3 zeta - chain in cd8 t cells , and inhibition of th17 cell differentiation [ 152 , 156 , 157 ] . an additional mechanism for ido stimulated immune suppression resides in the inhibitory effect of kynurenines on t cell and natural killer cell proliferation [ 158 , 159 ] . equally important was the observation that kynurenines stimulate the upregulation of treg cell proliferation that can further inhibit dc activation . in vitro cell culture experiments further demonstrate the immunosuppressive nature of ido showing that elevated ido activity can permit tumor cell escape from immune surveillance through depletion of l - trp in the dc and t cell microenvironment . in vivo experiments have shown that ido knockout mice experience acute rejection of transplanted mhc mismatched grafts , while wild - type mice with high tryptophan catabolism experienced long - term graft survival . further emphasizing the requirement for ido in suppression of dc activation , experimental reduction in the levels of pdcs in the pancreas of nod mice was shown to be accompanied by increased insulitis and a localized reduction in ido levels . together , this data confirms the role of ido as a strong immunosuppressive mediator in tissue - specific autoimmunity . dendritic cells were first observed to be a novel class of antigen presenting cells by ralph steinman almost a half century ago . since their discovery , however , considerable experimental data has accumulated describing their cytology and biological functions . resulting from these studies , an increasing number of dc subtypes have been identified that provide a broad diversity in antigen presentation to lymphocytes of the adaptive arm of the immune system . the unique properties of dc presentation of antigens to nave t cells and guidance of their differentiation into pro- or anti - inflammatory effector t cells have helped clarify the role of dcs as key mediators of protective immunity . assessment of dc functions in the initiation and prevention of autoimmunity will continue to reveal elements that contribute to their role in maintenance of immunological homeostasis . the development of promising dc - based therapeutic strategies will lead to more effective and safer prevention and treatment for an increasing number of autoimmune disorders . further analysis of mechanisms underlying dc activation and maturation will lead to a more complete understanding of how dcs function in the guidance of nave t cell differentiation into proinflammatory or anti - inflammatory lymphocytes that exacerbate or inhibit autoimmunity . finally , based on the present rate of accumulation of experimental data on dendritic cell cytology and functions , it is likely the number of dc subsets and our knowledge of their participation in the initiation and suppression of tissue - specific autoimmunity will continue to increase .

in this review , we explore the role of dendritic cell subsets in the development of tissue - specific autoimmune diseases . from the increasing list of dendritic cell subclasses , it is becoming clear that we are only at the beginning of understanding the role of these antigen presenting cells in mediating autoimmunity . emerging research areas for the study of dendritic cell involvement in the onset and inhibition of tissue - specific autoimmunity are presented . further , we compare tissue specific to systemic autoimmunity to demonstrate how development of dendritic cell - based therapies may be broadly applicable to both classes of autoimmunity . continued development of these research areas will lead us closer to clinical assessment of novel immunosuppressive therapy for the reversal and prevention of tissue - specific autoimmunity . through description of dendritic cell functions in the modulation of tissue - specific autoimmunity , we hope to stimulate a greater appreciation and understanding of the role dendritic cells play in the development and treatment of autoimmunity .

mathematical modelling continues to play a significant role in the development of our understanding of the growth and function of hyphal networks . one of the main problems that faces modellers is the choice of scale . in the study of fungal mycelia , scale is expressed in an extreme manner : the indeterminate growth habit of fungi ensures that models may have to incorporate processes operating over scales ranging from the ( sub ) micron to the kilometre . further complexity is added when modelling physical and nutritional heterogeneity of the host environment and the resultant multifarious sensing and response events that media hyphal growth and thus determine network architecture . most progress to date has been made by selecting a scale and developing appropriate models to address questions manifest at that chosen scale . however , as is now becoming apparent in other areas of biological research , a meaningful , systems - based understanding requires the transfer information across scales . this session show - cased state - of - the - art mathematical and computational approaches to both scale - specific and most importantly , cross - scale problems in modelling fungal physiology and function . the session was opened by the meeting organizer fordyce davidson , who gave an overview of the main challenges faced in attempting to link the increasing quantity and quality of genetic and sub - cellular experimental data to large - scale morphology and function . he detailed some of his group s ongoing work , which ranges from the bio - mechanics of hyphal growth and thigmotropism ( contact sensing ) to qualitative properties of colony growth . he finished by proposing that a multi - scale , quantitative ( mathematical modelling ) approach is ideal for and perhaps necessary to the efficient translation of molecular genetics to a deeper understanding of the organism as a whole and its role in the host environment ( davidson 2007 , 2010 , rosling et al . he presented a discrete - continuous hybrid approach to modelling a fungal mycelium developing in a planar environment . the model comprises a series of geometrically - unconstrained connected line segments used to represent individual hyphal , so that a mycelium is represented by a collection such series ( cohen 1967 , mekauskas et al . the resultant discrete network develops over a continuous and diffusing distribution of an external substrate , representing a combination of nutrients and minerals , and which is internalized by the network according to michael - menten dynamics . once internalized , substrate is translocated through the network , undergoing both multi - directional diffusion and directed advection toward the unconnected ends of line segments representing hyphal tips ( boswell et al . new line segments are created through processes analogous to subapical branching and hyphal tip extension . the orientation of new line segments are governed by local concentrations of a self - secreted inhibitor ( but additional tropisms , for example in response to toxic metals , are easily included ) and it is mathematically represented by a biased circular random walk and the related focker - planck equation ( plank & sleeman 2004 ) . the model was simulated in representations of homogeneous and heterogeneous nutrient configurations and it was shown that the simulated networks display numerous features that are in qualitative and quantitative agreement with experimental data ( fig . moreover , the model predicts that mycelial cords can develop through an initially stochastic but then self - reinforcing process ( cf . daniel hofstadler discussed a model for the development of hyphal networks where the key focus was on tip sensing of the environment . the model adopted the same core approach of that discussed by graeme boswell , but the main emphasis was on modelling tip movement in more detailed , with network formation based closely on the rule - based techniques of , for example , mekauskas et al . ( 2004 ) . a key novel feature of the model was tip - to - tip anastomosis that could be achieved by setting an appropriate tropism mediated by the internal supply of nutrients and spatio - temporal gradients of external autoregulators . a complementary study of network development was then presented by luke heaton , who discussed growth induced mass flows . he explained that the translocation of resources within fungal networks is ecologically critical , but it is much less well studied than transport in the other major multi - cellular kingdoms of life ( jennings 1987 ) . in particular , he noted that the relative roles of pressure driven mass - flows , diffusion and active transport remain poorly understood . the uptake of water and the maintenance of turgor pressure require an osmotic gradient between the hyphae and their environment , but because aqueous fluids are incompressible , mass flows can only take place when water is able to exit the translocation pathway through either localised exudation ( e.g. serpula lacrymans ) , evaporation , or by moving into a region of new growth ( cairney 1992 , lew 2005 ) . growth - induced mass flow can be used to describe the last of these phenomena ( heaton et al . the key idea here is that as the hyphal tips expand , the incompressibility of aqueous fluids ensures that in the supporting mycelium , there must be a net flow of fluid from the sites of water uptake to the sites of growth . to quantify the scale of growth - induced mass flows and gain insight into the developmental logic of fungal networks , a mathematical model based on circuit theory has been developed , and applied to three empirical networks of phanerochaete velutina . given a time series of networks where each edge has a measured volume , these currents reflect the minimal flow of material that is consistent with the measured changes in volume , under the assumptions that : ( 1 ) the inoculum is the sole source of water and nutrients ; ( 2 ) the conductance of cords is proportional to their cross sectional area ; and ( 3 ) the fluid follows the path of least resistance . cords that are predicted to carry larger currents were significantly more likely to increase in thickness than the other cords ( fig . over three replica experiments the spearman s rank correlation coefficient between the volumes predicted to have passed through the cords and the cord s cross - sectional areas was 0.51 . in contrast , the correlation coefficient between the age of cords and their cross - sectional areas was only 0.21 ( heaton et al . 2010 ) . the focus then shifted to lower spatial scales , but discussion of the processes of internal flow and network development continued . marcus roper discussed how fungal thalli grow by extension at hyphal tips , and by the flow of cytoplasm and nuclei into the spaces created at the extending tips ( rasmos - garcia et al . , he explained that hyphae grow too fast to be populated by nuclei produced by division at or near the tips . instead , nuclei are produced by mitosis distributed throughout the entire thallus , and migrate to the growing periphery of the thallus at speeds that may reach 10 m s or more ( roper et al . , unpubl . ) . by making heterokarya in which nuclei are histone h1 differentially fluorescently tagged , his group has been able to map the dynamics of these nuclear flows during thallus growth . they found complex multidirectional flows several centimeters behind the growing front of the colony , in fact much further from the tips than a simple transport of nuclei to fill spaces created by growth would require . it has long been known that nuclei within a single thallus may be genetically diverse ( caten & jinks 1966 , maheshwari 2006 ) . the complex and multidirectional paths taken by nuclei suggest that flows may facilitate a mixing of nuclei during their transport to the colony periphery , maximizing genetic diversity at the growing tips , and thus preventing e.g. sectoring . marcus presented simulations that compared nuclear flows in real thalli with idealized hyphal networks and discussed how this allowed his group to dissect how the network architecture might be designed to maximize mixing and accelerate the dispersal of new nucleotypes that arise e.g. by mutation . his talk detailed how after high - speed launches , air viscosity causes the rapid deceleration of fungal spores and limits their discharge distance . the fluid dynamics contained within stokes law posits a simple relationship between drag coefficient and velocity ( reynolds number , re ) . it provides an excellent fit to experimental measurements of the terminal velocity of free - falling spores and other instances of low reynolds number motion ( re<1 ) . for movements characterized by higher re , more complex models have been devised to determine drag but none have been evaluated for modelling the launch of fast moving fungal spores . in fischer et al . ( 2010 ) the first empirical test of stokes law and a commonly used complex drag model ( clift et al . 2005 , white 1974 , vogel 2005 ) was presented with launch speed data obtained from ultra - high - speed video recordings and experimental measurements of discharge distances ( pringle et al . 2005 , yafetto et al . they found that discharge distances predicted from launch speeds by the simpler stokes model provide a much better match to measured distances than the more complex drag model . stokes model works better over a wide range spore sizes , launch speeds , and discharge distances , from microscopic mushroom ballistospores discharged at < 1 ms over a distance of < 0.1 mm ( re=0.3 ) , to macroscopic sporangia of pilobolus that are launched at > 10 ms and travel as far as 2.5 m ( re=167 ) . even at reynolds numbers greater than 2000 as observed for sphaerobolus launches , stokes law models the relationship between launch speed and discharge distance much better than the more complex drag model ( fig .

this contribution is based on the six presentations given at the special interest group meeting on mathematical modelling of fungal growth and function held during imc9 . the topics covered aspects of fungal growth ranging across several orders of magnitude of spatial and temporal scales from the bio - mechanics of spore ejection , vesicle trafficking and hyphal tip growth to the form and function of mycelial networks . each contribution demonstrated an interdisciplinary approach to questions at specific scales . collectively , they represented a significant advance in the multi - scale understanding of fungal biology .

insulin - like growth factor 1 receptor ( igf1r ) is a transmembrane tyrosine kinase that plays an important role in proliferation , apoptosis , angiogenesis , and tumor invasion . mounting evidence has confirmed that igf1r is significantly upregulated in many types of cancer , and the upregulation usually confers resistance to different therapeutic interventions . these findings suggest that targeting and neutralizing igf1r can be an effective approach for cancer therapy . different therapeutic agents such as antibodies and tyrosine kinase inhibitors have been developed for this purpose and served as effective approaches in clinical trials . inhibition of igf1r is considered a favorable treatment for androgen - independent prostate cancer due to the fact that overexpression of igf1r leads to activation of androgen receptors in the absence of androgens . identification of igf1r expression level and relevant patient stratification are the prerequisites of a successful igf1r - targeted therapy . clinical data also demonstrated that treatment efficacy of igf1r antibody correlates with membrane expression of igf1r . noninvasive imaging of igf1r will provide invaluable information in three aspects : patient stratification where patients with high igf1r expression can be selected for igf1r targeted clinical trials ; treatment monitoring where noninvasive imaging of igf1r expression can indicate the therapeutic response ; and facilitating the drug development process through monitoring the therapeutic efficacy of various drugs that target the igf1r signaling pathway . at the same time , the expression level of igf1r in tumor is comparatively low ( 10 to 3 10 receptors per cell ) , and physiological igf1r expression exists in a number of tissues , such as colon , lung , pancreas , salivary gland , and stomach , although the expression level in those tissues is significantly lower than in tumor . the combination of low tumor expression and ubiquitous expression in normal tissues posed a challenge to acquiring accurate igf1r profiles . this dilemma was partially resolved with the utilization of radionuclide - based imaging techniques , including single - photon emission computed tomography ( spect ) and positron emission tomography ( pet ) , because these modalities can achieve superior sensitivity ( down to picomolar level ) and are highly quantitative . spect imaging studies of igf1r in prostate cancer were conducted using an affibody molecule ; however , there is no pet imaging of igf1r reported to date for prostate cancer . monoclonal antibodies ( mabs ) have long been considered promising candidates for targeted therapy and diagnostics due to their highly specific targeting ability : they offer unmatched utility by virtue of their ability to recognize essentially any target of interest . the goal of this study was to develop a mab ( named 1a2g11 ) for igf1r and investigate the in vitro and in vivo characteristics of cu - labeled 1a2g11 for pet imaging of igf1r in different prostate cancer models . we hypothesized that the uptake of cu - labeled 1a2g11 in these prostate tumors are dependent on their igf1r expression level . the production of igf1r mabs was carried out by neoclone biotechnologies international , llc ( madison , wi ) . briefly , the production procedure involved four major steps : ( 1 ) screening of peptidic immunogens for igf1r , ( 2 ) immunization in balb / c female mice with igf1r immunogens , ( 3 ) identification of igf1r - positive cell candidates through serial screening of different monoclonal colonies from hybridomas , and ( 4 ) bulk production of the most promising candidate . approximately 10 peptidic immuogens were screened using bioinformatical calculation based on the structure of human igf1r . balb / c female mice were inoculated with recombinant mouse protein with sequences of igf1r immunogens . enzyme - linked immunosorbent assay ( elisa ) was initially prepared with immobilized igf1r . ten to 20 clones of igf1r - positive cells with the highest read - outs in elisa were used to carry out flow cytometry assay . then , three clones with the highest cellular affinity for igf1r were radiolabeled with cu and applied for in vivo biodistribution assay . the antibody clone with the highest uptake in igf1r - positive tumors ( 1a2g11 ) was produced in a milligram scale . the binding affinity of different antibody clones for igf1r was evaluated with fluorescence - activated cell sorting ( facs ) analysis in mcf-7 human breast cancer cells ( igf1r - positive ) . cells were harvested and suspended in cold phosphate buffered saline ( pbs ) with 2% bovine serum albumin at a concentration of 5 10 cells / ml . the cells were incubated with supernatants from different cell clones ( total protein concentration : 50 g / ml based on bradford protein assay , which contains 0.5 g / ml of antibodies ) for 30 min at room temperature and washed three times with cold pbs at the end of the incubation period . they were then incubated with fitc - labeled rabbit antimouse igg ( 1 g / ml ) for 30 min at room temperature . subsequently , the cells were washed and analyzed using a bd facscalibur 4-color analysis cytometer , which is equipped with 488 and 633 nm lasers ( becton - dickinson , san jose , ca ) . mean fluorescence intensities of different antibody clones in the cells were computed using flowjo analysis software ( tree star , inc . , all animal studies were conducted under a protocol ( m02239 ) approved by university of wisconsin institutional animal care and use committee . tumors were established by subcutaneous injection of 5 10 du-145 ( igf1r - positive ) or lncap cells ( igf1r - negative ) suspended in 100 l of 1:1 mixture of pbs and matrigel ( bd biosciences , franklin lakes , nj ) into the right front flank of male nude mice purchased from harlan ( indianapolis , in ) . the tumor sizes were monitored every other day , and the mice were subjected to in vivo experiments when the diameter of tumors reached 68 mm ( typically 46 weeks after inoculation ) . pet scans were performed using an inveon micropet / microct rodent model scanner ( siemens medical solutions usa , inc . ) . each tumor - bearing mouse was injected with 510 mbq of the pet tracer via its tail vein and subjected to 515 min of static pet scans at various time points postinjection ( p.i . ) . the images were reconstructed using a maximum a posteriori ( map ) algorithm , with no attenuation or scatter correction . for each micropet scan , three - dimensional ( 3d ) regions - of - interest ( rois ) were superimposed on the tumor and major organs in the decay - corrected whole - body images by using a vendor software ( inveon research workshop [ irw ] ) . assuming a tissue density of 1 g / ml , the radioactivity in each roi volume was converted to mbq / g using a conversion factor , and then divided by the total administered radioactivity to obtain a percentage of injected dose per gram of tissue ( % id / g ) for each roi . to anatomically localize the radioactivity signal observed in pet a sample set of mice were transported to the microct gantry , positioned , and scanned at a voxel resolution of 210 m ( scanning time : 7 min ) . ct images were reconstructed using the vendor software ( inveon acquisition workshop ; siemens ) using fiducial markers for coregistration . the microct and micropet data sets were loaded into irw where the corresponding fiducial markers were coregistered to achieve roi location alignment . biodistribution studies were carried out to confirm that the quantitative tracer uptake values based on pet imaging truly represented the radioactivity distribution in tumor - bearing mice . after the last pet scans at 48 h p.i . , the mice were euthanized , and their blood , du-145/lncap tumors , and major organs / tissues were collected and wet - weighed . the radioactivity in each collected sample was measured using a wizard automatic gamma - counter ( perkinelmer ) and recorded as % id / g ( mean sd ) . the du-145/lncap tumors , liver , and spleen ( i.e. , tissues with significant uptake of cu - labeled antibodies ) were also frozen for histological analysis . frozen tissue slices of 5 m thickness were fixed with cold acetone for 10 min and air - dried at room temperature for 30 min . after rinsing with pbs and blocking with 10% donkey serum for 30 min at room temperature , the slices were incubated with antibody 1a2g11 ( 5 g / ml ) for 1 h at 4 c and visualized using fitc - labeled rabbit antimouse secondary antibody . the tissue slices were also stained for endothelial marker cd31 . after washing with pbs , the tissue slices were incubated with rat antimouse cd31 antibody ( 2 g / ml ) for 1 h , followed by cy3-labeled donkey antirat igg for 30 min . all images were taken with a nikon eclipse ti microscope . multiple rounds of screening were carried out on different monoclonal colonies to select antibodies with the highest affinity for igf1r on mcf-7 ( figure s1 , supporting information ) . candidate ranking was based on cellular fluorescence intensity levels from facs measurement in descending order ; the final candidates were 1a2g11 , 1g9c10 , and 3e5c9 ( figure 1a ) . the immunoglobin g ( igg ) contents from these three clones were purified and enriched to a final concentration of 0.10.5 mg / ml for nota conjugation and cu - labeling . cu - labeled 1a2g11 , 1g9c10 , and 3e5c9 were injected into du-145 tumor bearing mice ( n = 2 ) to evaluate their distribution pattern and tumor - targeting efficacy in vivo by gamma - counting measurement ( figure s2 , supporting information ) . a comparison of tumor - to - liver uptake ratio for the three candidates ( figure 1b ) indicated that 1a2g11 possesses the highest tumor - targeting capability ; therefore , it was selected for the subsequent studies . further enrichment and purification steps were undertaken to acquire 1a2g11 antibody at the final concentration of 1 mg / ml . ( a ) fluorescence enhancement of igf1r - positive mcf-7 cells measured by facs analysis after treatment with supernatants of different monoclonal colonies . ( b ) tumor - to - liver ratios of cu - labeled 1a2g11 , 1g9c10 , and 3e5c9 in du-145 tumor bearing mice ( n = 2 ) . cell binding assays confirmed that 50% inhibitory concentration ( ic50 ) of 1a2g11 for mcf-7 cells ( which expressed high level of igf1r ) was around 5 nm , confirming its high affinity for igf1r ( figure s4 , supporting information ) . data from facs analysis in mcf-7 suggested no observable difference in cellular binding affinity between 1a2g11 and nota-1a2g11 at the concentration of 5 g / ml . the binding to mcf-7 was antigen specific , as neither 1a2g11 nor nota-1a2g11 bound to igf1r - negative lncap cells , even at a much higher concentration of 20 g / ml ( figure 2 ) . in aggregation , facs analysis data indicated that nota conjugation did not alter the antigen binding affinity or specificity of 1a2g11 . flow cytometry analysis of 1a2g11 and nota-1a2g11 in mcf-7 ( igf1r - positive ) and lncap ( igf1r - negative ) cells at different concentrations . the cu - labeling procedure , including final purification using pd-10 columns , was completed within 100 10 min ( n = 7 ) . the estimated cu - nota-1a2g11 specific activity was 0.75 gbq / mg protein ( assuming full protein recovery after size - exclusion chromatography ) , a result based on the calculation of 25 g of nota-1a2g11 per 37 mbq of cu and an decay - corrected radiochemical yield of 51 15% . drawing on our prior experiences in pet imaging with cu - labeled antibodies , sample time points of 4 , 24 , and 48 h p.i . reconstructed coronal slices that contained the du-145 or lncap tumors are shown in figure 3a ; a representative pet / ct fused image of a du-145 tumor - bearing mouse at 24 h p.i . of cu - nota-1a2g11 quantitative data obtained from roi analysis at the 3 time points are graphed in figure 3c . blood pool activity was prominent at early time points and gradually declined thereafter ( 10.3 1.9 , 7.4 0.9 , and 4.1 0.6 % id / g at 4 , 24 , and 48 h p.i . , respectively , n = 3 ) . the liver uptake of cu - nota-1a2g11 in du-145 tumor - bearing mice displayed the same trend ( 20.2 2.1 , 15.2 0.8 , and 12.8 0.6 % id / , respectively , n = 3 ) . most significantly , the uptake of cu - nota-1a2g11 in du-145 tumor was clearly visible as early as 4 h p.i . and the level plateaued at around 24 h p.i . ( 2.8 0.7 , 10.2 2.6 , and 9.6 1.7 % id / g at 4 , 24 , and 48 h p.i . , respectively ; n = 3 ; figure 3a , c ) . serial pet imaging and roi quantifications of igf1r in du-145 and lncap tumor - bearing mice . ( a ) serial coronal pet images of du-145 and lncap tumor - bearing mice at 4 , 24 , and 48 h postinjection of cu - nota-1a2g11 . ( b ) representative pet / ct image of cu - nota-1a2g11 in du-145 tumor - bearing mice at 24 h p.i . ( c ) time activity curves of the tumor , liver , blood , and muscle upon intravenous injection of cu - nota-1a2g11 into du-145 or lncap tumor - bearing mice ( n = 3 ) . to further investigate igf1r specificity of cu - nota-1a2g11 , the lncap tumor , which does not express detectable level of igf1r , was selected as a control model . the uptake of cu - nota-1a2g11 in lncap was very low ( 1.7 0.8 , 2.8 0.7 , and 2.2 0.6 % id / g at 4 , 24 , and 48 h p.i . the liver uptake ( 18.2 1.8 , 16.7 1.4 , and 10.2 0.9 % id / g at 4 , 24 , and 48 h p.i . , respectively ) and the blood radioactivity ( 10.6 1.3 , 6.8 1.5 , and 3.5 0.7 % id / g at 4 , 24 , and 48 h p.i . respectively ; n = 4 ; figure 3a ) were similar to those in du-145 tumor model . an additional group of two mice were each injected with du-145 cells on the right flank and lncap cells on the left flank and subjected to serial cu - nota-1a2g11 pet scans . while significant uptake of cu - nota-1a2g11 in the du-145 tumors could be observed at 24 h p.i . , tracer uptake in the lncap tumors was at a significantly lower level ( figure s3 , supporting information ) . noninvasive detection of igf1r - positive tumors but not igf1r - negative tumors in the same mice further validated that cu - nota-1a2g11 can specifically target igf1r in vivo . all mice were euthanized after the terminal pet scans at 48 h p.i . for biodistribution and histology studies to validate the in vivo pet data . tumors , blood , liver , and spleen of the test subjects also had significant radioactivity accumulation at 48 h p.i . , an expected condition since a radiolabeled antibody typically has long circulation half - life and hepatic clearance ( figure 4 ) . appreciable tracer uptake ( 5%id / g ) in the kidney was also detected , an observation likely attributed to a small fraction of cu detaching from nota-1a2g11 and possible tracer degradation over time ( lower molecular weight species can undergo renal clearance ) . du-145/lncap tumor - bearing mice showed similar cu - nota-1a2g11 biodistribution profile in all the major organs / tissues except the tumor . uptake of cu - nota-1a2g11 in the du-145 tumor was significantly higher than in the lncap control model . comparisons of cu - nota-1a2g11 uptake levels in all major organs to that of the du-145 tumor have also suggested that excellent tumor contrast can be achieved in muscle tissue with a tumor / muscle ratio of 20.3 2.1 at 48 h p.i . biodistribution of cu - nota-1a2g11 in du-145 or lncap tumor - bearing mice at 48 h postinjection ( n = 3 ) . * p < 0.05 . immunofluorescence igf1r / cd31 staining revealed that igf1r expression was prominent on the du-145 tumor cells , but weak on the lncap cells ( figure 5 ) . igf1r staining of mouse liver / spleen / muscle all produced very low signal because these tissues do not express igf1r at a high level , and the binding affinity of 1a2g11 to murine igf1r is low . therefore , uptake of cu - nota-1a2g11 in mouse liver / spleen was largely unrelated to igf1r binding and more likely related to nonspecific capture by the reticuloendothelial system and hepatic clearance of the tracer . immunofluorescence igf1r / cd31 double - staining of the du-145 or lncap tumor , liver , and spleen tissue sections . 1a2g11 and fitc - labeled goat antimouse igg were used for igf1r staining ( green ) . subsequently , the tissue slices were stained with rat antimouse cd31 antibody and cy3-labeled donkey antirat igg ( red ) . all images were acquired under the same conditions and displayed on the same scale . mounting literature data have suggested a key role for igf1r in the development and metastasis of cancer . currently , several small - molecule inhibitors and antibodies directed against the igf-1r are being developed and tested in phase i and ii clinical trials . preliminary results of these studies have demonstrated the safety and tolerability of targeting igf1r . objective responses have been reported in patients treated with these new agents , but the relationship between treatment efficacy and igf1r expression is still poorly understood . in order to identify patients who may benefit from igf-1r target therapy , reliable biomarkers histology evaluation of tumor samples would be the most accurate approach to stratify patients for this purpose ; however , this method can not be used to measure the expression in different tumor lesions or at different regions within a tumor . this level of discernment would require multiple invasive sampling procedures . furthermore , the expression of igf1r may change with time as the result of tumor growth , therapy , or other circumstances . radionuclide imaging offers an alternative that allows repeated noninvasive evaluation of igf1r both for patient stratification and for monitoring expression - level changes in response to therapy . the present study developed a noninvasive imaging method that can be applicable for monitoring of the membranous igf-1r expression in prostate cancer . multiple groups have performed imaging of igf1r in cancer using agents ranging from peptides to antibodies , among which radiolabeled antibodies demonstrated highest tumor - to - background ratios . novel pet tracers for various purposes such as detection of receptor abundance or tumor environment , monitoring of therapy response , or locating apoptosis sites , are actively pursued by various research groups . over the past decade , a myriad of preclinical and clinical data has suggested that immunopet holds great potential for managing cancer in patients . the advantages of antibody - based tracers are high antigen specificity , high binding affinity , and absolute tumor uptake ; these are characteristics that make them suitable for internal radiotherapy applications and/or targeted delivery of anticancer drugs . major limitations of antibody - based imaging are slow tumor accumulation and high background signal in the reticuloendothelial system ; these drawbacks may be circumvented by employing peptide , small molecule , or antibody fragment - based tracers . although antibodies with high affinity for igf1r have already been developed ( e.g. , r1507 , imc - a12 , etc . ) , pet imaging with cu - nota-1a2g11 can monitor therapeutic responses of these antibodies in patients without the possible interference of blocking effects caused by excessive antibody in the bloodstream . in this study , we have successfully developed and characterized cu - labeled igf1r antibody ( cu - nota-1a2g11 ) for pet imaging of tumor igf1r expression in vivo . the main advantages of cu - nota-1a2g11 are its specific accumulation in the tumor and its comparatively rapid clearance from the circulation . in vitro experiments our data showed that cu - nota-1a2g11 specifically bound to igf1r - positive du-145 tumors but not to igf1r - negative lncap tumors , and the tumor uptake of cu - nota-1a2g11 was closely associated with igf1r expression level in the tumor . even on the same test subject , cu - nota-1a2g11 can delineate and differentiate tumors with different igf1r expression ( figure s3 , supporting information ) . such igf1r specificity in vivo makes cu - nota-1a2g11 a pet tracer with broad potential applications in many clinical situations . one limitation of cu - nota-1a2g11 lies in its moderate uptake in igf1r - positive tumors when compared with commercially available igf1r antibodies ( e.g. , r1507 , typically have an uptake of more than 20 % id / g in igf1r - positive tumors ) , which is partially due to its short circulation half - life . engineering of 1a2g11 ( e.g. , affinity maturation , multimerization , site mutation , etc . ) or further screening from the antibody library will bring hope to overcome this limitation in the future . to gain further insight on the long - term behavior of 1a2g11 in vivo , other long - lived isotopes ( e.g. , zr , which has a decay half - life of 3.3 days ) can be explored in future studies . since pet scanners detect the distribution of cu rather than the antibody itself , a key requirement for accurate pet imaging with cu - labeled antibodies is that the tracer should be sufficiently stable during the imaging period . nota is generally recognized as one of the best chelators for cu - labeling . a recent study that compared the effect of several bifunctional chelators on the biodistribution of a cu - labeled antibody has concluded that the thermodynamic stability of cu - chelator complexes did not significantly influence tumor uptake of the tracer , but did dramatically affect the normal tissue distribution . our studies have demonstrated the feasibility of cu - nota-1a2g11 pet for in vivo imaging of igf1r expression . however , these experiments were performed under optimal conditions because 1a2g11 does not cross react with the murine igf1r . in patients , radiolabeled 1a2g11 will also recognize igf1r that is widely expressed in normal tissues , including muscle , cartilage , and bone . future studies to optimize imaging of igf1r expression with 1a2g11 will be required in patients . herein we report the development , characterization , and in vivo investigation of cu - labeled igf1r mab ( 1a2g11 ) in mouse models of prostate tumors with different expression levels of igf1r . cu - labeled 1a2g11 exhibited rapid , prominent , and target - specific uptake in igf1r - positive tumors while demonstrating minimal interaction with igf1r - negative tumors . pet imaging can evaluate the pharmacokinetics , tumor targeting efficacy , and dose optimization of 1a2g11 , preparing it for future igf1r - targeted cancer therapeutics in the clinic .

insulin - like growth factor 1 receptor ( igf1r ) plays an important role in proliferation , apoptosis , angiogenesis , and tumor invasion . the expression level of igf1r is related to resistance to several targeted therapies . the goal of this study was to develop an immunopet tracer for imaging of igf1r in prostate cancer . murine antibodies against human igf1r were generated in balb / c mice , which were screened in igf1r - positive mcf-7 cells using flow cytometry as well as biodistribution studies with multiple 64cu - labeled antibody clones . the antibody production method we adopted could readily produce milligram quantities of anti - igf1r antibodies for in vivo studies . one antibody clone ( 1a2g11 ) with the highest affinity for igf1r was selected and conjugated to nota for 64cu - labeling . nota-1a2g11 maintained igf1r specificity / avidity based on flow cytometry . 64cu - labeling was achieved with good yield ( > 50% ) and high specific activity ( > 1 ci/mol ) . serial pet imaging revealed that uptake of 64cu - nota-1a2g11 was 2.8 0.7 , 10.2 2.6 , and 9.6 1.7 % id / g in igf1r - positive du-145 tumors at 4 , 24 , and 48 h postinjection , respectively ( n = 3 ) , significantly higher than that in igf1r - negative lncap tumors ( <3 % id / g at each time point ) except at 4 h postinjection . histology studies showed strong correlations between igf1r expression level in the prostate cancer tumor tissues and tumor uptake of 64cu - nota-1a2g11 . prominent , persistent , and igf1r - specific uptake of 64cu - nota-1a2g11 in igf1r - positive prostate tumors holds strong potential for future cancer diagnosis , prognosis , and therapy using this antibody .

the genes involved in the determination of the gonads comprise the wilms ' tumor suppressor gene wt1 , the orphan nuclear receptor sf-1 and the high mobility group family member sox9 ( parker et al . 1999 ) . after determination , the differentiation of the testis depends on the testis - determining factor sry ( goodfellow and lovell - badge 1993 ) . sry induces the sertoli cells to differentiate and to secrete the mullerian inhibiting substance , which suppresses the development of the female reproductive tract ( gustafson and donahoe 1994 ) . later , under the influence of sertoli cells , the leydig cells develop ( ge et al . the fetal leydig cells originate prenatally and produce androgens required for the masculinization during fetal and neonatal life . they regress thereafter and are substituted during puberty by the adult leydig cells , which supply the testosterone necessary for completion of spermatogenesis and maintenance of male reproductive function . adult leydig cells do not derive from preexisting fetal cells but from undifferentiated mesenchymal stem cells ( mclaren 1998 ) . the factors that regulate the commitment and early differentiation of stem cells to adult leydig cell lineage are unknown . pdgf - a , a high - affinity ligand for the receptor tyrosine kinase pdgf - r , is required for embryonic and postnatal development ( betsholtz and raines 1997 ) . pdgf - a deficient animals develop lung emphysema secondary to the failure of alveolar septation ( bostrm et al . 1996 ) , defective oligodendrogenesis ( calver et al . 1998 ; fruttiger et al . 1999 ) , and skin and hair defects ( karlsson et al . 1999 ) . previous studies have revealed that pdgf - a may play a role in testicular development ( gnessi et al . 1995 ) and that leydig cells express pdgf - r ( gnessi et al . 1992 deficient mouse generated by targeted gene disruption . from this study , we conclude that pdgf - a is an essential factor for adult leydig cells development . pdgf - a null mutant mice , heterozygous , and wild - type ( wt ) littermates were obtained from heterozygous crosses all bred as 129ola / c57 b16 hybrids ( bostrm et al . transgenic mice carrying the reporter gene -galactosidase ( lacz ) under control of a 6-kb regulatory domain of the murine pdgf - r promoter were generated as previously described ( reinertsen et al . nonradioactive in situ hybridization using pdgf - a and pdgf - r probes was performed as described ( bostrm et al . testes to be stained by periodic acid - schiff ( pas)/haematoxylin , bromodeoxyuridine ( brdu ) , tdt - mediated dutp biotin nick end labeling ( tunel ) , pdgf - a , pdgf - r , relaxin - like actor ( rlf ) , and gata-1 immunohistochemistry were fixed in 4% buffered paraformaldehyde and paraffin embedded . for routine histology , sections were stained by pas / haematoxylin according to standard protocols . for brdu labeling , brdu ( sigma - aldrich ) was injected intraperitoneally ( 100 mg / g body mass ) . injected animals were killed 2 h later and the fixed testes were processed as described ( karlsson et al . apoptotic cells were detected by tunel using the apoptag plus in situ detection kit ( oncor ) according to the instructions of the manufacturer . sertoli cells were detected using a gata-1specific antibody ( santa cruz biotechnology ) ; leydig cells were detected using a rat anti - rlf antibody ( generously provided by professor richard ivell , institute of hormone and fertility research , hamburg , germany ) ; pdgf - a and pdgf - rpositive cells were detected using rabbit anti - pdgf - a ( rdi inc . ) and rat anti - pdgf - r ( pharmingen ) antibodies . the antibodies ( anti - gata1 , anti - pdgf - a , and anti - pdgf - r at 1:100 dilution ; anti - rlf at 1:1,000 dilution ) were incubated overnight at 4c . the avidin - biotin immunoperoxidase system with 3-amino-9-ethylcarbazole as chromogen was used to visualize bound antibodies ( histostain - plus kit ; zymed laboratories ) . for lacz staining , transgenic animals were fixed and stained with x - gal for lacz activity , and 10-m cryostat testicular sections were treated as described ( reinertsen et al . serum testosterone and luteinizing hormone ( lh ) levels were determined by radioimmunoassays using diasorin and amersham assay systems according to the manufacturers ' instructions . pdgf - a null mutant mice , heterozygous , and wild - type ( wt ) littermates were obtained from heterozygous crosses all bred as 129ola / c57 b16 hybrids ( bostrm et al . transgenic mice carrying the reporter gene -galactosidase ( lacz ) under control of a 6-kb regulatory domain of the murine pdgf - r promoter were generated as previously described ( reinertsen et al . nonradioactive in situ hybridization using pdgf - a and pdgf - r probes was performed as described ( bostrm et al . testes to be stained by periodic acid - schiff ( pas)/haematoxylin , bromodeoxyuridine ( brdu ) , tdt - mediated dutp biotin nick end labeling ( tunel ) , pdgf - a , pdgf - r , relaxin - like actor ( rlf ) , and gata-1 immunohistochemistry were fixed in 4% buffered paraformaldehyde and paraffin embedded . for routine histology , sections were stained by pas / haematoxylin according to standard protocols . for brdu labeling , brdu ( sigma - aldrich ) injected animals were killed 2 h later and the fixed testes were processed as described ( karlsson et al . apoptotic cells were detected by tunel using the apoptag plus in situ detection kit ( oncor ) according to the instructions of the manufacturer . sertoli cells were detected using a gata-1specific antibody ( santa cruz biotechnology ) ; leydig cells were detected using a rat anti - rlf antibody ( generously provided by professor richard ivell , institute of hormone and fertility research , hamburg , germany ) ; pdgf - a and pdgf - rpositive cells were detected using rabbit anti - pdgf - a ( rdi inc . ) and rat anti - pdgf - r ( pharmingen ) antibodies . the antibodies ( anti - gata1 , anti - pdgf - a , and anti - pdgf - r at 1:100 dilution ; anti - rlf at 1:1,000 dilution ) were incubated overnight at 4c . the avidin - biotin immunoperoxidase system with 3-amino-9-ethylcarbazole as chromogen was used to visualize bound antibodies ( histostain - plus kit ; zymed laboratories ) . for lacz staining , transgenic animals were fixed and stained with x - gal for lacz activity , and 10-m cryostat testicular sections were treated as described ( reinertsen et al . serum testosterone and luteinizing hormone ( lh ) levels were determined by radioimmunoassays using diasorin and amersham assay systems according to the manufacturers ' instructions . most die within a few days , but some individuals survive for as long as 6 wk , eventually dying of pulmonary failure . due to the lethal phenotype of the homozygous mutants , our analysis was restricted to male mice recovered between postnatal day 10 ( p10 ) and p42 , killed before the first signs of respiratory failure were apparent . pdgf - a/ males had no defects in the external genitalia and testicular descent appeared normal . 1 a ) , however , the testicular size reduction in the mutants was more pronounced than the average reduction of the body size , and this feature increased dramatically with age ( fig testis histology revealed profound differences between pdgf - a null mice and wt ( fig . 1 , c j ) . at p10 , / animals showed absence of tubular lumen whose formation was starting in the controls ; no significant difference was observed in tubular diameter and germ cells number . sertoli cells , interstitial cells , peritubular myoid cells ( pmc ) and extracellular matrix layers appeared intact as well ( fig . , the mutant testes had reduced seminiferous tubule diameter and poor lumen formation but , likewise control littermates , spermatogenesis had proceeded up to pachytene spermatocytes ; sertoli cells and pmc were normal . however , the null mice showed an initial reduction in number of morphologically recognizable leydig cells , scattered in the interstitium among small undifferentiated spindle - shaped mesenchymal cells of fibroblastic appearance ( fig . , round spermatids were frequently found in the tubules of + /+ animals and the interstitium was filled with leydig cells ; no spermatids and a further reduction of the interstitial cells population were observed in the mutants ( data not shown ) . at p32 and p42 all stages of the spermatogenic cycle and abundant leydig cells were seen in control testis ( fig . the tubular diameter was greatly reduced and a spermatocytic arrest with further loss of leydig cells was observed ( fig . 1 h ) . p42/ testis displayed a dramatic decrease of spermatocytes , adluminal multinucleated giant cells , and an almost complete absence of leydig cells ( fig . immunostaining of p10-p42/ testicular sections with an antibody against the sertoli cell specific gene gata-1 ( yomogida et al . 1994 ) confirmed the presence of sertoli cells in the null testis , indicating that the lack of mature forms of germ cells in pdgf - a/ mice did not result from a loss of the supporting cells ( data not shown ) . we also performed an immunohistochemical analysis of rlf , which is a specific marker for the mature forms of fetal as well as adult leydig cells ( balvers et al . anti - rlf staining identified mature fetal leydig cells in both mutant and wt p10 testis ( fig . b ) . at p18 , concomitantly with the involution of fetal leydig cells and with the first appearance of the adult leydig cells progenitors , no rlf was expressed in / testis while sporadic punctate staining was seen in the interstitium of + /+ testis ( fig . 2c and fig . here there was no staining in the few interstitial cells of the ko testis , while the majority of the interstitial cells of the wt testis exhibited rlf - specific staining ( fig . f ) . by day 32 and onward , a more homogeneous cytoplasmic distribution of rlf positivity within the interstitial cells of the wt animals was observed while , concomitantly with the progressive depletion of the interstitium , rlf staining in the ko testis was absent ( data not shown ) . this rlf staining pattern indicates that the mutant prepubertal testis contains mature fetal leydig cells and confirms the absence of the adult leydig cell population in older / testis . to analyze whether the reduced size of the pdgf - a mutant testis and its histologic appearance could be due to decreased cell proliferation and/or increased apoptosis , brdu and tunel labeling were performed . at p18 , p25 , p32 , and p42 there was no difference in brdu incorporation of the actively proliferating germ cells between wt and mutant testes ( fig . 3 , a d ; data not shown ) . during the third week of postnatal life , the stem cell precursors of the adult leydig cell proliferate ( ge et al . accordingly , brdu staining was observed in some interstitial cells of p18 wt animals ( fig . 3 a ) ; on the contrary , no brdu labeling was seen in p18 pdgf - a/ littermates ( fig . regarding tunel , few apoptotic cells were seen in the tubules of both wt and / siblings between p10 and p32 ( data not shown ) . however , at p42 a large number of spermatocytes undergoing apoptosis was observed in null mice compared with control ( fig . 3e and fig . thus , an arrest of differentiation followed by increased apoptosis seem to be responsible for the seminiferous tubules aspect in the pdgf - a/ testes . the lack of brdu incorporation and the absence of tunel labeling in the intertitium of / testis , thus , we speculate that in the pdgf - a/ testis the normal pubertal replacement of the fetal leydig cells with the adult population of cells is deranged because of a deficiency in the commitment of the adult leydig cell precursors to proliferate and perhaps to differentiate . such a model requires the knowledge of the localization of pdgf - a and its receptor in normal testis . in situ hybridization for pdgf - a and pdgf - r in embryonic - day-17.5 ( e17.5)-p30 wt testis showed the mrnas expression in seminiferous epithelium and interstitial mesenchymal cells , respectively ( fig . later , some pdgf - a positive tubules and distinct populations of pdgf - rpositive interstitial cells could be seen . pdgf - r expression was moreover localized using transgenic mice carrying the reporter gene -galactosidase ( lacz ) under control of the murine pdgf - r promoter . according to the in situ data , the immunohistochemistry for pdgf - a confirmed the intratubular expression of the growth factor which , in agreement with previous results ( gnessi et al . 1995 ) , was mainly localized in the cytoplasm of the sertoli cells ( fig . 5 ) . at p18 , although with different intensities , all the tubules were stained ( fig . 5 a ) . at p42 , in line with the in situ findings , only the sertoli cells in some tubules , corresponding to spermatogenic stages ix - x ( russell et al . altogether , these results indicate that the pdgf - a expression in sertoli cells depends on the stage of maturation of the associated germ cells . at p42 5 b ) , which is consistent with the reported production of pdgf - a by adult leydig cells in vitro ( gnessi et al . concerning pdgf - r , in prepubertal animals the immunostaining was localized in the interstitial cells of both + /+ and / testis , although in the latter the interstitial cells were less in number ( fig . 5c and fig . the wt testis continued to show pdgf - rpositive cells between the tubules ( fig . 5 e ) , while the null testis showed a steadily decreasing number of positive cells , culminating in their complete absence at p42 ( fig . 5 f ) . the comparison between the testicular defects found in / mice and the normal expression pattern of pdgf - a and its receptor , indicates that paracrine pdgf - a / pdgf - r signaling constitutes a critical part of the epithelial - mesenchymal interaction , essential for adult leydig cells development . pdgf - a does not influence the fetal generation of leydig cells , as suggested by both the normal adrogenization and rlf - positive interstitial immunohistochemical staining of prepubertal / animals . due to the lack of specific markers , we could not produce direct evidence that among the interstitial pdgf - rpositive cells are indeed the precursors of the adult leydig cells , but the spatiotemporal distribution of pdgf - r-interstitial cells in normal mice seems to suggest this possibility . it is also reasonable to predict that a cell type specifically lost in pdgf - a/ mice should carry the receptor for pdgf - a , and should locate close to the source of the ligand , as is the case for the pdgf - rpositive interstitial mesenchymal cells described . however , given the chemotactic activity of pdgf - a ( heldin and westermark 1999 ) , we can not exclude that , similarly to what is described for other pdgf - dependent developmental processes ( bostrm et al . 1997b ; calver et al . 1998 ) , pdgf - a might influence the long - range migration of leydig stem cells , thus preventing their arrival in the gonads from primordial germ layer source besides proliferation and differentiation . regardless of whether pdgf - a influences the migration of the leydig cells precursors or their proliferation / differentiation , the main consequence of the lack of replacement of the fetal leydig cells with adult cells would be a progressive reduction of testosterone , followed by spermatogenic arrest and germ cells degeneration . the testicular phenotype of the null animals strongly indicates that this is the case . in / testis the initiation of spermatogenesis , which depends mainly on follicle - stimulating hormone , occurs normally , while its testosterone - dependent subsequent completion and maintenance ( sharpe 1994 ) are lacking . indeed , serum testosterone levels were similar in prepubertal / early pubertal ( p10-p18 ) wt ( 1.79 0.7 ng / ml ; n = 5 ) and mutant mice ( 2.0 0.89 ng / ml ; n = 3 ) , while in / older animals , circulating testosterone was not detectable , confirming that the spermatogenic arrest and germ line apoptotic regression in pdgf - a/ animals are mediated by androgen deficiency . they were similar in / ( 1.6 0.4 ng / ml ; n = 4 ) and + /+ ( 1.4 0.5 ng / ml ; n = 6 ) animals between p10 and p25 . in agreement with the testosterone reduction , in a null p42 animal the levels of lh ( 7.5 ng / ml ) were higher than in control littermates ( 3.8 0.3 ng / ml ; n = 4 ) . these data indicate that in pdgf - a null animals , the fate of the leydig cells can not be ascribed to an lh deficiency . in conclusion , although lh , androgens and igf-1 are recognized important elements required for the completion of leydig cell maturation , they can not be the factors that regulate the commitment of stem cells to adult leydig cell lineage ( benton et al . in fact , initial proliferation of stem cells can occur when lh is absent ( teerds et al . 1989 ) , and animals with androgen insensitivity ( murphy et al . 1994 ) or igf-1 gene deletion ( baker et al . 1996 ) develop leydig cells , suggesting that a separate factor regulates the earliest stage of adult leydig cell evolution . our findings indicate that pdgf - a may be this factor , and suggest that adult leydig cells arise from pdgf - r progenitors . the pivotal role played by pdgf - a in adult leydig cells development suggests that pdgf - a may be a potential target for the master genes involved in testicular organogenesis . in this respect , it is worth mentioning that wt1 , whose spatio - temporal sertoli cells expression profile ( pelletier et al . 1991 ; mundlos et al . 1996 ) is virtually superimposable on that of pdgf - a , can either repress or activate the pdgf - a gene ( wang et al . 1993b ; gashler et al . , 1992 ) , and has been involved in posttranscriptional processing within the sertoli cells ( larsson et al . the dramatic testicular phenotype of the pdgf - a/ animals is also of interest because recent studies have reported detection of pdgf - a and pdgf - r in the human testis ( basciani , s. , l. gnessi , m. arizzi , n. rucci , s. mariani , s. ulisse , e.a . proceedings of the 81st annual meeting of the endocrine society , san diego , ca . these considerations , coupled with our findings , may furnish a new approach for the understanding of the wt1-mediated mechanisms involved in testicular development and perhaps of wt1-dependent urogenital abnormalities and tumorigenesis ( hastie 1994 ; reddy and licht 1996 ; menke et al . 1998 ) . moreover , a local impairment of the pdgf - a/ pdgf - r system may provide a new conceptual framework for the comprehension of some forms of leydig cells hypoplasia , with high lh , low testosterone , and no mutations of the lh receptor gene ( zenteno et al . 1999 ) , and of cases of spermatogenetic failure , which are probably due to intratesticular testosterone deficiency in man ( sharpe 1994 ) .

platelet - derived growth factor ( pdgf)- a deficient male mice were found to develop progressive reduction of testicular size , leydig cells loss , and spermatogenic arrest . in normal mice , the pdgf - a and pdgf - r expression pattern showed positive cells in the seminiferous epithelium and in interstitial mesenchymal cells , respectively . the testicular defects seen in pdgf - a/ mice , combined with the normal developmental expression of pdgf - a and pdgf - r , indicate that through an epithelial - mesenchymal signaling , the pdgf - a gene is essential for the development of the leydig cell lineage . these findings suggest that pdgf - a may play a role in the cascade of genes involved in male gonad differentiation . the leydig cell loss and the spermatogenic impairment in the mutant mice are reminiscent of cases of testicular failure in man .

patients with rheumatoid arthritis ( ra ) are at increased risk to develop cardiovascular disease ( cvd ) [ 1 , 2 ] and experience cardiovascular mortality [ 3 , 4 ] . although treatments for ra have improved over time , cardiovascular mortality has remained relatively unchanged and is responsible for 50 percent of premature deaths in patients with ra [ 5 , 6 ] . traditional cardiovascular risk factors including smoking , hypertension , hyperlipidemia , obesity , and diabetes are important factors but do not adequately account for the excess burden of cvd in ra patients . there is growing evidence that serum uric acid ( sua ) may play a role in cvd in the general population [ 8 , 9 ] . indeed epidemiologic studies have found that hyperuricemia appears to be an independent risk factor for hypertension , heart failure ( hf ) , coronary artery disease , and stroke . experimental studies have also shown that uric acid is a functionally active molecule that can contribute to proatherogenic processes including inflammation , endothelial dysfunction , and oxidative stress . the role of hyperuricemia in ra has not been well studied and only a few papers have addressed the subject [ 15 , 16 ] . however , hyperuricemia is common in the general population with a prevalence of 68% among healthy adults and as many as 1 in 3 adults with uncontrolled hypertension and several cvd risk factors . given the excess burden of cardiovascular disease in patients with ra and the potential role of serum uric acid , the purpose of this study was to examine the effects of hyperuricemia on cvd specifically in patients with ra . an inception cohort of all cases of rheumatoid arthritis ( ra ) first diagnosed between january 1 , 1980 and december 31 , 2007 ( n = 813 ) among olmsted county residents 18 years of age was assembled as previously described . the incidence date was defined as the earliest date at which the patient fulfilled at least 4 of the 7 american college of rheumatology ( acr ; formerly , the american rheumatism association ) 1987 classification criteria for ra . a comparison cohort of subjects without ra with similar age , sex , and calendar year was also assembled . all patients in both cohorts were followed up longitudinally through their entire medical records until death , migration from olmsted county , or december 31 , 2008 . the study was approved by the institutional review boards of the mayo clinic and olmsted medical center . hyperuricemia was defined as serum uric acid ( sua ) > 8.0 mg / dl for males and > 6.1 mg / dl for females . clinically available data for traditional cardiovascular risk factors were collected , including age , gender , race , smoking history , body mass index ( bmi ) , diabetes mellitus , hypertension , and dyslipidemia . data from the medical record were also collected for factors that could affect sua levels including estimated glomerular filtration rate ( gfr ) , alcoholism , low - dose aspirin , and urate lowering therapy . cvd recorded for each patient included angina , myocardial infarction ( mi ; hospitalized or silent ) , revascularization procedures , and hf . noncardiac vascular diseases ( ncvd ) included stroke , thromboembolism , and peripheral vascular disease . descriptive statistics ( means , percentages , etc . ) were used to summarize the data . poisson regression models with smoothing splines were used to examine trends in uric acid levels according to time since ra incidence / index date with adjustment for age and sex . these methods are similar to kaplan - meier method with censoring of patients who are still alive at last follow - up . however , patients who die before experiencing hyperuricemia are appropriately accounted for to avoid the overestimation of the rate of occurrence of hyperuricemia , which can occur if such subjects are simply censored . patients who were diagnosed with hyperuricemia prior to the diagnosis of ra , or prior to the index date for subjects in the non - ra comparison cohort , were excluded from the analysis of cumulative incidence . a sensitivity analysis was performed to examine the possibility that differences in testing rates might influence cumulative incidence results . a subset of uric acid tests was randomly selected from patients with ra to mimic the testing rate in patients without ra for the sensitivity analysis . cox proportional hazards models were used to compare the rate of development of hyperuricemia between patients with ra and the non - ra comparison cohort . cox models were also used to assess the impact of hyperuricemia on the development of cvd , ncvd , or mortality among patients with ra and non - ra subjects . age was used as the time scale for these models to provide optimal adjustment for age under the assumption that age is likely the most important time determinate of cardiovascular disease . subjects entered the model at the age they met criteria for ra and remained in the model until the age of each cardiac or vascular disease event . subjects without events were censored at the age of death or last follow - up . traditional cardiovascular risk factors as well as factors that could affect both uric acid levels and the outcomes of interest ( e.g. , gfr , alcoholism , low - dose aspirin , and urate - lowering therapy ) were included in these models as adjustors . these time - dependent covariates allowed patients to be modeled as unexposed to the risk factor during the follow - up time prior to development of the risk factor and then change to exposed following development of the risk factor . the average age at ra incidence ( index date for the non - ra cohort ) was 55.9 ( sd 15.7 ) years and 556 ( 68% ) were female in each cohort ( table 1 ) . there was no difference in the presence of hyperuricemia at ra incidence / index date between cohorts ( p = 0.22 ) . rates of testing of sua were higher among patients with ra ( 0.77 tests per 1 person - year of follow - up ) than among non - ra subjects ( 0.50 per 1 person - year of follow - up ; p < 0.001 ) . there were no apparent assay changes in sua ranges by calendar years of interest ( figure not shown ) . examination of trends in serum uric acid according to time since ra incidence / index date revealed that the average serum uric acid decreases in ra patients prior to diagnosis of ra ( figure 1 ) . sua remains lower in ra on average than in non - ra for the first 10 years of ra disease duration . the cumulative incidence of hyperuricemia was higher in the ra patients compared to the non - ra subjects ( 18.1% 1.7% at 10 years in ra versus 11.5% 1.5% in non - ra ; p = 0.008 , table 2 ; figure 2(a ) ) . however , after randomly resampling ra patient data to mimic the testing rate of non - ra subjects , the difference in cumulative incidence of hyperuricemia between groups disappeared ( 12.9% 1.5% at 10 years in ra versus 11.5% 1.5% in non - ra ; p = 0.64 , table 2 ; figure 2(b ) ) . there was no difference in the rate of development of hyperuricemia among ra and non -ra by sex ( p = 0.61 for ra / non - ra by male / female interaction ) . the associations between hyperuricemia and cvd / mortality were more consistent in patients without ra than in patients with ra ( table 3 ) . however , hyperuricemia appeared to be more strongly associated with mortality among the ra ( hazard ratio [ hr ] : 2.00 ; 95% confidence interval [ ci ] : 1.51 , 2.64 ) than among the non - ra patients ( hr : 1.59 ; 95% ci : 1.12 , 2.27 ) , but this difference did not reach statistical significance ( interaction p = 0.32 ) . in the patients with ra , hyperuricemia was also associated with increased risk of peripheral arterial events ( hr : 2.62 ; 95% ci : 1.27 , 5.41 ; table 4 ) . in this population - based study we examined the role of hyperuricemia on cardiovascular disease in patients with and without ra . among subjects without ra , hyperuricemia was associated with an increased risk of hf and cvd . by contrast , among patients with ra , hyperuricemia was not associated with increased risk of mi , hf , or cvd . however , hyperuricemia was a predictor of peripheral arterial events in ra patients . these findings suggest that in patients with ra hyperuricemia may confer increased risk of peripheral arterial events and all - cause mortality but not necessarily cardiac disease . baseline serum uric acid levels in this study were similar to those reported for nationally representative data of adults in the united states . the prevalence of hyperuricemia among healthy us adults has been reported to be 68% but increased among persons with hypertension and cvd risk factors . similarly , in our study , a proportion of subjects had cardiovascular comorbidities and higher rates of hyperuricemia . baseline prevalence of hyperuricemia was similar between ra patients and non - ra subjects in our study . ra and gout cooccur infrequently . as a result , data are limited regarding the occurrence of hyperuricemia in patients with ra . we calculated the cumulative incidence of hyperuricemia in patients with and without ra and found that during follow - up , patients with ra appeared to develop hyperuricemia more frequently than their non - ra counterparts . however , when uric acid data for the ra cohort were randomly sampled at the same rate as the non - ra comparison cohort , the cumulative incidence of hyperuricemia in the resampled dataset was similar to that of the non - ra group . this suggests that excess hyperuricemia was incidentally found in ra patients due to higher rates of testing . a small study of healthy subjects found that it is not uncommon to detect transient hyperuricemia with repeated sua measurements . in many epidemiologic studies of the general population , hyperuricemia has been associated with increased risk of cardiac disease including myocardial infarction [ 26 , 27 ] , hf , and coronary heart disease . however , there are also studies which have suggested that uric acid is not an independent risk factor for heart disease [ 8 , 9 ] . for example , a recent mendelian randomization study by palmer et al . used a single nucleotide polymorphism of the slc2a9 urate transporter gene to evaluate the effects of hyperuricemia on ischemic heart disease and found no association . it is still debatable whether uric acid is a causal agent in cardiovascular disease pathogenesis or rather a biomarker of disease . subjects in the non - ra cohort of our study were reflective of the general population within olmsted county . in these patients , hyperuricemia was associated with increased risk of mi , hf , and cvd after adjusting for age , sex , and calendar year . increased risk for mi did not reach statistical significance , but this was likely due to a small number of observed events . upon further adjustment for traditional cardiovascular risk factors , hazard ratios for mi and cvd these associations were further attenuated after adjusting for reduced kidney function , alcoholism , low - dose aspirin , and urate - lowering therapy . in contrast , few studies have examined the potential effects of hyperuricemia on cvd in patients with ra . results from a cross - sectional study of 400 ra patients suggested that higher serum uric acid levels were associated with an increased likelihood of cvd prevalence even after adjusting for confounders ( or 1.36 per 1 mg / dl increase in serum uric acid ) . however , in our cohort of ra patients , hyperuricemia was not associated with increased risk of cardiac disease incidence , despite greater numbers of cardiac events compared to non - ra subjects . one possibility is that the null hypothesis , that hyperuricemia has no effect on cvd , could be true . however , some patients with transient hyperuricemia were likely included in the analysis due to a higher rate of testing among patients with ra . another consideration is that , on average , patients with ra started the study with lower serum uric acid levels and continued to have lower levels for the first 10 years of disease . the effect though is that ra patients may have had less exposure to uric acid prior to incidence of hyperuricemia and therefore experience fewer adverse cardiac outcomes related to the exposure . an alternative possibility is that hyperuricemia may have relatively less impact on cardiac processes in the context of disease - specific factors in ra ( e.g. , systemic inflammation ) , as has been suggested for some traditional cvd risk factors . in terms of noncardiac vascular outcomes , diseases of the peripheral arteries were examined as a composite outcome that included peripheral arterial disease ( pad ) , aortic aneurysm , renal artery stenosis , and arterial thromboembolism . few other studies have looked at the association of uric acid levels with peripheral arterial disease ( pad ) . one cross - sectional study of the us population found that higher sua was associated with pad . a case - control study reported an association between sua and increased risk of pad , though the relationship was not statistically significant . among patients in the ra cohort of our study , hyperuricemia was a predictor for the occurrence of peripheral arterial events even after adjusting for traditional cardiovascular risk factors , including smoking . mechanisms which may contribute to vascular disease in patients with hyperuricemia include uric acid 's ability to promote endothelial dysfunction , decrease nitrous oxide bioavailability , and increase oxidative stress . uric acid has also been associated with increased inflammatory markers such as c - reactive protein which have been implicated in peripheral vascular disease . such effects may interact synergistically with the high degree of systemic inflammation in ra to promote increased atherogenesis . sua has been found to be associated with all -cause mortality in many but not all studies . a recent meta - analysis found that across studies of the general population , sua in the highest level ( e.g. , quartile , quintile ) compared to the lowest level was associated with all - cause mortality ( pooled rr 1.24 ; 95% ci : 1.091.42 ) . interestingly , one study of high - functioning community - dwelling seniors found that inflammation was an important confounder in their analysis of sua and mortality in women but not in men . given the high levels of systemic inflammation that affect persons with ra , it is possible that underlying inflammation contributed to the increased risk of all - cause mortality associated with hyperuricemia in the ra cohort of our study . since data were retrospectively collected from the medical record , only clinically obtained data were available . detailed information about diuretic use was not available . also , the patient population consisted predominantly of caucasian patients , so results may or may not necessarily be generalizable to patients of other ethnicities . strengths of our study include that it was a population - based study with long follow - up duration . in summary , we evaluated hyperuricemia as a possible risk factor for cardiovascular disease in patients with ra . the results of our study suggest that hyperuricemia may not be a risk factor for excess cardiovascular disease in patients with ra . however , hyperuricemia was associated with increased risk of peripheral arterial events and all - cause mortality . further research should investigate other nontraditional risk factors of cardiovascular disease in patients with ra .

objective . to evaluate whether hyperuricemia is a risk factor for cardiovascular disease ( cvd ) in patients with rheumatoid arthritis ( ra ) . methods . a population - based inception cohort of patients diagnosed between 1980 and 2007 with adult - onset ra was assembled . a comparison cohort of age- and sex - matched subjects without ra ( non - ra ) was also assembled . all clinically obtained uric acid values were collected . cvd and noncardiac vascular events were recorded for each patient . cox proportional hazards models were used to assess the impact of hyperuricemia on development of cvd , mortality , and noncardiac vascular disease . results . in patients without ra , hyperuricemia was associated with heart failure ( hr : 1.95 ; 95% ci : 1.133.39 ) and cvd ( hr : 1.59 ; 95% ci : 0.992.55 ) . in patients with ra , hyperuricemia was not significantly associated with cvd but was significantly associated with peripheral arterial events ( hr : 2.52 ; 95% ci : 1.175.42 ) . hyperuricemia appeared to be more strongly associated with mortality among ra patients ( hr : 1.96 ; 95% ci : 1.452.65 ) than among the non - ra subjects ( hr : 1.57 ; 95% ci : 1.092.24 ) . conclusion . in patients with ra , hyperuricemia was a significant predictor of peripheral arterial events and mortality but not of cvd .

follicular carcinoma of thyroid gland is the second most common thyroid cancer after papillary thyroid carcinoma . clinically , differentiated thyroid cancer ( dtc ) commonly presents with a solitary thyroid nodule and/or cervical lymphadenopathy . distant metastasis at initial presentation , especially those involving bone , is seldom seen and associated with poor clinical outcomes . in those cases , the axial skeleton and in particular the spine and the ribs are preferentially involved resulting symptoms such as severe pain and compression fracture [ 1 , 2 ] . a pathological fracture of the upper limb as an initial symptom of a late diagnosis of dtc is therefore extremely rare and has almost never been reported in literature . in this paper , we present a case of a patient with follicular thyroid cancer who presented with pathological fracture of the humerus without preceding illness . a previously fit and well 38-year - old man was presented to the emergency department with immediate extreme pain in the left arm after twisting it at home that day . plain radiographs of the left upper limb showed a fracture of the left proximal humerus associated with a lytic lesion ( fig . 1 ) . figure 1:x - ray showed lytic lesion and pathological fracture of left proximal humerus . plain radiographs of the left . an urgent staging magnetic resonance imaging ( mri ) of the left humerus demonstrated a fairly well - defined destructive lesion of proximal left humerus , which measured ~6 cm craniocaudally ( fig . both the cortex and the marrow were diffusely infiltrated and there was a small extra - osseous tumour mass anteriorly . computed tomography ( ct ) scan of thorax / abdomen / pelvis with contrast could not identify a clear primary source of malignancy but it showed an enlarged right lobe thyroid gland with retrosternal extension ( fig . 3 ) . figure 2:mri left humerus showed pathological fracture of the neck and proximal shaft humerus , measure 6 2.5 2.8 cm in maximal dimension . an urgent staging magnetic resonance . figure 3:ct chest with contrast showed an enlarged right lobe thyroid gland with retrosternal extension causing compression and deviation of trachea ( arrow ) . mri left humerus showed pathological fracture of the neck and proximal shaft humerus , measure 6 2.5 2.8 cm in maximal dimension . an urgent staging magnetic resonance . ct chest with contrast showed an enlarged right lobe thyroid gland with retrosternal extension causing compression and deviation of trachea ( arrow ) . the thyroid lobe was investigated 6 years before where a multi - nodular goitre ( mng ) with a dominant solitary nodule on the right thyroid lobe was discovered . a fine needle aspiration cytology ( fnac ) specimen of the solitary nodule was reported as a thy2 with features consistent with a simple nodule . the investigations were arranged by the patient 's general medical practitioner who reassured the patient without the input of secondary services . following the finding of the ct scan , the patient was referred to the local ent department , where ultrasound of the neck and core biopsy of the thyroid nodule was conducted . the image confirmed a mng with largest nodule in the right lobe . and the core biopsy showed a follicular pattern with thyroid follicles of varying sizes set in a mixture of fibrous tissue . a ct guided biopsy of the left humerus after the initial presentation confirmed a metastatic thyroid cancer . histologically , the mass from the left proximal humerus demonstrated features consistent with metastatic thyroid carcinoma . a large thyroid lobe was found with no local invasion and four large level vi lymph nodes were excised . the pathologic findings confirmed complete excision of a 70 mm widely invasive follicular carcinoma with evidence of vascular invasion and metastasis of three out of the four central neck nodes . after thyroidectomy , the patient was seen by clinical oncology and radioiodine therapy was administered . the primary tumour is most likely found in breast and prostate followed by the thyroid gland . the bone metastases in dtc is osteolytic in nature and its distribution preferentially involves the weight - bearing skeletons such as vertebrae ( 52.5% ) , femur ( 20.4% ) , pelvis ( 16.0% ) and clavicle ( 13.6% ) . common manifestations of the osseous metastases include pain , fracture and spinal cord compression associated with lesion in the axial skeleton . in this case , we described an extremely rare scenario where pathological fracture of the upper limb was the initial presentation of metastatic dtc . of interest in our case is that the patient presented with a thyroid lump 6 years before which was diagnosed as a dominant nodule with a background of benign mng . in the absence of suspicious sonographic ultrasound features and benign pathocytological although ultrasound - guided fnac is an excellent tool for evaluating patients with solitary thyroid nodules , its utility in patients with mng has been questioned in the past . the british thyroid association ( bta ) recommends fnac should be carried out if ultrasound features are equivocal , indeterminate or suspicious of malignancy . in our case , fnac was performed although the nodule appeared benign and subsequent fnac result supported the findings that the nodule was benign in nature . despite this where fnac were conducted and proven to be benign in patients with non - functional , cervical mng the incidental thyroid cancer discovered at the time of thyroidectomy can be as high as 23% . male gender tends to be associated with an increased likelihood of cancer on final pathology [ 5 , 7 ] . in conclusion , despite the relative rarity of causing pathological fracture in the humerus , thyroid cancer should be considered during assessment of the patient with pathological fractures of unknown primary origin . although the gold standard for investigation thyroid nodule remains ultrasound - guided fnac , a benign fnac especially in male patients with a mng may need a more aggressive management due to the increased risk of an undetected thyroid cancer .

differentiated carcinomas of the thyroid gland usually have a good prognosis with prognosis often discussed in terms of 20 year survival . nevertheless its 10-year - survival rate decreases when accompanied by distant metastasis . follicular thyroid cancer ( ftc ) is the second most common thyroid cancer and usually presents with a solitary thyroid nodule with or without cervical lymphadenopathy . distance metastasis at initial diagnosis is seldom observed with incidence range from 1 to 9% . in cases of bone metastasis , the incidence is only 23% and weight - bearing skeleton is preferentially affected . in our case , we present a patient with ftc that metastasized to the upper limb causing severe pain and pathological fracture at the initial presentation .

a meningeal haemangiopericytoma ( hp ) is a mesenchymal tumour that makes up less than 1% of all cns tumours . when the disease is disseminated , chemotherapy produces a weak and short - lived response ; therefore , new drugs are needed . we describe the case of a 65-year - old woman with a 13-year history of recurrent hp . after local treatment with radiotherapy , she developed metastases that required systemic treatment , and treatment with sunitinib , an oral inhibitor of the vascular endothelial growth factor receptor and the platelet - derived growth factor receptor , was initiated . as a result , radiological stabilisation of the systemic disease was maintained for over 12 months . anti - angiogenic agents can be useful for treating disseminated hp , but further studies are needed to confirm their possible role in controlling metastatic disease . a meningeal haemangiopericytoma ( hp ) is a rare mesenchymal tumour that arises from the cells surrounding the capillary wall . its aetiology is unknown , and 25% of cases occur in the head or neck . meningeal hp makes up less than 1% of all cns tumours ; it is very aggressive and presents high rates of local recurrence and distant metastasis , most frequently in the lungs and the bones . surgery is the normal treatment , and no clear role has been established for either radiotherapy or chemotherapy . the hypothesis that this type of tumour could be sensitive to angiogenesis inhibitors is based on the fact that these tumours are highly vascularised and express platelet - derived growth factor receptor ( pdgfr ) and vascular endothelial growth factor receptor ( vegfr ) . however , few cases of patients treated with anti - angiogenic drugs have been reported . the low prevalence of this tumour prevents the implementation of clinical trials that could confirm our hypothesis . a 65-year - old woman with a personal history of arterial hypertension who had been treated for parotid adenoma came to our clinic in november 1997 . resectioning of the tumour was decided against in view of the likely outcome , and so the patient was given 17 gy of stereotactic radiotherapy in one fraction . the patient complained of pain , which had developed over months , in the right upper arm . radiography was performed and revealed a 70-mm permeative lesion in the middle third of the right humerus . mri of this area showed the lesion to be of a lytic nature , with an intermediate signal in t1 and t2 and perilesional oedema . an extension study was performed , involving a helical computerised axial tomography ( cat ) scan , which revealed a nodular image measuring 22 mm in the paraspinal muscles between the 11th and 12th ribs and two hepatic lesions in segments 3 and 7 , measuring 24 and 22 mm , respectively . a humeral biopsy was then performed , and the anatomopathological results revealed the existence of hp . with clinical evidence of hp metastatic relapse in the bone , soft tissues and liver , we decided to apply radiotherapy to the lesions in the humerus and the lower left dorsal paraspinal muscles due to the pain experienced in those regions and to obtain evolutive control of the hepatic lesions . after 4 months of monitoring , a cat re - evaluation was performed , and the diameter of the irradiated paravertebral lesion had decreased by 50% , while the hepatic lesions had enlarged to 38 and 25 mm , respectively ( fig . 1 ) . the cranial cat revealed a hyperechoic mass measuring 20 mm with a meningeal base at the apex of the petrous bone ; it could not be discerned whether this corresponded to residual remains or to a tumoural relapse . in june 2009 , progression to the liver suggested the need to begin treatment with 37.5 mg sunitinib daily . after 1 month of treatment with anti - vegfr , analysis revealed a two - fold increase in the values of got , gpt and ggt to 80 , 83 and 81 u / l , respectively , while ap levels rose to 169 u / l and ldh to 567 u / l . abdominal echography was performed , and the two hepatic lesions in segments 3 and 7 were observed to measure 30 and 70 mm , respectively . one of the lesions , thus , was smaller while the other was larger than at the time of the previous cat scan . we assumed that ischemia of the lesions might have been induced by the administration of sunitinib , and so the treatment was continued . in the control echography carried out after 1 month the patient showed an acceptable degree of tolerance , although with grade ii thrombocytopenia and grade iii neutropenia , with episodes of epistaxis and petechiae on the lower eyelids , it was necessary to reduce the dose of sunitinib to 25 mg daily for 4 weeks , followed by 2 weeks of rest . the image tests carried out from the start of anti - vegfr treatment in june 2009 until september 2010 revealed disease stabilisation ( fig . the patient therefore continued the same treatment , and no significant level of toxicity was observed . meningeal hp is a very rare tumour , but it is aggressive , with a tendency to recur and to metastasise . in studies by mena et al . of 94 patients and guthrie et al . of 44 patients with hp , recurrences were reported in 60.6 and 65% of the cases , and metastases were reported in 23.4 and 33% of the patients , respectively . the normal treatment for meningeal hp is surgical resection . in some clinics , adjuvant radiotherapy the utility of chemotherapy is unclear ; various chemotherapeutic agents have been used in the treatment of metastasic hp , but no consensus has been reached as to the most appropriate drug , dose , form of administration , scheme or response criteria , and so the results obtained should be interpreted with caution . adriamycin , alone or in combination , appears to be the most effective agent , with 6/12 ( 50% ) complete or partial responses and 4/12 ( 33% ) minor responses . other drugs , such as cyclophosphamide , vincristine , methotrexate , actinomycin and dtic , may exert some anti - tumoural activity , but due to the small number of patients under such treatment and because these drugs are often used in combinations , it is not possible to establish a protocol for application based on the results of previous studies [ 4 , 5 ] . the immunohistochemical expression of vegfr has been described in the tumoural cells of hp and in the endothelium , with overexpression of vegfr1 and vegfr2 only in the endothelium , suggesting that the autocrine and paracrine activation of the vegf - vegfr pathway may participate in the biology of hp . . showed that angiomatous meningiomas and hps frequently overexpress pdgfr and that this overexpression is detectable by immunohistochemistry . sunitinib is a tyrosine kinase inhibitor of vegfr , pdgfr and fibroblast growth factor , with antiproliferative and anti - angiogenic properties . among the anti - angiogenic treatments used for hp , interferon - a and/or thalidomide other patients have been treated with tyrosine kinase inhibitors such as sunitinib [ 9 , 10 ] , sorafenib or dasatinib as first - line or successive treatments . in some of these patients , disease stabilisation lasting over 6 months has been achieved ; others have enjoyed clinical benefits or even durable partial response ( table 1 ) . because hp is a tumour with a high probability of distant metastasis that shows little and short - lived response to chemotherapy , new drugs are needed to tackle disseminated disease . anti - angiogenic treatment , for example , utilising anti - vegfr drugs , could play a role in these new treatment regimens , but trials are needed to test this hypothesis because , to date , only individual cases have been reported .

introductiona meningeal haemangiopericytoma ( hp ) is a mesenchymal tumour that makes up less than 1% of all cns tumours . hps arise from pericytes and present high rates of recurrence and distant metastasis . the primary treatment option is surgery . when the disease is disseminated , chemotherapy produces a weak and short - lived response ; therefore , new drugs are needed.case presentationwe describe the case of a 65-year - old woman with a 13-year history of recurrent hp . after local treatment with radiotherapy , she developed metastases that required systemic treatment , and treatment with sunitinib , an oral inhibitor of the vascular endothelial growth factor receptor and the platelet - derived growth factor receptor , was initiated . as a result , radiological stabilisation of the systemic disease was maintained for over 12 months.conclusionsanti-angiogenic agents can be useful for treating disseminated hp , but further studies are needed to confirm their possible role in controlling metastatic disease .

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

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

the development of a first episode of schizophrenia is often preceded by prodromal states with variable symptoms . the factors determining the progression of disease from a prodromal state to a psychotic episode are of great interest . however , little is currently known about the factors influencing whether a prodromal proband progresses to psychosis or remains stable . we hypothesised that genetic variants play an important role in determining symptom progression . amongst the schizophrenia susceptibility genes identified in the past few years , we focused on a well characterised schizophrenia susceptibility locus , daoa / g72 , and queried whether variants in this locus might influence this progression . the daoa / g72 gene was originally discovered during the analysis of the schizophrenia susceptibility region on chromosome 13 . fine - mapping of the region on the long arm of chromosome 13 , 13q1213q34 led to the discovery of the new gene g72 . the g72 gene is transcribed in brain , and increased expression was found in the dorsolateral prefrontal cortex in schizophrenia compared with controls . to assess the function of this gene , a screen of binding proteins was performed , which showed that g72 binds to and activates d - amino acid oxidase . daoa / g72 is a very attractive schizophrenia candidate gene also due to the fact that it is a primate - specific gene . a recent meta - analysis of daoa / g72 in schizophrenia showed highly significant evidence of association between a number of markers in the daoa / g72 region and schizophrenia . these authors concluded that the association findings for the daoa / g72 gene and schizophrenia are amongst the most compelling in psychiatry . this led us to focus on the daoa / g72 gene , since we expected a sufficiently large effect to be visible in a prospective investigation . the main outcome measure of the present study was progression to a psychosis , namely the incidences of first episode schizophrenia and first episode psychosis . in a unique prospective multicenter approach combining psychopathology and genetics , prodromal subjects were followed for up to 2 years to prospectively identify those probands who would progress to psychosis . specifically , we assessed here whether common daoa / g72 variants might predict the conversion to a psychosis in this prospective multicentre study . we analysed a number of variants which are located in the coding region of daoa / g72 , but also variants in the 5 and 3 putative regulatory regions which are expected to have an important role in the function and expression of the daoa / g72 gene . the centres were located at the departments of psychiatry and psychotherapy at the universities of bonn , cologne , and dsseldorf . to aid in the recruitment of probands with prodromal syndromes , awareness programmes were put in place for primary and psychiatric health services , for the relatives of patients with schizophrenia , and were also aimed to inform youth support services as well as the general population . the aims of the programmes were to establish local mental health networks as well as the facilitation for persons at a risk to find help with the early intervention services . information was provided about early symptoms of schizophrenia as well as about the usefulness of early intervention . referrals of probands were from a number of settings , including from counselling services , youth support services , primary health care , and mental health professionals . the clinical study and the inclusion and exclusion criteria are described in more detail elsewhere . briefly , patients were eligible for inclusion in the trial if they met the criteria of the early or late initial prodromal state and did not fulfil exclusion criteria . the early initial prodromal state was operationally defined by either basic symptoms predicting psychosis or by a clinically relevant decline of functioning in combination with established risk factors ( risk and decreased functioning ) . basic symptoms required the presence of one or more of the following symptoms several times per week during the last 3 months : thought interferences , thought perseverations , thought pressure , thought blockages , disturbances of receptive language , decreased ability to discriminate between ideas and perception or between fantasy and true memory contents , tendency to delusion of reference with subject centrism , derealisation , visual perception disturbances , and acoustic perception disturbances . established risk factors were pre- or perinatal complications or a first - degree relative with a lifetime diagnosis of schizophrenia or a schizophrenia spectrum disorder . late initial prodromal state was defined by the presence of at least one of the following symptoms appearing several times a week : ideas of reference , odd beliefs or magical thinking , unusual perceptual experiences , odd thinking and speech , or suspiciousness or paranoid ideation ; or by the appearance of one of the following symptoms for less than 1 week and resolving spontaneously : hallucinations , delusions , formal thought disorder , or gross disorganised or catatonic behaviour . exclusion criteria of note were previous treatment with antipsychotics , present or past diagnosis of a schizophrenic , schizophreniform , schizoaffective , delusional , or bipolar disorder , present or past diagnosis of a brief psychotic disorder with a duration of more than 1 week or within the last 4 weeks regardless of its duration , organic brain disease , and alcohol or drug dependence within the last 3 months . during the study , probands were seen approximately once a month . progression to first episode psychosis was operationally defined by cut - off points on positive and negative syndrome scale ( panss ) subscales of 4 or more on hallucinations , 4 or more on delusions and 5 or more on conceptional disorganisation or formal thought disorder for at least 7 days and meeting criteria for schizophrenia ( icd-10 : f20 ) or acute schizophrenia - like psychotic disorder ( icd-10 : f23.2 ) . nine common variants spanning the daoa / g72 gene were identified from the ucsc genome browser ( genome.cse.ucsc.edu ) . single - nucleotide polymorphism nomenclature is based on the ncbi dbsnp build 129 ( april 2008 , updated june 2008 ) . moreover , the traditional m nomenclature as described by chumakov et al . daoa / g72 variants were genotyped by allele discrimination using taqman technology ; 12.5 ng of dna was used for taqman snp genotyping assays on demand , employing the applied biosystems protocol . following the completion of pcr reactions , fluorescence was determined on a tecan 384 ultra reader ( tecan usa , durham , nc ) . the probes were labelled with carboxyfluorescein ( fam ) , with an excitation wavelength of 485 nm and an emission wavelength of 535 nm . vic - labelled probes employed an excitation wavelength of 535 nm and an emission wavelength of 590 nm . the influence of daoa / g72 variants on progression of symptoms was assessed by tests , employing spss version 14.0 ( spss , chicago , usa ) . if cells had an expected frequency of < 5 , monte carlo simulation based on 10,000 permutations with the start value 92,208,573 was done . the level of significance was set at p < 0.05 ( two sided ) . to correct for multiple testing , the spectral decomposition method was employed to estimate the number of effective loci [ 14 , 17 ] . haplotype block structure was determined by the solid spine of ld method as implemented in haploview ( broad institute , mass , usa ) . famhap 17 yields a global significance value of the haplotype analysis employing the likelihood ratio test , as well as individual p values for each haplotype . the centres were located at the departments of psychiatry and psychotherapy at the universities of bonn , cologne , and dsseldorf . to aid in the recruitment of probands with prodromal syndromes , awareness programmes were put in place for primary and psychiatric health services , for the relatives of patients with schizophrenia , and were also aimed to inform youth support services as well as the general population . the aims of the programmes were to establish local mental health networks as well as the facilitation for persons at a risk to find help with the early intervention services . information was provided about early symptoms of schizophrenia as well as about the usefulness of early intervention . referrals of probands were from a number of settings , including from counselling services , youth support services , primary health care , and mental health professionals . the clinical study and the inclusion and exclusion criteria are described in more detail elsewhere . briefly , patients were eligible for inclusion in the trial if they met the criteria of the early or late initial prodromal state and did not fulfil exclusion criteria . the early initial prodromal state was operationally defined by either basic symptoms predicting psychosis or by a clinically relevant decline of functioning in combination with established risk factors ( risk and decreased functioning ) . basic symptoms required the presence of one or more of the following symptoms several times per week during the last 3 months : thought interferences , thought perseverations , thought pressure , thought blockages , disturbances of receptive language , decreased ability to discriminate between ideas and perception or between fantasy and true memory contents , tendency to delusion of reference with subject centrism , derealisation , visual perception disturbances , and acoustic perception disturbances . established risk factors were pre- or perinatal complications or a first - degree relative with a lifetime diagnosis of schizophrenia or a schizophrenia spectrum disorder . late initial prodromal state was defined by the presence of at least one of the following symptoms appearing several times a week : ideas of reference , odd beliefs or magical thinking , unusual perceptual experiences , odd thinking and speech , or suspiciousness or paranoid ideation ; or by the appearance of one of the following symptoms for less than 1 week and resolving spontaneously : hallucinations , delusions , formal thought disorder , or gross disorganised or catatonic behaviour . exclusion criteria of note were previous treatment with antipsychotics , present or past diagnosis of a schizophrenic , schizophreniform , schizoaffective , delusional , or bipolar disorder , present or past diagnosis of a brief psychotic disorder with a duration of more than 1 week or within the last 4 weeks regardless of its duration , organic brain disease , and alcohol or drug dependence within the last 3 months . during the study , probands were seen approximately once a month . progression to first episode psychosis was operationally defined by cut - off points on positive and negative syndrome scale ( panss ) subscales of 4 or more on hallucinations , 4 or more on delusions and 5 or more on conceptional disorganisation or formal thought disorder for at least 7 days and meeting criteria for schizophrenia ( icd-10 : f20 ) or acute schizophrenia - like psychotic disorder ( icd-10 : f23.2 ) . nine common variants spanning the daoa / g72 gene were identified from the ucsc genome browser ( genome.cse.ucsc.edu ) . single - nucleotide polymorphism nomenclature is based on the ncbi dbsnp build 129 ( april 2008 , updated june 2008 ) . moreover , the traditional m nomenclature as described by chumakov et al . daoa / g72 variants were genotyped by allele discrimination using taqman technology ; 12.5 ng of dna was used for taqman snp genotyping assays on demand , employing the applied biosystems protocol . following the completion of pcr reactions , fluorescence was determined on a tecan 384 ultra reader ( tecan usa , durham , nc ) . the probes were labelled with carboxyfluorescein ( fam ) , with an excitation wavelength of 485 nm and an emission wavelength of 535 nm . vic - labelled probes employed an excitation wavelength of 535 nm and an emission wavelength of 590 nm . the influence of daoa / g72 variants on progression of symptoms was assessed by tests , employing spss version 14.0 ( spss , chicago , usa ) . if cells had an expected frequency of < 5 , monte carlo simulation based on 10,000 permutations with the start value 92,208,573 was done . the level of significance was set at p < 0.05 ( two sided ) . to correct for multiple testing , the spectral decomposition method was employed to estimate the number of effective loci [ 14 , 17 ] . haplotype block structure was determined by the solid spine of ld method as implemented in haploview ( broad institute , mass , usa ) . famhap 17 yields a global significance value of the haplotype analysis employing the likelihood ratio test , as well as individual p values for each haplotype . eighty - two probands were enrolled in the prospective study of early prodromal states . they were followed longitudinally and were assessed for progression to psychosis . twenty - one probands experienced a progression of symptoms to schizophrenia or first episode psychosis . the mean duration for progression to psychosis was 29.7 weeks ( range 286 weeks).the mean age of those patients progressing to psychosis was 24.2 5.0 years and they were thus on an average slightly younger than those probands who remained stable ( 27.0 7.6 years ; f = 3.139 ; p = 0.083 by welch test ) . the gender distribution did not differ between the probands who progressed and those who remained stable ( = 1.320 ; df = 1 ; p = 0.251 ) . we then queried whether the likelihood of progression to psychosis is related to variants of the daoa / g72 gene . thus , for rs1341402 , 20.4% of probands with the tt genotype and 24.1% of heterozygous probands ( tc genotype ) progressed to psychosis ( table 1 ) . in contrast , all homozygous cc probands progressed to psychosis . this effect was statistically significant ( = 12.348 , df = 2 , p = 0.002 ) . this finding remained significant after the correction for multiple testing with the spectral decomposition method [ 14 , 17 ] : with an effective number of independent marker loci of 5 , the significance threshold is p = 0.01 . a weaker effect was seen for another daoa / g72 variant , rs778294 ( m19 ) . for the aa genotype , 50% of probands progressed to psychosis ( 5 out of 10 probands with the aa genotype ) . the increased likelihood of progression for the aa genotype was nominally significant ( = 7.027 , df = 2 , p = 0.030).table 1progression of prodromal probands to psychosis stratified according to daoa / g72 genotypepolymorphismprogression to psychosis according to genotypep valuers1935058tt : 9/26 ( 34.6%)tc : 7/38 ( 18.4%)cc : 5/18 ( 27.8%)2.1820.336rs1341402tt : 10/49 ( 20.4%)tc : 7/29 ( 24.1%)cc : 4/4 ( 100%)12.3480.002**rs2391191 ( m15)gg : 11/34 ( 32.4%)ga : 7/36 ( 19.4%)aa : 3/12 ( 25.0%)1.5320.465rs1935062aa : 12/39 ( 30.8%)ac : 6/30 ( 20.0%)cc : 3/13 ( 23.1%)1.0840.582rs947267 ( m18)aa : 3/20 ( 15%)ac : 10/41 ( 24.4%)cc : 8/21 ( 38.1%)2.9320.231rs778294 ( m19)gg : 5/38 ( 13.2%)ga : 11/34 ( 32.4%)aa : 5/10 ( 50.0%)7.0270.030*rs778293 ( m22)aa : 7/29 ( 24.1%)ag : 10/44 ( 22.7%)gg : 4/9 ( 44.4%)1.9010.387rs3918342 ( m23)cc : 8/23 ( 34.8%)ct : 10/39 ( 25.6%)tt : 3/20 ( 15.0%)2.1980.333rs1421292 ( m24)tt : 8/24 ( 33.3%)ta : 10/40 ( 25.0%)aa : 3/18 ( 16.7%)1.5150.469probands progressing to psychosis / number of prodromal probands with a given genotype are shownsingle - nucleotide polymorphism nomenclature is based on the ncbi dbsnp build 129 ( april 2008 , updated june 2008)the traditional m nomenclature as described by chumakov et al the nominal significance level is set at p < 0.05 ( the significancethreshold is 0.01 after correction for multiple testing according to nyholt 2004/li and ji 2005 ) * * df = 2 ; monte carlo simulation yielded a significance of p = 0.004 * df = 2 ; monte carlo simulation yielded a significance of p = 0.035 progression of prodromal probands to psychosis stratified according to daoa / g72 genotype probands progressing to psychosis / number of prodromal probands with a given genotype are shown single - nucleotide polymorphism nomenclature is based on the ncbi dbsnp build 129 ( april 2008 , updated june 2008 ) the traditional m nomenclature as described by chumakov et al . the nominal significance level is set at p < 0.05 ( the significance threshold is 0.01 after correction for multiple testing according to nyholt 2004/li and ji 2005 ) * * df = 2 ; monte carlo simulation yielded a significance of p = 0.004 * df = 2 ; monte carlo simulation yielded a significance of p = 0.035 a linkage plot of the nine variants analysed in the 82 prodromal probands is shown in fig . 1 . markers rs1341402 and rs778294 are located in the same haplotype block 1 . given a d of 0.95 between markers rs1341402 and rs778294 , we constructed a two - marker haplotype comprising these two variants . haplotype analysis of this two - marker haplotype revealed two haplotypes that had an impact on the progression of prodromal patients to psychosis ( table 2 ) . thus , haplotype c a is present in 35.7% of prodromal patients who progressed to psychosis , but only in 17.1% of prodromal subjects who remained stable ( p = 0.046 ; bonferroni corrected p value ) . g is present in 72% of prodromal subjects who remained stable compared with 50% of prodromal patients who progressed to psychosis ( p = 0.031 ; bonferroni corrected p value ) . thus , haplotype c a increases the susceptibility for progression to psychosis , while haplotype t g is protective . the global p value for this haploype analysis was p = 0.047.fig . haplotype block structure was determined in the 82 prodromal subjects employing the solid spine of ld method as implemented in haploview . the location of daoa / g72 is also showntable 2haplotype analysis of the two - marker haplotype rs1341402-rs778294 of daoa / g72 in prodromal subjectshaplotype ( rs1341402-rs778294)frequency in patients progressing to psychosis ( % ) frequency in probands remaining stable ( % ) p valuet g50.072.00.031*t a14.39.93>0.1c a35.717.10.046*the global significance of this haplotype analysis was p = 0.047 ( likelihood ratio test)p values are bonferroni corrected * p value significant after bonferroni correction haplotype block structure of the nine analysed snps spanning the daoa / g72 gene . haplotype block structure was determined in the 82 prodromal subjects employing the solid spine of ld method as implemented in haploview . the location of daoa / g72 is also shown haplotype analysis of the two - marker haplotype rs1341402-rs778294 of daoa / g72 in prodromal subjects the global significance of this haplotype analysis was p = 0.047 ( likelihood ratio test ) p values are bonferroni corrected * p value significant after bonferroni correction when haplotype analysis was performed on all six markers contained in haplotype block 1 , no significant association of the six marker haplotypes , with progression to psychosis was seen ( p = 0.21 ; likelihood ratio test ) . similarly , haplotype analysis of the markers in block 2 was not significant ( p = 0.295 ; likelihood ratio test ) . next , we assessed whether the positive and negative syndrome scale ( panss ) scores at study entry were related to the long - term progression of the disorder . panss scores at study entry were available for 75 out of the 82 study probands . positive symptom scores at study entry related with later progression : those probands who would progress during the study period had an initial score of 13.53 compared with 10.64 for those probands who remained stable ( f = 9.030 ; p = 0.004 ) . thus , probands with later progression had an initial negative symptom score of 15.0 compared with those who remained stable ( 12.34 ; f = 5.690 ; p = 0.02 ) . for total panss scores , the respective values were 60.37 ( later progression ) and 52.04 ( stable probands ; f = 6.196 ; p = 0.015 ) . finally , we queried whether daoa / g72 marker rs1341402 , which had the greatest influence on progression , also had an influence on panss scores at study entry . we found that the probands with the cc genotype , all of whom later progressed to psychosis , had higher panss scores at study entry compared with the other genotypes ( table 3 ) . for example , probands with the cc genotype had a positive panss score of 16.5 compared with 11.08 for the tt / ct genotypes ( p = 0.025 ) . a similar difference was found for negative symptoms : 17.25 versus 12.77 ( p = 0.033 ) . moreover , similar findings were seen in the haplotype analysis ( table 4 ) . thus , probands with two copies of the risk - haplotype c a had higher positive symptom scores at study entry ( 16.50 ; p = 0.014 ) , and probands lacking the protective haplotype t g also had higher positive ( 14.56 ; p = 0.012 ) and total ( 63.22 ; p = 0.03 ) panss scores at study entry compared with the other haplotypes ( table 4).table 3panss scores at study entry stratified according to daoa / g72 rs1341402 genotypepanss scalesrs1341402 ccrs1341402 tt / tcp value*positive symptoms16.5 5.0611.08 3.550.025negative symptoms17.25 3.3012.77 4.270.033global panss score34.75 7.6329.47 7.030.171total panss score68.5 14.4753.33 12.580.047 * mann whitney test with two - sided monte carlo significance . 10,000 simulations with a start value of 2,000,000 were donetable 4panss scores at study entry stratified according to daoa / g72 two - marker haplotype rs1341402rs778294panss scaleshaplotype t gp valuehaplotype c ap value012012positive symptoms14.56 3.8110.39 3.4111.48 3.790.01211.35 3.7710.60 3.1216.50 3.810.014negative symptoms15.11 4.7812.09 4.2013.36 4.220.14812.67 4.1912.96 4.5117.25 3.300.128global panss score33.56 5.6428.21 7.5630.27 6.710.11629.43 7.1229.56 7.0134.75 7.630.357total panss score63.22 12.1850.7 12.4655.12 12.830.03053.46 13.1453.12 11.7668.50 14.480.075panss scores are given for probands with 0 , 1 or 2 copies of each haplotypep values were calculated by one - way anova panss scores at study entry stratified according to daoa / g72 rs1341402 genotype * mann whitney test with two - sided monte carlo significance . 10,000 simulations with a start value of 2,000,000 were done panss scores at study entry stratified according to daoa / g72 two - marker haplotype rs1341402rs778294 panss scores are given for probands with 0 , 1 or 2 copies of each haplotype p values were calculated by one - way anova this is one of the first prospective studies of prodromal subjects that assess the contribution of genetic variants to the risk of progression to psychosis . in a multicenter study , we have identified probands with a prodromal state and followed them longitudinally to assess the progression of symptoms . two variants of daoa / g72 had an important influence on the progression of the prodromal subjects . thus , all homozygous carriers of the c allele of rs1341402 experienced a progression of symptoms . similarly , 50% of probands homozygous for the a allele of rs778294 ( m19 ) had a progression of symptoms . therefore , these variants of daoa / g72 identify probands at a very high risk of progression , despite the overall risk of progression of the probands in the prodromal state being much lower : in the overall sample , 21 out of 82 study subjects experienced a transition , corresponding to 25.6% of prodromal subjects . the relative risk for progression to psychosis was significantly increased in homozygous carriers of the rs1341402 c allele ( rr = 4.588 , 95% ci = 2.1754.588 , p = 0.003 ) . the increase in relative risk for subjects homozygous for the rs778294 ( m19 ) a allele failed to reach significance ( rr = 2.250 , 95% ci = 0.9673.987 , p = 0.114 ) . these results indicate that genetic variants in the daoa gene may substantially potentiate the risk for subsequent psychosis in subjects with prodromal symptoms , and may thus aid in treatment decisions . our findings raise the question of the mechanism by which daoa / g72 determines the increased risk for prodromal probands to develop schizophrenia . the daoa / g72 gene has since its discovery been described as the activator of d - amino acid oxidase ( dao ) . given that this enzyme oxidises d - serine , which is an agonist at glutamatergic nmda receptors , daoa / g72 has been linked to the glutamate dysfunction hypothesis of schizophrenia . thus , g72 was found to promote dendritic branching in hippocampal neurons by way of expression in the mitochondria . this effect was seen only during the period of active dendritogenesis in synaptically immature neurons . moreover , the expression of g72 protein in the amygdala was confirmed . it is currently not known which effect of the daoa / g72 gene , namely the d - serine / glutamate pathway or the mitochondrial function pathway , is mainly responsible for the role of daoa / g72 in schizophrenia . both mechanisms could contribute to the structural and functional brain alterations that may precede the onset of schizophrenia [ 16 , 18 , 21 ] . a meta - analysis of published association studies found highly significant associations between variants of daoa / g72 and schizophrenia as well as less significant evidence of an association with bipolar disorder , although a very recent meta - analysis showed weaker effects . thus , it remained unclear whether the associated markers all reflect the same functional variation or , more likely , multiple functionally important variants are present in a large region encompassing daoa / g72 . notably , markers rs778293 ( m22 ) , rs3913842 ( m23 ) , and rs1421292 ( m24 ) which we have previously shown to be associated with schizophrenia show no linkage disequilibrium with the other markers analysed . this further suggests that there is more than one functional allele contributing to schizophrenia susceptibility in the daoa / g72 region . interestingly , these three markers are located more than 50 kb downstream of the coding region of daoa / g72 . the only other gene in this area is a hypothetical gene called g30 , which is located on the opposite strand and might provide antisense regulation of the daoa / g72 transcript . these three markers are , therefore , likely to exert effect independent from the other daoa / g72 markers . in our study , these three markers did not influence progression of prodromal probands , in contrast to the markers located in the putative promotor region or in the coding region of daoa / g72 . we then queried whether the variants relating to symptom progression of prodromal patients may be functional . because common promotor variants frequently have major influence on the expression level of the gene product , rs1341402 is an excellent functional candidate variant in the promotor region of g72 . this variant is situated 15 bp upstream of the 5 end of a protein - coding exon of g72 . as chumakov et al . note this variant is part of the consensus sequence of an acceptor splice site and thus could potentially alter the splicing of g72 . this variant is in high linkage disequilibrium with the mostly associated variant rs1341402 , with a d value of 0.95 ( fig . 1 ) . a variant that does not relate to symptom progression of prodromal subjects is rs2391191 ( m15 ) . this is a coding variant of g72 , leading to a lysine / arginine exchange . because this is a conservative amino acid exchange , it is not surprising that this variant does not relate to symptom progression in prodromal subjects . progression of prodromal probands to schizophrenia has previously been investigated with regard to the neuregulin 1 ( nrg1 ) gene . the same variant was implicated in the development of psychosis in high - risk subjects for psychosis . the sample size of the study group in these two studies was similar to the present study ; with 67 prodromal probands and 79 high - risk subjects investigated , respectively , compared with 82 prodromal probands in the present study . in conclusion , daoa / g72 is one of the first genes identified to influence the risk of symptom progression of schizophrenia prodromal probands . we anticipate that other genes acting either alone or in concert with daoa / g72 and nrg1 will be identified in the future to also influence the risk of progression to schizophrenia . these other genes may act in the same pathophysiological pathways as daoa / g72 , or operate in different pathogenic biochemical pathways . thus , it may be feasible in the future to establish a risk profile of prodromal probands according to their combination of risk alleles and identify those probands in whom a prophylactic treatment with neuroleptics or other as yet to be discovered treatment modalities should be instituted immediately to prevent progression to schizophrenia .

the genetic factors determining the progression of prodromal syndromes to first episode schizophrenia have remained enigmatic to date . in a unique prospective multicentre trial , we assessed whether variants at the d - amino acid oxidase activator ( daoa)/g72 locus influence progression to psychosis . young subjects with a prodromal syndrome were observed prospectively for up to 2 years to assess the incidence of progression to schizophrenia or first episode psychosis . of the 82 probands with a prodromal syndrome , 21 probands experienced progression to psychosis within the observation period . assessment of nine common variants in the daoa / g72 locus yielded two variants with the predictive value for symptom progression : all four probands with the rs1341402 cc genotype developed psychosis compared with 17 out of 78 probands with the tt or ct genotypes ( 2 = 12.348 ; df = 2 ; p = 0.002 ) . the relative risk for progression to psychosis was significantly increased in the cc genotype : rr = 4.588 ( 95% ci = 2.1754.588 ) . similarly , for rs778294 , 50% of probands with the aa genotype , but only 22% of probands with a gg or ga genotype progressed to psychosis ( 2 = 7.027 ; df = 2 ; p = 0.030 ) . moreover , haplotype analysis revealed a susceptibility haplotype for progression to psychosis . this is one of the first studies to identify a specific genetic factor for the progression of prodromal syndromes to schizophrenia , and further underscores the importance of the daoa / g72 gene for schizophrenia .

obesity is a risk factor for five of the top ten causes of death in the united states ( cardiovascular disease , cancer , stroke , diabetes , and kidney disease ) and associated conditions such as hypertension , dyslipidemia , and sleep apnea . although weight gain is often discussed as a matter of individuals behavioral choices ( i.e. , diet , physical activity ) , the importance of environmental factors , including working conditions , is increasingly recognized . some studies have examined weight gain as a consequence of job strain , but results have been inconclusive especially for women . two cross - sectional studies reported that high job strain was associated with greater body mass index ( bmi ) for women , but the few large - scale longitudinal studies have not provided consistent evidence for the link between job strain and subsequent weight gain or obesity incidence . these inconsistent findings may be partly explained by the possibility that associations of job strain with weight gain are not uniform across the range of baseline bmi : in particular , a tendency of initially overweight people to gain weight under stress , countered by a tendency of people who were initially on the lower spectrum of bmi to lose weight under stress . using the whitehall ii data , kivimki et al . demonstrated that during a 5-year follow - up period , among those who reported high job strain at baseline , men with low baseline bmi ( < 22 kg / m ) were more likely to lose weight while those with high baseline bmi ( > 27 block et al . found similar evidence of effect modification by baseline bmi for both job demands and job control for men ; but for women , the association was significant only for job demands . in both studies , job strain and its components ( i.e. , job control , job demands ) were measured only once at baseline , and therefore they could not account for potential change in the exposure during the follow - up period . in the japan work stress and health cohort study , ishizaki et al . measured job strain twice and examined the association between the chance in job strain and change in bmi over 6 years among 1371 women and 2200 men . overall they found no significant associations , and the pattern of bmi gain and job strain exposure was quite similar across different levels of baseline bmi . this study further examines the association between job strain and bmi change by including the possibility of effect modification by baseline bmi as well as changes in job strain exposure over time . more specifically , we expect that the greater the baseline bmi is , the greater the effect of job strain on bmi gain . we used data from over 52 000 working women participating in the nurses health study ii to evaluate the relation between change in job strain and change in bmi over a 4-year period . this is a prospective analysis of data from the nurses health study ii ( nhsii ) , an ongoing cohort study started in 1989 with 116 430 female registered nurses , age 2542 at the time of enrollment . every two years since 1989 , these women have been providing a wide range of information on health , lifestyle , health behavior , and work characteristics through self - administered questionnaires . job strain was measured twice , in 1993 and 1997 ; this analysis uses the 1993 data as baseline and 1997 data as follow - up . at baseline , 87 021 women participated ; of those , 71 694 ( 82.4% ) women provided data also at follow - up . those who did not provide follow - up data were more likely to be obese ( 19% vs. 15% ) , african american ( 3% vs. 1% ) , a current smoker ( 14% vs. 10% ) , and reported high job strain in 1993 ( 22% vs. 20% ) . we excluded 2568 who developed cancer before 2001 because cancer may have affected their weight before diagnosis . those who were not working ( n=12 593 ) or who were pregnant ( n=2358 ) at either baseline or follow - up were excluded . in addition , those with missing data on job strain ( n=500 ) or body weight ( n=3273 ) were excluded ; so were women with a bmi value less than 15 or greater than 50 ( n=314 ) . after exclusions , 52 656 women ( 73.4% of those who were present at baseline and follow - up ) were included in the current analysis . those who were included and those who were excluded had nearly identical distributions of all study variables , including the baseline bmi and job strain exposure . body mass index ( bmi , weight ( kg)/height ( m ) ) was calculated using the data on height reported at the enrollment in 1989 and the current body weight reported in 1993 and 1997 . in the analysis , we subtracted the 1993 bmi from the 1997 bmi and used the change as a continuous outcome . a previous validation study confirmed that the self - reported measures of anthropometry were highly correlated with technician - measured data ( r = 0.98 ) in a validation subsample . psychological job demands and job control , the components of job strain , were assessed using karasek s job content questionnaire . psychological job demands are measured with five items , and job control with nine items . we calculated the mean score for each scale after applying reverse coding to make higher scores indicate stronger endorsement for experiencing job demands and job control . cronbach s alpha in this sample was .73 for job demands , and .80 for job control . scores were dichotomized at the median in order to define high job strain as a combination of high job demands ( i.e. , above median ) and low job control ( i.e. , below median ) . women were categorized according to their baseline job strain and changes in job strain during follow - up in accordance with previous studies . information on age , race / ethnicity , smoking and pregnancy history was obtained at baseline in 1989 . pregnancy history and smoking were updated in each follow - up questionnaire . average sleep duration over a 24-hour period ( < 5 hours , 5 hours , 6 hours , 7 hours , 8 hours , 9 hours , 10 + hours ) was recalled retrospectively in 2009 for each decade of the woman s life since age 25 . diet was assessed in 1991 and updated every 4 years using a previously validated semi - quantitative food frequency questionnaire ( ffq ) . responses to this questionnaire were used to calculate the alternative healthy eating index ( ahei ) , a summary score of dietary factors previously related to chronic disease risk and bmi . physical activity and sedentary activities were assessed in 1991 and updated in 1997 using a previously validated questionnaire . time spent in moderate to vigorous activities ( 4 metabolic equivalents ; mets ) was multiplied by the met value of each activity and their sum was expressed as met - hours - per - week . the type of employment was asked both at baseline and follow - up with the following response options : inpatient / emergency room , outpatient / community nurse , operating room , nursing education , nursing administration , other nursing occupation , or non - nursing employment . the 56 262 women who met inclusion criteria for this analysis had no missing data on job strain , bmi or age at either baseline or follow - up . all other study variables had less than 4% of missing data , except for the alternative healthy eating index ( 6.3% ) and average hours of sleep ( 18.7% ) . all missing values were coded as missing indicators so that all observations were retained in the regression analysis . we first present the sample characteristics adjusted for age and stratified by change in job strain exposure over time . then we show descriptive statistics of the bivariate relationship between the change in bmi and the baseline bmi category . informed by nyberg et al.s findings from the pooled analysis of 13 cohort studies , we examine the association between change in job strain and change in bmi . we model the change in bmi over 4 years as a function of the change in job strain exposure , adjusted for baseline bmi , baseline bmi squared , and demographic covariates ( i.e. , age , race / ethnicity , pregnancy history before baseline , and the number of pregnancies between baseline and follow - up ) . the square term of baseline bmi was added to the model because the descriptive statistics showed a strong quadratic curve . to test the hypothesis that the association of job strain change with bmi change would depend on baseline bmi , we included the interaction term between job strain change and baseline bmi ( model 1 ) . for clearer interpretations of the interaction , baseline bmi was grand - mean centered , and the square term was calculated using the centered baseline bmi value . this makes the main effect of job strain to be applicable to the women whose baseline bmi was the average value ( = 25.2 kg / m ) , and the interaction effect to be applicable to all others . in model 2 , we added baseline behavioral covariates ( i.e. , physical activity , tv watching hours , sleep , and smoking ) and the type of the job . changes in these behavioral covariates were not included in our analysis because they could reflect coping behaviors under job stress . this is a prospective analysis of data from the nurses health study ii ( nhsii ) , an ongoing cohort study started in 1989 with 116 430 female registered nurses , age 2542 at the time of enrollment . every two years since 1989 , these women have been providing a wide range of information on health , lifestyle , health behavior , and work characteristics through self - administered questionnaires . job strain was measured twice , in 1993 and 1997 ; this analysis uses the 1993 data as baseline and 1997 data as follow - up . at baseline , 87 021 women participated ; of those , 71 694 ( 82.4% ) women provided data also at follow - up . those who did not provide follow - up data were more likely to be obese ( 19% vs. 15% ) , african american ( 3% vs. 1% ) , a current smoker ( 14% vs. 10% ) , and reported high job strain in 1993 ( 22% vs. 20% ) . we excluded 2568 who developed cancer before 2001 because cancer may have affected their weight before diagnosis . those who were not working ( n=12 593 ) or who were pregnant ( n=2358 ) at either baseline or follow - up were excluded . in addition , those with missing data on job strain ( n=500 ) or body weight ( n=3273 ) were excluded ; so were women with a bmi value less than 15 or greater than 50 ( n=314 ) . after exclusions , 52 656 women ( 73.4% of those who were present at baseline and follow - up ) were included in the current analysis . those who were included and those who were excluded had nearly identical distributions of all study variables , including the baseline bmi and job strain exposure . body mass index ( bmi , weight ( kg)/height ( m ) ) was calculated using the data on height reported at the enrollment in 1989 and the current body weight reported in 1993 and 1997 . in the analysis , we subtracted the 1993 bmi from the 1997 bmi and used the change as a continuous outcome . a previous validation study confirmed that the self - reported measures of anthropometry were highly correlated with technician - measured data ( r = 0.98 ) in a validation subsample . job strain is defined as high job demands combined with low job control . psychological job demands and job control , psychological job demands are measured with five items , and job control with nine items . we calculated the mean score for each scale after applying reverse coding to make higher scores indicate stronger endorsement for experiencing job demands and job control . cronbach s alpha in this sample was .73 for job demands , and .80 for job control . scores were dichotomized at the median in order to define high job strain as a combination of high job demands ( i.e. , above median ) and low job control ( i.e. , below median ) . women were categorized according to their baseline job strain and changes in job strain during follow - up in accordance with previous studies . body mass index ( bmi , weight ( kg)/height ( m ) ) was calculated using the data on height reported at the enrollment in 1989 and the current body weight reported in 1993 and 1997 . in the analysis , we subtracted the 1993 bmi from the 1997 bmi and used the change as a continuous outcome . a previous validation study confirmed that the self - reported measures of anthropometry were highly correlated with technician - measured data ( r = 0.98 ) in a validation subsample . job strain is defined as high job demands combined with low job control . psychological job demands and job control , psychological job demands are measured with five items , and job control with nine items . we calculated the mean score for each scale after applying reverse coding to make higher scores indicate stronger endorsement for experiencing job demands and job control . cronbach s alpha in this sample was .73 for job demands , and .80 for job control . scores were dichotomized at the median in order to define high job strain as a combination of high job demands ( i.e. , above median ) and low job control ( i.e. , below median ) . women were categorized according to their baseline job strain and changes in job strain during follow - up in accordance with previous studies . information on age , race / ethnicity , smoking and pregnancy history was obtained at baseline in 1989 . pregnancy history and smoking were updated in each follow - up questionnaire . average sleep duration over a 24-hour period ( < 5 hours , 5 hours , 6 hours , 7 hours , 8 hours , 9 hours , 10 + hours ) was recalled retrospectively in 2009 for each decade of the woman s life since age 25 . diet was assessed in 1991 and updated every 4 years using a previously validated semi - quantitative food frequency questionnaire ( ffq ) . responses to this questionnaire were used to calculate the alternative healthy eating index ( ahei ) , a summary score of dietary factors previously related to chronic disease risk and bmi . physical activity and sedentary activities were assessed in 1991 and updated in 1997 using a previously validated questionnaire . time spent in moderate to vigorous activities ( 4 metabolic equivalents ; mets ) was multiplied by the met value of each activity and their sum was expressed as met - hours - per - week . the type of employment was asked both at baseline and follow - up with the following response options : inpatient / emergency room , outpatient / community nurse , operating room , nursing education , nursing administration , other nursing occupation , or non - nursing employment . the 56 262 women who met inclusion criteria for this analysis had no missing data on job strain , bmi or age at either baseline or follow - up . all other study variables had less than 4% of missing data , except for the alternative healthy eating index ( 6.3% ) and average hours of sleep ( 18.7% ) . all missing values were coded as missing indicators so that all observations were retained in the regression analysis . we first present the sample characteristics adjusted for age and stratified by change in job strain exposure over time . then we show descriptive statistics of the bivariate relationship between the change in bmi and the baseline bmi category . informed by nyberg et al.s findings from the pooled analysis of 13 cohort studies , we examine the association between change in job strain and change in bmi . we model the change in bmi over 4 years as a function of the change in job strain exposure , adjusted for baseline bmi , baseline bmi squared , and demographic covariates ( i.e. , age , race / ethnicity , pregnancy history before baseline , and the number of pregnancies between baseline and follow - up ) . the square term of baseline bmi was added to the model because the descriptive statistics showed a strong quadratic curve . to test the hypothesis that the association of job strain change with bmi change would depend on baseline bmi , we included the interaction term between job strain change and baseline bmi ( model 1 ) . for clearer interpretations of the interaction , baseline bmi was grand - mean centered , and the square term was calculated using the centered baseline bmi value . this makes the main effect of job strain to be applicable to the women whose baseline bmi was the average value ( = 25.2 kg / m ) , and the interaction effect to be applicable to all others . in model 2 , we added baseline behavioral covariates ( i.e. , physical activity , tv watching hours , sleep , and smoking ) and the type of the job . changes in these behavioral covariates were not included in our analysis because they could reflect coping behaviors under job stress . table 1 presents demographic characteristics of the study participants . at the baseline of this analysis , the age of the participants ranged from 29 to 46 years old , with an average age of 38.5 years ( sd = 4.6 ) . the average bmi at baseline was 25.2 kg / m ( sd = 5.4 ) . while a large majority ( 71% ) reported no job strain at either time , 7% reported high strain at both times . inpatient , emergency room , and operating room nurses were more likely to report high job strain at both baseline and follow - up . at baseline , mean bmi was slightly lower for the job strain remained low group compared with other groups . over the course of four years , 50.8% of the women gained > 5 lbs ( 2.27 kg ) , 15.4% lost > 5 lbs , and 33.8% maintained the weight within 5 lbs . the average change in bmi was 0.87 kg / m , which translates as 5.25 lbs ( 2.38 kg ) increase for the woman of the average height in this sample ( 5 feet 5 inches ( 1.65 m ) ) . figure 1 shows the median change in bmi by baseline bmi category ( underweight : bmi<18.5 ; normal : 18.524.9 ; overweight : 2529.9 ; class i obesity : 30.034.9 ; class ii and iii obesity : 35.0 ) . those who were lower and higher ends of the baseline bmi distribution tended to have a smaller average increase than those who were in the middle of the distribution . table 2 summarizes the regression analysis results by showing the mean difference in bmi change associated with baseline bmi , baseline bmi squared , job strain change categories , and the interaction between baseline bmi and job strain change . model 2 had additional adjustments for job types at both baseline and follow - up as well as behavioral covariates at baseline ; however , as shown in table 2 , including these additional covariates did not materially change the results . the main effect of job strain change was statistically significant : on average , those who experienced job strain change in either direction ( decreased or increased ) had a greater bmi gain ( b=0.063 0.077 , p=0.01 0.03 ) than those with no job strain exposure at either time point . women who experienced job strain at both times also gained bmi compared to women who experienced job strain at neither time , and the bmi gain for them was greater than for women who reported high job strain at only one of the two time periods . the interaction between baseline bmi and job strain change was statistically significant ( p=0.015 ) , which indicates that the relationship between job strain change and bmi change depends on each woman s baseline bmi . the positive and significant regression coefficient for the interaction between baseline bmi and job strain that remained high ( b=0.022 , p=0.001 ) indicates that the greater the baseline bmi , the greater the bmi gain associated with job strain that remained high . the nature of this interaction is illustrated in figure 2 as marginal means of bmi change by job strain change and baseline bmi . the differences in bmi change among the four job strain change groups are not prominent in the under- and normal weight range . however , among women whose baseline bmi was in the overweight or obese range , the difference between job strain that remained low ( gray solid line ) and job strain that remained high ( black solid line ) is greater ; that is , those who experienced job strain at both times gained more bmi than those whose job strain remained low , and the greater the baseline bmi , the greater the bmi gain associated with job strain that remained high . those who experienced high job strain only once , either at baseline ( job strain decreased , gray dotted line ) or at follow - up ( job strain increased , black dotted lines ) had a greater bmi gain , as described in the previous paragraph . the magnitude of bmi gain associated with increased or decreased job strain is uniform across the full range of baseline bmi ( i.e. , interaction was not significant ) . as a sensitivity analysis , we conducted the same regression analysis with those who did not change job types ( e.g. , emergency room nursing , operating room nursing , nursing management ) between baseline and follow - up because taking a different type of job could be a stressful event that impacts bmi change regardless of change in job strain that may or may not accompanied the job change . the results were similar to the full sample results , both the main effect and interaction , which suggest that change in job types did not impact the association between job strain and bmi change . this study examined the association between the change in job strain exposure and the change in bmi over 4 years among more than 52 000 working women . our specific focus was to test whether the effect of job strain on bmi gain would be greater for women whose baseline bmi was at the higher end of the spectrum . the analysis indicated that experience of job strain at baseline , follow - up , or both times were associated with a greater bmi gain . moreover , the higher the baseline bmi , the greater the bmi gain associated with job strain that remained high . this study clarifies previous inconsistent findings for women by using a large sample size and multiple measurements of both job strain and bmi as well as by accounting for a quadratic relationship between baseline bmi and bmi change over time . our data showed that the bmi gain associated with constantly high job strain is greater for women who had a higher bmi at baseline . three previous studies took similar approaches to examining the association between bmi change and job strain change , yet reported different findings . . followed 722 women over 9 years to examine the interaction between baseline bmi and a baseline measure of job demands . their results were similar to ours : the interaction was significant in that the bmi increase associated with job strain was greater for initially obese women than for normal and overweight women . a limitation of the block study is that because they measured job demands only once at baseline , the bmi change after 9 years was averaged across groups who may have experienced job demand changes during the 9 year follow - up period . ishizaki et al.s study of japanese workers had two measurements of job strain 6 years apart and categorized the change in job strain the same way we did in this study . among the 1370 women , however , ishizaki et al . did not find support for baseline bmi moderating the association between job strain change and bmi change . this could be due to the smaller sample size , the smaller proportion of overweight and obese women ( 14% in this japanese sample ) , and also a further loss of statistical power by categorizing the baseline bmi distribution . eek and stergren accounted for the baseline bmi in quartiles while they examined the associations between job strain change and bmi change over 5 years with nearly 5500 women in the scania public health cohort study . although they did not include the interaction term , their findings for middle - aged women are similar to the main effect of job strain change in our analysis . while cross - sectional studies and some prospective studies with job strain measured only at the baseline reported inconsistent findings , the current study together with eek and stergren provides stronger evidence for the link between job strain and women s weight gain over time . both physiological and behavioral paths from job strain exposure to weight gain have been suggested . exposure to chronic stress , including job strain , results in neuroendocrine dysregulation , such as increased cortisol production and disruption of diurnal cortisol curve , which is associated with greater bmi and waist circumference as well as higher intra - abdominal fat . this association is especially prominent among women , and there has been a report suggesting that obese women may be more vulnerable to stress - induced disturbance of cortisol production , which offers potential explanation for our findings . in addition to these physiological changes , exposure to job strain may also lead to unhealthy behaviors , but so far research findings are inconsistent . our results suggest that one explanation for previous inconsistent findings may be those with higher bmi being more prone to developing unhealthy behavioral habits under stress . although its methodological shortcomings have been pointed out , a recent pooled longitudinal analysis of 4 european studies showed that among those who did not have a healthy lifestyle at the baseline , high job strain exposure was associated with a lower likelihood of adopting healthy behaviors . important future research questions include whether job strain leads to unhealthy behaviors and whether the link is stronger for overweight and obese individuals . in our sample , women who reported high job strain only at baseline or follow - up had on average greater bmi increase than those who reported no job strain at either time . eek and stergren found a similar result . if the women had been exposed to high job strain for a long time before the baseline , the effect may linger even if the exposure diminished during the study period . those who reported high job strain only at baseline were more likely to have changed the types of jobs during the study period than those who reported no strain at both times ( 46.3% vs. 37.3% ) . potentially as a result of the job change , their job strain declined , but other job characteristics may have changed as well . for example , it is common for nurses who are new to the workplace to take less desirable shifts , which might result in weight gain . moreover , changing jobs in and of itself could be stressful , especially if the job change was not voluntary . however , our sensitivity analysis showed that job change was not a likely factor to alter the association between job strain and bmi change in this sample . this study has a number of strengths such as a large sample size , well - established measure of job strain , and prospective design . nevertheless , the interpretation of the results should take into account the following data limitations . obesity is strongly patterned by socioeconomic status ( ses ) , often measured by income , education , and occupation . for this analysis , household income was not available , although the participants were all registered nurses with roughly similar educational backgrounds . examining weight change in a socioeconomically somewhat homogeneous sample ( i.e. , a single - occupation sample ) limits generalizability of our findings ; however , single - occupation samples have its advantages as well because the homogeneous sample helps to separate the effect of job strain from that of ses . we did control for the type of the nursing job , which could be a rough proxy to ses ( e.g. , nurse administration , nursing education , er nurse ) . in our sample , both the job control and demands scales had a full - range , normal distribution . moreover , the mean job demands and control scores differ significantly across different types of nursing jobs : the mean job control score was the highest for nursing educators and administrators and the lowest for er and or nurses , whereas the mean job demands score was the highest for er and or nurses as well as nurse administrators . this mitigates the concern that a single occupation sample may limit the variability of the job demands and job control scores . although the job type had a significant association with bmi change ( data not shown ) , including the variable did not change the regression coefficients for the job strain main effect or interaction . the same was true for the sleep variable . because sleep was recalled retrospectively in 2009 , the validity of the data is questionable ; however , the presence or absence of this variable did not change the main results . all study variables , including height and weight , were collected via self - report , which is subject to various biases . in a validation subsample , the self - reported measures of anthropometry were highly correlated with technician - measured data ( r = 0.98 ) . in addition , because this analysis used the change in bmi as the dependent variable , the potential downward bias is unlikely to have affected our results . finally , we followed the standard formulation of job strain , which involves dichotomizing the job control and demands scores at the sample median score . this could lead to misclassification of exposure to those who scored on the median score and biases the results toward the null . we acknowledge this limitation , but in order to make comparisons with other studies easier , we followed this widely accepted procedure . future research should also examine other types of job stress such as effort - reward imbalance . this analysis of over 52 000 working women s weight change over 4 years showed that the association between job strain and weight gain is stronger for those with higher baseline bmi . this finding is directly applicable to over 3 million registered nurses in the united states . we contribute to job stress research by providing methodologically sound analysis of women s data in a sample large enough to detect interactions between variables . our findings identify that women with higher bmi are more vulnerable to weight gain if they were exposed to high job strain . future research focusing on mediating mechanisms between job strain and weight gain should explore the possibility of differential responses to job strain by initial weight . finally , our findings underscore the importance of an integrated approach toward obesity prevention in the workplace between health promotion ( traditionally focuses on individual health behaviors ) and occupational safety ( addresses changes in work characteristics , including job stressors , to reduce adverse health impacts ) as the national institute for occupational safety and health ( niosh ) advocates in its total worker health program ( http://www.cdc.gov/niosh/twh/ ) . occupational safety and health practitioners and health promoters should be aware of the different vulnerability to work stress by bmi levels , and intervention resources should be allocated more toward those who are already overweight and exposed to work stress .

objectivesthe relationship between job strain and weight gain has been unclear , especially for women . using data from over 52 000 working women , we compare the association between change in job strain and change in bmi across different levels of baseline bmi.subjects/methodswe used data from participants in the nurses health study ii ( n=52 656 , mean age = 38.4 ) , an ongoing prospective cohort study . using linear regression , we modeled the change in bmi over 4 years as a function of the change in job strain , baseline bmi , and the interaction between the two . change in job strain was characterized in four categories combining baseline and follow - up levels : consistently low strain [ low at both points ] , decreased strain [ high strain at baseline only ] , increased strain [ high strain at follow - up only ] , and consistently high strain [ high at both points ] . age , race / ethnicity , pregnancy history , job types , and health behaviors at baseline were controlled for in the model.resultsin adjusted models , women who reported high job strain at least once during the four - year period had a greater increase in bmi ( bmi=0.060.12 , p<0.05 ) than those who never reported high job strain . the association between the change in job strain exposure and the change in bmi depended on the baseline bmi level ( p=0.015 for the interaction ) : the greater the baseline bmi , the greater the bmi gain associated with consistently high job strain . the bmi gain associated with increased or decreased job strain was uniform across the range of baseline bmi.conclusionswomen with higher bmi may be more vulnerable to bmi gain when exposed to constant work stress . future research focusing on mediating mechanisms between job strain and bmi change should explore the possibility of differential responses to job strain by initial bmi .

cryptosporidium spp . are ubiquitous intracellular protozoan parasites that affect wide range of vertebrates like human and livestock . oocysts are infective stage of life cycle released via feces from infected hosts to environment in large amount . they are resistant to various environmental alterations and remain infective for long period in appropriate condition like surface of water and moist soils ; thus , they are liable for transmission of disease to animals and humans [ 2 , 3 ] . cryptosporidium as an opportunistic agent infects the alimentary tract of hosts and have wide spectrum of clinical symptoms in human ranging from self - limiting and asymptomatic in immunocompetent individuals to severe and life - threatening in immunocompromised persons [ 2 , 4 , 5 ] . for first time , human cryptosporidiosis was diagnosed in individuals who had severe watery diarrhea during 1970s . during past decades , numerous evidences have confirmed that small mammals such as rodents are considered as carrier or reservoir for several infectious agents . also role of rodents in transferring the helminth and protozoan infections are clear [ 710 ] . surveys in different zones of iran indicate the existence of various rodent species such as the house mice ( mus musculus ) , the black rat ( rattus rattus ) , the brown rat ( rattus norvegicus ) , and the himalayan rat ( rattus pectoris ) in country [ 8 , 10 , 11 ] . based on our previous survey in ahvaz district , southwest of iran , rodents could be considered as potential source of toxoplasma gondii infection for definitive hosts . small subunit - rrna ( ssu 18s rrna ) gene is being utilized for identification of cryptosporidium spp . according to ssu - rrna gene sequencing , at least 20 cryptosporidium species have been identified and more than sixty cryptosporidium genotypes have undeterminate status till now . approximately eight cryptosporidium species / genotypes including c. parvum , c. hominis , c. felis , c. meleagridis , c. ubiquitum , c. viatorum , c. canis , and c. cuniculus are the main species responsible for human infections , although c. hominis and c. parvum account for over 90% of human cryptosporidiosis worldwide [ 1 , 2 ] . numerous epidemiological surveys have been performed throughout the globe and prevalence of cryptosporidium infection in rodents was highly varied from 63% in uk , 32.8% in united states of america ( usa ) , 24.3% in italy , 7.6% in maryland , 11.5% in china , 25.8% in philippines , 8.2% in northern australia , 0% in northeast of iran ( mashhad city ) , and 27.3% in north of iran ( tehran city ) . due to lack of reports about cryptosporidium infection in rodents of southwest of iran till now , current study was aimed to determine the prevalence and molecular characterization of cryptosporidium spp . in this region . ahvaz city , capital of khuzestan province which is located in the southwest of iran ( 3150 n and 49 11 e ) , is ranked as the 7th largest city throughout the country and based on the latest census , its population was calculated at 1,395,184 in 352,128 families . weather temperature is highly variable throughout the year so that in summer temperature exceeds 50c whereas in winter it falls to 5c . also , annual average rainfall is approximately 230 mm . there is high density of rodents species and rat - man - domestic animals adjacency is remarkable in ahvaz city [ 9 , 11 ] . , mostly preyed on by cats and dogs , and , hence , could spread parasitic infections via other animals . ahvaz city initially divided into five geographical locations ( north , west , south , east , and center ) . in each location sherman live traps were placed outdoor at the entrance of rodent colonies and baited with favorite piece of foods ( including cucumber pieces , tomato , and roasted almonds ) . overall , 100 rodents were collected from three different species ( 6 m. musculus , 73 r. norvegicus , and 21 r. rattus ) . eventually trapped rodents were gathered and transferred to department of medical parasitology of ahvaz jundishapuruniversity of medical sciences . the trapped rodents were anaesthetized by putting the live traps in a thick transparent polythene bag and then a cotton swab was soaked in ether and placed near their nose . afterwards , the anaesthetized rodents were dissected and fecal samples gathered from rectum or large intestinal section . after sugar flotation ( sg 1.266 ; 128 g sucrose and 100 distilled water ) and modified ziehl - neelsen staining , the samples were examined to find the cryptosporidium spp . finally the samples were maintained at 2.5% potassium dichromate ( k2cr2o7 ) and kept in refrigerator ( 1 - 2 weeks ) until dna was extracted . dna extraction procedure was performed using qiaamp dna stool mini kit ( qiaamp dna stool mini kit , usa ) based on the manufacturer 's guideline . the extracted dna was kept in 20c for next tests . for nested - pcr method , we used two specific primers to detect 18s rrna gene whose length of produced fragments was 1325 bp and 830 bp . this dual stages technique was run using different primers that primary and secondary stages primers were as following : [ 4 , 20 , 23 , 24 ] . first stage primers forward ( f1 ) : 5-ttctagagctaatacatgcg-3reverse ( r1 ) : 5-cccatttccttcgaaacagga-3 forward ( f1 ) : 5-ttctagagctaatacatgcg-3 reverse ( r1 ) : 5-cccatttccttcgaaacagga-3 second stage primers forward ( f2 ) : 5-ggaagggttgtatttattagataaag-3reverse ( r2 ) : 5-ctcataaggtgctgaaggagta-3finally pcr products after loading on 1.5% agarose gel and electrophoresis for 1.5 hours were stained with ethidium bromide and then visualized under uv light using gel doc device ( uvidoc , gel documentation system , cambridge , uk ) . forward ( f2 ) : 5-ggaagggttgtatttattagataaag-3 reverse ( r2 ) : 5-ctcataaggtgctgaaggagta-3 rflp assay was done to determine the cryptosporidium spp . sspi and vspi endonuclease enzymes were employed for species recognition and genotyping , respectively , based on manufacturer 's protocol as earlier described . eventually visualization of the digested products was carried out under uv transilluminator after 1.5% agarose gel electrophoresis and ethidium bromide staining [ 20 , 26 ] . the nested - pcr positive samples were purified using bioneer kit corporation ( korea ) and were sequenced by the same corporation . nucleotide sequence data reported in current article are available in the genbank at accession numbers ab986579 , ab986580 , and ab986581 . three out of 100 samples were detected as positive for cryptosporidium spp . using modified ziehl - neelsen staining under light microscope . in addition , nested - pcr was done and showed 830 bp band which confirmed only 3 samples as cryptosporidium spp . so , overall prevalence of cryptosporidium infection in rodents of ahvaz city was calculated at 3% using both methods . all positive samples belonged to r. norvegicus ( 3/73 ) and from m. musculus ( 0/6 ) and r. rattus ( 0/21 ) no positive cases were observed . in order to determine the genotype , pcr - rflp technique using sspi and vspi restriction enzymes was utilized . with sspi restriction enzyme , three cuttings were seen in locations of 108 , 258 , and 421 bp visible on agarose gel after electrophoresis . also after using from vspi enzyme , three cuttings happened in locations 104 , 106 , and 600 bp ( figure 2 ) and indicate c. parvum pattern . the amplified 18s rrna genes from pcr - rflp products of three c. parvum were sequenced . after submitting the results to the ddbj / genbank at accession numbers ab986579 , ab986580 , and ab986581 , the nucleotide sequences were aligned with nucleotide sequences of c. parvum certified in genbank with accession number ab986578 ( figure 3 ) . based on findings , c. parvum was recognized as infectious agent of ahvaz rodents . rodents with maintaining the pathogens transmission cycle in surrounding regions play a key role in morbidity and mortality of human and livestock especially in areas with dense population . the routine method for diagnosis of cryptosporidium spp . is based on direct observation of oocysts in stool specimens using optical microscope . since this method has low sensitivity and needs expertise , also it is unable to distinguish between different species of parasites ; thus , molecular techniques like pcr have been used and developed recently . pcr - based techniques with high sensitivity , specificity , and rapidly features are capable of differentiating among species and genotypes in different specimens , that is , water , stool , and animal or human tissues , although they are expensive . previously , genotyping of cryptosporidium spp . has been done successfully by nested pcr - rflp method according to ssu - rrna ( 18s rrna ) gene [ 4 , 18 , 20 , 24 , 26 , 27 ] . according to previous epidemiological reports , prevalence and rate of infection of cryptosporidium spp . in rodents ranged from 0% to 63% and present investigation is the first report which focused on the prevalence and molecular detection of cryptosporidium spp . in wild rodents of ahvaz city , southwest of iran . we found 3% ( 3/100 ) prevalence by both direct microscopic observations with ziehl - neelsen staining and nested - pcr ( 830 bp ) , while , in bahrami et al . survey in tehran city ( capital of iran ) using these methods , the prevalence was reported at 13% and 27.3% , respectively . also all positive samples identified c. parvum by pcr - rflp using sspi and vspi restriction enzymes and were confirmed by sequencing that is in agreement with our study . differences between tehran and ahvaz findings could be justified with sample size , location of sampling , cities population , hygienic conditions , sewage systems , and so forth . it is worth mentioning that tehran as capital of iran is the most crowded city with highest density population over the country which is multifold than ahvaz . in mashhad city ( northeast of iran ) , prevalence of cryptosporidium spp . with 0% was lower than our study ( 3% ) , while higher prevalence was reported from usa 32.8% , china 11.5% , australia 8.2% , philippines 25.8% , and tehran city ( north of iran ) 27.3% . based on current study , 18s rrna gene of c. parvum was detected only in r. norvegicus ( 3/73 ) and from r. rattus and m. musculus no positive samples were isolated . it should be mentioned that r. norvegicus is the most abundant rodent in southwest of iran ( 73/100 ) and highest prevalence and rate of infection ( 4.1% , 3/73 ) allocate to this species which corresponds to previous investigations [ 9 , 11 ] . the obtained results of amplifying 18s rrna gene after sequencing were submitted to the ddbj / genbank under accession numbers ab986579 , ab986580 , and ab986581 and then compared with ab986578 as control . lv and colleagues studied the wild , laboratory , and pet rodents ( totally 723 rodents from 18 species ) in china and prevalence of cryptosporidium spp . was reported at 11.5% and c. parvum , c. muris , c. andersoni , and c. wrairi were identified , as well . prevalence in wild , laboratory , and pet rodents was 6.8% , 1.9% , and 21.8% , respectively . in another survey by ng - hublin et al . on 194 wild rats and mice from five species including the asian house rat ( r. tanezumi ) , the rice - field rat ( r. argentiventer ) , the pacific rat ( r. exulans ) , r. norvegicus , and m. musculus in philippines , overall prevalence was reported at 25.8% . in addition , based on sequencing and phylogenetic analysis of 18s rrna gene and actin locus , c. muris , c. parvum , c. scrofarum , c. suis - like genotype , and rat genotypes i iv were recognized . in current research , only c. parvum was identified using 18s rrna gene sequencing . throughout the world c. parvum have been isolated from numerous animals such as calves or cattle , some ruminants ( goats and sheep ) , horses , pigs , alpacas , some carnivores ( gray wolves and dogs ) [ 32 , 33 ] , reptiles , and rodents ( rat , hamster , mice , nutria , chipmunk , squirrel , and capybara ) [ 13 , 16 , 17 , 20 , 3437 ] . in addition predominant cryptosporidium species in iran is c. parvum that has been verified frequently , for example , 73.3% in humans and animals , 83.3% in tehran ( human samples ) , 100% in rodents of tehran , 100% in cattle of ilam , and 68.8% in ahvaz ( in immunocompromised patients and children ) . in rafiei investigation in southwest of iran on immunocompromised patients and children ( kidney transplant recipients , persons with hematological malignancies , hiv+ patients , and children less than 5 years old ) , 390 stool specimens were collected and examined . moreover , 3 different species were identified using pcr - rflp based on 18s rrna gene including 11 c. parvum , 4 c. hominis , and 1 c. meleagridis which was in consistent with our results . according to rafiei survey and our study , c. parvum was identified as the most common species in both rodents and individuals in ahvaz city . in past studies c. parvum transmission from rodents to human the present investigation was based on sampling of limited rodents species in limited areas . in future , for better understanding of exact burden of cryptosporidium spp . and genetic diversity , studies should be designed on wide spectrum of both wild and pet rodents ( rats , hamsters , mice , rabbits , etc . ) in vast regions like throughout the khuzestan province . present paper was the first report which focused on the prevalence and molecular characterization of cryptosporidium spp . in wild rodents of ahvaz city , southwest of iran ; that showed rate of infection in r. norvegicus is remarkable . the results can help public health care to pursue new strategies ( environmental sanitation , increasing the hygienic condition , increasing the hygienic condition , etc . ) . in future , adopting a suitable strategy for control and combat with rodents in order to decrease human cases is necessary and should be continued .

background . rodents could act as reservoir for cryptosporidium spp . specially c. parvum , a zoonotic agent responsible for human infections . since there is no information about cryptosporidium infection in rodents of ahvaz city , southwest of iran , hence , this survey was performed to determine the prevalence and molecular characterization of cryptosporidium spp . in this region . materials and methods . one hundred rodents were trapped from different regions of ahvaz city . intestine contents and fecal specimens of rodents were studied using both microscopy examination to identify oocyst and nested - polymerase chain reaction ( pcr ) technique for 18s rrna gene detection . eventually restriction fragment length polymorphism ( rflp ) method using sspi and vspi restriction enzymes was carried out to genotype the species and then obtained results were sequenced . results . three out of 100 samples were diagnosed as positive and overall prevalence of cryptosporidium spp . was 3% using both modified ziehl - neelsen staining under light microscope and nested - pcr ( 830 bp ) methods . afterwards , pcr - rflp was performed on positive samples and c. parvum pattern was identified . finally pcr - rflp findings were sequenced and presence of c. parvum was confirmed again . conclusions . our study showed rodents could be potential reservoir for c. parvum . so an integrated program for control and combat with them should be adopted and continued .

an avulsion fracture of the posterior cruciate ligament accounts for approximately 340% of all acute knee joint injuries of which 35% are displaced fractures.1 nonsurgical treatment has a high incidence rate of nonunion or malunion and can cause loss of ligament function , leading to further knee instability and traumatic arthritis.23 surgical methods include arthroscopic surgery or open reduction and internal fixation . arthroscopic surgery is less invasive ; however , because the end point of the posterior cruciate ligament is located deep on the posterior part of the tibial plateau , an arthroscopic operation would be relatively difficult and may result in avulsion of the posterior cruciate ligament , loose fracture suturing and problems in postoperative relaxation , which causes some patients to exhibit limited knee range of motion after the surgery.4 as open reduction and internal fixation have a clear surgical field exposure , the fractures can be reduced accurately under direct vision , with reliable fixation and early rehabilitation can be performed.5 the use of internal fixation materials such as hollow screws , absorbable screws , tension bands and sutures have many limitations ; defects such as insufficient fixation strength have also been reported , leading to fractures occurring easily.6 a stellate steel plate has a ratchet gear like design . each ratchet was designed based on a block structure , with a fixed length of gap between the gears , which has the advantages of firm fixation . this retrospective study analyzed avulsion fracture of the posterior cruciate ligament that were treated with a stellate steel plate at our hospital . 14 patients ( 9 men and 5 women ) with displaced avulsion fracture of the tibial insertion of the posterior cruciate ligament treated with a stellate steel plate between june 2009 and june 2011 were included in this study . the left knee was injured in five patients and the right knee was injured in nine patients . injury factors included traffic accident injuries in six patients , fall injuries in four patients , strike injury in one patient and sports related injuries in three patients . three patients had meniscus injuries and two patients had articular cartilage injuries ; no nerve injuries were reported in any patient . the fracture fragment diameter was < 5 mm in seven patients ; the fracture fragment diameter was 510 mm in four patients and the fracture fragment diameter was > 10 mm in three patients [ table 1 ] . all the patients were previously healthy had closed fractures , without any other injury except knee injuries and they were consecutively diagnosed and treated by the same group of surgeons . clinical details of patients all the patients underwent open reduction and internal fixation with a stellate steel plate ( depuy , raynham , ma 02767 , usa ) . the mean duration from the injury to surgery was 8.3 days ( range 615 days ) . after combined spinal epidural anesthesia , the operation was performed under tourniquet control in the prone position . the knees were flexed to approximately 20. using the modified burks - shaffer approach,7 a 2 cm curved incision was made from the transverse skin crease of the popliteal fossa inwardly exploring along the inner edge of the medial head of the gastrocnemius muscle 79 cm away and bluntly dissecting between the medial head of the gastrocnemius muscle and semimembranosus to arrive at the posterior joint capsule . the medial head of the gastrocnemius muscle and the popliteal vascular nerve were pulled to one side to expose the semimembranosus muscle insertion , popliteal muscle and posterior joint capsule . the joint capsule was split longitudinally along the joint space and complete removal of the hematoma and soft tissue was performed to reveal the posterior cruciate ligament and bone fragments in order to clean up the broken ends of the fracture and to achieve reduction of the fracture . during the operation , the type of avulsed bone block was determined . if the fracture block was > 5 mm , the patient underwent temporary kirschner wire fixation after reduction . the steel plate was sheathed in the kirschner wire and it was hammered to make the sharp steel teeth embed into the bone . if the fracture block was < 5 mm or crushed bones were observed , a simple fixation was first performed by using a nonabsorbable suture ( ethibond number 0 ) , and the steel teeth were then embedded at the binding site of the bone and ligament that underwent screw fixation . patients were encouraged to perform quadriceps contraction exercises in bed on the second postoperative day , knee flexion and extension 3 weeks later , walking was allowed with crutches after one month and walking without crutches after 2 months . patients were followed up at 2 weeks , 4 weeks , 3 months , 6 months , 9 months , 12 months , 18 months , 24 months and 32 months after the operation . the followup included radiographic examination , analysis of the patient 's knee joint activity , evaluation of pain and questions concerning subjective patient feelings . the score consists of eight factors including limp , joint locking , pain , stair climbing ability , usage of a walking aid , edema , squatting ability and stability . the maximum score is 100 points , in which a score of 95100 points is excellent , a score of 8494 points is good , a score of 6583 points score is medium , and a score of < 65 points is poor . after combined spinal epidural anesthesia , the operation was performed under tourniquet control in the prone position . the knees were flexed to approximately 20. using the modified burks - shaffer approach,7 a 2 cm curved incision was made from the transverse skin crease of the popliteal fossa inwardly exploring along the inner edge of the medial head of the gastrocnemius muscle 79 cm away and bluntly dissecting between the medial head of the gastrocnemius muscle and semimembranosus to arrive at the posterior joint capsule . the medial head of the gastrocnemius muscle and the popliteal vascular nerve were pulled to one side to expose the semimembranosus muscle insertion , popliteal muscle and posterior joint capsule . the joint capsule was split longitudinally along the joint space and complete removal of the hematoma and soft tissue was performed to reveal the posterior cruciate ligament and bone fragments in order to clean up the broken ends of the fracture and to achieve reduction of the fracture . during the operation , the type of avulsed bone block was determined . if the fracture block was > 5 mm , the patient underwent temporary kirschner wire fixation after reduction . the steel plate was sheathed in the kirschner wire and it was hammered to make the sharp steel teeth embed into the bone . if the fracture block was < 5 mm or crushed bones were observed , a simple fixation was first performed by using a nonabsorbable suture ( ethibond number 0 ) , and the steel teeth were then embedded at the binding site of the bone and ligament that underwent screw fixation . patients were encouraged to perform quadriceps contraction exercises in bed on the second postoperative day , knee flexion and extension 3 weeks later , walking was allowed with crutches after one month and walking without crutches after 2 months . patients were followed up at 2 weeks , 4 weeks , 3 months , 6 months , 9 months , 12 months , 18 months , 24 months and 32 months after the operation . the followup included radiographic examination , analysis of the patient 's knee joint activity , evaluation of pain and questions concerning subjective patient feelings . the score consists of eight factors including limp , joint locking , pain , stair climbing ability , usage of a walking aid , edema , squatting ability and stability . the maximum score is 100 points , in which a score of 95100 points is excellent , a score of 8494 points is good , a score of 6583 points score is medium , and a score of < 65 points is poor . two and half months after the operation , bone fracture healing was observed in 10 patients who had a negative posterior drawer test . six months after the operation , a radiographic image showed healed fractures and 12 patients achieved anatomic reduction of the fracture [ figure 1 ] . anteroposterior and lateral x - rays of knee joint showing avulsion fracture of posterior cruciate ligament fixed with stellate steel plate knee joint activity returned to normal in all , ( range of motion of 0130 ) , with no knee joint stiffness or knee instability . at the final followup , the mean lysholm - tegner knee function score was 97.1 3.7 . the posterior cruciate ligament is divided into the following two bundles : the anterolateral bundle and the posteromedial bundle.91011 the anterolateral bundle and the posteromedial bundle play a role in knee flexion and extension activities , respectively and they have a synergistic effect.12 a biomechanical experiment showed that the in vitro tensile strength of the posterior cruciate ligament is twice that of the anterior cruciate ligament.13 the posterior cruciate ligament is one of the most important structures for knee joint stability and its main role is to prevent tibial posterior displacement as well as external rotation and varus of the tibia.14 avulsion fracture of the posterior cruciate ligament is more common in sports related injuries which account for approximately 40% of these injuries.15 the mechanism of injury is mainly caused by direct impact of the tibial tuberosity and posterior tibial displacement , during which the posterior cruciate ligament is tensed . if anatomical reduction of the displaced fracture is not achieved , proper fracture union will not occur and the posterior cruciate ligament can loss function due to its misshapen form , leading to knee instability and traumatic arthritis as well as other problems,16 and this may seriously influence patient prognosis . therefore , many researchers such as nicandri et al.,17 believe that , regardless of the size of the displacement , an operation is necessary for the treatment of avulsion fractures of the posterior cruciate ligament . chen et al.4 believes that the arthroscopic surgery technique has improved quickly over the past 20 years , has little trauma and can effectively suture and fix posterior cruciate ligament avulsion fractures . however , some patients still have limited postoperative knee joint range of motion , which is related to postoperative posterior cruciate ligament relaxation and operation scarring . in addition , because the posterior cruciate ligament is located lateral to the tibial plateau and the location is deep , arthroscopic surgery is relatively difficult . owing to the fact that open reduction is a simple operation , with clear exposure , leading to open fracture reduction , reliable fixation and accurate and early exercise of joint function , it has become the first choice of most physicians.5618 commonly used internal fixations include hollow screws , absorbable screws , wires , suture , among others.19 the application of hollow screws and joint gaskets to cancellous bone screws achieved better fixation and could obtain better postoperative knee range of motion in acute injuries that had bigger fracture fragments , but it was not suitable for smaller fragment damages . a screw might result in small or crushed bone blocks and there are other problems such as not being firmly fixed , easy rotation , easily getting loosened and extracted and the possibility of entering into the joint cavity , which means that patients can not do functional exercises early . the strength of a wire suture fix during recovery of a fracture is poor , as the fracture is easily displaced again and the postoperative breaking time is longer , leading to limited knee range of motion . the absorbable screw has weak antirotation ability and it should be placed next to a screw with a larger diameter to meet the need of fixed strength ; it is not suitable for fixation of small or crushed fractures . nicandri et al.17 used an atlanto - odontoid dentoid plastic washer and screw fixation for avulsion fractures , with satisfactory results , but the fixed strength needs to be strengthened , and which was lack of long term followup . the curative effect is relatively satisfactory , but the operation is complex and auxiliary arthroscopy is needed . a stellate steel plate for the treatment of avulsion fracture of the posterior cruciate ligament has the advantages listed below . first , it will change the single screw force bearing point to a wide force bearing area , to avoid the bone block splitting again , which is suitable for fixation of different diameter bone blocks or crushed fractures . second , the surrounding spines unique gear type design can be embedded in the bone and fixed with hollow compression screws , leading to stronger fixation . mechanical experiments show that , compared with other types of implants , a stellate steel plate has significantly enhanced pulling resistance.21 third , each ratchet structure is designed with a barrier structure , and the gap between the gears can avoid compression of soft tissue , causing less vesicular damage as well as promoting fracture healing . all the fractures in the current study healed and a literature review revealed that the healing rate was significantly higher than that of other fixation methods.12 fourth , this technique has the advantages of being a simple operation , leading to effective shortening of the operation time as well as reducing the risk of infection and bleeding . fifth , because of firm fixation , early functional exercise of patients and shortened recovery time , the recovery of knee joint function was better . compared to patients treated with other implants reported in the literature,617 the functional scores and patient satisfaction of this group were significantly high , which supports our use of the treatment method . limitations of the current study include the small number of patients , the relatively short followup time , and the lack of a corresponding control group . the stellate steel plate can be used effectively to fix different types of avulsion fracture blocks , as it has the advantages of being a simple operation with short rehabilitation time and firm fixation .

background : the open reduction with internal fixation is an effective approach for treatment of avulsion fracture of posterior cruciate ligament . the previously used internal fixation materials including hollow screws , absorbable screw , tension bands and sutures have great defects such as insufficient fixation strength , susceptibility to re - fracture , etc . stellate steel plate is novel material for internal fixation which has unique gear - like structure design . we used stellate steel plate for treatment of displaced avulsion fractures of posterior cruciate ligament in this study.materials and methods:14 patients ( 9 men , 5 women ; aged , 1935 years ; mean age , 28 years ) with displaced avulsion fractures of the tibial insertion of the posterior cruciate ligament were retrospectively analyzed between june 2009 and june 2011 . the mean duration from injury to the operation was 8.3 days ( range 615 days ) . all the patients were treated with open reduction and internal fixation of a stellate steel plate ( depuy , raynham , ma 02767 , usa ) . the lysholm - tegner knee function score criteria were used to analyze results.results:the mean followup was 24.6 months ( range 1832 months ) . after 6 months , all the fractures healed and knee joint activity was normal , with no knee stiffness or instability . the lysholm - tegner scores were 97.1 1.7 points at the final followup.conclusion:owing to its unique gear structure , the stellate steel plate design can effectively fix an avulsion fracture block and it is a simple operation with short postoperative rehabilitation time and firm fixation .

diabetic cardiomyopathy ( dcm ) is a diabetes - associated ventricular dysfunction , resulted from abnormal ventricular structural alteration that is independent of other etiological factors such as hypertension . studies have shown that hyperglycemia - induced oxidative stress and the subsequent inflammation play critical roles in the development and progression of diabetic cardiomyopathy [ 2 , 3 ] . hyperglycemia - induced oxidative stress mainly results from increased production of reactive oxygen species ( ros ) with or without concomitantly damped antioxidant defense system [ 4 , 5 ] . our previous study found that the major endogenous antioxidant enzyme superoxide dismutase ( sod ) , which plays an important role in balancing ros generation and the overall tissue antioxidant capacity , was increased in both the plasma and heart tissue of rats at a relatively early stage ( 4 weeks ) of diabetes , but tissue and plasma levels of free 15-f2t - isoprostane , a specific marker of lipid peroxidation , were also increased . this indicates that the upregulation of sod is not sufficient to resist hyperglycemia - induced oxidative stress . of note , another important enzyme , heme oxygenase-1 ( ho-1 ) , a stress - inducible cytoprotective defense enzyme , has been shown to exert cytoprotective effect against oxidative insults . also , studies showed that enhancing myocardial ho-1 expression could attenuate diabetes - induced cardiac dysfunction . however , in contrast to the compensatory increase of sod during early diabetes mellitus , a number of studies showed that myocardial ho-1 expression was significantly decreased in the myocardium of diabetic rats [ 8 , 9 ] . thus , unveiling the underlying mechanisms governing the reduction of myocardial ho-1 in diabetes should lead to the development of novel therapies to upregulate ho-1 expression in diabetic heart . it has been reported that , in response to oxidative stress , brahma - related gene 1 ( brg1 ) is necessarily required in nuclear factor - e2 related factor 2 ( nrf2)/are - mediated induction of ho-1 . brg1 is the core atpases in the swi / snf complex , which plays a central role in the activation and transcription of genes in mammalian cells . the deficiency of brg1 results in the dissolution of discrete heterochromatin domains , aberrant mitotic progression , and genomic instability , which eventually induces cell death or cell apoptosis . a recent study showed that brg1 can bind to the promoters of antioxidant defense genes and protect cells from oxidative damage , which means that brg1 can exert antioxidative effect . furthermore , increasing evidence shows that brg1 can regulate gene expression during cardiac growth , differentiation , and hypertrophy [ 1416 ] . brg1 null mice embryos die when cardiomyocytes expansion and maturation begin , while in adult cardiomyocytes , brg1 is activated by cardiac stresses and assembles a chromatin complex to activate downstream signal transduction , including ho-1 and signal transducer and activator of transcription 3 ( stat3 ) [ 10 , 1618 ] , which are protective against ros - induced cardiomyopathy . studies by us and others found that myocardial ho-1 expression is reduced in diabetic rats [ 8 , 9 ] which is accompanied with reduced phosphorylation of stat3 ( p - stat3 , the activated status of stat3 ) . we postulated that reductions in myocardial ho-1 and stat3 in diabetes may be a consequence of reduction in cardiac brg1 expression subsequent to hyperglycemia - induced oxidative stress . accumulated evidence proves that therapies that can reduce oxidative stress are effective to attenuate the development of diabetic cardiomyopathy [ 20 , 21 ] . our previous study also found that treatment with the antioxidant n - acetylcysteine ( nac ) could attenuate the increase in inflammation factors tumor necrosis factor - alpha ( tnf - alpha ) and interleukin 6 ( il-6 ) and ameliorate myocardial dysfunction in diabetic rats . moderate levels of tnf - alpha or il-6 have been shown to initiate the activation of stat3 , a key protein in cardioprotective signaling pathway , whose activation is brg1 dependent [ 22 , 23 ] . therefore , the current study was designed to test the hypothesis that myocardial brg1 is reduced in diabetes and antioxidant nac may enhance cardiac brg1 expression and concomitantly increase cardiac stat3 activation and confer cardioprotection in diabetes . sprague - dawley male rats ( 220 20 g , 8 weeks of age ) were obtained from the laboratory animal service center ( university of hong kong ) . all rats were allowed to adapt in their houses and have free access to standard chow and water according to the principles of animal care of the university of hong kong . the experiment procedures were approved by the committee on the use of live animals in teaching and research ( culatr ) . diabetes was induced by a single tail vein injection of streptozotocin ( stz ) at the dose of 65 mg / kg body weight ( sigma - aldrich , st . louis , mo ) , freshly dissolved in 0.1 m citrate buffer ( ph 4.5 ) under anesthesia with sodium pentobarbital ( 65 mg / kg body weight ) , while control rats were given equal volume of citrate buffer alone . after three days of injection , blood glucose was measured using a glucose analyzer ( bayer healthcare , bayer ag , germany ) , and rats with blood glucose over 16.7 mm were considered diabetic . rats were randomly divided into three groups ( n = 6 per group ) : control ( c ) ; diabetes ( d ) ; diabetes treated with nac ( 1.5 nac was administered to the d + nac group dissolved in drinking water for 4 weeks after induction of diabetes starting one week after the onset of diabetes . upon completion of treatment , the rats were anticoagulated with heparin ( 1000 iu / kg ) and then anaesthetized with pentobarbital sodium ( 65 mg / kg body weight ) . blood samples were obtained from the inferior vena cava , and plasma was separated and stored at 80c for further analysis . rats were sacrificed after completion of echocardiographic assessment of cardiac function , and hearts were harvested and rinsed with ice - cold phosphate buffer saline , dried , and weighted . at the end of 4-week treatment , the rats were examined by echocardiography using a high resolution imaging system ( vevo 770 , visualsonics inc . , canada ) equipped with a 17.5-mhz liner array transducer . the following validated parameters were automatically calculated by the ultrasound machine : lv end - diastolic volumes ( lvvd ) , lv end - systolic volume ( lvvs ) , fractional shortening ( fs ) , ejection fraction ( ef ) , and stroke volume ( sv ) . m - mode images were recorded to detect heart rate ( hr ) , lv internal diameter in systole ( lvids ) and diastole ( lvidd ) , interventricular septal thickness in systole ( ivss ) and in diastole ( ivsd ) , and lv posterior wall thickness in systole ( lvpws ) and diastole ( lvpwd ) . lv mass was assessed by calculating the formula : lv mass = 1.053 [ ( lvidd + lvpwd + ivsd ) lvidd ] 0.8 . the peak velocities of early ( e ) and late ( a ) flows were obtained from the apical four - chamber view . the e / a ratio and the isovolumetric relaxation time ( ivrt ) were used as indices of lv diastolic function . all recordings were performed in rats that underwent inhalation of 3% isopentane in air throughout the whole process , and echocardiography was conducted by investigators who were blinded to the experimental group as we reported . free 15-f2t - isoprostane ( 15-f2t - isop ) , a specific marker of oxidative stress , was measured by using an enzyme - linked immunoassay kit ( cayman chemical , ann arbor , mi ) as described . plasma samples and homogenized heart tissue ( in pbs ) were purified using affinity sorbent and affinity column ( cayman chemical , ann arbor , mi ) , then processed for analysis , according to the protocol provided by the manufacturer . the values of plasma or cardiac free 15-f2t - isop were expressed as pg / ml in plasma or pg / mg protein in cardiac homogenates , respectively . plasma levels of tnf - alpha and il-6 were determined by using the commercially available rat elisa kit ( bender med , vienna , austria ) . frozen heart tissue was homogenized using lysis buffer ( 20 mmol / l tris - hcl ph = 7.5 , 50 mmol / l 2-mercaptoethanol , 5 mmol / l egta , 2 mmol / l edta , 1% np40 , 0.1% sodium dodecyl sulfonate ( sds ) , 0.5% deoxycholic acid , 10 mmol / l naf , 1 mmol / l pmsf , 25 mg / ml leupeptin , and 2 mg / ml aprotinin ) for 30 min and then sonicated and centrifuged at 12000 g for 20 min at 4c . protein concentrations were determined using the bradford assay ( bio - rad , usa ) . samples containing equal amounts were separated on a 10% sds - polyacrylamide gel , and then proteins were transferred to pvdf membrane overnight at 4c . membranes were blocked with 5% nonfat milk in tris - buffered saline ( tbs)-tween for 1 hour and were incubated with anti - brg1 ( abcam , usa ) or anti - stat3 , anti - phospho - stat3 ( t705 ) , anti - ho-1 antibodies ( cell signaling technology , beverly , ma , usa ) , and gapdh ( cell signaling technology , beverly , ma , usa ) at 1 : 1000 dilution for overnight at 4c . after washing with phosphate buffered saline - tween ( pbst ) three times for 30 min , membranes were then incubated with horseradish - peroxidase- ( hrp- ) conjugated anti - rabbit igg at 1 : 2000 dilution for 1 hour . protein bands were developed with enzymatic chemiluminescence , and images were measured by a densitometer with analysis software . data are presented as means standard error of the mean ( s.e.m . ) . the analysis was performed using statistical software package ( graphpad prism , san diego , ca , usa ) . administration of stz resulted in increased plasma glucose and food and fluid intake and reduced body weight gain as compared with age - matched control rats ( all p < 0.05 , table 1 ) . treatment with nac for 4 weeks significantly reduced food consumption and water intake in diabetic rats ( p < 0.05 , d + nac versus d ) but did not have significant effect on glucose levels and body weight gain . compared with the control group , the levels of free 15-f2t - isop were significantly increased in both the plasma and cardiac tissues of diabetic rats ( p < 0.01 or p < 0.05 versus c , table 2 ) . nac treatment reduced plasma and cardiac tissue 15-f2t - isop to a level comparable to that in the control ( p < 0.05 versus d , p > 0.05 d + nac versus c , table 2 ) . as shown in table 3 , lvm was much lower in diabetic rats ( p < 0.05 versus c ) , despite the fact that there were no significant differences in ivsd , ivss , lvids , lvpwd , and lvpws between the control and the diabetic rats . however , lvm to body weight ratio , an indicator of myocardial hypertrophy , was remarkably increased in diabetic rats ( p < 0.05 versus c ) . nac reduced lvm to body weight ratio to a level comparable to that in the control ( p < 0.05 , d + nac versus d ; p > 0.05 d + nac versus c , table 3 ) . the hrs of diabetic rats were significantly decreased as compared to those of the controls ( p < 0.05 versus c table 3 ) . lvvd and the e / a ratio in diabetic rats were significantly decreased , while ivrt increased ( all p < 0.01 versus c table 3 ) . this is indicative of compromised lv relaxation , which may contribute to the significantly reduced sv ( p < 0.05 d versus c , table 3 ) . nac treatment did not have significant effects on lvvd and ivrt nor did it improve sv ( p > 0.05 d + nac versus d , table 3 ) but remarkably increased e / a ratio which primarily resulted from a reduction in mva ( p < 0.05 d + nac versus d , table 3 ) . there was no difference in values of fs and ef between the control and diabetic rats . as shown in figures 1(a ) and 1(b ) , the plasma levels of tnf- and il-6 in diabetic rats were significantly increased as compared with the control group ( p < 0.05 ) . nac treatment reduced plasma tnf- and il-6 to a level comparable to that in the control ( p < 0.05 versus d ; p > 0.05 versus c ) although they were slightly higher than that in the control rats . to investigate whether the cardiac protein expression of brg1 is altered in diabetic rats at an early stage of the disease and whether or not it can be affected by antioxidants , we explored the effects of nac on cardiac levels of brg1 in stz - induced diabetic rats 4 weeks after the establishment of diabetes . as shown in figure 2(a ) , the protein expression of brg1 was significantly decreased in diabetic rats as compared to that of control group ( p < 0.01 ) . recent study demonstrated that brg1 is required to establish chromatin accessibility at stat3 binding targets , which is essential to enable these sites to respond to downstream signaling . therefore , in addition to exploring the changes of myocardial brg1 protein in diabetes , we also investigated the myocardial protein levels and phosphorylation / activation status of stat3 in diabetic heart . as shown in figures 2(c ) and 2(d ) , the protein expression of p - stat3 ( tyr705 and ser727 ) but not total stat3 was significantly reduced in the myocardium of diabetic rats , accompanied with concomitant reduction of cardiac protein expression of ho-1 ( all p < 0.05 versus c , figure 2(b ) ) , an important signaling protein downstream of brg1 . nac completely restored myocardial p - stat3 at site tyr705 and ho-1 protein expression and partially but significantly enhanced p - stat3 at site ser-727 in diabetic rats ( p < 0.01 versus d ; p < 0.05 , p < 0.01 versus c ) . consistent with our previous studies , we have shown in the current study that oxidative stress increased in the early stage ( at 4 weeks ) rats with stz - induced type 1 diabetes as indicated by a significant increase in both plasma and heart tissue levels of free 15-f2t - isoprostane , a specific index of oxidative stress . enhanced levels of oxidative stress were accompanied by increased tnf - alpha and il-6 . in the current study , we further discovered that diabetic rat hearts exhibited decreased expression of brg1 , which was coincident with decreased cardiac expressions of p - stat3 and ho-1 and compromised cardiac diastolic function as assessed by echocardiography . effective antioxidant treatment with nac evidenced as complete prevention of hyperglycemia - induced increases in plasma and heart tissue free 15-f2t - isoprostane significantly attenuated the reduction of myocardial brg1 protein expression , subsequently significantly enhanced myocardial p - stat3 and ho-1 , and improved cardiac relaxation in diabetic rats . to our knowledge , this is the first study to explore the changes of cardiac brg1 in diabetic rats and the effectiveness of antioxidant treatment on brg-1 cardiac expression in diabetes . oxidative stress occurs in diabetes as a consequence of hyperglycemia - induced abnormalities , including glucose autooxidation , the formation of advanced glycation end products , and impairment of antioxidant defense system . we previously reported that the heart tissue sod activity was compensatorily increased , but both the plasma and heart tissue levels of free 15-f2t - isoprostanes were still increased in diabetic rats at the early stage of 4-week diabetes , which indicates that , during early stage of diabetes , compensatory increase in myocardial sod was not sufficient to combat hyperglycemia - induced oxidative stress . while in our current study conducted in the same model of early stage of 4-week diabetes , we showed that the protein expression of ho-1 , another important antioxidative enzyme , was decreased significantly in the myocardium of diabetic rats , which suggests that a decrease in ho-1 expression may be a major contributor to hyperglycemia - induced oxidative stress in early diabetes . it has been shown that , in response to oxidative stress , ho-1 expression can be induced through the nrf2/are signaling pathway , while , effective nrf2/are signaling needs the participation of brg1 . however , as we showed in the current study , cardiac brg1 expression in rats at the 4th week after stz - induced type 1 diabetes was significantly reduced , which might be the major reason why cardiac protein expression of ho-1 was significantly decreased . antioxidant nac normalized cardiac free 15-f2t - isoprostanes in diabetic rats and enhanced myocardium brg1 expression , leading to full restoration of cardiac ho-1 expression . this finding suggests that enhancing brg1 may represent a novel mechanism whereby nac confers its antioxidant protection at least in early diabetes . consistent with our recent study findings , we showed in the current study that ventricular dysfunction occurs during early stage of diabetes manifested as abnormal relaxation function that was coincident with significant reduction in myocardial brg1 protein expression . this finding is similar in nature to a previous study which showed that mice with cardiac - specific deletion of brg1 developed impaired cardiac relaxation evidenced as reduction in e / a ratio as determined by ultrasound . the findings by us and others point out the importance of brg1 in the maintenance of normal cardiac function . in our study , nac treatment mediated improvement in cardiac diastolic function manifested as significant elevation of e / a ratio in diabetes which may be contributable to enhancement of cardiac brg1 protein expression . the risk of progression to heart failure after myocardial ischemia and reperfusion was significantly higher in diabetes compared with nondiabetes . myocardial stat3 is an important transcription factor in the safe pathway ( i.e. , jak2/stat3 signaling cascade ) , especially during myocardium ischemia reperfusion injury , but cardiac p - stat3 is reduced in diabetes . further , stat3-deficient mice spontaneously develop a form of dilated cardiomyopathy similar to that which occurred in diabetic mice , indicating that reduced stat3 activation may lead to myocardial remodeling . brg1 is necessarily required to establish chromatin restructure for the activation of stat3 , especially stat3 phosphorylation at site try705 in various cells such as cancer cell and macrophagocyte , and for stat3 signaling transduction . in the current study , we also found that stat3 phosphorylation at both ser727 and try705 was dramatically reduced in the hearts of diabetic rats , which was concomitant with decreased brg1 expression . nac treatment increased the expression of brg1 and consequently enhanced p - stat3 at both try705 and ser727 in the myocardium of diabetic rats . based on the fact that brg1 is a needed component for stat3 activation , our findings suggest that brg1 may play a key role in the transcriptional induction of cardiac stat3 , especially at try705 in diabetic rats . nac may have increased stat3 phosphorylation in diabetes through enhancing brg1 expression , although further study is needed to confirm this hypothesis . inflammation has been considered as an important process in the progression of diabetes [ 31 , 32 ] . elevation of tnf - alpha and il-6 was detected in diabetes , which was related to the progression of diabetic complications . in the current study , we showed that plasma levels of tnf - alpha and il-6 were remarkably elevated in diabetic rats at 4 weeks after the establishment of diabetes , and nac treatment decreased them , indicating that nac can inhibit the inflammation reaction in diabetes . studies found that moderate levels of tnf - alpha or il-6 can increase the phosphorylation of stat3 at try705 , and this is brg1 dependent [ 22 , 23 ] . in our study , we found that the levels of tnf - alpha and il-6 in nac - treated diabetic rats were significantly lower than that in the untreated diabetes , but still slightly higher than that in nondiabetic rats . these remaining slight elevations in tnf - alpha and il-6 after nac treatment should have contributed to the enhancement of cardiac p - stat3 as seems to be in the nac treated group , which added to the effect of brg1 in enhancing cardiac brg1-mediated stat3 activation as previously mentioned . this explains why nac treatment did not completely restore cardiac brg1 protein expression in diabetic rats but completely restored cardiac p - stat3 at site try705 . in summary , we first report that the expression of brg1 was decreased significantly in the myocardium of diabetic rats , which may be responsible at least in part for the reduced expressions of ho-1 and p - stat3 and impairment of cardiac diastolic function as summarized in the schematic diagram ( figure 3 ) . enhancement of cardiac brg1 expression may thus represent a novel mechanism whereby nac enhanced cardiac ho-1 and p - stat3 expressions and attenuated cardiac diastolic dysfunction in diabetes .

brahma - related gene 1 ( brg1 ) is a key gene in inducing the expression of important endogenous antioxidant enzymes , including heme oxygenase-1 ( ho-1 ) which is central to cardioprotection , while cardiac ho-1 expression is reduced in diabetes . it is unknown whether or not cardiac brg1 expression is reduced in diabetes . we hypothesize that cardiac brg1 expression is reduced in diabetes which can be restored by antioxidant treatment with n - acetylcysteine ( nac ) . control ( c ) and streptozotocin - induced diabetic ( d ) rats were treated with nac in drinking water or placebo for 4 weeks . plasma and cardiac free15-f2t - isoprostane in diabetic rats were increased , accompanied with increased plasma levels of tumor necrosis factor - alpha ( tnf - alpha ) and interleukin 6 ( il-6 ) , while cardiac brg1 , p - stat3 and ho-1 protein expression levels were significantly decreased . left ventricle weight / body weight ratio was higher , while the peak velocities of early ( e ) and late ( a ) flow ratio was lower in diabetic than in c rats . nac normalized tissue and plasma levels of 15-f2t - isoprostane , significantly increased cardiac brg1 , ho-1 and p - stat3 protein expression levels and reduced tnf - alpha and il-6 , resulting in improved cardiac function . in conclusion , myocardial brg1 is reduced in diabetes and enhancement of cardiac brg1 expression may represent a novel mechanism whereby nac confers cardioprotection .

a 6 - 8 hours fasting period for solids and liquids is the traditional management in the preoperative preparation process . prospective randomized studies demonstrated that reducing the preoperative fasting to 2 hours along with ingestion of a maltodextrin solution provides emotional and especially metabolic benefits , besides not determining anesthesia - associated risk for pulmonary aspiration . there are many references to this concept in publications by the european group eras ( enhanced recovery after surgery ) , and by our research group acerto , ( acelerao da recuperao total ps - operatria ) . the ingestion of liquids with maltodextrin , especially in medium and large surgeries , modulates the metabolic response to trauma by lowering insulin resistance , thus attenuating nitrogen ( protein ) consumption and consequently reducing the loss of strength and function of skeletal muscles . a positive interference with the nitrogen balance implies preservation of muscular strength and reducing postoperative complications . cholecystolithiasis is an endemic disease that usually requires elective surgery and may present with pulmonary complications . in the immediate postoperative period , there is often a restrictive respiratory disorder usually leading to reduced forced vital capacity ( fvc ) , and reduced forced expiratory volume in the first second ( fev1 ) . there are few data in literature about the influence of a short - time preoperative fast with maltodextrin solution on muscle strength and lung function . to evaluate pulmonary function and functional capacity in the immediate preoperative period and 24 hours after surgery in patients undergoing elective cholecystectomy by laparotomy , with either prolonged or shortened fasting . this was a prospective randomized clinical trial including 92 patients treated at the surgical outpatients clinic of hospital santa helena , in cuiab ( mt ) , brazil , from january to december 2011 . the study was approved by the research ethics committee of the hospital universitrio jlio mller , universidade federal de mato grosso , protocol number 783/cep - hujm/10 . the inclusion criteria were female patients with an indication for cholecystectomy by laparotomy and spinal anesthesia , aged 18 to 60 years , with body mass index ( bmi ) ranging from 18.5 to 29.5kg / m , american society of anesthesiology ( asa ) i and ii , and subjective global assessment ( sga ) a. we excluded patients requiring cholecystectomy with bile duct exploration , patients who underwent surgery with operative time longer than 120 minutes or whose anesthesia had to be converted to general anesthesia , patients requiring another immediate surgical procedure , patients with postoperative complications , pregnant patients , and those suffering from diabetes , hypertension , chronic obstructive pulmonary disease , lung disease , heart disease , arthritis , and gastroesophageal reflux disease . g1 was composed of patients who underwent shortened fasting , with restriction of solids for 8 hours and ingestion of tea with addition of 12.5% maltodextrin in 400ml and 200ml , 6 and 2 hours , respectively , before the scheduled time for surgical procedure . in g2 , the patients followed the prolonged fasting time , with no intake of solids or liquids for a period of at least 8 hours before the operation . the surgical procedure was performed by the same professional team and with the same standard surgical technique . spinal anesthesia was used and all patients were sedated with intravenous midazolam at a dose of 0.01 to 0.08mg / kg . analgesia was initiated in the recovery room , consisting of 2 g of dipyrone and 75 mg of sodium diclofenac . subsequently , dypirone 1 g was prescribed every 6 hours . prophylactic antibiotic therapy with cefazolin was prescribed . data collection was carried out in two stages : in the preoperative period , 2 hours before surgery ; and 24 hours after starting time of surgery . the incentive spirometer one flow fvc hs clement clarke international ( harlow , essex , england ) unit , with a disposable nozzle as interface between patient and spirometer , was used for all measurements . the patients remained seated , with their heads in neutral position , without bending the neck and using a nose clip . maximal inspiration was made until they reached their inspiratory capacity , followed by a blow as fast and as hard as possible reaching the vital capacity . three reproducible maneuvers were done with a 1 minute interval between them . the best value curve , as elected by the spirometer software , the variables for muscle strength were dominant hand grip strength ( dhgs ) and non - dominant handgrip strength ( ndhgs ) , measured by a hydraulic dynamometer ( saehan corporation , masan , korea ) . measurement was performed with the patient seated on a chair with a straight back and no support for the arms , shoulder adducted and neutrally rotated , elbow flexion at 90 , forearm and wrist in neutral position between zero and 30 of extension , and zero and 15 of ulnar deviation . three measurements were performed , and the arithmetic mean of the two highest readings of each hand was used for analysis . all variables were normally distributed after applying the kolmogorov - smirnov test , thus justifying the use of parametric statistical methods . individual variables , clinical characteristics , spirometry and functional capacity values were compared according to the preoperative fasting and postoperative evaluation , initially by student 's t test for independent variables . later , the student 's t test was used with dependent groups ( paired ) for intragroup comparison ( pre - and postoperatively ) . the arithmetic means and standard deviation results were used for all analyses , as displayed on the tables . the level of significance was set at 5% , using the epi - info , version 3.5.2 , and statistical package for the social science ( spss ) , version 13.0 , for windows software . one hundred and six patients were considered for randomization , in that , 53 patients for each group . fourteen patients were eliminated by the exclusion criteria ( manipulation of the biliary tract , previous intraoperative cholangiography , operative time > 120 minutes , and requirement of general anesthesia ) . the final sample consisted of 92 patients , there were no postoperative complications , and no patient dropped out or refused compliance with the protocol . there were also no statistical differences regarding the time of surgery and size of the incision , in addition to variables related to the preoperative evaluation ( pef , fev1 , fvc , dhgs and ndhgs ) , problably because the good adjustment when applying the inclusion criteria . all study variables ( pef , fev1 , fvc , dhgs and ndhgs ) were similar in both groups in the preoperative evaluation . however , postoperatively these variables showed statistically significant differences , with higher values in g1 than in g2 ( table 2 ) . however , in g2 , all variables showed pre- and postoperative differences ( table 3 ) . the results of this study showed that shortened fasting with maltodextrin preserved muscle strength in the dominant hand . another finding of this study was that there was loss of muscle function after surgery . comparing the types of fasting , the best values the results of this study demonstrated that shortened fasting using maltodextrin brought benefits to lung function and preserved muscle strength in the dominant hand . therefore , it contributed to a better clinical outcome , since reduced lung function and reduced muscle strength are associated with postoperative complications . this result is in agreement with several other studies that showed safety and benefits of shorter fasting time . several studies demonstrated that a shortened fasting had favorable emotional effects on the patients , such as reducing hunger , sadness , thirst , anxiety , malaise , and irritability . in terms of metabolism , this shortened fasting also positively modulates the response to trauma , by determining a better organic response to surgical stress . in 2010 it was shown that a prolonged preoperative fasting diminishes the substrate reserves , thus affecting mitochondrial function of mononuclear cells . additionally , this is associated with a lower muscular rnam and reduced pyruvate dehydrogenase kinase , and the latter is the mechanism that makes the ingestion of a solution containing maltodextrin cause attenuation of the development of postoperative insulin resistance . the strengths of this study were evaluation of the respiratory and muscular functions as the endpoint variables of the two different preoperative fasting approaches . we did not find in the literature any study evaluating the effect of a shorter fasting on lung and muscular functions assessed in the same patient . this is the first study to use this approach and indicates the need of further research with this methodology . the decreased respiratory function found in this study was similar to what reported by some authors in this same type of surgery . . observed that a shortened fasting time in lung operations led to lower pulmonary complication rates . furthermore , they found lower fev1 values in this group , which is similar to our results . our results indicated that a shortened fasting using maltodextrin was beneficial to the respiratory dynamics , probably due to maintenance of muscular strength . another finding of this study was that there was a loss of muscle function postoperatively . the preservation of dhgs in the shortened fasting group probably reflects the behavior of the skeletal muscles as a whole . some studies showed that low levels of hgs in the preoperative period are strong indicators of postoperative complications , besides predicting a worsening in functional status during hospitalization , with a consequent increase in mortality rate of these patients . in a pioneer study on dynamometry and postoperative complications , observed that 95% of patients who developed complications had a hgs 85% lower than that of healthy individuals . another study conducted in an intensive care unit revealed that the lower the hgs , the greater association with hospital mortality ( or=4.5 ; 95%ci 1.5 - 13.6 ; p=0.007 ) . a study by bragagnolo et al . demonstrated that thickness of the abductor muscle of the thumb and hgs index are safe methods of nutritional assessment , and that hgs of the dominant hand is a good prognostic indicator in patients who are candidate for large digestive surgeries . this most often occurs as the result of the fasting required for tests , diagnostic procedures , or surgeries . in these cases , hgs is a relevant prognostic method , proving that the adequate intake of food during the first week of hospitalization leads to increased grip strength bilaterally . perrone et al . found that the intake of nutrients ( carbohydrate and protein ) in the immediate preoperative period is seen as a beneficial factor to minimize the organic response to surgical stress and improve welfare of patients . the supply of a fluid containing 12.5% carbohydrate and protein , 6 and 2 hours before surgery in patients undergoing colorectal surgery , decreased peripheral resistance to insulin and unpleasant manifestations , such as thirst and hunger , which resulted in enhanced wellbeing and less anxiety and stress , without causing gastric stasis . the patients submitted to laparoscopic cholecystectomy showed a significant decrease in lung volumes and respiratory muscle strength on the first postoperative day . however , when compared with data from literature , their return to preoperative values was quicker in laparoscopic surgery than in open abdominal surgery . the supply of carbohydrates before surgery reduces the organic metabolic effects associated with surgical trauma . it can be inferred that in greater magnitude surgeries , in which the organic response is more intense , this routine can promote a lower metabolic impact and therefore , provide important benefits to the patients . in this study , 2 hours fasting was safe in 95% of cases due to the strict control of fluid supply in the preoperative period . however , a limitation was the fact that the participants in our study were not blinded . another limitation was the size of surgeries ( medium size ) , since in such procedures there is less metabolic aggression , in addition to less postoperative complications , thus producing smaller impacts of short - time fasting on the body . the present study adds new data to the literature on the advantages of shortened fasting , now focusing on preservation of muscle and respiratory function ; it thus allows inferring that this change in the protocol may represent a positive impact on the clinical evolution of the patient . the shorter preoperative fasting time , with supply of a fluid containing maltodextrin before elective medium - sized operations was beneficial to the lung function and preserved the dominant hand grip strength of patients . avaliar a alterao da funo respiratria e da capacidade funcional , conforme o tipo de jejum pr - operatrio . ensaio clnico prospectivo randomizado , com 92 pacientes do sexo feminino , submetidas colecistectomia por laparotomia , observando jejum convencional ou abreviado de 2 horas com maltodextrina . as variveis foram : pico de fluxo expiratrio , volume expiratrio no primeiro segundo , capacidade vital forada , fora de preenso palmar dominante e fora de preenso palmar no dominante . as avaliaes foram realizadas 2 horas antes da induo anestsica e 24 horas aps a operao . os dois grupos foram semelhantes quanto s caractersticas demogrficas , clnicas e em todas as variveis estudadas , quando avaliadas no pr - operatrio . no entanto , no ps - operatrio , o grupo abreviado apresentou valores maiores que o grupo convencional para pico de fluxo expiratrio ( 128,762,5 versus 115,759,9 ; p=0,040 ) , volume expiratrio no primeiro segundo ( 1,50,6 versus 1,20,5 ; p=0,040 ) , capacidade vital forada ( 2,31,1 versus 1,80,9 ; p=0,021 ) , fora de preenso palmar dominante ( 24,96,8 versus 18,47,7 ; p=0,001 ) e fora de preenso palmar no dominante ( 22,96,3 versus 17,07,8 ; p=0,0002 ) . na avaliao intragrupo , houve diminuio nas variveis ao se compararem os valores do pr - operatrio em relao ao ps - operatrio , exceto para fora de preenso palmar dominante ( 25,26,7 versus 24,96,8 ; p=0,692 ) no grupo de jejum abreviado . a abreviao do tempo de jejum pr - operatrio com soluo contendo maltodextrina beneficia a funo pulmonar e preserva a fora de preenso palmar dominante . um perodo de jejum de 6 a 8 horas de slidos e lquidos a conduta tradicional no processo de preparao pr - operatria . estudos prospectivos randomizados tm demonstrado que a reduo do tempo de jejum pr - operatrio para 2 horas mais a ingesto de uma soluo contendo maltodextrina , alm de no determinar um risco de broncoaspirao associada anestesia , tambm fornece benefcios emocionais e , especialmente , metablicos . h muitas referncias a este conceito em publicaes do grupo europeu eras ( enhanced recovery after surgery ) e do nosso grupo de pesquisa acerto ( acelerao da recuperao total ps - operatria ) . a ingesto de lquidos com maltodextrina , especialmente em cirurgias de porte mdio e grandes , modula a resposta metablica ao trauma , por meio da diminuio da resistncia insulina , atenuando , assim , o consumo de nitrognio ( protena ) e , consequentemente , reduzindo a perda de fora e funo de msculo esqueltico . a interferncia positiva no balano do nitrognio implica na preservao da fora muscular e na reduo de complicaes ps - operatrias . colecistolitase , uma doena endmica , que geralmente resulta em cirurgia eletiva , pode apresentar complicaes pulmonares . no perodo ps - operatrio imediato , frequentemente h um distrbio respiratrio restritivo , que geralmente origina uma reduo da capacidade vital forada ( cvf ) e do volume expiratrio forado no primeiro segundo ( vef1 ) . h poucos dados na literatura sobre a influncia do jejum pr - operatrio por curto tempo com soluo de maltodextrina sobre a fora muscular e a funo pulmonar . avaliar a funo pulmonar e capacidade funcional no perodo pr - operatrio imediato e 24 horas aps a cirurgia em pacientes submetidos colecistectomia eletiva por laparotomia com jejum convencional ou de curto perodo . este foi um estudo clnico prospectivo randomizado que inclui 92 pacientes tratados na unidade cirrgica ambulatorial do hospital santa helena , em cuiab ( mt ) , de janeiro a dezembro de 2011 . o estudo foi aprovado pela comisso de tica em pesquisa do hospital universitrio jlio mller , universidade federal de mato grosso , protocolo nmero 783/cep - hujm/10 . todos os participantes assinaram um termo de consentimento livre e informado . os critrios de incluso foram pacientes do sexo feminino com indicao de colecistectomia por laparotomia e raquianestesia , com idades entre 18 e 60 anos , ndice de massa corporal ( imc ) que variava entre 18,5 e 29,5kg / m , american society of anesthesiology ( asa ) i e ii , e subjective global assessment ( sga ) a. o estudo excluiu pacientes que necessitavam de colecistectomia com explorao do ducto biliar , pacientes que se submeteram cirurgia com tempo operatrio maior que 120 minutos ou cuja anestesia teve de ser convertida para anestesia geral , pacientes que necessitaram de uma nova interveno cirrgica imediata , pacientes com complicaes no ps - operatrio , grvidas , e aquelas com diabetes , hipertenso , doena pulmonar obstrutiva crnica , doena pulmonar , doena cardaca , artrite e doena do refluxo gastresofgico . no grupo g1 , as pacientes foram submetidas a um jejum mais curto , com restrio de slidos por 8 horas e ingesto de ch com adio de 12,5% de maltodextrina , nos volumes de 400 e 200ml , 6 e 2 horas antes do horrio marcado para o procedimento cirrgico , respectivamente . no grupo g2 , estavam aquelas que seguiram o tempo convencional de jejum , sem ingesto de slidos ou lquidos por um perodo de pelo menos 8 horas antes da cirurgia . o procedimento cirrgico foi feito pela mesma equipe de profissionais e com a mesma tcnica cirrgica padro . a tcnica anestsica foi raquianestesia e todas as pacientes foram sedadas com midazolam intravenoso na dose de 0,01 a 0,08mg / kg . a analgesia foi iniciada na sala de recuperao , consistindo de 2 g de dipirona e 75 mg de diclofenaco de sdio . a coleta de dados foi feita em dois estgios : no pr - operatrio , 2 horas antes da cirurgia ; e 24 horas aps o incio da cirurgia . a unidade do espirmetro de incentivo one flow fvc hs clement clarke international ( harlow , essex , inglaterra ) , com bocal descartvel de interface entre paciente e espirmetro , foi usada para todas as mensuraes . as pacientes permaneceram sentadas , com as cabeas em posio neutra , sem dobrar o pescoo e usando uma pina nasal . a inspirao mxima foi feita at que alcanassem sua capacidade inspiratria , seguida de um assopro o mais rpido e forte possvel , chegando capacidade vital . a melhor curva conforme o software do espirmetro foi includa para anlise . as variveis de fora muscular foram fora de preenso palmar da mo dominante ( da fppd ) e fora de preenso palmar da mo no dominante ( fppnd ) , medidas por um dinammetro hidrulico ( saehan corporation , masan , corea ) . a medida foi feita com a paciente sentada em uma cadeira espaldar reto e sem suporte para os braos , com ombros em aduo e rotao neutra , cotovelo flexionado em 90 , antebrao e punho em posio neutra entre zero e 30 de extenso , e zero e 15 de desvio ulnar . trs medidas foram feitas , e a mdia aritmtica das duas maiores de cada mo foi usada para anlise . todas variveis foram distribudas normalmente aps aplicao do teste de kolmogorov - smirnov , justificando , assim , o uso de mtodos estatsticos paramtricos . valores de variveis individuais , caractersticas clnicas , espirometria e capacidade funcional foram comparados conforme jejum no pr - operatrio e avaliao ps - operatria , inicialmente pelo teste t de student para variveis independentes . depois , o teste t de student foi usado em grupos dependentes ( pareados ) para comparao intragrupo ( no pr- e ps - operatrio ) . o nvel de significncia foi estabelecido como 5% , usando epi - info , verso 3.5.2 , e statistical package for the social science ( spss ) , verso 13.0 , ambos para windows . cento e seis pacientes foram consideradas para randomizao , sendo 53 pacientes para cada grupo . catorze pacientes foram rejeitadas pelos critrios de excluso ( manipulao da rvore biliar , colangiografia intraoperatria anterior , tempo de cirurgia > 120 minutos e necessidade de anestesia geral ) . a amostra final consistiu de 92 pacientes , sendo que o g1 contou com 47 pacientes e o g2 , com 45 . no houve nenhuma complicao ps - operatria , e nenhum paciente abandonou ou recusou cumprir o protocolo . nossos resultados no foram diferentes em relao s variveis demogrficas e clnicas entre os dois grupos . tambm no houve diferenas estatsticas com respeito ao tempo de cirurgia e o tamanho da inciso , alm das variveis relacionadas avaliao pr - operatria ( pef , vef1 , cvf , fppd e fppnd ) , o que pode ser justificado pelo bom ajuste promovido pela aplicao dos critrios de incluso . todas as variveis do estudo ( pef , vef1 , cvf , fppd , e fppnd ) foram similares em ambos os grupos na avaliao pr - operatria . no entanto , no ps - operatrio , essas variveis mostraram diferenas estatisticamente significantes nos valores maiores em g1 que em g2 ( tabela 2 ) . na anlise intergrupos , no houve diferena em g1 exceto quanto varivel da fpp . no entanto , em g2 , todas as variveis mostraram diferenas pr- e ps - operatrias ( tabela 3 ) . os resultados deste estudo mostraram que a abreviao do tempo de jejum com maltodextrina preservou a fora muscular na mo dominante . comparando os tipos de jejum , os melhores valores foram observados no grupo com jejum abreviado . os resultados deste estudo demonstraram que o tempo curto de jejum usando maltodextrina promoveu benefcios para a funo pulmonar e preservou a fora muscular na mo dominante . assim , contribuiu para um melhor desfecho clnico , j que a reduo da funo pulmonar e a reduo de fora muscular esto associadas a complicaes ps - operatrias . este resultado est em concordncia com vrios outros estudos que mostraram segurana e benefcios de um tempo mais curto de jejum . vrios estudos demonstraram que um jejum abreviado teve efeitos emocionais favorveis sobre as pacientes , tais como a reduo da fome , tristeza , sede , ansiedade , mal - estar e irritabilidade . em termos de metabolismo , esse jejum mais breve tambm modula , de forma positiva , a resposta ao trauma , pela determinao de uma melhor resposta orgnica ao estresse cirrgico . em 2010 , foi mostrado que o jejum pr - operatrio prolongado diminui as reservas de substratos , afetando , assim , a funo mitocondrial de clulas mononucleares . ademais , isso est associado a um menor nvel de rnam muscular e reduo da desidrogenase pirvica quinase ; o ltimo mecanismo faz a ingesto de uma soluo contendo maltodextrina causar atenuao no desenvolvimento de resistncia insulina no ps - operatrio . os pontos fortes deste estudo estiveram na avaliao das funes respiratrias e musculares como as variveis de desfechos de duas abordagens de jejum pr - operatria . na literatura , no foi possvel encontrar nenhum estudo que avaliasse o efeito de um tempo de jejum mais curto sobre as funes do pulmo e do msculo no mesmo paciente . este estudo , sendo o primeiro a usar essa abordagem , apontou para a necessidade de usar esse enfoque e de novas pesquisas usando essa metodologia . a funo respiratria diminuda encontrada neste estudo foi similar ao que foi identificado por alguns autores nesse mesmo tipo de operao . mostraram uma regresso a valores pr - operatrios no segundo dia ps - operatrio ( cvf e fev1 ) . muehling et al . observaram que um tempo encurtado de jejum em cirurgias do pulmo levou a menores taxas de complicaes pulmonares . ademais , foram encontrados valores menores de vef1 neste grupo , o que similar aos nossos resultados . nosso estudo indicou que um jejum mais curto usando maltodextrina foi benfico para a dinmica respiratria , provavelmente em funo da manuteno da fora muscular . outro achado deste estudo foi de que houve uma perda ps - operatria de funo muscular . na comparao dos tipos de jejum , o grupo do jejum mais curto mostrou os melhores valores . a preservao de da fppd no grupo de jejum encurtado provavelmente reflete o comportamento dos msculos esquelticos como um todo . alguns estudos mostram que baixos nveis de fpp no perodo pr - operatrio so fortes indicadores de complicaes ps - operatrias , alm de predizerem uma piora do estado funcional durante a hospitalizao , com crescente aumento na mortalidade desses paciente . em um estudo pioneiro sobre dinamometria e complicaes ps - operatrias , observaram que 95% das pacientes que desenvolveram complicaes tinham fpp 85% menor do que a de indivduos sadios . outro estudo conduzido em uma unidade de terapia intensiva revelou que quanto mais baixa a fpp , maior a associao com mortalidade hospitalar ( or=4,5 ; ic95% : 1,5 - 13,6 ; p=0,007 ) . demonstrou que a espessura do msculo abdutor do polegar e o ndice da fpp so mtodos de avaliao nutricional , e que a fpp da mo dominante um bom indicador do prognstico em pacientes candidatos a grandes cirurgias digestivas . isso ocorre com maior frequncia devido ao jejum requerido para exames , procedimentos diagnsticos , ou cirurgias . nestes casos , a fpp um mtodo prognstico importante , e prova que a ingesto adequada de alimentos durante a primeira semana de hospitalizao propicia maior fora de preenso bilateralmente . encontraram que a ingesto de nutrientes ( carboidratos e protenas ) no perodo pr - operatrio imediato vista como fator benfico que minimiza a resposta orgnica ao estresse cirrgico e melhora o bem - estar da paciente . o suprimento de uma bebida lquida com 12,5% carboidrato e protena , 6 e 2 horas antes da cirurgia , em pacientes submetidos cirurgia colorretal , diminuiu a resistncia perifrica insulina e as manifestaes desagradveis , como sede e fome , o que resultou num melhor bem - estar e menores ansiedade e estresse , sem causar qualquer estase gstrica . as pacientes que se submeteram a colecistectomia laparoscpica mostraram significante diminuio nos volumes pulmonares e na fora muscular respiratria no primeiro dia ps - operatrio . entretanto , quando comparadas com dados da literatura , seu retorno aos valores pr - operatrios foi mais rpido na cirurgia laparoscpica do que na cirurgia abdominal aberta . o suprimento de carboidratos antes da cirurgia reduz os efeitos metablicos orgnicos associados ao trauma cirrgico . pode - se inferir que , em operaes de maior magnitude , em que a resposta orgnica amplificada , essa rotina pode promover um menor impacto metablico e , assim , fornecer importantes benefcios s pacientes . neste estudo , o jejum de 2 horas foi seguro em 95% dos casos devido ao rgido controle de oferta de lquidos no pr - operatrio . no entanto , uma limitao foi o fato de que o estudo no foi cego para as participantes . outra limitao foi o tamanho das cirurgias ( de mdio porte ) , j que , nesses casos , h menor agresso metablica , alm de menos complicaes ps - operatrias , produzindo , assim , menores impactos do jejum mais curto sobre o corpo . de qualquer modo , um ponto forte do estudo foi o tamanho satisfatrio da amostra . literatura sobre as vantagens do jejum de curto prazo , focando agora na preservao das funes muscular e respiratria . desta forma , permite a inferncia de que esta alterao no protocolo pode representar um impacto positivo sobre a evoluo clnica da paciente . a abreviao no tempo de jejum pr - operatrio com o suprimento de uma bebida com maltodextrina antes de operaes eletivas de tamanho mdio foi benfica para a funo pulmonar e preservou a fora de preenso muscular na mo dominante das pacientes .

abstractobjective : to evaluate the change in respiratory function and functional capacity according to the type of preoperative fasting.methods:randomized prospective clinical trial , with 92 female patients undergoing cholecystectomy by laparotomy with conventional or 2 hours shortened fasting . the variables measured were the peak expiratory flow , forced expiratory volume in the first second , forced vital capacity , dominant handgrip strength , and non - dominant handgrip strength . evaluations were performed 2 hours before induction of anesthesia and 24 hours after the operation.results:the two groups were similar in preoperative evaluations regarding demographic and clinical characteristics , as well as for all variables . however , postoperatively the group with shortened fasting had higher values than the group with conventional fasting for lung function tests peak expiratory flow ( 128.762.5 versus 115.759.9 ; p=0.040 ) , forced expiratory volume in the first second ( 1.50.6 versus 1.20.5 ; p=0.040 ) , forced vital capacity ( 2.31.1 versus 1.80.9 ; p=0.021 ) , and for muscle function tests dominant handgrip strength ( 24.96.8 versus 18.47.7 ; p=0.001 ) and non - dominant handgrip strength ( 22.96.3 versus 17.07.8 ; p=0.0002 ) . in the intragroup evaluation , there was a decrease in preoperative compared with postoperative values , except for dominant handgrip strength ( 25.26.7 versus 24.96.8 ; p=0.692 ) , in the shortened fasting group.conclusion:abbreviation of preoperative fasting time with ingestion of maltodextrin solution is beneficial to pulmonary function and preserves dominant handgrip strength .

insulin - dependent diabetes mellitus ( iddm ) is associated with autonomic neuropathy and/or cardiovascular dysregulation , which are major complications of diabetes mellitus ( dm ) . type 1 dm patients have a fourfold to eightfold risk of coronary heart diseases , compared with the general population1 . in a recent study , 69% of children with type 1 dm were found to have one or more cardiovascular risk factors2 . abnormal cardiac autonomic nervous system ( ans ) is recognized as one of the early symptoms of cardiac autonomic neuropathy3 and has been demonstrated in type 1 diabetic children4,5,6,7 . low ans activity may also be associated with symptoms of coronary heart disease and related - risk factors in type 1 dm8 . in our previous study9 , ans activity levels were significantly decreased in type 1 diabetic children in comparison with children without diabetes . regular physical activity and/or exercise training play a critical role in treatment and management of type 1 dm . regular physical activity reduces the blood glucose level and blood pressure , helps control weight , improves lipoprotein profile and insulin sensitivity , and decreases the risk of dm complications10,11,12,13 . these benefits of physical activity remain controversial in type 1 dm patients . to the best of our knowledge , there has not been a study investigating whether 12 weeks of exercise training can modify ans activities and cardiovascular risk profiles of type 1 dm children . thus , this present study focuses on alterations in cardiac ans activities and cardiovascular risk profiles following regular exercise training in children with type 1 dm . the study was approved by the medical center institutional review board of dong - a university for use of human subjects . fifteen type 1 dm children ( all boys ; 13.01.0 years of age ) volunteered for this study . all experimental procedures were explained in detail to all subjects and their parents , who then signed a statement of written informed consent . we considered children with type 1 dm and no predominant neuropathy , cardiovascular diseases , stroke , or retinopathy as potential subjects for the study . we also excluded subjects having kidney dysfunctions ( creatinine > 2.0 mg / dl , glycohemoglobin > 10% , and fasting blood sugar > 200 mg / dl ) , hypoglycemia symptoms with consciousness disorder over at least 3 months , hypertension , or ketonemia . body composition of the subjects was measured by means of bioelectrical impedance ( venus 5.5 , jawon medical co. , ltd . , was undertaken in compliance with the medical center institutional review board of dong - a university approval of the experiment for use of human subjects . in order to determine cardiorespiratory capacity and exercise intensity of each subject for exercise training , a progressive exercise test was performed on a treadmill using the modified balke protocol . maximal oxygen consumption ( vo2max ) of each subject was measured using a metabolic gas analyzer ( quark b2 , cosmed , rome , italy ) by the breath - by - breath method . following warm - up exercise at 4.0 speed for 3 min , the exercise load at a walking speed of 4.8 kph was increased by increasing the grade by 2% every 2 min . maximal heart rate ( hrmax ) was automatically recorded when the vo2max value of each subject was measured . children with type 1 dm took part in the walking exercise program 3 times per week for a total of 12 consecutive weeks . we also determined the exercise duration required for each individual to expend 250 kcal per exercise session by calculating vo2 per min and calorie expenditure per min14 . our power spectral analysis procedures have been completely described elsewhere15,16,17 . in brief , the analog value of the electrocardiogram ( ecg ) monitor ( life scope , nihon kohden ) was digitized through a 13-bit analog - to - digital converter ( transera htb 420 ) at a sampling rate of 1,024 hz . the digitized ecg signal was differentiated , and the resultant qrs spikes and r - r intervals of the ecg were stored continuously in our computer . ecg measurements were conducted before the exercise training started , and after the 12 weeks of exercise training . the stored r - r interval data were shown and aligned sequentially with a sampling frequency of 2 hz and displayed on a computer screen for examination , before power spectral analysis was conducted . then , the dc component and trend were fully erased by digital filtering for the band between 0.007 and 0.5 hz . low filtering at 0.007 hz was chosen to obtain the frequency components associated with thermogenic function . the root mean square value of the ecg r - r interval was calculated and represented the mean amplitude . after passing the data through the hamming - type data window , ecg r - r interval analysis by means of fast fourier transform was performed on consecutive 512-sec time series of r - r interval data obtained during the experiment18 . to examine ans activity , we analyzed very low - frequency ( vlf ) power ( 0.0070.035 hz ) , which expresses the thermogenic sympathetic activity ; low - frequency ( lf ) power ( 0.0350.15 hz ) , which expresses the sympathovagal activity ; the high vagal component ( 0.150.5 hz , hf ) associated with parasympathetic activity , and total power ( 0.0070.5 hz , total ) which expresses all ans activity . the average heart rate of each 512-sec period was also obtained with the standard error ( se ) . blood samples ( 10 ml ) were collected from subjects via the antecubital vein for examination of serum samples after an 8-h overnight fast . each blood sample was centrifuged for 10 min at 3,000 rpm at 4c . serum total cholesterol ( tc ) , high - density lipoprotein cholesterol ( hdl - c ) , and triglyceride ( tg ) concentrations were assayed using an enzyme - linked immunosorbent assay ( elisa ) kit . indexes of cardiovascular lipid profiles were used to calculate the tc / hdl - c ( mg / dl ) and tg / hdl - c ( mg / dl ) ratio . this has completely been published in the previous study for the frequency power spectral analysis of ultrasound signal19 . to evaluate the doppler - shifted fourier pulsatility index ( pi ) analysis , a 5 mhz continuous wave doppler ( vasculab d10 ) set was used to measure forward blood flow velocity ( flow ) in the radial artery . to present a single standardized numeric index expressing the degree of flow braking , a fourier pi introduced in previous literature20 , 21 was used and was expressed as follows : , where an means the amplitude of the fourier harmonic and ao expresses the mean value . for this experiment , the doppler signal was band - pass filtered ( 100 to 10 khz ) and digitized at a sampling rate of 8,192 hz . the digitized data were processed with the hamming window function and 512-point fft to obtain the frequency power spectra , the mean flow , and the fourier pi . all statistical analyses were performed using a commercial software package ( spss version 11.5 for windows , spss inc . , statistical differences for physical characteristics , cardiac autonomic nervous activities , and blood samples between the before and after exercise training were analyzed with the nonparametric mann - whitney u test . the relationships with cardiac autonomic activities and cardiovascular risk profile levels were examined with the pearson correlation coefficients . a p value of < 0.05 was considered statistically significant . the physical characteristics of the subjects before and after exercise training are presented in table 1table 1 . physical characteristics of the subjects between before and after exercise trainingtype 1 dm ( n=15)variablesprepostage ( yr)13.0 1.013.0 1.0height ( cm)156.5 7.1157.7 7.0weight ( kg)49.3 4.950.3 5.1%fat ( % ) 13.3 2.613.7 2.7sbp ( mm hg)111.4 6.1105.6 4.1dbp ( mm hg)66.4 3.263.4 2.1vo2max ( ml / kg / min)41.4 3.143.1 3.0dd ( month)36.7 8.6-values represent means se . sbp , systolic blood pressure ; dbp , diastolic blood pressure ; dd , diabetic duration . a significant difference was observed in height , weight , systolic blood pressure ( sbp ) , and diastolic blood pressure ( dbp ) after exercise training . sbp , systolic blood pressure ; dbp , diastolic blood pressure ; dd , diabetic duration . the changes of cardiac ans activities from hrv power spectral analysis in type 1 diabetic childrenvariablespreposttotal ( ms)998.46232.571587.47449.25lf ( ms)541.26187.59942.44397.84vlf ( ms)128.1158.66389.44198.55values represent means se , * p<0.05 . total , total power ; lf , low - frequency power ; vlf , very low - frequency power shows the alterations of cardiac autonomic nervous activities after exercise training in our subjects . total power , representing overall ans activity , was enhanced significantly after exercise training ( p<0.05 ) . lf power , which is associated with sympathetic and parasympathetic balance , and vlf power , which represents the thermogenic sympathetic activity , were also increased significantly after exercise compared with before exercise ( p<0.05 ) . however , the global sympathetic nervous activity index , sympathetic nervous system ( sns ) index , and hf power , representing the parasympathetic nervous activity , were not significantly different between before and after exercise training . total , total power ; lf , low - frequency power ; vlf , very low - frequency power cardiovascular risk profiles of the subjects with exercise intervention are represented in table 3table 3 . the alterations of cardiovascular risk profiles of type 1 diabetic children variablespreposttc ( mg / dl)179.9 2.6164.4 11.4tg ( mg / dl)137.9 38.8124.2 19.5hdl - c ( mg / dl)64.7 4.759.6 4.5tc / hdl - c ( mg / dl)2.9 0.3 2.8 0.2tg / hdl - c ( mg / dl)2.4 1.0 2.1 0.5hba1c ( % ) 8.2 0.4 8.5 0.5fourier pi5.13 0.35.11 0.3flow34.09 3.1034.41 tc and hdl - c levels were significantly different in children with type 1 dm before and after exercise training . however , tg values were not significantly different after exercise training . in the anti - atherosclerosis profile indexes , the tc / hdl - c and tg / hdl - c ratios of the subjects were also not significantly different despite intervention in the form of exercise training . the hba1c levels , which reflect glycemic control , were also not significantly different after the intervention of 12 weeks of exercise training . fourier pi index and flow , predictors of arterial stiffness , were not also different after walking exercise training . values represent means se , * * p < 0.01 in the present study , we also examined the correlation with cardiac ans activities and cardiovascular risk profiles after exercise training . the pearson correlation coefficients for the cardiac ans activities and cardiovascular risk profiles of the type 1 diabetic children are described in table 4table 4 . the correlation matrix of cardiac ans activities and cardiovascular risk profiles measured after exercise intervention in type 1 diabetic childrenpisbpdbptotalvlflfhfsns indextctghdltc / hdltg / hdlhba1cpi10.250.010.060.100.140.050.220.200.040.280.100.020.16sbp10.380.360.140.200.360.160.120.390.550.520.430.05dbp10.690.020.310.770.420.020.410.310.420.290.32total10.620.770.790.060.120.360.230.390.060.49vlf10.900.080.430.490.390.080.420.030.27lf10.220.330.360.410.050.430.010.42hf10.400.160.150.310.190.090.34sns index10.180.010.080.120.300.14tc10.550.590.380.300.28tg10.290.920.890.16hdl10.520.400.37tc / hdl10.820.07tg / hdl10.40hba1c1values represent means se . * p<0.05 ; * * p < 0.01 ; * * * p < 0.00 . the results showed that the total power of heart rate variability ( hrv ) was correlated with hba1c levels , but the difference did not reach statistical significance ( p>0.066 ) . the vlf power also showed the correlation with tc levels , but there was no significant difference ( p>0.063 ) . however , total and hf power were significantly correlated with the dbp value ( p<0.01 , respectively ) . values represent means se . * p<0.05 ; * * p < 0.01 ; * * * this study evaluated the influence of a long - term walking exercise program on cardiac ans activities and cardiovascular disease risk profiles including a metabolic control factor ( hba1c level ) . cardiac autonomic neuropathy can be described as a result of dysfunction of sympathetic nervous system ( sns ) activity , parasympathetic nervous system ( psns ) activity , or a combination of both22 and is an important complication of type 1 dm since it is associated with increased risk to the cardiovascular system . abnormal hrv in diabetes also shows an increased risk for ventricular arrhythmias and total cardiovascular morbidity and mortality23 . although hrv reduction is recognized as one of the early symptoms of cardiac autonomic neuropathy6 , little is known about the mechanism by which type 1 dm causes hrv reduction . previous studies have suggested that hrv reduction and autonomic neuropathy are associated with impairment of psns activity24 , poor metabolic control5 , 24 , age25 , and diabetes duration5 , 26 . regular physical activity can improve lipoprotein levels , blood pressure , insulin sensitivity , and blood glucose level in the general population . additionally , regular exercise training has beneficial effects on cardiac ans activity in healthy30 , 31 and obese children32 , 33 . however , its effects still remain controversial in type 1 dm . in this study , cardiac ans activities improved significantly after exercise training . on the results for the hrv activities suggest that exercise intervention may play a primary role in management and treatment of autonomic neuropathy among type 1 dm children . since our most recent review of cardiovascular risk factors and physical activity in children with type 1 dm , the present study investigated the effects of exercise training on cardiovascular risk factors in pediatric patients with type 1 dm . a previous study34 reported that increased physical activity in children with type 1 dm is associated with lower lipoprotein levels , a lower dbp , and better glycemic control . in the present study , we observed that tc levels and sbp and dbp values had significantly reduced after exercise training , which was consistent with findings from the previous study34 . other studies have also revealed contradictory results of exercise training with regard to the hba1c value35,36,37,38 . the differences in findings might be due to differences in study designs35 , 37 or because of differences in patient age . in these regards , the present study did not show any changes in hba1c values after exercise training . further studies are needed to evaluate whether exercise training reduces hba1c levels in children with type 1 dm . in adults with type 1 dm , atherosclerotic cardiovascular disease is the most common cause of mortality and morbidity29 . one possible mechanisms for this may be vascular stiffness , which may be an independent risk factor for atherosclerosis39 , 40 . arterial vascular alterations begin in childhood and adolescence41 . in our previous study19 , fourier pi measured by ultrasound technique was significantly associated with lipid profiles in healthy adults . it is suggested that a damping of the blood velocity waveform and increased arterial stiffness might accompany unfavorable distributions of lipids and lipoprotein . the present study investigated the relationship between two diagnosis indices of peripheral arterial disease and ans activities . however , no correlation was found between the fourier pi results and abnormal cardiac ans activity in type 1 dm children . these findings might have derived from the small number of subjects and the ages of the subjects . although diabetes mellitus increases the progression of arterial diseases such as arterial stiffness and arteriosclerosis , arterial stiffness is a prominent marker of the aging process in humans42 including cardiovascular disease mortality and morbidity . nevertheless , there has been no study , to the best of our knowledge , that has investigated the correlation among long - term exercise training , cardiac autonomic dysfunction , and cardiovascular risk factors in children with type 1 dm . from this point of view , the present study may provide valuable information , although no significant correlation was found among them . in conclusion , our results suggest that a regular walking exercise program may play an essential role in regulating diabetes complications such as subclinical autonomic neuropathy and cardiovascular disease risk in children with type 1 dm . regular exercise intervention should be recommended in the management of children with type 1 dm .

[ purpose ] we investigated the effect exercise training has on cardiac autonomic nervous system ( ans ) and cardiovascular risk profiles in children with type 1 diabetes mellitus ( dm ) . [ subjects ] fifteen type 1 dm children ( all boys ; 13.01.0 years of age ) were enrolled in the study . [ methods ] the subjects received exercise training three times a week in a 12-week program . each child was asked to walk on a treadmill to achieve an exercise intensity of vo2max 60% . ans activity was measured by power spectral analysis of the electrocardiogram ( ecg ) . blood samples were obtained for serum lipid profiles . to evaluate doppler - shifted fourier pulsatility index ( pi ) analysis , a 5-mhz continuous wave doppler ( vasculab d10 ) set was used to measure forward blood flow velocity ( flow ) in the radial artery . [ results ] total and low - frequency ( lf ) power of heart rate variability increased significantly after exercise intervention . total cholesterol ( tc ) levels were significant lower after exercise intervention . total and high - frequency ( hf ) power were significantly correlated with higher tc levels , but diastolic blood pressure and hf was significantly correlated with lower tc levels . [ conclusion ] regular exercise intervention should be prescribed for children with type 1 dm .

rheumatoid arthritis ( ra ) is a chronic inflammatory disease of polyarticular arthritis affecting approximately 1% of adults worldwide [ 1 , 2 ] . it typically leads to deformity and destruction of the joints and systemic disorders throughout the body as well . although the etiology and pathogenesis of ra remain unknown , immunological hyperreactivity caused by large numbers of t cells , mostly cd4 , and plasma cells is generally considered to be important to contribute its development . the clinical diagnosis of ra is based on several criteria , including physical symptoms , joint radiographs , and serological tests . in the acr / eular 2010 classification criteria , definite ra is defined based on the confirmed presence of synovitis in at least 1 joint , absence of an alternative diagnosis that better explains the synovitis , and achievement of a total score of 6 or greater ( of a possible 10 ) from the individual scores in 4 domains : number and site of involved joints ( score range 05 ) , serologic abnormality ( score range 03 ) , elevated acute - phase response ( score range 0 - 1 ) , and symptom duration ( 2 levels ; range 0 - 1 ) . multiple studies have revealed ra - related autoantibodies and numerous biomarkers including cytokines / chemokines as well as erythrocyte sedimentation rate ( esr ) and c - reactive protein ( crp ) orchestrate pathological processes in ra [ 46 ] . during the course of the disease , ra patients need to be diagnosed very early , possibly before diagnostic criteria are fulfilled or maybe even before clinical symptoms are apparent . early identification of patients with ra will help improve clinical outcomes with early treatments . finally , screen tests for prediction of response to therapy and progression of the diseases are also necessary . a biomarker , also known as biological marker , generally refers to a measured characteristic which may be used as an indicator of some biological state or condition . this term occasionally refers to a substance whose presence indicates the existence of living organisms . biomarkers play pivotal roles in disease diagnosis and interventions at early stage and are also helpful in knowing the state of treatment and how body is acting or responding to the medication . therefore , exploring and measuring biologic markers in blood or in joint fluids may serve as not only indicators of diagnosis but also indicators of prognosis and the subsequent response to therapy . a good biomarker can be used to measure the disease progress and the treatment effectiveness . , we discuss the biomarkers and pathogenic - related cytokines involved in clinic , pathogenesis , and prospection in ra . the biomarkers currently used for the diagnosis of ra are mostly clinical . now there are several useful clinical biomarkers , including various autoantibodies , such as rheumatoid factors ( rf ) , anti - perinuclear factor ( apf ) , anti - keratin antibodies ( aka ) , anti - filaggrin antibodies ( afa ) , and anti - cyclic citrullinated peptide antibodies ( anti - ccp ) . erythrocyte sedimentation rate ( esr ) and c - reactive protein ( crp ) also have associations with ra . rheumatoid factor ( rf ) , an antibody to the igg fc region , is a classic feature of the disease with sensitivity of 6080% , but its specificity is not very high since it can also be detected in other autoimmune diseases , such as systemic lupus erythematosus ( sle ) and sjogren 's syndrome ( ss ) . the key pathogenic markers are igm and iga rheumatoid factors in ra . showing perinuclear fluorescence , apf is probably an antibody of the 7s gammaglobulin type against keratohyalin granules in buccal mucosa cells and is high in ra patients compared to the healthy people . aka , a naturally occurring antibody that reacts with the keratinized tissue , is found in the serum of around 60% ra patients but not in healthy people . autoantibodies induced by citrullination , conversion of peptidyl - arginine to peptidyl - citrulline , can be measured efficiently by using cyclic citrullinated peptides ( ccp ) as antigens [ 11 , 12 ] . the detection of anti - ccp antibodies , as well as rfs , particularly iga - rf , has a strong predictive value for diagnosis in early ra [ 12 , 13 ] . not only esr but also anti - ccp is an important predictor of early bone mineral density loss . similar to rf , antibodies against citrullinated proteins ( acpa ) have correlation with bone destruction , but the latter is associated with larger bone erosions by directly initiating the differentiation of bone - resorbing osteoclasts [ 15 , 16 ] . moreover , the bone loss caused by acpa occurs before disease onset in acpa - positive patients [ 17 , 18 ] . the knowledge on acpa made great advancement in the field of ra in the past decade and this biomarker has already been included into the acr / eular 2010 diagnostic criteria . using both rf and acpa may be helpful in diagnosis and classification of ra [ 19 , 20 ] . furthermore , anti - mutated citrullinated vimentin ( mcv ) antibodies have been reported as a fairly sensitive serological marker of ra and were significantly higher in early ra patients . in clinical management of ra , crp and esr are commonly ordered tests to guide diagnosis of ra besides the measurements of the mentioned autoantibodies . increased crp was detected before the onset of ra and also indicated the activity of the disease during the course of the disease . erythrocyte sedimentation rate ( esr ) is a nonspecific measure of inflammation and can be useful not only in diagnosing autoimmune diseases , such as rheumatoid arthritis , but also in monitoring the disease process . the combination of esr and crp may improve sensitivity and specificity of the diagnosis of ra [ 23 , 24 ] . what is more , levels of serum calreticulin ( crt ) are now detected to be increased in patients with ra compared with those controls and have a significant correlation with disease activity in ra . likewise , there is an autoantibody system that discriminates between citrulline- and homocitrulline - containing antigens in the sera of ra patients . anti - carbamylated protein ( anti - carp ) antibodies igg and iga were observed in ra sera . interestingly , these anti - carp antibodies are also present in around 20% of the acpa - negative ra patients and have association with more severe joint damage , indicating they could serve as a unique and relevant serological marker for acpa - negative ra . important associations between the above clinical markers and severity of ra have been noted and used in aiding diagnosis of ra , monitoring disease progress , and assessing prognostic in patients with established disease . besides the clinically used biomarkers , scientists have been engaging in investigating potential biologic markers for many years . cytokines serve as molecular messengers between cells and there are multiple studies revealing that numbers of cytokines have been involved in the pathogenesis of ra by triggering or regulating the inflammatory responses ( figure 1 ) [ 28 , 29 ] . overexpressions of certain cytokines , such as il-1 , il-6 , il-8 , il-17 , il-21 , tumor necrosis factor ( tnf)- and granulocyte - macrophage colony - stimulating factor ( gm - csf ) , were observed in ra patients [ 3035 ] . these cytokines could promote synovial membrane inflammation and osteocartilaginous resorption via stimulation of osteoclastic mediators . autoreactive t cells , such as th1 and th17 cells , are thought to play important roles in autoimmune pathology of ra [ 3638 ] . the percentage of interferon ( ifn)- expressing cells was slightly increased in peripheral blood mononuclear cells ( pbmc ) but highly increased in synovial fluid mononuclear cell ( sfmc ) of ra patients . il-12 , a th1 cytokine , was increased in serum and synovial fluid in ra and its level correlated with disease activity score . th17 profile therefore has shown pathogenic role in ra [ 4042 ] . increased il-17 was reported in the sera and synovial fluid of ra patients [ 4245 ] . neutralizing il-17 treatment reduced the severity and slowed the progression of collagen - induced arthritis ( cia ) , a mouse model of human rheumatoid arthritis . secreted by th17 , il-21 and il-22 were also observed at a high level in ra patients compared to the healthy controls or osteoarthritis ( oa ) patients [ 43 , 47 ] . in addition , il-21 autoregulated its own production in human cd4 + t cells and enhanced th17 proliferation , leading to the expression of rorc production , and was strongly associated with the levels of auto - antibodies and disease severity as well [ 31 , 48 ] . il-23 is known to promote il-17 production and was detected in ra synovial fibroblasts , but , like a loop , it can be upregulated by il-17 in ra synovial fibroblasts via pi3-kinase / akt- , nf-b- , and p38-mapk - mediated pathways . il-23 directly induced osteoclast differentiation by upregulation of receptor activator of nf-b ( rank ) in mouse myeloid precursor cells and rank ligand ( rankl ) in human fibroblast - like synoviocytes [ 51 , 52 ] . rank / rnakl signaling regulates the formation of multinucleated osteoclasts from their precursors as well as their activation and survival in normal bone remodeling and in a variety of pathologic conditions . in ra synovial tissue , p19 subunit and rankl have positive correlation and might contribute to bone destruction in ra [ 54 , 55 ] . il-1 is produced by a variety of cells that are part of the innate system and mediates bone resorption and cartilage destruction . the il-1-nf-b axis is central in the production of proinflammatory mediators in the inflamed synovium [ 56 , 57 ] . nf-b activation by il-1 induced the gene expressions of matrix metalloproteinases ( mmps ) that are major products of cytokine stimulated fibroblast - like synovial cells ( fls ) and efficiently degrade the collagenous components of cartilage and bone , leading to the joint deformity and pain in the patient with ra . additionally , proinflammatory cytokine , il-6 , has important effects on the differentiation and activation of b and t cells , macrophages , osteoclasts , chondrocytes , and endothelial cells and broad effects on hematopoiesis in the bone marrow . together with tgf- , il-6 is the key inducer of th17 via signal transducer and activation of transcription-3 ( stat-3 ) and its level was significantly associated with clinical symptoms and levels of clinical biomarkers . additionally , tnf- has shown its contribution to the pathogenesis of ra at early stage . it is produced locally in the joint by synovial macrophages and lymphocytes infiltrating the joint synovium . tnf has been recognized as a key pathogenic cytokine that drives a pathogenic cytokine milieu leading to tissue damage [ 61 , 62 ] . other cytokines / chemokines , such as il-8 , il-9 , il-15 , leukemia inhibitory factor ( lif ) , granulocyte colony - stimulating factor ( g - csf ) , chemoattractant protein-1 ( mcp-1 ) , growth - related oncogenes gro- and gro- , ccl19 , cxcl12 , and cxcl13 , were upregulated in preclinical or clinical period of ra development by induction of il-17 or tnf- and il-1 synergically [ 4 , 5 , 63 , 64 ] . therapeutic strategies that specifically block these investigational inflammatory cytokines are expected to be highly effective in treating ra patients . the biological therapeutics include tnf inhibitors , il-1 inhibitors , il-6 inhibitors , and il-17 inhibitors , as well as b cell depletion , costimulatory blockers , and jak inhibitors [ 20 , 35 , 65 ] . on one hand these approaches neutralize the inflammatory mediators and show effectiveness in clinical trial ; on the other hand , their effect can be evaluated by measuring the concentration of the inflammatory cytokines combined with clinical biomarkers , such as rf , acpa , esr , and crp . recently , several interleukins involved in inflammatory joint diseases have been identified and characterized ( figure 1 ) . il-7 , which is an il-2 cytokine family member , involves t cell - driven autoimmunity , inflammation , and tissue destruction [ 67 , 68 ] . in addition , il-7 and il-7r were coexpressed on ra synovial tissue lining and sublining macrophages and endothelial cells . this cytokine can stimulate tnf- and ifn- production and its level correlates with disease activity , suggesting it has value as a diagnostic biomarker predicting the progression to ra [ 71 , 72 ] . the overproduction of il-33 , a newly identified inflammatory cytokine , was found in synovial fluids of ra patients . it could be upregulated by hypoxia - inducible factor-1 ( hif-1 ) which was also increased in ra synovium through the activation of p38 and erk pathways . and these two participants exacerbate the disease severity and may serve as new therapeutic targets in biologic treatment . il-34 , expressed by giant cell tumours of bone , plays a critical role in macrophage differentiation and osteoclastogenesis [ 74 , 75 ] . in ra patients , the secretion of il-34 was upregulated in fls by tnf- or il-1 induction and was mediated by the transcription factor nuclear factor b ( nf-b ) and activation of c - jun n - terminal kinase ( jnk ) , indicating its osteoclastogenic role in ra [ 76 , 77 ] . increased il-36 ( one form of il-36 ) expression was found in ra synovium and could lead to il-6 and il-8 production by synovial fibroblasts through p38/nfb activation in vitro . il-36 , known as il-1f8 , was found to be expressed in synovial membrane in cia and synovial membrane biopsies from ra patients [ 66 , 79 ] . another member of il-1 family which was detected in various inflammatory diseases including ra is il-37 which interacted intracellularly with smad3 [ 66 , 80 ] . in contrast to the inflammatory cytokines , il-35 is an inhibitory cytokine that contributes to regulatory t ( treg ) cells function and suppresses the pathogenic cells , such as th1 and th17 cells [ 55 , 81 , 82 ] . il-35 belongs to il-12 family and is mainly produced by cd4+foxp3+treg cells [ 55 , 83 ] . il-27 is a novel member of il-12 family and has two sides ' properties , pro- or anti - inflammatory in ra . significantly higher concentration of il-27 was found in plasma and synovial fluid of ra patients than in oa patients [ 86 , 87 ] . expressions of icam-1 , vascular cell adhesion molecule , inflammatory chemokines , such as il-6 , ccl2 , cxcl9 , cxcl10 , and mmp-1 , in ra fls were induced under il-27 stimulation . although il-27 shows its pathogenic role , it was also found to have suppressive function in macrophage responses to tnf- and il-1 . furthermore , il-27 also promotes cd4+foxp3-t regulatory cell to produce il-10 and downregulate il-6 and il-1 in early cia , which resulted in suppression of th17 and monocyte migration and vascularization [ 89 , 90 ] . although numerous studies revealed the new candidates participating in development of ra , their exact mechanisms by which these molecules modulate the proinflammatory or anti - inflammatory response still require elucidation . these emerging players not only give us new evidences in complicated pathogenesis of ra but also suggest new strategies of therapeutic intervention in ra since current biotherapies are not effective for all ra patients . ra is a chronic and systemic autoimmune disease that affects many tissues and organs , especially flexible joints . biomarkers , not only already clinically used but also under investigation , play important roles in the development of chronic human diseases which include ra . they may serve as indicators for clinical observation on the disease progression and the therapeutic effect . furthermore , some of the biomarkers can also be or has already been a therapeutic target for the treatment . we expect that targeting specific biomarkers with the least side effects will bring hope to ra patients .

rheumatoid arthritis ( ra ) is a common autoimmune disease with unknown etiology and pathogenesis . although major therapeutic advances have been made in recent years , there is no cure for the disease . current medications mainly reduce inflammation in order to relieve pain and slow joint damage , but many have potentially serious side effects . therefore , to find specific biomarkers will benefit both ra patients to find relief from the disease and physicians to monitor the disease development . a number of biomarkers have been discovered and used clinically , and others are still under investigation . the autoantibodies , which are widely used in diagnosis and prognosis , novel biomarkers , which reflect clinical disease activity , and newly found biomarkers and pathogenic - related cytokines are discussed in this review .

the lifetime risk of elderly undergoing at least one surgery for prolapse or incontinence by age of 80 years is 11% . pelvic organ prolapse ( pop ) is rampant in a remote village called shillai , district sirmour , himachal pradesh . a project prolapse was introduced by the department of obstetrics and gynaecology , santparmanand hospital in 2009 . mobile surgical camps in this area were organized to address the surgical needs of these poor women . these camps have been conducted in remote , unreached villages and not in regular well - equipped hospital . the current study endeavors to look in to the epidemiology , pop quantification ( popq ) staging and surgeries in these camps . to study the epidemiological factors responsible for prolapse in village womento do popq staging and evaluate most common type of prolapseto study the variety of surgeries conducted and their outcome . to study the epidemiological factors responsible for prolapse in village women to do popq staging and evaluate most common type of prolapse to study the variety of surgeries conducted and their outcome to study the epidemiological factors responsible for prolapse in village womento do popq staging and evaluate most common type of prolapseto study the variety of surgeries conducted and their outcome . to study the epidemiological factors responsible for prolapse in village women to do popq staging and evaluate most common type of prolapse to study the variety of surgeries conducted and their outcome this is a retrospective analysis of surgeries conducted in five camps in shillai in himachal pradesh from 2009 to 2013 . funds for the camps were raised from government national rural health mission or nongovernment ( ongc , friends of sant parmanand hospital ) organizations . a group of general surgeon , gynecologist , anesthetist , and ophthalmologist hires a local team of paramedical staff , cook and driver from a city nearest to the village . all the enrolled patients are admitted 2 days prior to the local government or private hospital and subjected to the thorough examination , preanesthetic checkup and investigations as hemoglobin , platelet count , blood urea , liver function test , urine routine , random blood sugar , hiv , hbsag , chest x - ray , and electrocardiography . only american society of anesthesiologist ( asa-1 ) , . visual inspection of the cervix and not pap smear is done because of logistic reason . the meals medication and lodging is provided free to all patients , and they are discharged on day 3 usually with discharge medication and to and fro transport fare in their hand . in last five camps , a total of 490 surgeries conducted in five camps , out of which 192 were done for gynecological problems . eighty - two percent of these surgeries were conducted for pop [ table 1 ] . the minimum and maximum age of the patients was 30 and 70 years with a mean age being 47.2 years . early marriage was documented in 92% patients with a mean age of marriage being 18.2 years . all women had unassisted home deliveries in the cow shed sitting in a squatting position on two bricks as told by the elderly women in the house . the mean postpartum rest period was only 20 days except one patient who rested for 1 year before restarting the strenuous physical work and sexual activity . smoking was a habit in 33.3% subjects and 54.7% women were used to carrying heavy weights of wood and grass on their backs uphill . staging of the cases was done on standard popq system as shown in table 4 . the most common type of surgery done was vaginal hysterectomy with anterior repair with culdoplasty . manchester repair was done in one patient , 3.6% patients were subjected to vault suspension with sacrospinous fixation and 7.56% patients with posthysterectomy cystocele were managed by cystocele repair with mesh . one patient had intra - operatively diagnosed bladder injury , which was repaired and seven patients had significant intra - operative and postoperative complication as shown in table 7 . all these patients were managed in the camp setting only since all of them were asa-1 and thin built . a total of 490 surgeries conducted in five camps , out of which 192 were done for gynecological problems . eighty - two percent of these surgeries were conducted for pop [ table 1 ] . the minimum and maximum age of the patients was 30 and 70 years with a mean age being 47.2 years . early marriage was documented in 92% patients with a mean age of marriage being 18.2 years . all women had unassisted home deliveries in the cow shed sitting in a squatting position on two bricks as told by the elderly women in the house . the mean postpartum rest period was only 20 days except one patient who rested for 1 year before restarting the strenuous physical work and sexual activity . smoking was a habit in 33.3% subjects and 54.7% women were used to carrying heavy weights of wood and grass on their backs uphill . staging of the cases was done on standard popq system as shown in table 4 . the most common type of surgery done was vaginal hysterectomy with anterior repair with culdoplasty . manchester repair was done in one patient , 3.6% patients were subjected to vault suspension with sacrospinous fixation and 7.56% patients with posthysterectomy cystocele were managed by cystocele repair with mesh . one patient had intra - operatively diagnosed bladder injury , which was repaired and seven patients had significant intra - operative and postoperative complication as shown in table 7 . all these patients were managed in the camp setting only since all of them were asa-1 and thin built . the all india poverty ratio declined from 37.2% in 2004 - 2005 to 29.8% in 2009 - 2010 , but still in rural areas about 33.8% population is designated below the poverty line . she is malnourished often not sent to school , eats her brothers left over , is married off early , has frequent unattended child births with limited access to health facilities . once her productive years are over she is typical elderly recluse in the family with no one to give her an ear and not spare penny to spend on her . these are the women who are targeted in mobile surgical camps under project prolapse . reducing maternal morbidity , which causes untold suffering to millions of women , is not accorded comparable priority . for every maternal death , an estimated 6 - 15 women face debilitating morbidity , which goes higher in remote villages of india . a woman who has never moved out of her village in her lifetime , shall die quietly with her disease in her village only . hence , organization of such camps to reach the unreached is the moral duty of each doctor and government or nongovernment organizations involved in social responsibility programs . early marriage , multiparty , malnutrition , unattended deliveries , carrying heavy weights on their backs with a full bladder is probably responsible for the anterior compartment prolapse in these women confirming similar results from other studies too . smoking - induced activation of vaginal macrophage elastase had been suggested in the pathogenesis of prolapse in women who smoke . on statistical analysis a positive correlation was found between degree of prolapse and parity / early resumption of postpartum work , which means carrying heavy weight on back uphill , but the r was low suggesting need for more number of observations . the community health providers and female health volunteers must propagate against early marriage , encourage family planning practices , and promote nutritious diet , antenatal care , and delivery by skilled birth attendants . they should teach kegel 's exercises and should be able to detect early presentations of prolapse and manage them with nonsurgical interventions before surgery is mandatory . the midwives should be trained for correct conduct of labor using pelvic infant models or projectors and should be trained to teach postpartum exercises . women should be counseled to avoid strenuous physical work is postpartum period . in patients of prolapse with completed family , vaginal hysterectomy is the patient evaluated efficient 1 time treatment . manchester repair in 0.01% , non descent vaginal hysterectomy in 4.1% and total abdominal hysterectomy in 10.9% and cervical biopsy 0.01% . histopathology of the entire postoperative specimen was showing benign lesion except one patient who had cancer cervix on cervical biopsy and was referred for radiotherapy . a recurrence of vault prolapse is seen in 0.2 - 12.8% subjects as per literature and that observed in our study was 3.6% who were managed by vault suspension or anterior colporrhaphy with mesh in patients with cystocele . all the surgeries were conducted by senior specialists , so the complications were negligible 2.5% . one intra - operative bladder injury and two cases of hemorrhage were managed efficiently by early detection . mounted gauze used to push bowel away was lost in one patient who required laparotomy and one diabetic patient had posthysterectomy wound infection . complication rate in hysterectomies in camps as reported in literature is 15.1% . the duration of suffering from prolapse both vaginal and rectal was as long as 50 years in one patient [ figure 1 ] which suggests dearth of surgical facilities in our remote villages . one mentally disturbed socially neglected widow with huge prolapse and impacted big urethral stone was a shame on humanity [ figure 2 ] . the improved quality of life of these women postsurgery encourages the team to conducted many more such camps . in each camp , parallel counseling of women and dais for prevention of prolapse and training of postgraduate gynecologists for prolapse surgery is thought to go in long run to do away with rampant prolapse in this area in remote village of himachal pradesh poor nutrition , multiparty , unattended births , heavy workload , and smoking were believed to be the contributing factors toward high incidence of prolapse . anterior compartment prolapse was the most common as per popq staging and was thought to be secondary to uphill tasks with heavy weights on back with full bladder . vaginal hysterectomy with repair and culdoplasty was an effective 1 time treatment for these women . free surgical camps targeting an unreached population of women with rampant pop are a helpful intervention to improve overall health and quality - of - life of these women . let their suffering not end with the sunset of their lives , let us be of some help to others before the sunset of our lives

aims : the aim was to study the epidemiological factors responsible for pelvic organ prolapse ( pop ) in poor women of the remote village shillai , do their pop quantification staging , to study the variety of surgeries conducted in mobile surgical camps in this area.materials and methods : retrospective analysis of surgeries conducted in five mobile surgical camps in shillai , himachal pradesh from 2009 to 2013 , under project prolapse.results:a total number of surgeries conducted in five camps from 2009 to 2013 were 490 including 192 gynecological surgeries . eighty - two percent of gynecological surgery was conducted for pop . poor nutritional status ( mean weight 41.1 kg ) , multiparty ( mean 3.5 ) , early marriage ( mean age 18.2 years ) , unassisted home deliveries ( 100% ) , premature bearing down ( 23.8% ) , early postpartum resumption of strenuous activity ( 54.7% ) and smoking ( 33% ) contribute to the high incidence of pop . anterior compartment prolapse was seen in 99% of patients undergoing surgery while posterior compartment prolapse was seen in 4% of patients . vaginal hysterectomy with anterior repair with culdoplasty was the most common procedure performed ( 73.4% ) , and vault suspension was done in 3.6% subjects . the complication rate was negligible.conclusion:uterovaginal prolapse is not only socially embarrassing and disabling ; its surgical treatment is complex and costly too . the free mobile surgical camps under project prolapse in shillai , himachal pradesh has provided relief to old neglected , disabled women suffering from prolapse in this remote village . parallel counseling of women and dais for safe hospital delivery and training subordinates in prolapse surgery may help in addressing the problem of pop in this area in the long run .

previous reports suggest that about two thirds of middle - aged men and women suffer from nocturia [ 1 - 4 ] . according to one study , the prevalence of nocturia increased at an annual rate of 7.3% in men and 3.5% in women . generally , the prevalence of nocturia is thought to be higher in men than in women , especially in the elderly population . however , nocturia is also one of the most bothersome lower urinary tract symptoms ( luts ) in women as well as in men . newman and koochaki showed that 72% of women with overactive bladder ( oab ) reported that nocturia was very or extremely bothersome among their luts . the results of a meta - analysis of the prevalence of nocturia showed that 20.4% to 43.9% experienced nocturia of more than 1 episode , and 4.4% to 18% experienced nocturia of more than 2 episodes . in older women , the incidences were increased : 74.1% to 77.1% of older women showed nocturia of more than 1 episode and 28.3% to 61.5% of women showed nocturia of more than 2 episodes . in addition , research on the association between age and nocturia in korean women showed that the most common cause of nocturia was nocturnal polyuria as shown by use of 3-day frequency volume charts . also , the incidence of nocturnal polyuria tended to increase as the women grew older . desmopressin is a synthetic analogue of antidiuretic hormone that has been used to reduce nocturia , especially nocturnal polyuria , which is an overproduction of urine at night . the clue to the use desmopressin is that secretion of antidiuretic hormone is decreased in elderly people compared with young adults ; therefore , replacement of antidiuretic hormone can reduce nocturnal voids . studies of the effect of desmopressin have shown significant decreases in the number of nocturia episodes , nocturnal urine volume , and nocturnal diuresis . as a result , the first sleep period was longer and sleep quality was improved after medication with desmopressin . adverse effects such as hyponatremia were similar between patients who received placebo and those who were treated with desmopressin according to randomized controlled trials [ 10 - 12 ] . for women , desmopressin can also help to reduce the nocturia caused by nocturnal polyuria , which is the most prevalent cause of nocturia in elderly women . in addition , nocturia is related to various voiding problems that are common in old - age women . for example , most nocturia is associated with oab - related storage symptoms ; therefore , reduced functional bladder capacity is regarded as one of the causes of nocturia . as a result , nocturia can also be improved by resolving the voiding problems causing nocturia . we analyzed concomitant voiding dysfunction occurring with nocturia and the effect of desmopressin on nocturia retrospectively . we reviewed the medical records of 84 women who were treated with desmopressin 0.1 or 0.2 mg and had reported more than 2 nocturnal voids on a pretreatment frequency volume chart in the urologic outpatient clinic between january 2008 and december 2011 . all patients underwent a physical examination with a comprehensive history taking , physical examination , urinalysis , international prostate symptom score ( ipss ) , and a 3 day frequency volume chart . patients who had neurologic diseases , previous radical pelvic surgeries , or pelvic organ prolapses were excluded from the study . numbers and volumes of voids over 24 hours were calculated as an average over the 3 days of the frequency volume chart . nighttime urine production , maximal functional bladder capacity , and nocturnal index ( nocturnal polyuria index [ npi ] and nocturnal bladder capacity index [ nbci ] ) were calculated according to the international continence society . the mean age of the women was 66.8 years old ; therefore , we defined nocturnal polyuria as a nighttime urine volume of more than 33% of the total daily urine volume ( npi>0.33 ) . reduced nocturnal bladder capacity was defined as nbci greater than 1 . mixed - type nocturia was defined as a combination of nocturnal polyuria and reduced nocturnal bladder capacity . after 1 month , the dose of desmopressin was increased to 0.2 mg in patients who showed no effect with desmopressin 0.1 mg . the treatment effect was evaluated in the patients who needed dose escalation after 1 month to desmopressin 0.2 mg . after treatment with desmopressin , a reduction by more than half in the number of nocturnal voids compared with baseline was regarded as effectiveness . improvement was defined as a reduction of nocturnal voids regardless of the number compared with baseline . the data were analyzed by using the mann whitney u - test , and p - values < 0.05 were considered statistically significant . the mean patient age was 66.810.2 years ( range , 39 to 72 years ) . eight patients had diabetes mellitus and 1 had a total hysterectomy owing to uterine myoma . the mean number of nocturia episodes was 3.71.3 ( range , 2 to 5 ) . mean ipss storage and voiding subscores were 10.14.4 and 9.37.1 , respectively . among 84 women , 60.7% ( 51/84 ) complained of concomitant oab symptoms such as frequency , urgency , or urge incontinence , and they were treated with anticholinergic agents . a total of 10.7% ( 9/84 ) took an alpha - blocker or cholinergic agent owing to concomitant female voiding dysfunction ( fvd ) . a total of 13.1% ( 11/84 ) of patients had previously undergone anti - incontinence surgery owing to stress urinary incontinence . according to the analysis of the 3-day frequency volume chart , 70.2% ( 59/84 ) of the women had nocturnal polyuria , 7.1% ( 6/84 ) had reduced nocturnal bladder capacity , and 22.6% ( 19/84 ) had mixed type nocturia ( table 1 ) . after 1 month of treatment with desmopressin , the percentage of women who needed dose escalation of desmopressin from 0.1 to 0.2 mg was 39.3% ( 33/84 ) . after medication with desmopressin , 73 women ( 86.9% ) showed improvement of nocturia and the mean number of nocturia episodes ( 1.41.5 ) was significantly reduced compared with baseline ( 3.71.3 ) ( p<0.05 ) . among the women who showed improvement , a reduction by more than half in the number of nocturnal voids compared with baseline was observed in 41 women ( 48.8% ) . eleven women ( 13.1% ) did not show improvement of nocturia after medication with desmopressin ( fig . the 41 women in whom the number of nocturnal voids was reduced by more than half had a lower baseline urgency grade ( 3.31.2 ) by the urinary sensation scale than did the 32 women who showed improvement in nocturia but not by more than half ( 4.21.1 ) ( p<0.05 ) ( fig . among the women treated with desmopressin in this study , oab was observed in 60.7% of women and fvd in 10.7% . nocturia was reported by 13.1% of the women after a transobturator tape procedure . according to the 3-day frequency volume chart , 70.2% of the women had nocturnal polyuria , 7.1% had reduced nocturnal bladder capacity , and 22.6% had the mixed type of nocturnal polyuria combined with reduced nocturnal bladder capacity . overall , the mean number of nocturia episodes was significantly reduced compared with before treatment ; however , a reduction in the number of nocturia episodes by more than half was observed in 41 of the 73 women who showed improvement after medication . furthermore , a significantly lower baseline urgency grade was observed in the women in whom nocturia was reduced by more than half than in the women showing improvement of nocturia but not by more than half after treatment with desmopressin . in general , nocturia is well known to be a multifactorial disease that is also related to luts . the prevalence is reported to be 16% to 54.5% among community - dwelling women , and the symptom of nocturia is included in the symptoms complex of oab [ 14 - 16 ] . in this study , the reason for this relatively high ratio might be the inclusion of patients who visited a tertiary hospital . the cause of nocturia related with oab is related to impairment of the storage function of the bladder ; therefore , anticholinergic therapy to relieve oab symptoms can decrease nocturia . however , some patients do not benefit from oab treatment alone . in this study , 60.7% of women had oab and they also complained of nocturia of more than 2 times even after anticholinergic treatment . we suspect that this finding may be because the majority of patients with oab had concomitant nocturnal polyuria . in addition , we observed that the portion of nocturnal polyuria is greater if women with both nocturnal polyuria only and those with the mixed type are included . nocturnal polyuria can occur as the result of excessive nighttime urine production despite normal 24-hour urine output in association with impaired secretion of arginine vasopressin . the prevalence of nocturnal polyuria has been reported to be up to 82.9% , and it is regarded as the most common cause of nocturia [ 19 - 21 ] . according to a study of age - related changes in nocturia in women over 20 years , 40.8% of the women showed nocturnal polyuria , and the ratio in women over 60 years was 46.7% . compared with the previous study , more women ( 66.7% ) in the present study we studied older women ( mean age , 66.810.2 years ) than in the previous report ( 57.211.8 years ) and included women complaining of more frequent nocturia ( more than 2 voids per night ) compared with the previous study ( at least 1 void per night ) . these findings suggest that nocturnal polyuria is a common cause of nocturia , especially in an older population . desmopressin is an effective medicine for relieving nocturia due to nocturnal polyuria . according to a long - term study by lose et al . , the percentage of the study group showing a reduction in nocturia of more than half of the baseline number was 46% at 3 weeks , and this figure increased to 67% at 12 months of desmopressin treatment . another study examined the effect of desmopressin in women with nocturia of more than 2 times and a nocturia index > 1 according to a frequency volume chart . a score of > 1 on the nocturia index suggests increasing nocturnal urine volume ; therefore , the cause of nocturia in patients showing an increase in the nocturia index can be regarded as nocturnal polyuria . after 3 weeks of medication , the authors observed that nocturia was reduced by half or more in 46% of the women . in the present study , 48.8% ( 41/84 ) of women experienced a reduction in the number of nocturia episodes of more than half after desmopressin 0.1 or 0.2 mg , and nocturnal polyuria was observed in 58.5% ( 24/41 ) of them . in addition , 40.7% ( 24/59 ) of the women with nocturnal polyuria showed a reduction in nocturia of more than half after medication with desmopressin . therefore , we also noticed the effect of desmopressin on women with nocturnal polyuria . on the other hand , 38.1% ( 32/84 ) of the women showed a reduction in nocturia that was not more than half of the baseline value after treatment with desmopressin . the difference between these women and those in whom nocturia was reduced by more than half seemed to be the baseline luts , especially urgency . the women who showed a reduction in nocturia that was not more than half of baseline complained of more severe urgency , which may have been the reason for the decrease in the treatment effect . this result suggests that attention should be paid to other luts to increase the treatment effect in patients with nocturia , because common voiding dysfunction like oab is usually combined with nocturia . in addition , there were women who complained of voiding problems other than oab in this study . the women complaining of voiding symptoms , low maximal flow rate , and increased postvoid residual urine volume were regarded as having fvd . therefore , physicians should consider fvd if women with nocturia have symptoms and signs of voiding difficulties . among the women treated with desmopressin , concern about nocturia seems to be necessary after treatment of stress urinary incontinence , because nocturia can be persistent or newly developed regardless of the stress urinary incontinence . although the results of this study might indicate an influence of luts on the treatment outcome of desmopressin in women with nocturia , this study had some limitations . first , it was an uncontrolled , retrospective study and thus the therapeutic effect of desmopressin was not confirmed in the combined medication or anti - incontinence surgery group . second , the number of subjects in this study was small , and therefore further evaluation is necessary in a large number of women with nocturia and other voiding dysfunction . the majority of women showed nocturia and other voiding dysfunction such as oab concurrently . regardless of the type of nocturia as shown by the frequency volume chart , treatment with desmopressin can effectively reduce nocturia . therefore , consideration of luts other than nocturia is needed to increase the treatment effect of desmopressin on nocturia in women .

purposeto investigate the type of nocturia and concomitant voiding dysfunction ( vd ) and the effect of desmopressin treatment on nocturia in women.materials and methodswe reviewed 84 women who experienced more than 2 nocturia episodes as recorded on a pretreatment frequency volume chart and who were treated with desmopressin . all patients underwent history taking , physical examination , urinalysis , international prostate symptom score assessment , completion of a urinary sensation scale , and completion of a 3 day frequency volume chart . nocturia was divided into nocturnal polyuria ( np ) , reduced nocturnal bladder capacity ( rnbc ) , and mixed type . after treatment with desmopressin , a reduction in nocturia of over 50% compared with baseline was regarded as effective.resultsamong 84 women , the most common concomitant vd was overactive bladder ( oab , 60.7% ) . np was observed in 70.2% ( 59/84 ) of the women , rnbc in 7.1% ( 6/84 ) , and mixed type in 22.6% ( 19/84 ) . after medication with desmopressin , 73 women ( 86.9% ) showed a significantly reduced number of nocturia episodes ( 1.41.5 ) compared with baseline ( 3.71.3 , p<0.05 ) . eleven women ( 13.1% ) did not show improvement . of the 73 women who showed improvement , 41 women showed a reduction of more than 50% over baseline , and these women had a lower baseline urgency grade.conclusionsin the majority of women , nocturia coexisted with other vd such as oab . treatment with desmopressin effectively reduced the nocturia . however , other lower urinary tract symptoms ( luts ) such as urgency may reduce the effect of desmopressin . therefore , consideration of concomitant luts seems to be necessary to increase the treatment effect of desmopressin on nocturia in women .

the isoniazid - monoresistant and multidrug - resistant tuberculosis ( mdr - tb ) isolates were obtained from the patients ' source hospital or mru . all isolates were identified as belonging to the same strain of m. tuberculosis ( rapet or is6110 typing ) , and all drug - susceptibility testing was carried out at mru according to standard procedures . the wildtype control isolate used for these studies was m. tuberculosis , h37rv ( atcc , 9360 national collection of type culture , london , uk ) . three isolates , 018 , 483 , and 915 , were from patients in whom mdr - tb developed as a result of poor compliance with therapy , whereas isolates 604 , t7 , and 371 were from patients who contracted primary mdr - tb . dna was prepared from the 6 isolates of m. tuberculosis by emulsifying 23 colonies in 400 l tris - edta buffer and heating the suspension in a water bath for 40 min at 80c . polymerase chain reaction ( pcr ) was performed on the extracted dna with 6 sets of primers designed to amplify 6 overlapping fragments of the rpob gene from the 6 m. tuberculosis isolates ( table ) . ten microliters of dna was added to the pcr mix containing 81.4 l pcr - quality water , 10 l potassium chloride buffer ( bioline ltd . , london , uk ) , 0.4 l of each primer ( 100 mmol ) ( sigma - genosys ltd . , haverhill , uk ) , 3 l deoxynucleoside triphosphates ( 5 mmol ) , and 1 l taq ( bioline ) . the amplification was performed on a techgene thermal cycler ( techne , princeton , nj , usa ) . pcr products were separated by gel electrophoresis on a 1.5% agarose gel , and dna bands were stained with ethidium bromide . primers and excess nucleotides were removed from the amplified dna with a pcr clean - up kit ( qiagen , inc . , the amount of dna in the cleaned - up product was quantified by comparing the intensity of the band to bands of known intensity in a hyperladder marker ( bioline ) . * denotes a primer position in the sequence before the start ( ) or after the end ( + ) of the rpob gene sequence . forward and reverse cycle sequencing reactions were performed with the big dye terminator cycle sequencing ready reaction dna sequencing kit ( applied biosystems , inc . , foster city , ca , usa ) . briefly , 40 ng of cleaned - up dna was added to 10.8 l pcr - quality water , 3 l buffer , 3.2 l of forward or reverse 1 mmol primer , and 1 l of cycle sequencing ready reaction mix . the labeled dna was precipitated by adding 14.5 l pcr - quality water , 62.5 l 95% ethanol , and 3 l sodium acetate solution ( 2.3 mol / l ) and centrifuging ( 13,000 g , 15 min , 4c ) . the supernatant was removed with a fine - tipped pipette , and the pellet was cleaned with 200 l 70% ethanol and then recentrifuged ( 13,000 g , 15 min , 4c ) . again the supernatant was removed , and the pellet was dried at 37c for 30 min . four microliters of formamide and 1 l of dextran loading buffer were added to each pellet , and 1.5 l of sample was added to each well of the sequencing gel . the 6 fragments of the rpob gene from each of the 6 isolates were then sequenced with an abi 377 applied biosystems sequencer . the sequences obtained from each isolate were joined together to form a continuous whole gene sequence , aligned with clustalw ( http://www.ebi.ac.uk/clustalw/ ) and compared to the wildtype to identify base - pair mismatches . in addition , all 6 isolates were tested with a line probe resistance determining hybridization assay , inno - lipa ( innogenetics belgium ) , as described in the manufacturer 's instructions . briefly , an 81-bp region of the rpob gene was amplified with biotinylated primers , which yielded a biotinylated target sequence , and hybridized with specific oligonucleotide probes immobilized on a parallel strip . inno - lipa consists of 10 oligonucleotide probes ( 1923 bases in length ) , encompassing the 81-bp region ( rrdr ) of the rpob gene . one is specific for m. tuberculosis complex , whereas the other 5 partially overlapping wildtype probes ( s1s5 ) cover the region from positions 507 to 534 of the rpob gene . these s - probes hybridize to the wildtype ( rifampicin - sensitive ) dna sequence . failure of any of these s - probes to hybridize indicates that a mutation has occurred . four other probes ( r2 , r4a , r4b , and r5 ) are specific for amplicons carrying the most common rpob mutation that confers rifampicin resistance . the results of the inno - lipa assay showed 5 of the 6 isolates , which were phenotypically rifampicin - resistant , were negative . the target dna hybridized to all 5 wildtype s - probes and none of the r - probes , which demonstrated that the assay failed to detect rifampicin resistance in these 5 isolates . failure to detect rare mutations within the rrdr may be caused by nonspecific hybridization of the wildtype s - probes because of slight fluctuations in temperature during the hybridization process . isolate 483 showed a weak dna hybridization reaction with the s4 probe , indicating that a mutation was present on codon 451 ( codon 526 in escherichia coli ) . analysis of the sequence data identified specific mutations in rpob in all 6 strains studied ( figure 1 ) . three had mutations within the rrdr , and of these , 2 were c - to - g mutations at codon 456 , inducing a serine to tryptophan amino acid conversion ( s456w ) . the third was an a - to - g mutation at codon 451 , resulting in a change from histidine to arginine ( h451r ) ( figure 1 ) . no other mutations were identified in the dna sequences outside the rrdr in these 3 isolates . the 3 other isolates had the mutation g to t at codon 176 , outside the rrdr , which caused a change from valine to phenylalanine ( v176f ) . neither rrdr mutations nor any other mutations were found in the rpob gene sequences of these 3 isolates . wt , wildtype ; t7 , 018 , 604 v176f ( g to t , highlighted in blue ) ; 483 , h526r ( a to g , highlighted in yellow ) ; 915 , 317 , s531w ( c to g , highlighted in pink ) . of the mutations found within the rrdr , h526r occurs in < 4% , and s531w occurs in 1.4% of all rifampicin - resistant isolates , respectively ( 7 ) . mutations outside the rrdr account for < 4% of rifampicin resistance , and few have been described ( 79 ) . in a previous study ( 7 ) , v176f was found in 5 of 18 isolates with no mutations in the rrdr from asia ( 9,10 ) and africa ( 11,12 ) . to our knowledge , this is the first time this type of mutation has been detected in strains isolated in the united kingdom . three different mutations in the rpob gene , v176f , h526r and s531w , were detected in the isolates associated with the isoniazid - resistant outbreak . given that all the mutations found in this study are rare and that mdr - tb developed during treatment in 3 patients ( 018 , 483 , and 915 ) ( figure 2 ) , the rifampicin - resistance mutations observed in these 3 patients likely occurred on 3 independent occasions , as a result of poor compliance with therapy . two of these patients , 018 and 915 , were contacts of the 3 patients who subsequently had primary mdr - tb ( patients t7 and 604 [ v176f ] and 317 [ s531w ] ) ( figure 2 ) . suggested epidemiologic relationship between 6 cases of multidrug - resistant tuberculosis ( mdr - tb ) . resistance to rifampicin developed in patients 018 , 483 , and 915 while on therapy , whereas patients t7 , 604 , and 317 contracted primary mdr - tb . the type of mutation present in each patient 's strain of mdr - tb is highlighted in bold . our study highlights problems associated with using a line probe hybridization assay to detect rifampicin resistance in m. tuberculosis . the inno - lipa assay can not detect mutations outside the rrdr , and failure to detect rare mutations within the rrdr may be caused by nonspecific hybridization of the wildtype s - probes because of slight fluctuations in temperature during the hybridization process . in this study , all the rare mutations , inside and outside the rrdr , were detected by sequencing the entire rpob gene . the inability to identify mdr - tb isolates results in treatment failure and increased risk for transmission of resistant disease in the community . we recommend that , when the index of suspicion for mdr - tb is high and the line probe assays fail to detect mutations conferring rifampicin resistance , the entire rpob gene should be sequenced to prevent unnecessary delay in diagnosing mdr - tb .

mycobacterium tuberculosis isolates cultured from 6 patients associated with an isoniazid - resistant m. tuberculosis outbreak acquired rifampicin resistance . the rpob gene sequence showed that resistance was associated with rare mutations in each isolate . three isolates had a mutation outside the rifampicin resistance determining region .

the compartmentalization involved in viewing the mouth separately from the rest of the body is not acceptable anymore , because oral health affects general health by causing considerable pain and discomfort and by affecting mastication , speech and the person s quality of life and well - being . considering high expenses of dental care procedures , it seems that prevention of oral diseases would be a cost effective solution to this issue . thus , strengthening of preventive public health programs that incorporate oral health into need assessments and target the common risk factors for health promotion are urgently needed . community health workers ( called as behvarz in iran ) are male or female staff of the health houses who deliver primary health care services to the population in first level of health care network . due to integration of oral health care into health care network , community health workers are responsible for providing oral health care to the target groups , including oral hygiene education , periodic examination of the teeth , supervision of sodium fluoride mouth rinsing , referrals to the higher levels and follow up of the outcome.[5 - 6 ] since health care personnel including community health workers are supposed to play a substantial role in preventive oral health care activities , their own oral health knowledge should be good and their oral health behavior should confirm their professional recommendations . 7 with proper knowledge and attitude toward oral health , they can play an important role in the health education of individuals and groups,[8 - 10 ] and act as role models for lay people and the community in large scale . the aim of this study was to determine knowledge and attitude of community health workers toward oral health in east azerbaijan , which is one of the northwest provinces of iran . for this cross - sectional study , a questionnaire was designed according to previous studies and the references used in continuing education courses for community health workers.[11 - 12 ] subsequently , it was checked for face and content validity through a panel discussion with six dental specialists experienced in continuing education programs . the questionnaire was pretested on 20 subjects representative of the study population and the reliability coefficient ( cronbach alpha ) , ease of comprehension and relevance to the intended topics were evaluated . the final version comprised three parts : a ) demographic data including age , gender , education level , work experience and number of continuing education courses that the subject has taken . b ) knowledge about oral health care principles that included multiple choice questions on sequence and time of tooth eruption , signs of tooth decay , bacterial plaque , characteristics of mixed dentition , role of fluoride in caries prevention , role of sugar in caries , fissure sealant , dental treatment for pregnant women , target groups of community oral health care , integrating oral health care in primary health care and oral hygiene instruction to children . c ) attitude section including polar questions about importance of dental care in oral health , preventability of tooth decay , relationship between foods and caries , preference of prosthesis to natural teeth , bleeding on probing as a sign of gingival disease and complementary role of dental floss and toothbrush . the researchers visited the health houses during fall 2015 and asked all 1170 community health workers to fill in anonymous questionnaires . 1150 of the participants returned the completed questionnaires for a response rate of 98% . independent - samples t - test was used to compare knowledge and attitude scores between two gender groups . the mean knowledge and attitude scores of different educational levels and different time lengths passed since the last session of continuing education were compared using one - way anova . bivariate analysis of the data was performed by pearson correlation coefficient and p < 0.05 was considered as the level of significance . table 1 summarizes the demographic data and the differences observed among two gender groups regarding the number , mean age and years of work experience . table 2 shows community health workers knowledge and attitude towards oral health care according to their different educational levels . 82.5% of men and 88% of women were well informed about oral health care principles . on the other hand , increase in age of the participants was in negative correlation with their level of knowledge . ( pearson correlation coefficient = - 0.22 and p= 0.016 ) although the knowledge of community health workers increased with higher level of education , this increase was not statistically significant ( p= 0.08 ) . moreover , higher level of knowledge was in correlation with fewer years of practice ( p= 0.0083 ) . in other words , older subjects and the ones with longer years of service obtained lower grades of knowledge . demographic data of the study population by gender mean score of knowledge and attitude according to level of education of participants the average number of the continuing education courses that community health workers had taken part was about 8 sessions . the average time passed since the last session in 92.5% of the cases was less than one month . pearson correlation coefficient was -0.21 ( p= 0.012 ) indicating an inverse relation between number of the continuing education courses and the level of knowledge , suggesting that the more the frequency of participation in continuing education courses the less the knowledge regarding oral health care ( p= 0.023 ) . the mean level of knowledge in 1 , 6 and 12 months periods after the last session of continuing education course did not differ significantly ( p= 0.148 ) . however , it was not affected by the gender of the participants ( p= 0.08 ) . considering the information in table 2 , it appears that higher level of education does not influence attitude of community health workers ( p= 0.156 ) . pearson correlation analysis failed to determine any relationship between attitude and age or years of work experience or number of continuing education sessions . comparing the mean weight of questions related to attitude of community health workers in 1 , 6 and 12 months from the last continuing education session indicated that their attitude did not differ significantly with time ( p=0.43 ) . attitude and level of knowledge of community health care workers were not associated ( p=0.06 ) . community health workers are key members of integrated public health system , involved in primary prevention and their knowledge and attitude may be important determinants of expected preventive behavior . this study investigated the knowledge and attitude of community health workers in east azerbaijan . in our research , knowledge was defined as what community health workers know about oral and dental health and attitude meant to gauge the prevailing beliefs and ideas regarding oral health topics . poorhashemi , there was no statistically significant relationship between gender and the level of knowledge in the present study . although poorhashemi did not discuss this difference , it seems that the greater number of female subjects than males ( nearly twice ) would be a justified explanation . in our study , the mean age of women was less than men ( p= 0.001 ) . while this might have caused the higher level of knowledge in females , the equal work experience would explain the similarity in level of knowledge in two gender groups . in other words , it appears that the time passed since the graduation of community health workers is a more important determinant than their age in level of knowledge . our results showed that there is not a relationship between higher levels of education with more knowledge regarding oral health care . this might be due to lack of structured instruction on oral health care principles in the educational curriculum from 5 grade to the time of graduation from high school . even the two subjects with associates degree , were educated in the field of public health without any instruction related to oral health care , resulting in a knowledge level similar to other community health workers . this result was in accordance with the study performed by poorhashemi , which did not confirm a similar relation in female group . the present study revealed that there was an inverse relationship between knowledge of community health workers and their age and also their work experience . similar results were obtained in the investigations of poorhashemi and taghavi et al . the more time passed since graduation the less the ability of community health workers to remember the material learnt about oral health care and the less incentive and opportunity to consider studying them again . these results are somehow similar to that of petrson et al . , which suggested the younger people were more aware of oral health principles and their own status of oral health than the elderly that indicated adherence to mouth hygiene and awareness of its importance is stronger in younger age group . the unanticipated fact of an inverse relationship between the number of continuing education courses and oral health knowledge may indicate the ineffectiveness and impracticality of the offered information . this also may be because these courses are being attended irregularly or as a compulsory need for score of sessions . nevertheless , the age of community health workers may be considered as a confounding factor , since the older community health workers have participated in more continuing education courses . the mean attitude score of community health workers in this study was 5.5 , which is graded as a good level . attitude was associated neither with the age , gender , educational level , number of continuing education courses nor with the level of knowledge . in other words , the level of knowledge is not necessarily the only effective factor on the attitude and it is rather influenced by individual concepts and understanding of the person . it may be concluded that overall knowledge and attitude of community health workers in east azerbaijan is in an acceptable level . however the lower scores of older and more experienced community health workers suggest the need for more rigorous plan for continuing education courses to maintain and increase the level of knowledge and attitude efficiently .

statement of the problem : prevention is the key factor in acquiring dental and oral health . community health workers , as a part of health care networks in iran , play an important role in delivering primary care and their knowledge and attitude directly affect the population whom they interact with in their service scope . purpose : the aim of this research was to evaluate the knowledge and attitude level of community health workers regarding oral health . materials and method : this descriptive analytical study was carried out on 1170 community health workers who were employed in health offices in east azerbaijan to evaluate their knowledge and attitude level about oral health . data were acquired through filled out questionnaires and were analyzed by spss software . results : there was no significant statistical relationship between knowledge and gender ( p= 0.063 ) , level of education ( p= 0.08 ) and the period spent from the last continuing education course ( p= 0.148).however , by increasing age ( p= 0.016 ) , work experience ( p=0.083 ) and number of attended continuing education courses ( p= 0.023 ) , the knowledge scores were reduced . no statistically significant relationships were found between attitude and any of research variables . conclusion : the level of knowledge and attitude of community health workers in east azerbaijan regarding oral health was good . there was a reverse relationship between age , work experience , and frequency of participation in continuing education courses and knowledge scores which emphasizes the necessity of continuous training and revising the method of training in education of community health workers and other staffs of health care system .

lung cancer is the fourth most common cancer and the leading cause of cancer deaths in korea .the platinum - based regimen using paclitaxel , gemcitabine and docetaxel has shown high response rates and a survival benefit for patients with advanced non - small cell lung cancer ( nsclc ) . no significant difference in survival was observed among four commonly used regimens , but the combination regimen of paclitaxel and carboplatin had a lower rate of toxic effects than the other regimens . when considering toxicity profile , a combination chemotherapy of paclitaxel and carboplatin is an appropriate option in advanced or metastatic nsclc . previously , paclitaxel ( taxol , bristol - myers squibb co. , princeton , nj ) was obtained using a semisynthetic process from taxus baccata , but another paclitaxel , genexol ( samyang genex co. , seoul , korea ) was produced from taxus chinensis using plant cell culture technology . this technology enables the manufacture of genexol with a high purity , low purification cost , constant yield , and without environmental problems . in a phase ii trial with advanced nsclc patients , combination chemotherapy using genexol and cisplatin showed no less response than a previous study and had no significant toxicity . we conducted a phase ii trial to evaluate the safety and efficacy of a combination treatment of another paclitaxel ( genexol ) plus carboplatin in patients with advanced nsclc . the eligibility criteria were as follows : 1 ) histologically - confirmed inoperable , locally advanced or metastatic nsclc , 2 ) patients between 18 and 75 years of age , 3 ) no previous chemotherapy , 4 ) an eastern cooperative oncology group ( ecog ) performance status of 0 - 2 , 5 ) no serious or uncontrolled concurrent medical illness , 6 ) at least one radiographically measurable lesion , and 7 ) adequate baseline hematologic function , defined as ( an absolute neutrophil count [ anc ] 1.510/l , and a platelet count 10010/l ) , adequate hepatic function ( serum bilirubin 1.25upper normal limit [ unl ] , serum aspartate aminotransferase alanine aminotransferase 2.5unl and serum alkaline phosphatase 5.0unl ) , and adequate renal function ( serum creatinine 1.5 mg / dl ) . 8) all patients gave their written informed consent to participate in the study , which was approved by the local institutional review boards . the exclusion criteria included the following : 1 ) pre - existing peripheral neuropathy , 2 ) concurrent or prior malignancy , except curatively resected cervical carcinoma in situ or squamous cell carcinoma of the skin , 3 ) active infection , 4 ) brain metastasis , 5 ) pregnancy confirmed , and 6 ) concurrent treatments that interfered with the study 's evaluation . genexol was supplied by samyang genex co. as a concentrated sterile solution for intravenous ( iv ) administration in a 5 ml vial containing 30 mg of paclitaxel in polyoxyethylated castor oil and dehydrated alcohol ( cremophor el / ethanol ) . the patients received genexol , 225 mg / m as a 3-hour iv infusion followed by carboplatin , area under the concentration - time curve ( auc)=6 , as a 1-hour iv infusion on day 1 every 3 weeks . the genexol was given with premedication as follows : dexamethasone 10 mg ( or its equivalent ) iv administered 12 hours and 30 minutes before the genexol , chlorpheniramine maleate 4 mg ( or its equivalent ) iv , ranitidine 50 mg ( or its equivalent ) iv , 30 minutes before the genexol. antiemetic prophylaxis was given according to a local protocol . in the absence of progressive disease or intolerable toxicity , the patients were treated for a maximum of six cycles . patients who achieved a complete or partial response ( pr ) could receive two additional cycles of chemotherapy after best response , to a maximum 8 cycles . each cycle was started if the anc and platelet counts on the day of treatment were 1.510/l and 10010/l , respectively . in the case of any grade 3 or greater toxicity , except alopecia , chemotherapy was delayed 1 week , then restarted after recovery . the patients were treated at three dose levels ( 0 , -1 , -2 ) as follows : enrolled patients were started at dose level 0 , in which they received genexol , 225 mg / m , plus carboplatin , auc=6 . if grade 3/4 thrombocytopenia or febrile neutropenia , grade 4 neutropenia , or grade 3/4 non - hematologic toxicity were observed in the previous cycle , the dose of test drugs was decreased to level -1 ( genexol , 180 mg / m , plus carboplatin , auc=5 ) . if the above toxicities occurred after dose reduction , the dose of drugs was decreased to level -2 ( genexol , 135 mg / m , plus carboplatin , auc=4 ) . if a recovery was still not achieved by day 35 of treatment , the patient was withdrawn from the study . a pretreatment evaluation was performed within 2 weeks of the therapy initiation , including full history , physical examination , complete blood cell counts ( cbc ) with the differential count , serum biochemistry , chest x - ray , chest computed tomography ( ct ) , brain ct and site - specific imaging . the response to treatment was assessed according to the response evaluation criteria in solid tumors ( recist ) criteria . all the patients who received at least one cycle of chemotherapy were considered evaluable for toxicity . after every 2 cycles of chemotherapy , ct scans were performed to determine the response of the disease , or sooner if there was evidence of any clinical deterioration . cbc with the differential count and serum biochemistry were repeated just before the start of each chemotherapy cycle , and the toxicity was graded according to the national cancer institute common toxicity criteria ( nci - ctc version 2.0 ) . the trial was conducted according to the group sequential design with the response rate as the primary efficacy endpoint . the number of patients required for the study was calculated based on a targeted response rate of 45% and a minimum response rate of 25% , with error of 0.1 and error of 0.2 . analysis of the clinical efficacy was performed according to an intent - to - treat analysis . all patients who received at least two cycles of treatment , or progressed after one course of chemotherapy , were assessable for response and patients who received at least one course were assessable for toxicity . the time to progression ( ttp ) was calculated from the 1st day of chemotherapy to the date of disease progression or the last examination , and the overall survival ( os ) from the first day of chemotherapy to the date of death or the last follow - up . the kaplan - meier method was used to analyze the ttp and os , and the 95% confidence interval ( ci ) for the median time to event was computed . the actual delivered dose - intensity ( di ) was calculated as the ratio of the total dose ( expressed in mg ) per m actually received by the patient , divided by the actual total treatment duration expressed in weeks . the relative di was calculated as the ratio of the actual delivered di to the di planned by the protocol . the spss ver . 14.0 ( spss inc . , chicago , il ) were used for the statistical computation . the eligibility criteria were as follows : 1 ) histologically - confirmed inoperable , locally advanced or metastatic nsclc , 2 ) patients between 18 and 75 years of age , 3 ) no previous chemotherapy , 4 ) an eastern cooperative oncology group ( ecog ) performance status of 0 - 2 , 5 ) no serious or uncontrolled concurrent medical illness , 6 ) at least one radiographically measurable lesion , and 7 ) adequate baseline hematologic function , defined as ( an absolute neutrophil count [ anc ] 1.510/l , and a platelet count 10010/l ) , adequate hepatic function ( serum bilirubin 1.25upper normal limit [ unl ] , serum aspartate aminotransferase alanine aminotransferase 2.5unl and serum alkaline phosphatase 5.0unl ) , and adequate renal function ( serum creatinine 1.5 mg / dl ) . 8) all patients gave their written informed consent to participate in the study , which was approved by the local institutional review boards . the exclusion criteria included the following : 1 ) pre - existing peripheral neuropathy , 2 ) concurrent or prior malignancy , except curatively resected cervical carcinoma in situ or squamous cell carcinoma of the skin , 3 ) active infection , 4 ) brain metastasis , 5 ) pregnancy confirmed , and 6 ) concurrent treatments that interfered with the study 's evaluation . genexol was supplied by samyang genex co. as a concentrated sterile solution for intravenous ( iv ) administration in a 5 ml vial containing 30 mg of paclitaxel in polyoxyethylated castor oil and dehydrated alcohol ( cremophor el / ethanol ) . the patients received genexol , 225 mg / m as a 3-hour iv infusion followed by carboplatin , area under the concentration - time curve ( auc)=6 , as a 1-hour iv infusion on day 1 every 3 weeks . the genexol was given with premedication as follows : dexamethasone 10 mg ( or its equivalent ) iv administered 12 hours and 30 minutes before the genexol , chlorpheniramine maleate 4 mg ( or its equivalent ) iv , ranitidine 50 mg ( or its equivalent ) iv , 30 minutes before the genexol. antiemetic prophylaxis was given according to a local protocol . in the absence of progressive disease or intolerable toxicity , the patients were treated for a maximum of six cycles . patients who achieved a complete or partial response ( pr ) could receive two additional cycles of chemotherapy after best response , to a maximum 8 cycles . each cycle was started if the anc and platelet counts on the day of treatment were 1.510/l and 10010/l , respectively . in the case of any grade 3 or greater toxicity , except alopecia , chemotherapy was delayed 1 week , then restarted after recovery . the patients were treated at three dose levels ( 0 , -1 , -2 ) as follows : enrolled patients were started at dose level 0 , in which they received genexol , 225 mg / m , plus carboplatin , auc=6 . if grade 3/4 thrombocytopenia or febrile neutropenia , grade 4 neutropenia , or grade 3/4 non - hematologic toxicity were observed in the previous cycle , the dose of test drugs was decreased to level -1 ( genexol , 180 mg / m , plus carboplatin , auc=5 ) . if the above toxicities occurred after dose reduction , the dose of drugs was decreased to level -2 ( genexol , 135 mg / m , plus carboplatin , auc=4 ) . if a recovery was still not achieved by day 35 of treatment , the patient was withdrawn from the study genexol was supplied by samyang genex co. as a concentrated sterile solution for intravenous ( iv ) administration in a 5 ml vial containing 30 mg of paclitaxel in polyoxyethylated castor oil and dehydrated alcohol ( cremophor el / ethanol ) . the patients received genexol , 225 mg / m as a 3-hour iv infusion followed by carboplatin , area under the concentration - time curve ( auc)=6 , as a 1-hour iv infusion on day 1 every 3 weeks . the genexol was given with premedication as follows : dexamethasone 10 mg ( or its equivalent ) iv administered 12 hours and 30 minutes before the genexol , chlorpheniramine maleate 4 mg ( or its equivalent ) iv , ranitidine 50 mg ( or its equivalent ) iv , 30 minutes before the genexol. antiemetic prophylaxis was given according to a local protocol . in the absence of progressive disease or intolerable toxicity , the patients were treated for a maximum of six cycles . patients who achieved a complete or partial response ( pr ) could receive two additional cycles of chemotherapy after best response , to a maximum 8 cycles . each cycle was started if the anc and platelet counts on the day of treatment were 1.510/l and 10010/l , respectively . in the case of any grade 3 or greater toxicity , except alopecia , chemotherapy was delayed 1 week , then restarted after recovery . the patients were treated at three dose levels ( 0 , -1 , -2 ) as follows : enrolled patients were started at dose level 0 , in which they received genexol , 225 mg / m , plus carboplatin , auc=6 . if grade 3/4 thrombocytopenia or febrile neutropenia , grade 4 neutropenia , or grade 3/4 non - hematologic toxicity were observed in the previous cycle , the dose of test drugs was decreased to level -1 ( genexol , 180 mg / m , plus carboplatin , auc=5 ) . if the above toxicities occurred after dose reduction , the dose of drugs was decreased to level -2 ( genexol , 135 mg / m , plus carboplatin , auc=4 ) . if a recovery was still not achieved by day 35 of treatment a pretreatment evaluation was performed within 2 weeks of the therapy initiation , including full history , physical examination , complete blood cell counts ( cbc ) with the differential count , serum biochemistry , chest x - ray , chest computed tomography ( ct ) , brain ct and site - specific imaging . the response to treatment was assessed according to the response evaluation criteria in solid tumors ( recist ) criteria . all the patients who received at least one cycle of chemotherapy were considered evaluable for toxicity . after every 2 cycles of chemotherapy , ct scans were performed to determine the response of the disease , or sooner if there was evidence of any clinical deterioration . cbc with the differential count and serum biochemistry were repeated just before the start of each chemotherapy cycle , and the toxicity was graded according to the national cancer institute common toxicity criteria ( nci - ctc version 2.0 ) . the trial was conducted according to the group sequential design with the response rate as the primary efficacy endpoint . the number of patients required for the study was calculated based on a targeted response rate of 45% and a minimum response rate of 25% , with error of 0.1 and error of 0.2 . analysis of the clinical efficacy was performed according to an intent - to - treat analysis . all patients who received at least two cycles of treatment , or progressed after one course of chemotherapy , were assessable for response and patients who received at least one course were assessable for toxicity . the time to progression ( ttp ) was calculated from the 1st day of chemotherapy to the date of disease progression or the last examination , and the overall survival ( os ) from the first day of chemotherapy to the date of death or the last follow - up . the kaplan - meier method was used to analyze the ttp and os , and the 95% confidence interval ( ci ) for the median time to event was computed . the actual delivered dose - intensity ( di ) was calculated as the ratio of the total dose ( expressed in mg ) per m actually received by the patient , divided by the actual total treatment duration expressed in weeks . the relative di was calculated as the ratio of the actual delivered di to the di planned by the protocol . the spss ver . 14.0 ( spss inc . , chicago , il ) were used for the statistical computation . between january 2003 and january 2005 , 28 patients with a median age of 63 years ( range , 45 to 73 years ) from seoul , bucheon , and cheonan soonchunhyang university hospital were studied . there were 20 men and 8 women , and 10 patients ( 35.7% ) had an ecog performance status of 2 . twenty patients ( 71.4% ) had primary metastatic disease and 8 patients ( 28.6% ) had stage iiib disease . twenty - five of the enrolled 28 patients were evaluable for response according to the recist criteria . three patients were excluded from the response analysis because these patients declined continuation of treatment for voluntary withdrawal after one cycle . ten patients ( 35.7% ) achieved a pr , 7 patients ( 25.0% ) had stable disease , and 8 patients ( 28.6% ) progressed during the course of treatment . the median follow - up duration was 8.1 months ( range , 0.4 to 23.5 months ) . the ttp and os were 3.2 months ( 95% ci , 1.5 to 4.9 months ) ( fig . 1 ) and 8.2 months ( 95% ci , 4.1 to 12.3 months ) ( fig . the main non - hematologic toxicity was peripheral neuropathy , which was observed in 12 patients ( 42.9% ) . grade 3/4 neuropathy occurred in 8 patients ( 28.6% ) , and three patients ( 10.7% ) discontinued the treatment because of peripheral neuropathy . a dose reduction was required in 12 patients ; eight patients , due to peripheral neuropathy , and four patients , due to grade 4 neutropenia . the relative dose intensities of genexol and carboplatin were 74.4% and 77.2% of planned doses , respectively . between january 2003 and january 2005 , 28 patients with a median age of 63 years ( range , 45 to 73 years ) from seoul , bucheon , and cheonan soonchunhyang university hospital were studied . there were 20 men and 8 women , and 10 patients ( 35.7% ) had an ecog performance status of 2 . twenty patients ( 71.4% ) had primary metastatic disease and 8 patients ( 28.6% ) had stage iiib disease . twenty - five of the enrolled 28 patients were evaluable for response according to the recist criteria . three patients were excluded from the response analysis because these patients declined continuation of treatment for voluntary withdrawal after one cycle . ten patients ( 35.7% ) achieved a pr , 7 patients ( 25.0% ) had stable disease , and 8 patients ( 28.6% ) progressed during the course of treatment . the median follow - up duration was 8.1 months ( range , 0.4 to 23.5 months ) . the ttp and os were 3.2 months ( 95% ci , 1.5 to 4.9 months ) ( fig . 1 ) and 8.2 months ( 95% ci , 4.1 to 12.3 months ) ( fig . the median number of cycles was 4 ( range , 1 to 8) . table 2 summarizes the incidence of the main toxicities . the main non - hematologic toxicity was peripheral neuropathy , which was observed in 12 patients ( 42.9% ) . grade 3/4 neuropathy occurred in 8 patients ( 28.6% ) , and three patients ( 10.7% ) discontinued the treatment because of peripheral neuropathy . a dose reduction was required in 12 patients ; eight patients , due to peripheral neuropathy , and four patients , due to grade 4 neutropenia . the relative dose intensities of genexol and carboplatin were 74.4% and 77.2% of planned doses , respectively . platinum - based combination chemotherapy has shown a high response rate and survival benefit for patients with advanced or metastatic nsclc , but none of the combination chemotherapy regimens ( paclitaxel+cisplatin vs. gemcitabine+cisplatin vs. docetaxel+cisplatin vs. paclitaxel+carboplatin ) have shown a significant advantage over the others for the treatment of advanced nsclc . . the use of carboplatin instead of cisplatin may reduce the occurrence of peripheral neurotoxicity , and by using the auc , myelotoxicity can be manageable . based on this evidence , we investigated the activity and safety of a combination treatment of genexol plus carboplatin in patients with advanced nsclc . combination chemotherapy with genexol plus cisplatin showed no less response than the previous study , with no considerable toxicity . however , the result of a genexol plus carboplatin combination regimen for treating nsclc was not published . the results of this study showed that the regimen is an effective combination chemotherapy as first - line treatment for advanced nsclc . the response rate , ttp and os of this trial were 35.7% , 3.2 months and 8.2 months , respectively . these results were within the range of previous trials , where platinum - based chemotherapy was used for treatment of advanced nsclc . a severe hypersensitivity reaction caused by paclitaxel has been reported to develop in 1 to 3% of patients during a 3-hour infusion , however , we observed no patient who experienced a grade 3/4 of hypersensitivity reaction in this trial . myelotoxicity was not greater than in the previous report and no toxic death was noticed . neutropenia was the most frequent hematologic toxicity , although no febrile neutropenia was seen . except for neurotoxicity , non - hematologic problems were mild and manageable . it was observed in 12 patients ( 42.9% ) , and grade 3/4 neuropathy occurred in 8 patients ( 28.6% ) . three patients ( 10.7% ) discontinued the treatment because of peripheral neuropathy , however , all these patients achieved a partial response . a dose reduction was required in 8 patients due to peripheral neuropathy , however , based on univariate analysis , grade 3/4 neuropathy was not a significant factor affecting os and response . of the 8 patients who required dose reduction , 5 patients ( 62.5% ) had an ecog performance status of 2 . although we used carboplatin , instead of cisplatin to reduce neurotoxicity , the rate of grade 3/4 neuropathy was relatively high compared to other studies . perhaps the rate of toxicity reflected the relatively high dose of genexol. a higher dose of paclitaxel ( 225 mg / m vs. 175 mg / m ) combined with carboplatin ( auc=6 ) prolongs the median time to progression but causes more neurotoxicity and neutropenia [ 8 - 10 ] . however , to make such an assumption , more evidence should be provided , i.e. , 175 mg / m vs. 225 mg / m of genexol in caucasian and asian patients . although our study was not a randomized trial comparing paclitaxel plus carboplatin combination chemotherapy , genexol and carboplatin is an effective regimen for patients with advanced or metastatic nsclc , and our results indicate that , when used with this schedule , genexol dose not appear to be safe from a neurotoxicity

purposethis phase ii clinical trial was conducted to evaluate the activity and safety of a combination treatment of paclitaxel ( genexol ) plus carboplatin in patients with advanced non - small cell lung cancer.materials and methodschemotherapy - nave patients having histologically confirmed advanced or metastatic non - small cell lung cancer were enrolled . genexol was administered at 225 mg / m2 intravenous ( iv ) infusion over 3 hours , followed by carboplatin ( area under the concentration - time curve=6 ) iv on day 1 every 3 weeks.resultstwenty-eight patients were enrolled between january 2003 and january 2005 . a total of 110 cycles of chemotherapy were given . the median number of chemotherapy cycles was 4 . a total of 25 study patients were evaluable . on an intent - to - treat basis , there were ten partial responses ( response rate 35.7% ) . the median time - to - progression was 3.2 months ( 95% confidence interval [ ci ] , 1.5 to 4.9 ) and the median overall survival was 8.2 months ( 95% ci , 4.1 to 12.3 ) . the main hematologic grade 3/4 toxicity was neutropenia , which was observed in 14 ( 50.0% ) patients . the main non - hematologic toxicity was peripheral neuropathy , which was observed in 12 patients ( 42.9% ) . grade 3/4 neuropathy occurred in 8 patients ( 28.6% ) and three patients discontinued treatment because of neuropathy.conclusionin this trial , the combination of genexol and carboplatin showed significant activity as first line treatment for patients with advanced or metastatic non - small cell lung cancer . however , a modest dose reduction of genexol is needed due to sensory neuropathy .

osteoporosis is a systemic metabolic bone disease which produces a reduction in bone density together with abnormal structure ( 1 , 2 ) . currently , both the diagnosis and therapy of osteoporosis focus on determining the individual risk for bone fracture ( 3 ) . this approach requires collecting information on the occurrence of dietary and non - dietary risk factors for bone fractures . it is commonly thought that adequate calcium intake improves bone mineral density ( 5 ) . dietary calcium intake and dairy product consumption are the most important during childhood and adolescence ( 5 ) . non - dietary factors increasing bone fracture risk include female sex , white race , advanced age , menstruation disorders , early menopause , previous fractures , hip fractures in parents , low bmi , low bone mass , administration of corticosteroids , rheumatoid arthritis , low physical activity , poor self - evaluated health , smoking and alcohol abuse ( 1 , 3 ) . individuals with a family history of osteoporosis reveal a tendency to a more intense bone degradation process . therefore , the probability of osteoporosis in a woman whose mother had this condition , as well as in a woman who inherited tall height or slight build bone from her mother is high ( 10 ) . it is estimated that heredity determines 7085% of the mineral density of axial skeleton bones and proximal heads of femurs and 5060% of the appendicular skeleton ( 11 ) . the rate of bone density loss and inherited tendency to develop osteoporosis can be reduced by a change of modifiable factors of bone fracture risk , including an improvement in calcium intake ( 12 ) . the family environment affects the food intake by members of the family ( 13 , 14 ) . since women are more sensitive to environmental stimuli , positive attitudes of mothers towards health can have an effect on more favourable dietary patterns of the daughter and their better nutritional status ( 15 , 16 ) . in polish teenagers , jeewska - zychowicz ( 14 ) revealed that positive eating habits occurring in the family , hospitality and openness to knowledge were conductive to more appropriate frequency of consumption of milk , dairy products , fruit and raw and cooked vegetables . in american youth the frequency of having common family meals was positively related to the intake of fruit , vegetables , cereal products and calcium rich food , and negatively related to the consumption of soft beverages ( 17 ) . thus a strong positive correlation between family meal consumption and the quality of the diet was showed . mutual relations between genetic and non - genetic factors determine higher intensification of osteoporosis in some families and lower intensification in others . the improvement of public health and prevention of osteoporosis requires better understanding of family environment factors . an important element of this understanding is to determine the burden of dietary and non - dietary risk factors for bone fractures and osteoporosis in mothers and daughters . the aim of the study was to demonstrate similarities and differences between mothers and daughters regarding risk factors for bone fractures and osteoporosis . the study was carried out in 20072010 within the scientific project analysis of mother - daughter dairy products dietary patterns in relation to bone mineral status and calcium deficiency and osteoporosis risk among women . a recruitment details and inclusion / exclusion criteria can be found elsewhere ( 15 ) . the study included mothers ageing under 60 years and daughters ageing above 12 years and before 21 years . finally , the study included 712 mothers and daughters ( table 1 ) . in the total sample , the dietary study was carried out and bone fractures risk factors were collected . in the subsample ( 170 family pairs ) bone mineral density ( bmd ) was measured and some other osteoporosis risk factors was collected . for the subsample all mothers and daughters were chosen living in one typical area of poland . notes : ( ) in the brackets indicated the number of mothers and daughters description of the total sample and sub - sample of mothers and daughters tested with bone densitometry mean standard deviation ; significant differences between total sample and sub - sample at p<0.05 as follows : for daughters was given present educational level in family ; ( ) in the brackets indicated minimum - maximum range ; for 417 mother - daughter family pairs with complete physical activity data the research received permission from : ( i ) the bioethics committee of the regional medical chamber in olsztyn in 2001 , on june 27 , 2001 , resolution no . 49/2001 and ( ii ) the bioethics committee of the faculty of medical sciences , university of warmia and mazury in olsztyn on june 17 , 2010 , resolution no . the respondents were asked about their date of birth , place of residence and education level ( elementary , secondary or higher ) . two closed - questions were used for description of the family economic situation : self - declared economic situation with four answers : bad , satisfactory , good and very good, self - declared situation of the household with six answers ( based on the polish central office of statistics ) : ( i ) we live very poorly we do not have enough resources even for the cheapest food and clothing , ( ii ) we live poorly we do not have enough resources for housing fees , ( iii ) we live modestly we have enough resources only for food and clothing , ( iv ) we live very thriftily , ( v ) we live relatively thriftily , ( vi ) we live very well we can afford everything without limitations self - declared economic situation with four answers : bad , satisfactory , good and very good , self - declared situation of the household with six answers ( based on the polish central office of statistics ) : ( i ) we live very poorly we do not have enough resources even for the cheapest food and clothing , ( ii ) we live poorly we do not have enough resources for housing fees , ( iii ) we live modestly we have enough resources only for food and clothing , ( iv ) we live very thriftily , ( v ) we live relatively thriftily , ( vi ) we live very well we can afford everything without limitations the consumption of dairy products was determined using a validated semi - quantitative food frequency questionnaire ( acronym ados - ca ) ( 16 ) . calcium intake from each dairy product and daily diet was calculated . briefly , the data was collected on the frequency of consumption and portion size usually consumed ( during the last six months ) of 11 groups of dairy products . the calcium intake ( mg / day ) from dairy products was calculated by summing up the calcium intakes from all groups of dairy products . finally , the calcium intake from the daily diet was calculated on the basis of calcium intake from dairy products according to the formula worked out in the validation study ( 16 ) . the number of mothers and daughters ( in % ) who met the polish calcium intake recommendations and did not meet calcium intake recommendations was calculated ( 18 ) using the probability method . the established cut - off points ( i.e. , z - values of individual calcium intake < 1sd or > 1sd ) produced conclusions with a probability of 0.85 . information on risk factors influencing bone mass and presence of osteoporosis was collected . a questionnaire with closed - questions a comprehensive list of dietary and non - dietary risk factors was prepared . in total , information regarding 21 risk factors was collected , including dietary ( 6 items ) and non - dietary risk factors ( 15 items ) ( 2 , 3 , 5 ) . low bmi was not included in any subgroup because of the difficulty of classifying it as a non - dietary risk factor , although weight and bmi are strongly correlated with diet . information about the customary consumption of ready - made calcium enriched cereal products and calcium enriched juices was collected as well as everyday consumption of dairy products at pre - school and school age ( yes , no , i do nt know ) . during the interview information about non - dietary risk factors affecting bone weight and occurrence was collected , such as : occurrence of bone pains ( yes , no , i do nt know),current smoking ( no , occasionally 5 pcs / day , > pcs / day),past fractures ( yes , no , i do nt know),calcium supplement use ( yes , no , i do nt know),incidence of other chronic diseases than tthyroid diseases and rheumatoid arthritis , e.g. lung diseases , cardiac diseases , vascular diseases , cancer , diabetes mellitus , stroke ( 19 ) ( yes , no),menstruation ( yes regularly , yes irregularly , i havent started menstruating , i have stopped menstruating),incidence of osteoporosis in the family ( yes , no , i do nt know),physical activity ( low , moderate , high),incidence of thyroid diseases ( yes , no),hormonal contraceptive use ( yes , no),glucocorticosteroid use ( yes , no),incidence of rheumatoid arthritis ( yes , no),sun exposure frequency ( i avoid sun exposure , rarely , often , very often),health status in own assessment ( very good , good , quite good , poor),consumption of significant amounts of alcohol ( yes , no , i do nt know ) . occurrence of bone pains ( yes , no , i do nt know ) , current smoking ( no , occasionally 5 pcs / day , > pcs / day ) , past fractures ( yes , no , i do nt know ) , calcium supplement use ( yes , no , i do nt know ) , incidence of other chronic diseases than tthyroid diseases and rheumatoid arthritis , e.g. lung diseases , cardiac diseases , vascular diseases , cancer , diabetes mellitus , stroke ( 19 ) ( yes , no ) , menstruation ( yes regularly , yes irregularly , i havent started menstruating , i have stopped menstruating ) , incidence of osteoporosis in the family ( yes , no , i do nt know ) , physical activity ( low , moderate , high ) , incidence of thyroid diseases ( yes , no ) , hormonal contraceptive use ( yes , no ) , glucocorticosteroid use ( yes , no ) , incidence of rheumatoid arthritis ( yes , no ) , sun exposure frequency ( i avoid sun exposure , rarely , often , very often ) , health status in own assessment ( very good , good , quite good , poor ) , consumption of significant amounts of alcohol ( yes , no , i do nt know ) . the physical activity level was determined by using a validated international physical activity questionnaire ( ipaq ) , a long version last 7 days , expressed as a standard metabolic energy turnover in met - minutes / week ( 20 , 21 ) . according to the procedure made by the ipaq research committee , 295 family pairs were excluded ( activity time > 960 minutes/24h ) . three levels of physical activity were classified : low ( < 600 met - minutes / week ) , moderate ( 6002999 ) , high ( 3000 or more ) . bone mineral density ( bmd , g / cm ) was measured with a pdexa densitometer with a dual - energy x - ray absorptiometry ( dxa ) in the distal part of the radius and the ulna ( 2 ) . three subgroups were created according to bmd terciles ( bottom , middle , upper ) for mothers ( mt1bmd , mt2bmd , mt3bmd ) and separately for daughters ( ct1bmd , ct2bmd , ct3bmd ) . weight and height of the respondents were measured and body mass index was calculated ( bmi , kg / m ) . the average intake of calcium of dairy products , age , bmi , bmd and t - score bmd were expressed with the mean ( x ) and the standard deviation ( sd ) . the spearman s rank correlation for calcium in - take from dairy products and dietary calcium in - take of mothers and daughters were calculated ( 22 ) . lists of dairy products according to their diminishing share in calcium supply in the daily diet were created separately for mothers and daughters . similarity of the calcium source ranking in diets of mothers and daughters as well as similarity of dietary and non - dietary bone risk factor ranking was verified using a spearman rank order test . spearman s rank correlation for bmd and three fracture risk factors with numerical data ( age , dietary calcium intake , physical activity ) were calculated separately for mothers and daughters . before the analysis , bmd ( in g / cm ) , dietary calcium in - take ( in mg / day ) and physical activity ( in met - minutes / week ) were converted to sd scores by using values obtained for the participants . the relationship between bmd mothers and bmd daughters in a complex system was evaluated with a correspondence analysis using burt s panels . we analyzed the relations between bmd mothers and bmd daughters divided into terciles ( mt1bmd , mt2bmd , mt3bmd , dt1bmd , dt2bmd , dt3bmd ) and obtained a high level of explained inertia in this model ( 56% ) . the statistical analysis was performed with statistica 10.0 pl software ( statsoft , poland ) . the study was carried out in 20072010 within the scientific project analysis of mother - daughter dairy products dietary patterns in relation to bone mineral status and calcium deficiency and osteoporosis risk among women . a recruitment details and inclusion / exclusion criteria can be found elsewhere ( 15 ) . the study included mothers ageing under 60 years and daughters ageing above 12 years and before 21 years . finally , the study included 712 mothers and daughters ( table 1 ) . in the total sample , the dietary study was carried out and bone fractures risk factors were collected . in the subsample ( 170 family pairs ) bone mineral density ( bmd ) was measured and some other osteoporosis risk factors was collected . for the subsample all mothers and daughters were chosen living in one typical area of poland . notes : ( ) in the brackets indicated the number of mothers and daughters description of the total sample and sub - sample of mothers and daughters tested with bone densitometry mean standard deviation ; significant differences between total sample and sub - sample at p<0.05 as follows : for daughters was given present educational level in family ; ( ) in the brackets indicated minimum - maximum range ; for 417 mother - daughter family pairs with complete physical activity data the research received permission from : ( i ) the bioethics committee of the regional medical chamber in olsztyn in 2001 , on june 27 , 2001 , resolution no . 49/2001 and ( ii ) the bioethics committee of the faculty of medical sciences , university of warmia and mazury in olsztyn on june 17 , 2010 , resolution no . the respondents were asked about their date of birth , place of residence and education level ( elementary , secondary or higher ) . two closed - questions were used for description of the family economic situation : self - declared economic situation with four answers : bad , satisfactory , good and very good, self - declared situation of the household with six answers ( based on the polish central office of statistics ) : ( i ) we live very poorly we do not have enough resources even for the cheapest food and clothing , ( ii ) we live poorly we do not have enough resources for housing fees , ( iii ) we live modestly we have enough resources only for food and clothing , ( iv ) we live very thriftily , ( v ) we live relatively thriftily , ( vi ) we live very well we can afford everything without limitations self - declared economic situation with four answers : bad , satisfactory , good and very good , self - declared situation of the household with six answers ( based on the polish central office of statistics ) : ( i ) we live very poorly we do not have enough resources even for the cheapest food and clothing , ( ii ) we live poorly we do not have enough resources for housing fees , ( iii ) we live modestly we have enough resources only for food and clothing , ( iv ) we live very thriftily , ( v ) we live relatively thriftily , ( vi ) we live very well we can afford everything without limitations the consumption of dairy products was determined using a validated semi - quantitative food frequency questionnaire ( acronym ados - ca ) ( 16 ) . briefly , the data was collected on the frequency of consumption and portion size usually consumed ( during the last six months ) of 11 groups of dairy products . the calcium intake ( mg / day ) from dairy products was calculated by summing up the calcium intakes from all groups of dairy products . finally , the calcium intake from the daily diet was calculated on the basis of calcium intake from dairy products according to the formula worked out in the validation study ( 16 ) . the number of mothers and daughters ( in % ) who met the polish calcium intake recommendations and did not meet calcium intake recommendations was calculated ( 18 ) using the probability method . the established cut - off points ( i.e. , z - values of individual calcium intake < 1sd or > 1sd ) produced conclusions with a probability of 0.85 . information on risk factors influencing bone mass and presence of osteoporosis was collected . a questionnaire with closed - questions a comprehensive list of dietary and non - dietary risk factors was prepared . in total , information regarding 21 risk factors was collected , including dietary ( 6 items ) and non - dietary risk factors ( 15 items ) ( 2 , 3 , 5 ) . low bmi was not included in any subgroup because of the difficulty of classifying it as a non - dietary risk factor , although weight and bmi are strongly correlated with diet . information about the customary consumption of ready - made calcium enriched cereal products and calcium enriched juices was collected as well as everyday consumption of dairy products at pre - school and school age ( yes , no , i do nt know ) . during the interview information about non - dietary risk factors affecting bone weight and occurrence was collected , such as : occurrence of bone pains ( yes , no , i do nt know),current smoking ( no , occasionally 5 pcs / day , > pcs / day),past fractures ( yes , no , i do nt know),calcium supplement use ( yes , no , i do nt know),incidence of other chronic diseases than tthyroid diseases and rheumatoid arthritis , e.g. lung diseases , cardiac diseases , vascular diseases , cancer , diabetes mellitus , stroke ( 19 ) ( yes , no),menstruation ( yes regularly , yes irregularly , i havent started menstruating , i have stopped menstruating),incidence of osteoporosis in the family ( yes , no , i do nt know),physical activity ( low , moderate , high),incidence of thyroid diseases ( yes , no),hormonal contraceptive use ( yes , no),glucocorticosteroid use ( yes , no),incidence of rheumatoid arthritis ( yes , no),sun exposure frequency ( i avoid sun exposure , rarely , often , very often),health status in own assessment ( very good , good , quite good , poor),consumption of significant amounts of alcohol ( yes , no , i do nt know ) . occurrence of bone pains ( yes , no , i do nt know ) , current smoking ( no , occasionally 5 pcs / day , > pcs / day ) , past fractures ( yes , no , i do nt know ) , calcium supplement use ( yes , no , i do nt know ) , incidence of other chronic diseases than tthyroid diseases and rheumatoid arthritis , e.g. lung diseases , cardiac diseases , vascular diseases , cancer , diabetes mellitus , stroke ( 19 ) ( yes , no ) , menstruation ( yes regularly , yes irregularly , i havent started menstruating , i have stopped menstruating ) , incidence of osteoporosis in the family ( yes , no , i do nt know ) , physical activity ( low , moderate , high ) , incidence of thyroid diseases ( yes , no ) , hormonal contraceptive use ( yes , no ) , glucocorticosteroid use ( yes , no ) , incidence of rheumatoid arthritis ( yes , no ) , sun exposure frequency ( i avoid sun exposure , rarely , often , very often ) , health status in own assessment ( very good , good , quite good , poor ) , consumption of significant amounts of alcohol ( yes , no , i do nt know ) . the physical activity level was determined by using a validated international physical activity questionnaire ( ipaq ) , a long version last 7 days , expressed as a standard metabolic energy turnover in met - minutes / week ( 20 , 21 ) . according to the procedure made by the ipaq research committee , 295 family pairs were excluded ( activity time > 960 minutes/24h ) . three levels of physical activity were classified : low ( < 600 met - minutes / week ) , moderate ( 6002999 ) , high ( 3000 or more ) . bone mineral density ( bmd , g / cm ) was measured with a pdexa densitometer with a dual - energy x - ray absorptiometry ( dxa ) in the distal part of the radius and the ulna ( 2 ) . three subgroups were created according to bmd terciles ( bottom , middle , upper ) for mothers ( mt1bmd , mt2bmd , mt3bmd ) and separately for daughters ( ct1bmd , ct2bmd , ct3bmd ) . weight and height of the respondents were measured and body mass index was calculated ( bmi , kg / m ) . the average intake of calcium of dairy products , age , bmi , bmd and t - score bmd were expressed with the mean ( x ) and the standard deviation ( sd ) . the spearman s rank correlation for calcium in - take from dairy products and dietary calcium in - take of mothers and daughters were calculated ( 22 ) . lists of dairy products according to their diminishing share in calcium supply in the daily diet were created separately for mothers and daughters . similarity of the calcium source ranking in diets of mothers and daughters as well as similarity of dietary and non - dietary bone risk factor ranking was verified using a spearman rank order test . spearman s rank correlation for bmd and three fracture risk factors with numerical data ( age , dietary calcium intake , physical activity ) were calculated separately for mothers and daughters . before the analysis , bmd ( in g / cm ) , dietary calcium in - take ( in mg / day ) and physical activity ( in met - minutes / week ) were converted to sd scores by using values obtained for the participants . the relationship between bmd mothers and bmd daughters in a complex system was evaluated with a correspondence analysis using burt s panels . we analyzed the relations between bmd mothers and bmd daughters divided into terciles ( mt1bmd , mt2bmd , mt3bmd , dt1bmd , dt2bmd , dt3bmd ) and obtained a high level of explained inertia in this model ( 56% ) . the statistical analysis was performed with statistica 10.0 pl software ( statsoft , poland ) . the women from the total sample and in the sub - sample did not significant differ in social and demographic parameters as well as bmi in either mothers or daughters ( table 1 ) . calcium intake from all dairy products by mothers and daughters was correlated ( p<0.05 ) ( table 2 ) . the correlation coefficients ranged from 0.29 for cheese for spreading to 0.17 for cream . the correlation coefficient for the dietary calcium intake of mothers and daughters was 0.25 in the total sample and 0.23 in sub - sample ( both p<0.05 ) ( table 2 ) . comparison of calcium intake from dairy products and dietary calcium intake by mothers and daughters in the total sample ( mg / day ; meanstandard deviation ) significance of differences at : a high correlation ( r=0.92 ) between mothers and daughters was found for calcium sources and their share in diet ( fig . 2 ) . the main sources of calcium in the daily diet were in mothers : milk , rennet cheese , fruit yoghurt and natural yoghurt and in daughters : milk , rennet cheese and fruit yoghurt . mothers supplied in their daily diet significantly less calcium than daughters ( 596 vs. 723 mg / day ) ( table 2 ) , both intakes were extremely below the polish national recommendation ( rda for calcium : 10001300 mg / day ) ( 18 ) . daughters , in comparison to mothers , consumed significantly more calcium from rennet cheese , fruit yoghurt , ice - creams , processed cheese and homogenized cheese . comparison of the calcium source ranking similarity according to calcium sources share in daily diet of mothers and daughters in the total sample ( mean value in % calcium ) . notes : r correlation coefficient for dietary calcium source ranking ; significance of differences at : * p < 0.05 the presence of risk factors for bone fractures in mothers and daughters was significantly correlated ( table 3 ) . the spearman rank coefficients were significant for dietary factors of fracture risk : 0.87 in whole sub - sample , 0.94 in bottom and 0.82 in middle tercile of bmd , and for non - dietary factors of fracture risk : 0.83 in whole sub - sample , 0.86 in bottom , 0.93 in middle , 0.65 in upper tercile of bmd . comparison of the occurrence of fracture risk factors in mothers and daughters by bmd terciles in the sub - sample mean standard deviation ; used cut - off points produced conclusions with a probability of 0.85 and , because of this , the number respondents who met or did not meet calcium intake recommendations do not sum up to 100% ; other chronic diseases than thyroid diseases and rheumatoid arthritis , e.g. lung diseases , cardiac diseases , vascular diseases , cancers , diabetes mellitus , stroke ; low physical activity : < 600 met - minutes / week ; mt1bmd bottom tercile bmd by mothers , mt2bmd middle tercile bmd by mothers , mt3bmd upper tercile bmd by mothers ; dt1bmd bottom tercile bmd by daughters , dt2bmd middle tercile bmd by daughters , dt3bmd upper tercile bmd by daughters ; significance of differences between mothers and daughters in whole sub - sample at : p<0.001 ; significance of differences at p<0.05 : between mothers bmd terciles between daughters bmd terciles , between mothers and daughters bmd terciles ; mothers from the bottom tercile of bmd were significantly older than mothers from the middle or upper tercile of bmd ( table 3 ) . daughters from the bottom tercile of bmd were significantly younger than daughters from the middle or upper tercile of bmd . bmd of mothers was significantly higher than daughters in the whole sub - sample ( 379 vs. 337 mg / cm , respectively ) as well as in each bmd tercile ( table 3 ) . more mothers than daughters consumed dairy products daily during pre - school and school periods . in comparison to daughters , fewer mothers consumed ready - made cereal products enriched with calcium . significantly more mothers than daughters suffered from bone pains , smoked , suffered from chronic diseases , menstruation disorders , thyroid diseases , rheumatoid arthritis , poor health condition , used glucocorticosteroids avoided sun exposure . the total physical activity was significantly higher in mothers than daughters in the whole sub - sample as well as in the bottom and upper bmd terciles . no significant correlation was found between bmd and dietary calcium intake or bmd and physical activity in the total sub - sample and each bmd tercile in mothers as well as daughters ( table 4 ) . in daughters whole sub - sample , the correlation between age and bmd was significant ( r=0.30 ) . correlation coefficients for bone mineral density and fracture risk factors in mothers and daughters by bmd terciles within the sub - sample with spearman s rank test values were converted to sd scores ; mt1bmd bottom tercile bmd by mothers , mt2bmd middle tercile bmd by mothers , mt3bmd upper tercile bmd by mothers ; dt1bmd bottom tercile bmd by daughters , dt2bmd middle tercile bmd by daughters , dt3bmd upper tercile bmd by daughters ; significance of differences at : the same terciles of bmd were stated in 39% of mother - daughter pairs , in the bottom tercile of bmd in 16% of pairs , in the middle tercile of bmd in 11% pairs and in the upper tercile of bmd in 12% pairs ( table 5 ) . the correspondence analysis demonstrated the existence of the strong relation between bmd of mothers and bmd of daughters . the s1 cluster was made up by the middle bmd tercile of mothers and the upper bmd tercile of daughters . the s2 cluster was made up of the upper bmd tercile of mothers and the middle bmd tercile of daughters . the s3 cluster was made up of the both bottom bmd terciles of mothers and daughters . distribution of mothers and daughters by bmd terciles ( ) in the brackets percentage of mother - daughter pairs graphic presentation of the relationship between bone mineral density in mothers and daughters in the sub - sample ; ( ) in brackets are the given explained inertia in two dimensions ; areas marked by rectangles and signed s1 , s2 and s3 point three clusters grouping features correlated with each other . notes : mt1bmd bottom tercile bmd by mothers , mt2bmd middle tercile bmd by mothers , mt3bmd upper tercile bmd by mothers ; dt1bmd bottom tercile bmd by daughters , dt2bmd middle tercile bmd by daughters , dt3bmd upper tercile bmd by daughters the study showed the existence of a similarity between mothers and daughters , both in the occurrence of dietary and non - dietary factors of fracture risk . differences between mothers and daughters concerned the level of threat with risk factors . in comparison to daughters , mothers were more burdened with adverse factors increasing bone fracture risk , but they revealed more favourable dietary habits reducing the risk of bone fractures . a strong similarity in bmd concerned mothers and daughters with low bmd . we used a unique approach by grouping subjects before the statistical analysis , but agreement with previous studies may be found . authors who used correlation analysis showed a significant association between mothers and daughters in bmd ( 2325 ) , which partially confirms our results . there are three possible explanations for the similarity of low bmd in mothers and daughters . mothers with the lowest bmd were older ( by 23 years ) and daughters with the low bmd were younger ( by 23 years ) than those with moderate and high bmd . thus , our findings in older women group might result from the intensifying of bone resorption and in younger girls might result from the slowing of bone mineralisation within 23 years after menarche ( 26 ) . secondly , the daughters low bmd might be inherited from mothers . there is evidence of the occurrence of similar skeleton features among family members ( 28 ) . a high correlation of bmd was observed among the three female mexican generations ( 23 ) . in japanese daughter - mother pairs , bmd was significantly correlated , but no significant correlation was found in mother - grandmother or daughter - grandmother pairs ( 24 ) . a family similarity concerning bone strength in girls and women probably is established in the first year of life , but the familial correlation of bmd might disappear due to a menopause - associated estrogen deficiency ( 24 , 25 ) . the next explanation of low bmd similarity in mothers and daughters might involve environmental factors ( 17 , 27 ) . some of these factors may be inherited such as lifestyle , habits and behaviors osteoporotic environment , defined as the sum of influences which depend on the surroundings , opportunities , or conditions of life , may promote osteoporosis in certain families . in our study , a significant correlation between mothers and daughters in the occurrence of both dietary and non - dietary fracture risk factors was observed . it should be underlined that correlation coefficients were higher in mothers and daughters with low or moderate mineral density ( from 0.82 to 0.94 ) than those with high bone mineral density ( below 0.66 ) . non - dietary risk factors for bone fractures which might be under the influence of family behaviours and create an osteoporotic environment in the family include : past fractures , calcium supplementation , chronic diseases , osteoporosis in the family and hormonal contraceptive use . surprising , we found significant differences ( lack of similarity ) between mothers and daughters in the occurrence of low physical activity . swee and win ( 29 ) also found no correlation in physical activity in pairs of premenopausal mothers and their daughters aged 1019 years . teenage girls are more likely to have a sedentary lifestyle than boys at the same age ( 30 , 31 ) . it may be supposed that a lack of similarity in physical activity between mothers and daughters resulted from girls specific lifestyle , so the impact of family environment was weaker . family similarity between mothers and daughters was reflected by the occurrence of similar dietary factors of fracture risk . mothers and these were mainly milk , cheese and fruit yoghurt , which supplied over 50% of dietary calcium . a study by harris ( 32 ) of 902 families found a strong association between the number of servings of dairy foods consumed by the adolescents and their parents . a similar relation was observed in american ( 33 ) and malay mother - daughter pairs ( 29 ) with conclusion that mothers play an important role in influencing the dietary habits of their daughters which , in turn , may affect their bone health status the similarity between mothers and daughters was also reflected by lack of significant correlation between bmd and dietary calcium intake or physical activity . our results are compatible with some previous findings ( 34 , 35 ) . however , there are some contradictory findings ( 3639 ) . in contrast to our findings , physical activity is clearly recognized as factor positively associated with an increase in bone mass during a developmental period ( 6 ) . this was confirmed in subjects reporting recreational exercise ( 40 , 41 ) . in our study , mothers with low or high bmd were significantly more physically active than mothers with moderate bmd , and all of them were under the influence of many different fracture risk factors . the impact of environmental factors on bmd is complex , since it is difficult to explain the association between bone mineral density and a single risk factor ( 1 , 2 , 5 , 42 ) . we showed that mothers were more burdened with factors increasing bone fracture risk than daughters . the main five non - dietary risk factors occurred ( in decreasing order ) in 75% to 22% of the mothers , and 56% to 16% of the daughters . we can not identify which factors ( from 21 which were assessed ) were more effective than others in low bmd . based on previous findings , it may be assumed that past fractures , osteoporosis in the family , current smoking and low physical activity in mothers or daughters were the most important factors increasing the risk of bone fractures ( 4245 ) . we found more protective dietary habits in mothers reduced the risk of bone fractures . in the past , during pre - school and school periods , more mothers than daughters consumed dairy products ( and calcium ) daily . this might have resulted from generation transformation and changes in food supply as well as food consumption in poland over the past fifty years . mothers were in pre - school and school age when dairy consumption in poland was relatively high , and peaked in the 1980s ( on average , 260280 litres / person / year after recalculation to milk ) ( 46 ) . daughters were in pre - school and school age when dairy consumption decreased significantly by 30% , but at the same time increased the consumption of fast - food and highly - prepared food and the traditional polish diet became westernized ( 31 ) . therefore , we documented that the occurrence of protective factors of bone fracture may be irrespective of age . secondly , we showed the limitation of inheritance dietary habits shared with family members . our findings demonstrate that family habits in food consumption are also influenced by the external impact such as food supply on domestic market . bone densitometric measurements were carried out in a medium - sized subsample , which was non - randomly selected from the general population . however , in mothers / daughters from a large - sized basic sample and sub - sample , a similarity in dietary calcium intake was revealed the correlation coefficient for dietary calcium intake of mothers and daughters was nearly equal . this allows generalization of results obtained in the sub - sample and stronger conclusions to be drawn . the study strength is the assessment of the occurrence of dietary and non - dietary factors of fracture risk ( 13 ) . an advantage of this research is the use of validated tools to assess dietary calcium intake ( 16 ) and physical activity ( 20 ) . bone densitometric measurements were carried out in a medium - sized subsample , which was non - randomly selected from the general population . however , in mothers / daughters from a large - sized basic sample and sub - sample , a similarity in dietary calcium intake was revealed the correlation coefficient for dietary calcium intake of mothers and daughters was nearly equal . this allows generalization of results obtained in the sub - sample and stronger conclusions to be drawn . the study strength is the assessment of the occurrence of dietary and non - dietary factors of fracture risk ( 13 ) . an advantage of this research is the use of validated tools to assess dietary calcium intake ( 16 ) and physical activity ( 20 ) . in mothers and daughters , similarity was found in the occurrence of dietary and non - dietary factors of fracture risk . differences between mothers and daughters concerned the level of threat with risk factors . in comparison to daughters , mothers were more burdened with factors increasing bone fracture risk , but they revealed more favourable dietary habits reducing the risk of bone fractures . our results demonstrate the stronger effect of adverse factors of family environment than protective factors on bone fracture risk and osteoporosis . improvement of public health in poland requires the elimination of adverse dietary and non - dietary habits in women and the development and strengthening of health - promoting habits in girls . ethical issues ( including plagiarism , informed consent , misconduct , data fabrication and/or falsification , double publication and/or submission , redundancy , etc . ) have been completely observed by the authors .

background : the aim of this study was to demonstrate similarities and differences between mothers and daughters regarding dietary and non - dietary risk factors for bone fractures and osteoporosis.methods:the study was carried out in 20072010 on 712 mothers ( 2959 years ) and daughters ( 1221 years ) family pairs . in the sub - sample ( 170 family pairs ) bone mineral density ( bmd ) was measured for the forearm by dual - energy x - ray absorptiometry ( dxa ) . the consumption of dairy products was determined with a semi - quantitative food frequency questionnaire ( ados - ca ) and calcium intake from the daily diet was calculated.results:the presence of risk factors for bone fractures in mothers and daughters was significantly correlated . the spearman rank coefficient for dietary factors of fracture risk was 0.87 ( p<0.05 ) in whole sub - sample , 0.94 ( p<0.05 ) in bottom tercile of bmd , 0.82 ( p<0.05 ) in middle tercile of bmd , 0.54 ( p>0.05 ) in upper tercile of bmd and for non - dietary factors of fracture risk was 0.83 ( p<0.05 ) in whole sub - sample , 0.86 ( p<0.05 ) in bottom tercile of bmd , 0.93 ( p<0.05 ) in middle tercile of bmd , 0.65 ( p<0.05 ) in upper tercile of bmd.conclusions:our results confirm the role of the family environment for bone health and document the stronger effect of negative factors of the family environment as compared to other positive factors on bone fracture risk .

symptoms of irritable bowel syndrome ( ibs ) are among the most common gastrointestinal complaints and lead to millions of consultative visits each year . while the third iteration of the rome consensus conference provided yet another approach to positively diagnose functional illnesses , many physicians continue to see and approach ibs as a diagnosis of exclusion . once a diagnosis is made , we have to think about therapy , which may range from antibiotics for presumed small bowel bacterial overgrowth to serotonergic agonists or antagonists , depending on predominant symptoms and physician or patient preference . with spiraling healthcare costs and a highly prevalent syndrome , we need to ask ourselves whether we should standardize care and , in doing so , perhaps deliver more for less . this call for standardized approaches and quality benchmarks led to development of guidelines that typically rely on evidence - based medicine and its primary tool , the meta - analysis . mechanistic studies showing the influence of anxiety and/or hypervigilance on ibs symptoms may provide a rationale for this approach . yet , prior studies have shown conflicting results , which could arguably be due to limited power and thus be clarified with a meta - analysis . a team of investigators thus compiled data from all published studies ( identifying 13 studies that compared antidepressants with placebo ) and concluded that antidepressants are effective in the treatment of ibs . although statistically convincing , a purely numerical approach computing an average across very different studies will gloss over any remaining conceptual concerns . one of the studies supporting the utility of these agents came from iran , an area with male predominance in ibs , quite distinct from what physicians see in the us and european countries . the dosages of tricyclics vary five- to ten - fold , with the best effects seen in studies with the lowest dose . so , can we truly attribute such effects to the pharmacology of a medication and recommend a specific intervention ( drug ) if the effects can not be explained by known mechanisms of action ? this question becomes even more important when we look at negative results of recent trials examining the effects of citalopram in non - depressed adults with ibs or of amitriptyline in children with functional disorders . both trials were negative , not showing a difference between the active agent and placebo . interestingly , pediatric patients showed impressive improvements in both arms ( amitriptyline and placebo ) , demonstrating the power of placebo . recently , a study did suggest that imipramine may be effective in the treatment of ibs , however , another demonstrated no significant benefit for paroxetine , further highlighting the differences in the results within this field . so , why do we see discrepancies between these studies and the meta - analysis ? perhaps if rigor went beyond methodological approaches and transparency to include conceptual assessments , less disparity would be seen . it may be a clinically useful label , but the term describes a syndrome , not disease mechanisms . rather than relying on algorithmic approaches that presume similar disease mechanisms and applying similar treatments to every person , no matter whether diarrhea , constipation , bloating or pain predominate , we need to practice , one may well use antidepressants , basing decisions on the relative contribution of affect and choosing an agent that fits best in terms of effect and side - effect profile . the study by saps and colleagues highlighted the placebo effect , which in most large ibs trials is lower than in this study , but still ranges around 40% . the importance of placebo responses has triggered investigations into the different components of this powerful effect . the training we go through largely focuses on the content of our discussions : what is going on , what treatments we recommend and why we suggest them , rather than how we relay that information to the patient . as part of a trial on acupuncture in ibs , a team of investigators paired sham treatment with different communicative strategies . one group met a qualified therapist who explained the treatment plan , the other talked to a person who employed empathy , active listening and instilled confidence . augmented the placebo effect of acupuncture alone , and therefore may matter more than the technical competence that we can claim as certified specialists . the results remind us that therapy starts even before the first pill has been taken . such findings may well confirm what we already thought , yet , even if the results may not surprise us , we as healthcare providers should more effectively employ these communicative skills , as patients seemingly continue to leave our offices not feeling listened to , not understanding their illness or not feeling understood . in a small study , collins et al . examined whether expectations were met during consultative visits with gastroenterologists . despite a focus on physicians with special interest and expertise in functional bowel diseases , many patients remained unsure or unconvinced about their diagnosis . halpert and colleagues chose a different approach and surveyed ibs patients using the internet . most participants looked for education , explanation , empathy and engaged , active listening from physicians , mirroring the very components that contribute to the placebo response . perhaps these studies give us a benchmark for quality improvements that are more applicable than those one can obtain from meta - analyses . if the patients or physicians do not want to use pharmacotherapy , there are other options available , ranging from herbal remedies to dietary changes and psychologically - based interventions . when considering food intake , high - fiber diets seem to be indelibly linked to improvements in ibs , especially ibs coexisting with constipation , with many reviews and books recommending it as one of the pillars of ibs management . but does it truly improve patients quality of life , which should be our primary goal in persons with functional gastrointestinal disorders ? a recent study demonstrated that insoluble fiber ( bran ) was not better than placebo , and although soluble fiber ( psyllium ) transiently helped , this benefit was lost at the end of the 3-month study . the minor and brief statistical difference does not translate into clinical relevance , especially considering the chronic nature of ibs . the results should prompt us to look for correlations between a patient s symptoms and their intake and output ( or outcome ) before automatically recommending changes in fiber intake . fiber intake is only one of the many complex components that make up our diet , so we should not stop obtaining information about other food and drink intake , or promoting healthy nutrition , and should perhaps even think about fiber as a prebiotic. yet , this study should lead us to question the seemingly automatic assumption that increasing fiber intake will improve ibs . besides fiber , intake of other foods can also affect ibs symptoms , with many patients reporting intolerances or trigger foods. a group of italian investigators tried to find an objective correlate for such subjective intolerances and examined the role of food allergies in ibs . using elimination diets and food challenges , about one - third of their patients reacted to cow milk or wheat protein . roughly half of these patients may have suffered from a true food allergy , which was operationally defined by activation of basophils in vitro . thus , food allergies may not be very common in ibs patients , but this study at least suggests that they may be more common than other illnesses often associated with ibs , such as celiac disease . the basophil activation assay is not widely available and confirmatory studies are certainly needed . however , if proven to be effective , we may need to make use of strategies such as this assay because patient history , effects of elimination diets and food challenge , as well as serum ige levels against suspected antigens , poorly predict the potential allergic basis of food intolerance . if carbohydrates from fructose and gluten - containing substances do indeed significantly contribute to ibs symptoms , as this and some other studies of elimination diets suggest , one could take a different approach and eliminate carbohydrates completely . in a small , uncontrolled study , austin and colleagues asked patients with refractory functional diarrhea to follow a strict carbohydrate exclusion diet . the rigid approach was difficult , required implementation through a clinical research center , prompted several drop outs and may not be desirable as it is , by definition , nutritionally imbalanced . however , it also led to improvement in more than two - thirds of those who complied . while the popularity of the atkins and south beach diets may have dropped in recent years , less stringent strategies may not be quite as effective but could be more easily implemented , still resonate with many patients and may well provide secondary benefits in diarrhea - predominant ibs patients by targeting the spreading obesity epidemic . in 2010 , we may need to split more and look to original data , rather than lumping and considering only the meta - analyses . paying attention to the patient s primary symptoms and their main concerns should be made . the contribution of dietary or other lifestyle choices to their symptoms should be considered , as should the role of affect . while trying to obtain this information , physicians need to focus on more than just the content of their discussion and hone in on communicative strategies that may not only increase patients satisfaction , but also improve outcome . clinical trials have to use rigor and systematic strategies to test hypotheses and understand and/or treat possible mechanisms of disease . in poorly understood complex syndromic illnesses with divergent clinical manifestations and likely divergent underlying mechanisms such as ibs , the clinician may need to abandon evidence - based medicine and rely instead on individualized approaches aimed at increasing quality of life .

treatment of functional gastrointestinal disorders remains difficult with many very different approaches showing similar response rates , regardless of whether they target luminal contents ( e.g. , presumed bacterial overgrowth ) , signaling within the gut wall ( e.g. , serotonin agonists or antagonists ) or processing in the brain ( e.g. , cognitive behavioral therapy ) . discrepancies between recent clinical trials and a meta - analysis have forced us to re - examine the use of antidepressants . other studies have looked beyond the traditional drug therapies and have suggested other options such as dietary interventions and communication strategies that address relevant disease mechanisms and enable us to understand patient concerns , with the ultimate goal being to individualize and thus improve treatment outcomes .

in the last 3 decades of medical development , transcatheter treatment of coarctation of aorta with balloon angioplasty and stent implantation has now become an acceptable alternative to surgical repair . however these procedures may result in complications , such as intimal tearing , neo - intimal hyperplasia , restenosis of the lesion , aneurysm formation , dissections , and , rarely , even fatal aortic rupture.1 - 3 ) when the usual balloon expandable stents are used , some degree of vascular wall injury may be inevitable during the forced immediate dilatation of the stenotic lesion . however , a nitinol self - expandable stent , when deployed at the coarctation site , produces low constant radial force , which may result in a gradual widening ( stretching ) of the stenotic lesion , instead of tearing vascular wall structures ( ' stretching rather than tearing ' ) , leaving less tissue injury.4)5 ) and when the result of the self - expansion of the stent is not enough , further in - stent balloon dilatation can be done easily immediately or after an appropriate interval . we report our experience with a nitinol self - expandable stent for the treatment of native aortic coarctation in an adolescent patient . an 18-year - old female , height of 165 cm and body weight of 55 kg , was diagnosed with a native aortic coarctation . her blood pressure ( bp ) measurements were 123/64 mm hg and 123/62 mm hg at her right and left arm , while 86/47 mm hg and 83/49 mm hg at her right and left ankle , showing 50 mm hg higher bp in the upper extremities ( table 1 ) . on echocardiographic study , discrete aortic coarctation was diagnosed with the peak pressure gradient across the stenotic lesion of 50 mm hg by doppler study ( fig . ct angiogram a discrete narrowing at the isthmic portion of the aortic arch due to a posterior shelf lesion was seen ( fig . the narrowest dimension of the stenotic lesion was 4 - 5 mm , and the dimensions of the pre- and post - coarctation descending aorta were 16 mm and 19 mm , respectively ( fig . the distance from the origin of left subclavian artery to the coarctation site was 24 mm . a 20 mm self - expandable nitinol stent ( hercules vascular stent 2060 mm , s&g biotech , seongnam , korea ) was deployed across the coarctation site , without previous balloon dilatation procedure ( fig . no further balloon dilatation was done immediately after the stent deployment to avoid unnecessary tissue injury . immediately after the procedure , the peak pressure gradient across the coarctation site decreased from 50 mm hg to 20 mm hg ( table 2 ) , and the dimension of the stenotic lesion shown by the stent waist somewhat increased from 4 - 5 mm to 8 mm on angiography and plain chest x - ray images ( fig . three months after the procedure , the dimension of the stent waist had enlarged gradually up to 13 mm on plain x - ray and 11.5 mm on ct images ( fig . further interventional procedure , such as instent low - pressure balloon dilatation , was suggested to trim the remnant waist of the stent . but the patient and her parents were happy with the present status and refused any further procedures . one year after the stent deployment , the dimension of the stent waist further increased slightly up to 14 mm ( fig . the bp measurements in the upper extremities somewhat decreased , while the bp measurements in the lower extremities increased significantly up to higher than those of the upper extremities ( table 1 ) . on echocardiographic study , the exact doppler pressure gradient across the pervious stenotic site was not available because the images of the previous stenotic lesion were poor and definite flow turbulence was not found , suggesting the remnant stenosis not significant . subjectively , as she felt no significant difficulty in her physical activity before the stent insertion , she felt not so much difference in her physical activity after the stent insertion . however , she said she felt more warmness in her lower extremities after the stent insertion than before , having more tolerance for cold weather when she was wearing skirts . a nitinol self - expandable stent exerts low constant radial force after the deployment in the stenotic lesion and results in gradual widening of the stenotic lesion . it is flexible and easy to deploy and because the radial force of the self - expandable stent is not so strong , self - expansion ability of the stent depends on the compliance of the stenotic lesion to the radial force . in the native virgin state of coarctation of aorta , where no intimal injury was made by the previous surgery or interventional procedures , the use of nitinol self - expandable stent can be desirable to avoid tearing injury on the vascular wall structure . in the restenosis lesions after the previous balloon dilatation or surgery where the compliance of the stenosis lesion to the radial force is not good , the self - expansion ability of the stent may not be enough . in these cases , further in - stent balloon dilatation or balloon expandable stent insertion can be considered . in using a self - expandable stent in a native coarctation patient , we do not recommend any previous balloon dilatation prior to the stent insertion or any further balloon dilatation immediately after the stent insertion to avoid unnecessary tissue injury . if self expansion of the stent does n't look sufficient after several months ' observation period , in - stent low pressure balloon dilatation procedure can be easily done to relieve the remnant stenosis . mechanical properties of stress ( external force inducing deformation of the stent ) and strain ( degree of deformation , change in the diameter of the stent ) of the self - expandable stent showed a kind of linear relation pattern in a limited range of the stent dimension . as the loaded stress increases , the strain increases in linear relationship initially , but after the stress ( external force ) reaches a certain level , a much larger strain ( deformation ) is observed at a small change of the stress . and some different curves were observed at the time of stress loading and stress unloading ( fig . 5).4)5 ) the resistance force of the stent measured during the external concentric force ( stress ) was being loaded was larger than the expansion force of the stent measured during the external concentric force was being unloaded . while unloading the stress , as the diameter of the stent ( strain ) is recovered , the measured stress ( radial force exerted by the stent ) decreased rather steeply . in an experiment of concentric compression and relaxation of the self - expandable nitinol stent ( s&g biotech , seongnam , korea ) , the radial forces of the stent the 20 mm diameter stent was tightened gradually by a ringed device to the diameter of 5 mm and , thereafter , loosened gradually to the diameter of 20 mm ( fig . generally , the smaller the diameter the stent was reduced to , the stronger the radial force exerted by the stent was observed . as the diameter of the stent recovered , the radial force exerted by the stent decreased steeply . when the diameter of the stent increased to a certain level , the radial force exerted by the stent ( equivalent to the stress ) became quite weak not enough to expand the stent further significantly . when a self - expandable stent of 20 mm diameter was inserted into the 5 mm stenotic lesion in this case , the radial force produced by the stent might be enough to gradually widen the stent waist during the early period after the stent insertion . it might be reasonable to wait and observe the self - expansion of the stent over a several month period . as the diameter of the self - expandable stent increased over several months , the radial force exerted by the stent decreased steeply to an insignificantly low level , allowing no significant further self - dilatation of the stenotic lesion . at this point for the discrete stenotic lesion of the descending aorta in this case , the stent with a shorter length ( 40 - 45 mm ) was sought first . however , for the diameter of 20 mm , only 60 mm length stent was available from the product company then . we decided the use of the 60 mm length stent could be an acceptable option in the adult sized patient of this case . in conclusion , for an 18-year female with native aortic coarctation , a self - expandable nitinol stent was used successfully . no balloon dilatation procedure was done before or immediately after the stent insertion . over the following 3-month period , gradual self - expansion of the stent resulting in gradual widening ( stretching ) of the stenotic lesion to an acceptable dimension was observed . for some patients with native aortic coarctation , where the compliance of the stenotic lesion is well maintained , the use of a nitinol self - expandable stent can be the best treatment option to relieve the stenosis avoiding unnecessary vascular wall injury .

transcatheter treatment of aortic coarctation , with balloon angioplasty or stent implantation , is now an acceptable alternative to surgical repair . however these procedures may result in complications , such as vascular wall injury and re - stenosis of the lesion . a nitinol self - expandable stent , when deployed at the coarctation site , produces low constant radial force , which may result in a gradual widening of the stenotic lesion leaving less tissue injury ( ' stretching rather than tearing ' ) . for an adolescent with a native aortic coarctation , a self - expandable stent of 20 mm diameter was inserted at the discrete stenotic lesion of 5 mm diameter without previous balloon dilatation procedure . no further balloon dilatation was done immediately after the stent insertion . with the self - expandable stent only , the stenosis of the lesion was partially relieved immediately after the stent deployment . over several months after the stent insertion , gradual further widening of the stent waist to an acceptable dimension was observed .

the online version of this article ( doi:10.1007/s40119 - 016 - 0060 - 8 ) contains supplementary material , which is available to authorized users . inwardly rectifying potassium channels ( kir ) allow greater potassium ion ( k ) flow into the cell than in the outward direction . the penetration efficiency of nanoparticles may reach approximately 75% , especially for those with anisotropic shapes or asymmetric surface decoration . the identification of new types of molecules targeting ion channels is of significant interest in biological research . therefore , the aim of our work was to explore the mechanistic evidence of using novel materials such as undecagold , as ion channel blockers . gold nanoparticles can block the function of a channel by fitting into its pore and preventing further conformational change . the key elements in g - loop gating transition involve the movement of the n - terminus and c - linker that removed constraining intermolecular interactions . negatively charged gold nanoparticles ( 0.8 nm ) with spermidine are able to enter the cell and block the human kir channel at its intracellular face . the g loops are located towards the cytoplasmic site , playing a role in g - loop disrupted gating or inward rectification . the g loop is the narrowest portion of the cytoplasmic domain in the kir3.1 ion pore . this ion pore which has a diameter of approximately 8 nm has never been permeated by negatively charged undeca - gold . the cytoplasmic ion - permeation pathways are occluded by four cytoplasmic loops that form a girdle around the central pore ( g - loop ) . kir channels , similar to other k channels , have a tetrameric architecture , but , unlike other channels , have a long ion permeation pathway that consists of both a transmembrane and a cytosolic portion . the gating of kir channels has been shown to involve the binding of divalent cations and polyamines to its cytoplasmic domain . g - protein - gated k channels ( kir3.1kir3.4 ) control electrical excitability , regulating the heart rate by opening a g - loop gate in the cytoplasmic domain . g loop crystal structures revealed two possible conformations of the cytoplasmic domains , one with a constricted cytosolic gate and another with a dilated g - loop gate . in cryo - electron microscopy ( em ) images , undecagold - spermidine complex and negative charged gold nanoparticles ( 0.8 nm ) bind ionically to two nh and one nh portions of the spermidine molecules . spermidine ( 1.2 nm ) is a suitable candidate for introducing gold nanoparticles into the cytoplasmic vestibule ( 3 nm diameter ) of the receptors . spermidine passes through the pore domain of the kir channel with the incapacity for blocking other channels . kir3.1 normally functions as a heteromultimer with other members of the kir3 family , whereas the chimera are homomultimer . the chimera used in this study might lack proper coupling between the cytoplasmic and transmembrane pores . however , single channel activity has yet to be demonstrated for the prokaryotic kir channels . we have purified the cytoplasmic domain of the kir3.1 receptor from e. coli ( figs . 1 , 2 , 3.).fig . 1control ( kir receptor cytoplasmic domain ) : a purified protein of the cytoplsmic domain of the kir channel have collapsed lumens . 2two dimensional tem images show the tetramer of kir3.1 cytoplasmic domain with collapsed lumen of the ion pore . tem transmission electron microscopy , k ir inward - rectifier potassium ion channelfig . 3mass spectra of the tetramer of kir3.1 cytoplasmic domain from the maldi set - up shows the peptide fragments of the protein . k ir inward - rectifier potassium ion channel , maldi matrix - assisted laser desorption / ionization control ( kir receptor cytoplasmic domain ) : a purified protein of the cytoplsmic domain of the kir channel have collapsed lumens . k ir inward - rectifier potassium ion channel two dimensional tem images show the tetramer of kir3.1 cytoplasmic domain with collapsed lumen of the ion pore . tem transmission electron microscopy , k ir inward - rectifier potassium ion channel mass spectra of the tetramer of kir3.1 cytoplasmic domain from the maldi set - up shows the peptide fragments of the protein . k ir inward - rectifier potassium ion channel , maldi matrix - assisted laser desorption / ionization the 0.8 nm negatively charged gold nanoparticles were purchased from nanoprobes , inc ( new york , ny , usa ) . the ionic bond was induced by vortexing two samples with different concentrations of positively charged spermidine ( i.e. , 10 mm and 100 nm ) , 1 mg of meso-2.3-dimercaptosuccinic acid ( dmsa ) powder , and 50 nm of negatively charged gold nanoparticles for approximately 15 min followed by mixing with a pipette for 15 min . the negatively charged coo particles were individually coated with a thin film of three nanoparticle molecules with dmsa , each bound to one nh2 and two nh1 spermidine molecules ( supplementary video clip ) . the spermidine hydroxide solution and the dmsa were obtained from sigma aldrich ( st . louis , mo , usa ) . undecagold has a core of 11 gold atoms only 0.8 nm in diameter and is ideal for ultra - high - resolution em work such as cryo - em , scanning transmission em . in this study , we present the structure , obtained by single particle analysis with cryoem of the cytoplasmic kir domain containing undeca - gold inside the complex ( supplementary figs . 1 , 2 ) . purified protein solutions were diluted to 10 g ml in a buffer solution containing 20 mm tris hcl ( ph 8.0 ) , 100 mm nacl , and the corresponding detergent . a diluted protein solution of the cytoplsmic domain of the kir channel was added at a volume ratio of 1:10 to reach a concentration of ~5 g ml . protein complexes were adsorbed for 1 min onto glow - discharged , 400 mesh carbon - coated grids . micrographs were taken at a low dose on a jem-2100 operated at 120 kv ( jeol inc , peabody , ma 01960 , usa ) . tilt pairs were recorded at 0 and 45 or 0 and 60 , at a nominal magnification of 69000 on a gatanultrascan 4 k ccd ( gatan inc . , spermidine0.8 nm gold nanoparticles complexes , which is not determined by a transmission em ( tem ) of jem-2100 ( figs . 4 , 5 , 6 ) with energy dispersive x - ray spectroscopy ( eds ) , surface - coated with dmsa ( figs . 7 , 8) , have been successfully used to produce a high signal in tem images in the ion pore or surrounding tetramer components.fig . 4treated by gold nanoparticles : single particle analysis with cryoem shows the cytoplasmic kir domain containing undeca - gold inside the complexfig . 5two - dimensional tem images show the tetramer of kir3.1 cytoplasmic domain with dilated lumen of the ion pore by gold nanoparticles . tem transmission electron microscopy , k ir inward - rectifier potassium ion channelfig . 6mass spectra of tetramer of kir3.1 cytoplasmic domain from the maldi set - up shows the peptide fragments of the protein containing gold peaks ( red arrow ) . k ir inward - rectifier potassium ion channel , maldi matrix - assisted laser desorption / ionizationfig 8two - dimensional tem images of tetramer of kir3.1 cytoplasmic domain after treating with undecagold cating with dmsa spermidine complex shows the hyperdense dmsa coating in the ion pore or adjacent tetramer . tem transmission electron microscopy , k ir inward - rectifier potassium ion channel , dmsa meso-2.3-dimercaptosuccinic acid treated by gold nanoparticles : single particle analysis with cryoem shows the cytoplasmic kir domain containing undeca - gold inside the complex two - dimensional tem images show the tetramer of kir3.1 cytoplasmic domain with dilated lumen of the ion pore by gold nanoparticles . tem transmission electron microscopy , k ir inward - rectifier potassium ion channel mass spectra of tetramer of kir3.1 cytoplasmic domain from the maldi set - up shows the peptide fragments of the protein containing gold peaks ( red arrow ) . k ir inward - rectifier potassium ion channel , maldi matrix - assisted laser desorption / ionization treated by gold nanoparticles coating with dmsa - spermidine complex . dmsa meso-2.3-dimercaptosuccinic acid two - dimensional tem images of tetramer of kir3.1 cytoplasmic domain after treating with undecagold cating with dmsa spermidine complex shows the hyperdense dmsa coating in the ion pore or adjacent tetramer . tem transmission electron microscopy , k ir inward - rectifier potassium ion channel , dmsa meso-2.3-dimercaptosuccinic acid dimercaptosuccinic acid can form chelates with gold nanoparticles via the two sulfur atoms . dmsa is water soluble with extracellular distribution and can not penetrate the lipid bilayer , but is able enter to the cytoplasmic domain of kir ion pore accompanied by spermidine molecules , already coated by the gold nanoparticles . moreover , in atomic force microscope observations , the point less than 0.8 nm can not be counted as the gold nanoparticles because of the substrate roughness originating from the natural convex point . evidence of the existence of gold nanoparticles in the ion pore interaction has been supported by high density surface - coated with dmsa in tem images . energy dispersive x - ray spectroscopy was also used to confirm the presence of the gold deposits in the cytoplasmic domain of the kir receptor ( figs . x - ray photoelectron spectroscopy measurements were performed using an ultrahigh vacuum chamber with a base pressure below 510 torr . xps data was collected using an x - ray photoelectron spectrometer phi 5400 ( physical electronics ) with a nominal energy resolution of 0.7 ev . spectra were acquired using a photon beam of 1486.6 ev , selected from a conventional ( nonmonochromatic ) al / mg dual - anode x - ray source . data was collected at room temperature under vacuum , and samples were analyzed as soon as they were prepared , without any extra cleaning procedure . the eds spectrum of the receptors containing the gold nanoparticles indicates the presence of c and o in the receptor molecules . the small gold peak evidenced in the eds spectrum confirms that a validated amount of the 0.8 nm gold nanoparticle is present in the ion pore of the kir channels .fig . 9eds by stem of the cytoplasmic domain of the receptor treated by gold nanoparticles : transmission em ( tem ) with surface - coated with dmsa have been successfully used to produce a high signal in tem images in the ion pore or surrounding tetramer components . 10two - dimensional stem images for the eds shows hyperdense gold nanoparticle ( red arrow ) in the lumens of the tetramer of the cytoplasmic domain . 11the eds spectrum of the receptors containing the gold nanoparticles in it indicates the presence of c and o from the receptor molecules . the small gold peak evident in the eds spectrum confirms that a validated amount of the 0.8 nm gold nanoparticle is present in the ion pore of kir channels . eds energy dispersive x - ray spectroscopy , k ir inward - rectifier potassium ion channel eds by stem of the cytoplasmic domain of the receptor treated by gold nanoparticles : transmission em ( tem ) with surface - coated with dmsa have been successfully used to produce a high signal in tem images in the ion pore or surrounding tetramer components . eds energy dispersive x - ray spectroscopy , stem scanning transmission electron microscopy two - dimensional stem images for the eds shows hyperdense gold nanoparticle ( red arrow ) in the lumens of the tetramer of the cytoplasmic domain . stem scanning transmission electron microscopy , eds energy dispersive x - ray spectroscopy the eds spectrum of the receptors containing the gold nanoparticles in it indicates the presence of c and o from the receptor molecules . the small gold peak evident in the eds spectrum confirms that a validated amount of the 0.8 nm gold nanoparticle is present in the ion pore of kir channels . eds energy dispersive x - ray spectroscopy , k ir inward - rectifier potassium ion channel gold nanoparticles ( 0.8 nm ) belonging to a new class of ion channel inhibitors directly block the human kir channel i(kach ) when accompanied by the antagonist spermidine . blocking of the i(kach ) channel by the gold nanoparticle - spermidine complex has been shown to occur in atrial cardiomyocytes . a reduced k current delays the repolarization of the complex in patients with atrial fibrillation ( af ) . this complex is a new class of three anti - arrhythmic agents that may potentially treat af . the complex is designed to be a selective i(kach ) blocker in contrast to multiple class 1 and 3 anti - arrhythmic agents with less proarrhythmic potential . a limitation of this study was that the authors could not identify the 3-d images of the gold nanoparticles in the ion pore because of technical limitations . in conclusion , we could identify undeca - gold in the ion pore of the kir3.1 channel in order to clarify its direct blocking effect in the kir ion pore by undecagold . below is the link to the electronic supplementary material . supplementary material 1 ( doc 4184 kb)supplementary material 2 ( avi 91922 kb ) supplementary material 1 ( doc 4184 kb ) supplementary material 2 ( avi 91922 kb ) this article does not contain any new studies with human or animal subjects performed by any of the authors . this article is distributed under the terms of the creative commons attribution - noncommercial 4.0 international license ( http://creativecommons.org/licenses/by-nc/4.0/ ) , which permits any noncommercial use , distribution , and reproduction in any medium , provided you give appropriate credit to the original author(s ) and the source , provide a link to the creative commons license , and indicate if changes were made .

introductionin our previous study , we found that negatively charged gold nanoparticles with spermidine have the potential of blocking inwardly rectifying potassium channels ( kir ) , both at the cellular and the tissue level.methodsfor the purpose of the present study , we purified the cytoplasmic domain of the kir 3.1 receptor from escherichia coli . using single particles with surface coating by transmission electron microscopy , we identified the gold nanoparticles at the cytoplasmic side of the human kir channel.resultsenergy-dispersive x - ray spectroscopy showed the presence of the gold deposits in the cytoplasmic domain of the kir receptor.conclusionin conclusion , we could identify undecagold in the ion pore of the kir3.1 channel in order to clarify its direct blocking effect in the kir ion pore by undecagold.electronic supplementary materialthe online version of this article ( doi:10.1007/s40119 - 016 - 0060 - 8 ) contains supplementary material , which is available to authorized users .

celiac sprue , also termed as celiac disease is characterized by small intestinal malabsorption of nutrients after ingestion of wheat gluten or related proteins from rye and barley a characteristic , though not specific villous atrophy of the small intestinal mucosa ; prompt clinical and histological improvement after adherence to strict gluten free diet and clinical relapse when gluten is reintroduced . celiac disease is an important cause of chronic diarrhea , failure to thrive , and anemia in children . this triad of symptoms was classical of celiac disease reported earlier . over the time early recognition and suspicion introduction of serology for screening of celiac disease has enabled to recognize celiac disease in asymptomatic patients , its atypical manifestations and in its latent form . diverse problems such as dental anomalies , osteopenic bone disorders , lactose intolerance , infertility , refractory anemia may sometimes be the presenting manifestations of celiac disease . at least , 10% of children with celiac disease have associated conditions including selective iga deficiency , dermatitis herpetiformis , diabetes mellitus type 1[6 , 7 ] and down 's syndrome . study was conducted to determine the mode of clinical presentation of a large group of patients with celiac disease and whether there has been a change in the presentation with the time . study population and design : all the prospective patients coming to celiac disease clinic in division of pediatric gastroenterology , post graduate institute medical education and research , chandigarh , india were enrolled for study , starting from july 1 2006 to december 31 2007 . inclusion criteria were pediatric patients of celiac disease as diagnosed by revised espghan ( european society for pediatric gastroenterology , hepatology and nutrition ) criteria . a verbal assent was taken from the children and a written informed consent was obtained from legal guardian of each subject . all patients underwent hemoglobin , total leucocytes count , differential leukocytes count , platelet count , reticulocyte count and peripheral smear for the type of anemia , liver function test including serum bilirubin , sgot / sgpt , serum alkaline phosphatase , serum proteins , serum calcium , serum phosphorus , iga anti - tissue transglutaminase ( anti - ttg ) and serum zinc levels . this is an indirect non - competitive enzyme immunoassay for determination of ttg antibodies in human sera or plasma . reference cut off of anti - ttg iga by above kit was 15 u / ml . in case of clinical setting but serology endoscopic markers were evaluated for grooving / scalloping of the duodenal folds , mosaic pattern of the mucosa , nodularity , and reduction and thinning in the numbers ( no more than three folds in straight line on endoscopic vision or absence of kerkring folds at maximum insufflations ) . biopsy specimens comprising of at least three fragments with a forceps ( open cup 6 mm ) were taken . biopsies were embedded in paraffin wax , cut in sections and stained with hematoxylin and eosin . statistical analysis : statistical analysis was performed with statistical package for the social sciences software ( spss version 13.0 , chicago , illinois , usa ) . in total study group , for continuous data mean , standard deviation and range were calculated . for categorical data , number and one hundred and thirty four children diagnosed as having celiac disease who came for regular follow up were included in the study group . age and sex distribution of the study group : the number of cases present in age group of < 5 years were 60 ( 45% ) , followed by 55 cases ( 41% ) in 510 years of age group and 19 cases ( 14% ) in more than 10 years of age group . mean age of onset of symptoms was 3.4 years ( 6 months-11.5 years ) and patients presented at a mean age of 6.2 years ( 114 ) with mean period of delay in diagnosis of 33.4 months ( 0132.0 ) . mean age at cereal introduction was 7.2 ( 229 ) months ( table 1 ) . in 77% of patients , symptoms appeared early i.e. below 5 years of age ( mean age 3.36 years ) but 55.2% presented to the hospital late i.e. after 5 years of age ( mean age 6.19 years ) with mean delay in diagnosis of 33.4 months . demographic profile of the study group ( n=134 ) rural and urban distribution of study group : distribution noted in the study group was urban 57% and rural 43% . the urban group presented early ( 59.6% of patients < 5 years of age ) than rural patients ( 40.4% of patients < 5 years of age ) . presenting symptoms of study group : diarrhea was presenting symptoms in 54.5% , failure to thrive in 52.2% , features suggestive of anemia were seen in 40.3% , short stature in 20.9% , abdominal pain in 19.4% , vomiting in 15.7% and constipation in 2.2% of cases . history of seizures was elicited in 2 cases but in one case these were post traumatic and in other they were related to past history of tuberculous meningitis . one patient had features suggestive of down 's syndrome and one was already a known case of juvenile rheumatoid arthritis . family history of celiac disease was noted in 7 cases out of which 5 were siblings and 2 were paternal aunts . physical examination of study group : pallor was present in 100% of cases , short stature in 60.4% , clubbing and edema in 15.7% and 3% of cases at the time of diagnosis , respectively . evidence of rickets , b complex group of vitamin deficiency and vitamin a deficiency was seen in 23 , 9 and 1 case respectively . no abnormality was seen in any case with regards to respiratory , cardiovascular and nervous system examination . weight and height at presentation : at presentation , the mean weight was 14.65.5 kg . 14.2% of the patients had weight > 80% of 50 percentile of nchs for that age and sex at the time of diagnosis . 24.6% of patients had weight for age less than 60% of normal ( grade iii and iv or severe pem according to the iap classification ) . 60.4% of patients had length / height below 3 percentile of nchs for that age and sex . anemia was microcytic hypochromic in 60.4% of patients , dimorphic in 20.9% and normocytic normochromic in rest of patients ( table 2 ) . laboratory investigations in the study group ( data expressed as mean sd ; n=134 ) sgot : serum glutamic oxaloacetic transaminase ; sgpt : serum glutamic pyruvic transaminase , sap : forty nine patients ( 36.6% ) had thrombocytosis at the time of diagnosis . serum trans- aminases were raised in 38.8% of cases but rise was mild in all ( <3 times the normal ) except those with associated liver disease ( table 2 ) . anti tissue transglutaminase iga ( ttg ) levels in study group : the mean serum anti ttg level of study group at presentation was 164.24u / ml ( ranging from 0 to 749 u / ml ) . 15 patients were negative for the serology but 8 out of them had iga deficiency and all of these had histopathology suggestive of celiac disease . histological categorization of small intestinal biopsy in study group : 86 patients ( 64.2% ) had subtotal villous atrophy ( marsh iiib ) , 30 ( 22.4% ) had partial villous atrophy ( marsh iiia ) and only one patient had evidence of total villous atrophy on small intestinal biopsy . levels of iga anti ttg correlated with the severity of small intestinal damage on biopsy ( table 3 ) . this prospective study was undertaken to study the clinical and investigative patterns of presentation in patients of celiac disease . mean age at onset of symptoms , mean age at diagnosis , mean period of delay in diagnosis and mean age at cereal introduction correlates with other indian studies as reported earlier[1114 ] . in majority of studies , conducted in western countries , the classical age of presentation is 918 months and the diagnosis is usually made within 6 months of onset of symptoms[1517 ] . in the study conducted by baudon et al in france , the first symptoms / signs occurred before one year of age in 73% of the cases , during the second year of life in 20.5% and after 3 in only 6.5% . the diagnosis was made before 2 years of age in 77% of the cases and after 3 in only 23% . in contrast to this , in the present study , no patient presented before one year of age though symptom onset occurred in 23.8% . symptom onset during the second year of life occurred in 19% and in 35% of patients the symptoms occurred in 25 years of age . no patient was diagnosed before one year of age and only 5.2% between 12 years of age and more than half of cases ( 55.2% ) were diagnosed after 5 years of age . the higher mean age of onset of symptoms in our patients may be attributed to prolonged breast feeding practices , delayed weaning and late introduction of gluten in the diet . the delay in diagnosis is probably because in developing countries like india , infection as a cause of chronic diarrhea is very common and there is lack of awareness about celiac disease . in the present study , early diagnosis in urban patients may be because of their direct presentation to tertiary care hospitals while delayed diagnosis in rural population shows that celiac disease is still not being diagnosed at primary health care centers . in the present study , the major symptoms at presentation were diarrhea , failure to thrive , abdominal distension , while pain abdomen , vomiting and constipation were relatively less common symptoms . the main differences in the clinical features of celiac disease as highlighted by the present study and other studies from northern india[1114 ] as compared to west are the higher incidence of failure to thrive and anemia in india ( table 4 ) . these two clinical manifestations are features of more severe disease and correlate with delay in diagnosis in our country . with increasing awareness about the varied manifestations of cd and with the availability of reliable noninvasive markers ( celiac serology ) of the disease , it is now being recognized in the atypical form with early diagnosis as compared to earlier studies ( table 4 ) . comparison of clinical features of children with celiac disease at presentation in various studies the mean iga anti ttg level at time of presentation was 164.24 u / ml which was higher than the study conducted by tursi et al . iga deficiency was confirmed on investigation in 6% of cases which is higher than previously noted[1 , 20 ] . tursi et al showed that the mean serum value of anti ttg ranged from 3.6 u / ml in marsh i lesions to 74.95 u / ml in marsh iiic lesions , respectively . the higher mean anti ttg levels in our study correspond with severe intestinal damage ( marsh iiib - c lesions ) . 22.4% had partial villous atrophy ( marsh iiia ) and only one patient had evidence of total villous atrophy on small intestinal biopsy at the time of presentation . young et al reported total villous atrophy in all his patients at the time of presentation . anemia is a frequent feature of celiac disease and present in all the cases at the time of presentation . iron deficiency , microcytic hypochromic anemia was the most common type of anemia recorded in our study which is in conformity with the results of other studies . thrombocytosis was detected in 37% of our patients at diagnosis . in the study done by patwari et al , thrombocytosis was present in 60% of the cases at the time of diagnosis . ogrady et al documented evidence of hyposplenism of unknown cause with thrombocytosis and splenic atrophy in 50% of adults with celiac disease . in most of the cases the evidence of hyposplenism disappeared with elimination of gluten from the diet . serum transaminases were raised in 38.8% of our cases but rise was mild in all ( <3 times the normal ) except those with associated liver disease . chronically elevated transaminases levels in the range of 1.5 to twice normal values have been reported in 940% of patients with untreated celiac sprue . in our study associated liver disease was present in 2 cases ; down 's syndrome in one case , nephrotic syndrome in one and juvenile rheumatoid arthritis in one case . out of these selective iga deficiency was present in 3% of children , dermatitis herpetiformis and diabetes mellitus type i each in 2% of cases ; chronic active hepatitis , psoriasis , cardio - vascular disease and down 's syndrome each in 1% of children with celiac disease . vitiligo , cystic fibrosis , fibrosing alveolitis , renal tubular defects , 1 anti - trypsin deficiency , henoch - schnlein purpura were also noted as associated disorder in children with celiac disease . there was some referral bias also as the study center is a tertiary care referral center . classical presentation of celiac disease is less commonly encountered these days in india in accordance to apparent changing trend worldwide . appreciation of the more occult presentations of celiac disease together with the more widespread use of serologic testing will probably result in an increase in the rate of diagnosis of celiac disease in india .

objectiveceliac disease is an important cause of chronic diarrhea , failure to thrive , and anemia in children . mode of presentation of celiac disease has changed in last few years . study was conducted to determine the mode of clinical presentation of a large group of patients with celiac disease and whether there has been a change in the presentation with the time.methodsa prospective study was conducted on 134 children diagnosed to be having celiac disease in the pediatric gastroenterology , pgimer , chandigarh , from july 1st 2006 to december 31st 2007 . their detailed clinical profile was recorded on a pretested proforma and all patients underwent hemogram , liver function tests , iga anti - tissue transglutaminase ( anti ttg ) , and upper gastro - intestinal endoscopy.findingsmajor symptoms at presentation were diarrhea ( 54.5% ) , failure to thrive ( 52.2% ) , abdominal distension ( 41% ) , anemia ( 40% ) , pain abdomen ( 19.4% ) , vomiting ( 15.7% ) and constipation ( 2.2% of cases ) . 60.4% of patients had short stature . anemia was microcytic hypochromic in 79.1% of patients , and dimorphic in 20.9% . serum transaminases were raised in 38.8% of cases . the mean serum anti ttg level was 164.24u / ml ( range 0 - 749 u / ml ) and levels correlated with the severity of small intestinal damage on biopsy . 15 patients were negative for the serology but 8 out of them had iga deficiency and all had histopathology suggestive of celiac disease.conclusionclassical presentation of celiac disease is less commonly encountered these days probably related to the more widespread use of serologic testing and early recognition of atypical manifestations of celiac disease .

knee osteoarthritis ( oa ) is an important cause of pain and disability in old population , and it is becoming increasingly prevalent worldwide due to its association with an aging population and due to a growing prevalence of obesity . to investigate the prevalence and risk factors of knee osteoarthritis , the etiology of knee oa is believed to be multifactorial , and the following risk factors have been identified : heredity [ 24 ] , obesity [ 58 ] , injury [ 811 ] , and physical workload [ 1215 ] . however , most of the large - scale studies were conducted in north america and europe , with scant information from less developed regions reference , and most of the study participants were based on urban residents or workers , while people in rural areas , especially farmers , were rarely investigated . in fact , farmers form a large proportion of the population in less developed regions , most of whom have to endure heavy physical workload until an old age . the prevalence and risk factors of knee oa among these people might be different . we conducted a population - based cross - sectional survey in wuchuan county , a remote rural region in inner mongolia of northern china . we found that the prevalence of severe radiographic and symptomatic knee oa was much higher than that reported from urban regions of china or in the framingham cohort . we estimates the risk of knee osteoarthritis related to the following factors : age , sex , bmi , knee injury , physical activity level , and physical activity exposure at work including walking , walking roughly , cycling , standing , digging , kneeling , squatting , climbing , and lifting of loads . to allow valid comparisons with the beijing oa study and the framingham cohort , the same survey instruments and comparable radiographic protocols were used the study design has been described in detail in a previous publication . briefly , a total of 1165 individuals reporting to be aged 50 years and older were identified in 762 randomly selected households in wuchuan county , inner mongolia . of these , 27 subjects were excluded from further study participation . of eligible participants , 90% completed the home interview and attended the radiographic examination , and these subjects were included in the analysis . a total of 1025 participants ( 520 women and 505 men ) were involved in the analysis , over 90% living on farming as reported . the survey questionnaire focusing on joint symptoms and possible risk factors for knee oa was completed under the supervision of the intensively trained interviewers who went door to door to enumerate and interview all men and women . a posterior - anterior weight - bearing semi - flexed view radiographs were taken of both knees , strictly according to a validated acquisition protocol . radiographs were read by the chief investigator xk using the oarsi atlas , and kellgren - lawrence grades ( range 04 ) were also assigned . radiographic knee oa was defined as having a kellgren - lawrence grade 2 in one or both knees . similarly , severe radiographic knee oa was defined as having a kellgren - lawrence grade 3 in one or both knees . we defined medial and lateral radiographic oa , respectively , if a knee had a kellgren - lawrence grade 2 and medial or lateral joint space narrowing score 1 . logistic regression analysis for the parameters sex , age , bmi , knee injury , physical activity level , and physical activity exposure at work including walking , walking roughly , cycling , standing , digging , kneeling , squatting , climbing , and lifting of loads was performed for radiographic tibiafemoral knee parameters that have significant influence were included in the regression equations , and odds ratios ( or ) and 95% confidence intervals ( ci ) for these parameters were calculated . then we calculated the adjusted or ( adjusted for the parameters included in the equations ) and 95% ci for the rest of parameters which were excluded in the regression equations . for better analysis , the parameter age was divided into four grades : 50 to 52.5 , > 52.5 to < 60 , 60 to < 70 , and 70 years old , and bmi was divided into four grades , too : < 18.5 , 18.5 to < 25.0 , 25.0 to < 28.0 , and 28.0 kg / m . the characteristics of the participants are presented in table 1 . as described in the previous publication . at the time of data analysis , it was discovered that 24 men and 26 women were actually younger than 50 years ( either 48 or 49 years ) . almost all participants ( 91% ) were farmers or had been engaged in farming as their main occupation , with the remaining 9% reporting their main occupation as businessman or shopkeeper . at the time of the survey , 85% of all participants were still working . most of participants ( 91% ) reported that the occupation they had held the longest involved heavy physical work . the prevalence rates of radiographic tibiofemoral knee oa , lateral compartment knee oa , and medial compartment knee oa are presented in table 2 . age , sex , bmi , and knee injury were included in the regression equation for the radiographic tibiofemoral knee oa risks . a steep dose - response relationship between age and the diagnosis of knee osteoarthritis was found : compared with persons at least 70 years old ( group 4 ) , persons about 50 years old ( group 1 ) had a much lower knee osteoarthritis risk ( or 0.065 ; 95% ci 0.0340.123 : table 3 ) . the mean bmi was also strongly associated with knee osteoarthritis : compared to persons with a bmi at least 28.0 kg / m , persons with a bmi of less than 18.0 kg / m only had an or of 0.15 ( 95% ci 0.060.38 ) . assuming a linear dose - response relation , the risk would halve for each 4.2 kg / m decrease in the bmi . males had an or of 0.45 compared with females ; this means females have more than a 2-fold radiographic tibiofemoral knee oa risk than males . participants without knee injury showed an or of 0.43 ( 95% ci 0.230.81 ) for the risk of knee osteoarthritis , in comparison to persons with knee injury . we calculated the adjusted ors for the parameters not in the regression equation ( adjusted for age , sex , bmi , and knee injury ) ( table 3 ) . compared with heavy physical level , participants with moderate physical level had a lower adjusted or ( 0.81 , 95% ci 0.441.48 ) . persons cycling or digging less than 2 hours per day had a lower radiographic tibiofemoral knee oa risk in comparison to persons cycling or digging at least 2 hours per day ( or 0.71 or 0.72 , 95% ci 0.441.16 or 0.481.08 ) , indicating that cycling and digging increase the risk of radiographic tibiofemoral knee oa despite that the influence was not statistically significant ( p > .05 ) . kneeling elevated the risk slightly with an adjusted or of 0.93 ( 95% ci 0.641.35 ) for kneeling less than 30 minutes per day compared with at least 30 minutes per day . however , lifting or moving objects weighing 10 kilograms or more did not increase the radiographic tibiofemoral knee oa risk with an adjusted or of 0.99 ( 95% ci 0.551.80 ) , while squatting even decreased the risk of radiographic tibiofemoral knee oa with an adjusted or of 1.25 ( 95% ci 0.851.84 ) for kneeling at least 30 minutes per day compared with squatting at least 30 minutes per day . age , sex , knee injury , and squatting were included in the regression equation for the lateral compartment knee oa risk ( table 3 ) . a dose - response relationship between age and the lateral compartment knee oa was found , too , although the correlation was not as significant as that of age and radiographic tibiofemoral knee oa . compared with radiographic tibiofemoral knee oa , similar results were found for sex and knee injury in influencing the risk of the lateral compartment knee oa . unexpectedly , squatting decreased the lateral compartment knee oa risk with an adjusted or of 2.67 ( 95% ci 1.504.76 ) for kneeling at least 30 minutes per day compared with squatting at least 30 minutes per day , and it was statistically significant ( p < .05 ) . no significant effect on the lateral compartment knee oa risk was found for bmi . we calculated the adjusted ors for the parameters not in the regression equation ( adjusted for age , sex , bmi , and squatting ) ( table 3 ) . participants digging less than 2 hours per day had a lower lateral compartment knee oa risk in comparison to the ones digging at least 2 hours per day ( or 0.71 or 0.72 , 95% ci 0.441.16 or 0.481.08 ) ; this means that digging increases the lateral compartment knee oa risk . as showed in table 3 , subjects who did not lift or move objects weighing 10 kilograms or more had an adjusted or of 0.76 ( 95% ci 0.311.86 ) , and the adjusted or for kneeling less than 30 minutes per day was 0.83 ( 95% ci 0.471.47 ) compared with at least 30 minutes per day . cycling elevated the risk slightly with an adjusted or of 0.93 ( 95% ci 0.432.01 ) for cycling less than 2 hours per day compared with at least 2 hours per day . bmi did not show a definite trend for the risk of lateral compartment knee oa . compared with bmi 28.0 kg / m , the adjusted ors of bmi < 18.5 , 18.5 to < 25.0 , and 25.0 to < 28.0 kg / m were 1.69 ( 0.377.59 ) , 1.09 ( 0.313.81 ) , and 2.36 ( 0.628.95 ) , respectively . however , we should note that the number of participants with lateral compartment knee oa in bmi < 18.5 kg / mgroup and bmi 28.0 kg / m group were too small , only 6 and 3 , respectively , to make the analysis result inconvincible . a heavier physical work level did not lead to a higher risk of lateral compartment knee oa , though , with an adjusted or 1.30 ( 95% ci 0.572.99 ) for moderate physical work compared with heavy physical work . age , sex , and bmi were included in the regression equation for the medial compartment knee oa risk ( table 3 ) . a dose - response relationship between age , bmi , and the medial compartment knee oa was found too , similar with radiographic tibiofemoral knee oa . compared with persons at least 70 years old ( group 4 ) , persons at 50 to 52.5 , > 52.5 to < 60 , and 60 to < 70 years old had a lower medial compartment knee oa risk with ors ( 95% ci ) of 0.037 ( 0.0170.082 ) , 0.089 ( 0.0470.17 ) , and 0.39 ( 0.220.70 ) , respectively . the mean bmi was also strongly associated with medial compartment knee oa compared to persons with a bmi 28.0 kg / m , persons with a bmi < 18.5 , 18.5 to < 25.0 , and 25.0 to < 28.0 kg / m had ors ( 95% ci ) of 0.13 ( 0.0430.36 ) , 0.20 ( 0.0950.42 ) , and 0.44 ( 0.191.00 ) , respectively . females had a higher medial compartment knee oa risk : the risk of females was 1.5-fold higher than that of males . the adjusted ors for the parameters not in the regression equation ( adjusted for age , sex , and bmi ) were calculated as showed in table 3 . the medial compartment knee oa risk of participants with knee injury was almost double that of participants without knee injury ( adjusted or ( 95% ci ) : 0.52 ( 0.241.10 ) ) , and it was statistically significant ( p = .085 ) . heavier physical workload , cycling , and digging lead to a higher medial compartment knee oa risk . lifting or moving objects weighing 10 kg or more also increased the medial compartment knee oa risk slightly . persons kneeling and squatting did not show a higher medial compartment knee oa risk , though . squatting even decreased the risk , with the adjusted or ( 95% ci ) of 1.15 ( 0.731.80 ) for kneeling less than 30 minutes per day compared with squatting at least 30 minutes per day . our results showed that age , sex , and previous knee injury were strongly associated with radiographic tibiofemoral knee oa , lateral compartment knee oa , and medial compartment knee oa . age had a steep dose - response relationship with the risk of all of the three kinds of knee oa . females or participants with previous knee injury approximately had a 2-fold risk compared with males or those without . bmi had a definite dose - response relationship with the risk of radiographic tibiofemoral knee oa and medial compartment knee oa , while the relationship between bmi and lateral compartment knee oa had an inexact trend . however , we should note that the number of participants with lateral compartment knee oa in bmi < 18.5 kg / m group and bmi 28.0 kg / m group was too small , only 6 and 3 respectively , to make the analysis result inconvincible . excluding these two groups , we found that persons of bmi 25.0 to < 28.0 kg / m had more than a 2-fold risk of lateral compartment knee oa compared with persons of bmi 18.5 to < 25.0 kg / m , indicating that elevated bmi increased the risk of lateral compartment knee oa , too . heavier physical workload showed a positive association with radiographic tibiofemoral and medial compartment knee oa , but a negative association with lateral compartment knee oa . one explanation is that the number of participants with lateral compartment knee oa in moderate physical workload level group was small , only 9 . in our study , no one reported that the physical activity level was sedentary ; 7 persons reported it was light physical activity level , and we excluded this group for analysis ; nearly 8% subjects ( 84 persons ) reported it was moderate physical activity level , while more than 91% persons reported the physical activity level was heavy . thus , the influence of physical workload on the risk of knee oa was hard to be highlighted , as most of the participants had heavy physical activity . this may explain the result that physical activity exposure such as cycling , digging , lifting or moving objects , and kneeling only had a slight or moderate effect on the risk of knee oa . another explanation for this result is that we did not make detailed levels for these kinds of physical activity exposure , we just roughly classified them into two levels . for instance , digging was only divided into digging 2 hours or more per day or less than 2 hours . and we did not estimate the accumulative time of exposure . in this case , the influence of physical activity exposure was hard to stand out . in addition , squatting decreased the risk of radiographic tibiofemoral , lateral compartment , and medial compartment knee oa . this effect was even statistically significant for lateral compartment knee oa ( p = .001 ) . this result could be explained by the biomechanical role of loading on the pathogenesis of knee oa . during deep knee flexion , such as kneeling and squatting these high sagittal movements would be expected to increase loads across the patellofemoral joint . while repeated squatting and kneeling may well have consequences for the tibiofemoral joint , the part of the knee most likely to be exposed to the highest load during such activities is the patellofemoral joint . amin et al . found frequent squatting / kneeling lead to a greater likelihood for worse cartilage morphology scores at the patellofemoral joint , while had no significant influence on the tibiofemoral joint . however , other studies [ 19 , 20 ] showed that squatting increased the risk of tibiofemoral knee oa , while the association with patellofemoral knee oa was weaker or doubtful . as noted in the previous publication , the overall prevalence of radiographic knee oa in the compared age group was similar to that demonstrated in the beijing oa , despite lower bmi ( 22 - 23 kg / m versus 25 - 26 kg / m ) , which is a known risk factor for knee oa . the data on the correlation between physical activity level / exposure and knee oa would suggest that it was likely to be the heavier physical activity in wuchuan osteoarthritis study that counteracted the bmi gap . but it is hard to confirm this hypothesis by internal comparison , as most of participants had a high physical activity level . however , although the comparison between these two cohorts was unavailable , this result reflected the effect of physical workload on the risk of knee oa . measured knee alignment of participants without knee oa from the beijing osteoarthritis study and the framingham osteoarthritis study , respectively . they found that this there was a more valgus alignment in chinese cohorts , and that possibly explains why the chinese had a higher lateral compartment oa with lower prevalence of obesity compared with caucasian communities . using the similar methods as the beijing osteoarthritis study and the framingham osteoarthritis study , the wuchuan osteoarthritis study showed a similar prevalence of lateral compartment disease with the beijing osteoarthritis study , and it was two to three times higher in both chinese cohorts compared with estimates from the framingham oa study . we measured the femoral tibial angle ( fta ) of subjects in our study , and calculated the mean fta ( sd ) of knees ( n = 1737 ) without radiographic oa . the knee alignment ( fta ( sd ) : 4.9 ( 3.1 ) ) of wuchuan cohorts without oa was a little more valgus than the beijing cohorts ( anatomic axis angle : 4.32 ) , and more obviously valgus than the framingham cohorts ( anatomic axis angle : 2.59 ) . the more valgus alignment of the knee in the chinese would serve to shift the mechanical loading towards the lateral compartment , and provide a possible explanation why the chinese have a higher prevalence of lateral compartment oa . most of the participants were living on farming , and more than 90 percent persons had heavy physical work on the job . this made it hard to highlight the effects of physical activity level and exposure on the knee oa risk as the lack of lower physical activity population . another is that we just investigated the rough hours of those physical activity exposure ( e.g. , digging < 2 hours / day or 2 hours / day ) instead of the exact number of hours , thus it is hard to perform correlation analysis of the activity and knee oa , which is better to reflect the relationship of physical activity exposure and knee oa . we also have to mention that the average age of the participants in the study was only 55 to 57 years old , which might have a lot to do with the relatively low overall prevalence rate of knee oa . in addition , our knee alignment ( fta ) measures were taken manually with goniometer with 1 degree precision instead of measuring on full - length radiographs . the values of fta and anatomic axis angle would be similar theoretically , but this similarity needs to be further verified and there might be an error of manual measurement with goniometer . further this is a cross - sectional analysis and the relations between potential risk factors and knee oa need to be further examined in longitudinal samples . we found that aging , female gender , obesity , and knee injury had a strong association with radiographic tibiofemofal knee oa risk , and similar association existed for medial or lateral compartment knee heavier physical activity increased the risk of radiographic tibiofemofal knee oa , lateral compartment knee oa , and medial compartment knee oa . physical activity exposure such as cycling , kneeling , lifting , and digging had a light to moderate effect on the elevation of knee oa risk . although this effect was not that obvious via internal comparison , it was likely to be the heavier physical activity in wuchuan osteoarthritis study that counteracted the bmi gap compared with the beijing and the framingham oa studies . we verified that the chinese had a more valgus alignment of the knee compared with caucasian population , and this provides a possible explanation why chinese have a higher prevalence of lateral compartment oa . future work in this area should focus on the longitudinal study of these potential risk factors and their relationship to the development of oa .

objective . to investigate the risk factors of radiographic tibiafemoral knee osteoarthritis ( oa ) . methods . a population - based cross - sectional survey was conducted in wuchuan county . a questionnaire and bilateral weight - bearing posterior - anterior semi - flexed knee radiographs were completed and read for kellgren and lawrence ( k / l ) grade and joint space narrowing ( jsn ; 03 scale ) in each compartment . an logistic regression analysis was performed for radiographic tibiafemoral , lateral compartment , and medial compartment knee oa , respectively . adjusted odds ratios ( or ) and 95% confidence intervals ( ci ) were calculated . results . age , sex , and knee injury were strongly associated with tibiafemoral , lateral and medial compartment knee oa . bmi also had a dose - response relationship with them . physical activity level , and physical activity exposure at work , not significantly though , were associated with an elevated risk for this three kinds of knee oa . conclusions . physical activity exposure increased the risk of knee oa . it was likely to be the heavier physical activity in wuchuan osteoarthritis study that counteracted the bmi gap compared with the beijing and the framingham oa study . we verified that chinese had a more valgus alignment of the knee compared with caucasian population , and this provide a possible explanation why chinese have a higher prevalence of lateral compartment oa .

to test our hypothesis , we studied the invasion dynamics of the wns fungus by sampling bats of 5 species at 2 hibernacula in central illinois , usa . we collected samples twice each winter for 2 years ( 201213 and 201314 ) . the hibernacula were moderately sized ( 510 hectares , 25 m high ) abandoned limestone mines that bats use for fall mating and hibernation from september through april . during each visit , we counted all visible bats at each site , which produced complete census data for 4 of the 5 species . accurately collecting census data for bats of the remaining species ( eptesicus fuscus ) was difficult because these bats , unlike those of other species , roosted primarily behind crumbling slabs of rock around mine entrances , which were dangerous and difficult to survey . during each site visit we sampled 1520 bats of each species by epidermal swabbing ( 7 ) . we also sampled the wall or ceiling of hibernacula under , near ( 1020 cm ) , and far from ( > 2 m ) roosting bats by using the same swabbing technique . samples were tested for p. destructans by using real - time pcr ( 9 ) ; according to a serial dilution experiment , the limit of detection was 50 conidia . we obtained 611 samples from bats and 444 from substrate . in early winter of 201213 , only 1 individual ( myotis septentrionalis ) of 129 bats of 5 species sampled was positive for p. destructans , and none of the 46 substrate samples were positive ( figure 1 , panels a , c , e ) . just 4 months later , in march 2013 , prevalence was > 85% for bats of 2 species ( m. septentrionalis , m. lucifugus ) , 40%75% for 2 species ( e. fuscus , myotis sodalis ) , and 15%60% for 1 species ( perimyotis subflavus ) at the 2 sites ( figure 1 , panel a ) . the prevalence of p. destructans on the substrate under these bats varied from 0% to 67% , and substrate prevalence paralleled fungal prevalence for bats of each species ( figure 1 , panel c ) . despite widespread apparent infection of bats at this time , none of the 36 substrate samples taken just 1020 cm from bats were positive for p. destructans ( figure 1 , panel e ) . pseudogymnoascus destructans prevalence ( 1 se , calculated from the variance of a binomial distribution sample ) over 2 winters , 201213 and 201314 , at 2 sites ( diamonds and triangles ) in illinois , usa , on bats of 5 species ( a , b ) ; prevalence of p. destructans on substrate under bats of each species ( c , d ) , and prevalence of p. destructans under , near ( 1020 cm ) , and far from ( > 2 m ) bats ( e , f ) . lines join observed mean prevalence for each species ( solid circles ) to facilitate presentation but do not indicate trajectories between time points . prevalence of species or substrate means indicated by the same letter did not differ significantly ( p>0.05 ) in a logistic regression analysis with either species and site as fixed effects at each sampling point ( a , b ) or substrate sample type at each sampling point ( c f ) ; effect of site was not significant in any of these comparisons . in early winter of the next year ( late november 2013 ) , patterns differed markedly from those of the previous early winter . p. destructans was already widespread in the environment , found in 70% of samples from under bats , 22% of samples 1020 cm from bats , and 14% of samples > 2 m from bats ( figure 1 , panels d , f ) . prevalence among bats of 4 species was already > 70% , and prevalence among bats of 1 of these species ( p. subflavus ) , for which prevalence at the end of the previous winter had been lowest , was already 85%100% ( figure 1 , panel b ) . by the end of the second winter , 109 ( 98% ) of 111 bats were positive for p. destructans , and p. destructans was present throughout the hibernacula ( in 91% of samples from under bats , 66% of samples near bats , and 44% of samples far from bats ) ( figure 1 , panels b , d , f ) . over these 2 years , the effect of wns on bat populations mirrored the patterns of p. destructans prevalence . during the first winter , declines were limited at the larger site and moderate ( 50%75% ) at the smaller site ( figure 2 ) . in contrast , over the second winter , counts of m. septentrionalis bats declined by 95%99% and m. lucifugus bats by 81%88% ( 20,000 bats of this species disappeared ) ( figure 2 , panel a ) . populations of bats of the 2 other species also experienced moderate to severe declines in the second year ( m. sodalis , 16%96% ; p. subflavus , 47%73% ) ( figure 2 , panel b ) . declines probably resulted from disease - related deaths because high hibernacula site fidelity makes emigration unlikely ( 10 ) and substantial numbers of dead bats were observed at both sites . complete population counts on a log scale of 4 species of bats at 2 sites in illinois , usa , over 2 winters , 201213 and 201314 . early in the first winter studied , prevalence of p. destructans was very low , and although transmission resulted in most bats harboring p. destructans by winters end , declines in bat populations were limited . in contrast , early in the second winter , fungal prevalence among bats was already high and severe communitywide declines occurred over the next 4 months . the earlier timing of exposure in the second year would be expected to increase the effects of wns because by winter s end most bats would have been infected and in hibernation for at least 70100 days ( the approximate time between infection and death ; 5 ) . few would be able to survive until spring , when bats cease hibernating and clear the fungus ( 7 ) . patterns of p. destructans distribution in the environment mirrored prevalence among bats and population declines . early in the first year , when p. destructans was rare on hibernacula substrates , most bats were not infected in early winter , and 4 months later , p. destructans was not detectable in one third of bats of 3 species . however , by the end of the first winter , p. destructans was present on hibernacula substrate under bats , probably resulting from bats shedding p. destructans into the environment . at the beginning of the following winter , p. destructans was widespread in the environment , and nearly all bats had fungus on them . the widespread occurrence of p. destructans in the environment at this time may have contributed to higher prevalence among bats because most bats clear infections during the summer , when their body temperature is too high for p. destructans growth ( 7,11 ) . long - term persistence of p. destructans in the absence of bats ( 8,12 ) suggests that an environmental reservoir of p. destructans may contribute to wns persistence , as occurs for other diseases , such as cholera ( 13 ) . wns continues to spread south , west , and north from new york , where it was first detected in 2006 , and continues to cause widespread bat population declines . potential control strategies include development of probiotic treatments ( 14 ) and alteration of hibernacula microclimates to make them cooler and drier ( 3,15 ) . our results suggest that if p. destructans invasion in other sites is similar to what we documented in illinois , interventions must be implemented proactively , or quickly after p. destructans invasion , to prevent collapse of bat communities . reduced bat populations will probably have a negative effect on humans because bats play a useful role in ecosystems by consuming disease vectors and many forest and agricultural insect pests .

white - nose syndrome has devastated bat populations in eastern north america . in midwestern united states , prevalence increased quickly in the first year of invasion ( 201213 ) but with low population declines . in the second year ( 201314 ) , environmental contamination led to earlier infection and high population declines . interventions must be implemented before or soon after fungal invasion to prevent population collapse .

intestinal atresia is commonly seen as one of the four types described in any of the currently existing pediatric surgery specialty literature . none of the classifications in vogue today , include long gap bowel atresias like a near - total / total jejuno - ileal atresia . though rare , long gap bowel atresias are difficult to treat . however , existence of a certain minimum length of small bowel is the most crucial factor affecting the outcome of repair , overall salvage of the infant and long - term prognosis of such cases . a female neonate with birth weight of 1.7 kg , born to a healthy adolescent primigravida by preterm vaginal delivery at 34 weeks , was referred to us on d2 of life because of non - passage of meconium . erect x - ray of the abdomen showed two dilated loops in the upper abdomen ; with paucity of gas in the pelvis [ figure 1 ] . erect x - ray of the abdomen showing two dilated loops in the upper abdomen ; with the paucity of gas in the pelvis on exploratory laparotomy , there was evidence of high jejunal atresia . entire small bowel , 2 - 3 cm beyond the duodeno - jejunal ( dj ) flexure was atretic . the duodenum was massively dilated and tortuous and had a length of approximately 7 - 8 cm [ figure 2 ] . the jejunum was blind ending and had a length of about 2 - 3 cm . distally , only 3 - 4 cm of ileum was present proximal to the ic junction [ figure 3].the colon was unused but , could be distended by saline infilling . operative photograph showing massively dilated , tortuous duodenum and atretic jejuno - ileum schematic representation of the atresia to preserve the small bowel length as much as possible , single layer extramucosal end - to - back anastomosis was performed between the dilated duodenum and spatulated end of ileum ( duodeno - ileal anastomosis ) using 5 - 0 polyglactin . enterotomy and web excision was done for type - i atresia at the dj flexure . dye study carried out on the post - operative day 20 showed free flow of contrast across the anastomosis , though it was albeit slow . the baby received total parenteral nutrition ( tpn ) for about 3 weeks . the tpn was gradually tapered , when formula feeds were well - tolerated to meet the child 's daily requirement . however , the family could not afford special elemental diet for long and the baby had to be put on expressed breastmilk ( ebm ) feeds . the ebm feeds were progressively increased upto 140 ml / day , when she was discharged . however , the baby had very little weight gain and eventually succumbed to intractable sepsis at 14 weeks of life . the currently existing classifications in standard international literature viz . the grosfeld modification of louw classification ( 1979 ) describe four types of intestinal atresias : type i or mucosal web , type ii or fibrous cord , type iiia or mesenteric gap defect , type iiib or applepeel atresia and type iv or multiple atresias . intestinal atresias were initially thought to be produced by a failure of recanalization of the solid cord stage of the bowel ( tandler1900 ) . later work confirmed the theory of spiggs that the origin of intestinal atresias is mechanical accidents , including vascular occlusions . this theory was confirmed further by louw and barnard ( 1955 ) who obtained intestinal atresias by inducing mesenteric vascular accidents in puppies . they confirmed that intrauterine clinical instances of intestinal volvulus , intussusception , internal hernia or even constriction of the intestines in a tight gastroschisis could lead to intestinal atresia . none of the studies published so far mentioned the possibility of getting a near - total / total jejuno - ileal atresia . however , it can be postulated that an insult of the jejunal and ileal branches of the superior mesenteric artery or a very large intrauterine midgut volvulus and resultant resorption of the entire small bowel during early intrauterine life could be the possible reasons for such extensive atresia . the standard treatment for commonly occurring jejuno - ileal atresias is exploratory laparotomy upon initial hemodynamic stabilization . the grossly dilated aperistaltic terminal portion of the proximal bowel is excised and anastomosed end - to - back to spatulated distal atretic segment . recovery of bowel function depends upon the degree of dilatation of the proximal bowel , residual length of the small bowel and presence / absence of the ic valve . the outcome of neonates born with jejuno - ileal atresias has improved considerably in recent years due to many new acquisitions in neonatal intensive care and anesthesia , operative procedures and use of tpn . have reported use of gastroduodenal intraluminal silicone tube stents across five successive anastomoses in a case of apple peel atresia with ultimate salvage of the baby . the bianchi technique or the serial transverse enteroplasty procedure all these procedures started from some available small gut , but for a near - total jejuno - ileal atresia , as the one described in this case , the only logical and life - saving option would be an intestinal transplantation , a method available in only very few institutions in the world . though the results of intestinal transplantation in 6 children with intestinal atresia as reported by nishida et al . have been encouraging , for lack of highly advanced neonatal health - care infrastructure , it is still not possible in many centers including ours. prognosis of long gap bowel atresias is universally dismal . have applied the waterston classification to identify three risk groups in long gap bowel atresias . they have reported 0% survival rate for group c with high jejunal atresia ( within 15 cm of dj flexure ) . wilmore 's review of 50 infants with significant small bowel resection suggests that though most infants with > 35 cm of residual intestine and intact ic valve survive , survival drops to 50% with bowel length of 15 - 25 cm and decreases further to 0% in infants with < 15 cm residual small bowel even with intact ic valve . child in the current report being < 1.8 kg , belongs to group c of the waterston classification . furthermore , the total small bowel length in this neonate from pylorus to ic junction ( including the duodenum ) was < 10 cm ; which is incompatible with survival with traditional methods of treatment . this case is reported for it 's extreme rarity , dismal prognosis and the management challenge it presents even today . we strongly propose an addition of near - total jejuno - ileal atresia , as an offshoot to the presently existing classifications of small bowel atresia . by collecting the database of this extremely rare form of jejuno - ileal atresia , in near future we might be able to formulate management guidelines to salvage these neonates for whom we can offer no definitive solution today .

a 2-day - old female neonate with the clinical picture of proximal small bowel atresia , on exploration , turned out to have intestinal atresia of a rare variety , i.e. , a near - total jejuno - ileal atresia . the baby had total small bowel length of less than 10 cm . she survived for 3 months on enteral feeding after end - to - back duodeno - ileal anastomosis and thereafter succumbed to septicemia . the case is presented for it 's extreme rarity and consideration of this extreme form of small bowel atresia as an offshoot of the existing classifications of jejuno - ileal atresia since it has dismal prognosis and presents as a management challenge even today .

the rainbow trial , reported in 2014 , was a randomized , placebo - controlled , double - blind , phase 3 trial comparing paclitaxel plus ramucirumab with paclitaxel plus placebo in patients with previously treated advanced gastric or gastroesophageal junction adenocarcinoma . this study indicated the combination of ramucirumab and paclitaxel could serve as a standard second - line treatment for patients with unresectable advanced gastric cancer with an acceptable frequency and severity of adverse events . however , the reported incidence of gastrointestinal perforation in the ramucirumab plus paclitaxel group was higher than in the placebo plus paclitaxel group . here we report a case of intra - abdominal abscess following gastric perforation during the second cycle of chemotherapy with ramucirumab plus paclitaxel for advanced gastric cancer treated with endoscopic ultrasound ( eus)-guided drainage . a 67-year - old man was referred to our hospital for treatment of a gastric tumor detected by esophagogastroduodenoscopy ( egd ) . he reported abdominal discomfort , occasional vomiting , and weight loss of 10 kg during the 4 months preceding his presentation . following admission to our hospital , we performed an egd that showed copious gastric food debris and a tumor involving the entire stomach ( fig 1 ) . because definite stenosis caused by the tumor was absent , the accumulation of gastric debris appeared to result from reduced peristalsis due to tumor extension throughout the stomach . the histological diagnosis was human epidermal growth factor receptor 2 ( her2)-negative well - differentiated gastric adenocarcinoma . abdominal computed tomography ( ct ) showed cancer invasion of the pancreas and metastatic lymph nodes near the aorta . the patient 's performance status was sufficiently good , and chemotherapy was indicated . in japan , for patients with her2-negative gastric cancer , s-1 plus cisplatin combination chemotherapy is the standard of care . however , our patient sometimes vomited due to delayed gastric emptying ( dge ) , and oral treatment was difficult . after the chemotherapy was started , the patient 's oral intake gradually increased , and he ceased vomiting . he was discharged in satisfactory condition after finishing the first cycle of chemotherapy . the patient continued chemotherapy as an outpatient but developed sudden severe upper abdominal pain on the seventh day of the second cycle of the chemotherapy . a ct examination showed perforation at the lesser curvature of the gastric body ( fig 2a ) , and he was readmitted to our hospital . because he had been taking a proton pump inhibitor , we did not think that the perforation was caused by acid - induced ulceration , and we attributed the perforation to ramucirumab . the patient 's condition improved with conservative treatment . on the 15th hospital day , his ct scan showed improvement of the gastric perforation , and we removed the nasogastric tube . he started to take a liquid diet but developed a fever ( 39.0c ) on the 24th hospital day . his laboratory data showed an increased white blood cell count and elevated c - reactive protein level . a ct examination on the 27th hospital day showed enlargement of the fluid collection outside the stomach ( fig 2b ) . we considered an intra - abdominal abscess following the gastric perforation and performed eus - guided fine - needle aspiration of the fluid collection using a 19-gauge needle ( sonotip pro control ; medi - globe gmbh , achenmhle , germany ) . because malodorous yellowish - white pus was aspirated , we diagnosed the lesion as an intra - abdominal abscess and conducted eus - guided drainage ( fig 3a ) . the procedure was performed using a convex - type echoendoscope ( gf - uct260 ; olympus medical systems , tokyo , japan ) . a 0.025-inch guidewire ( visiglide , olympus medical systems ) the fistula was dilated using a dilation catheter ( es dilator ; zeon medical , tokyo , japan ) , and a 6-fr pigtail nasal biliary catheter ( nasal biliary drainage set ; cook medical , bloomington , in , usa ) was subsequently placed ( fig 3b ) . gram staining and culturing of an aspirate sample confirmed anaerobic bacteria ( prevotella melaninogenica and peptostreptococcus micros ) . meropenem ( 3 g / day ) was administered because the antibiotic sensitivity test showed that all the causative bacteria were sensitive to meropenem and it had the lowest minimum inhibitory concentration . the external drainage catheter placed via the nose was irrigated once daily with 1020 ml of sterile saline . the clinical course after drainage was uneventful and the external drainage catheter was removed on the 34th hospital day . the patient had not vomited since he started oral intake on the 36th hospital day , and we observed that his dge was improved by the first cycle of the chemotherapy with ramucirumab plus paclitaxel . therefore , we started chemotherapy with s-1 plus cisplatin on the 41st hospital day , and the patient was discharged after the first cycle of this chemotherapy was finished . japanese gastric cancer treatment guidelines suggest that s-1 plus cisplatin combination chemotherapy is a standard first - line treatment for patients with her2-negative unresectable gastric cancer . the combination of ramucirumab and paclitaxel is regarded as a standard second - line treatment . because our patient had dge and tolerated oral medications poorly , we started intravenous chemotherapy with ramucirumab plus paclitaxel . the patient 's symptom of dge - induced vomiting improved during the first cycle of chemotherapy , and the treatment was effective . however , gastric perforation occurred during the second cycle of chemotherapy and , as the patient was taking a proton pump inhibitor , we believe this perforation was caused by ramucirumab . gastrointestinal perforation is a serious adverse event reported in approximately 1% of patients treated with ramucirumab plus paclitaxel . although the mechanism by which ramucirumab causes gastrointestinal perforation is not completely understood , this adverse event is associated with most antiangiogenic treatments . in our case , the ct scan revealed that the perforation occurred in the portion of the stomach affected by the tumor rather than in normal tissue . due to our concern that a perforation through cancerous tissue would not close naturally , we considered surgical treatment . however , antiangiogenic medications may cause a failure to heal , and gastrectomy was impossible because the tumor had invaded the pancreas . these considerations led us to avoid surgery . fortunately , the perforation closed with conservative treatment and the patient 's condition improved although the fluid collection outside the stomach developed an abscess . eus - guided drainage has been reported to be a safe and effective method not only for patients with a peripancreatic fluid collection but also for those with an abdominal abscess [ 3 , 4 ] . we performed eus - guided transgastric drainage for this patient with advanced malignancy because eus - guided drainage is less invasive than surgical drainage . in conclusion , we present a case of intra - abdominal abscess following gastric perforation during the second cycle of chemotherapy with ramucirumab plus paclitaxel . eus - guided drainage improved the patient 's condition allowing s-1 plus cisplatin combination chemotherapy to be started . eus - guided drainage is a treatment option for patients with intra - abdominal abscess following gastric perforation due to ramucirumab . all procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation ( institutional and national ) and with the helsinki declaration of 1964 and later versions . this article does not contain any studies with human or animal subjects performed by any of the authors .

gastrointestinal perforation is a serious adverse event that occurs in approximately 1% of patients receiving ramucirumab and paclitaxel . a 67-year - old man with unresectable advanced gastric cancer was admitted to our hospital and treated with ramucirumab and paclitaxel . gastric perforation occurred during the second cycle of chemotherapy . although the patient 's condition improved without surgery , an abscess developed in the intra - abdominal fluid collection resulting from the perforation . we performed endoscopic ultrasound - guided abscess drainage . the patient improved and was discharged in satisfactory condition . endoscopic ultrasound - guided drainage is a treatment option for patients with intra - abdominal abscess following gastric perforation due to ramucirumab .

peripheral arterial disease ( pad ) is a severe manifestation of atherosclerosis that may be asymptomatic or lead to critical ischemia of the lower limbs , and is also associated with increased risk of death from cardiovascular disease.1 some known risk factors for pad include smoking , diabetes mellitus , familial history of atherosclerotic event , hypertension , and hypercholesterolemia.2 the diagonal lobular crease ( dlc ) , described by frank,3 and anterior tragal crease ( atc ) , by miot et al.4 ( figure 1 ) , were identified as factors independently associated with coronary artery disease,5 - 7 but they have not yet been studied in patients with pad . detection of external signs associated with pad can assist in risk stratification and identification of patients who would benefit from early intervention or modification of risk factors related to disease progression . we studied the prevalence of ear creases in patients with pad of the lower limbs , in comparison with patients without documented atherosclerotic disease . a crosssectional study consisted of interviews with adult male patients treated at the university hospital . cases were selected from claudication outpatients , with pad of the lower limbs confirmed by arteriography . the control group was matched for gender and age and was recruited among dermatologic outpatients , who had not been revascularized ( legs or ankle ) or who had an anklebrachial index not < 0.9 or > 1.3 ( arterial noncompliance ) . patients who presented any of the following conditions were excluded from the study : immunosuppression , ear deformity , earring use or arterial obstruction due to other etiologies that could not be attributed to pad . the main dependent variable was the presence of pad in the lower limbs , and the main independent variable was the presence of bilateral auricular creases . other covariates were diabetes mellitus , previous smoking , age , familial and personal past history of atherosclerotic vascular disease ( cerebral or myocardial atherosclerotic event ) , dyslipidemia , body mass index , and hypertension . patients who did not know their family history were considered to have a negative history . categorical variables are given as frequencies and bivariately compared by the test or fisher 's exact test . continuous variables are represented by mean and standard deviation and compared by the student ttest . the association between variables is expressed as an odds ratio from the bivariate analysis subsequently adjusted by conditional logistic regression , comprising covariates with p<0.2 . sample size was defined according to the final multiple logistic model with = 0.05 and power = 85%.8 this study was approved by the institutional ethics committee . the main clinical and demographic characteristics of patients and controls are listed in table 1 . the bivariate analysis disclosed the higher frequencies of known pad risk factors among patients and also identified their positive association with ear creases . dlc and atc were also more prevalent among cases than controls in all age groups ( data not shown ) . multivariate analysis of the frequency of ear creases adjusted for age , diabetes mellitus , hypertension , myocardial infarction , dyslipidemia , and tobacco reinforced the association between pad and ear creases ( table 2 ) . these creases may represent an external sign of microangiopathic lesions in terminal circulation of the ear that occur at the same time as pad since atherosclerosis is a systemic disease . in other series of patients , overall mortality and sudden death were significantly higher in the group with dlc.9,10 male patients were selected to minimize the effect that earrings might have had on the ears , and because men are more affected by pad and atherosclerosis than women . as the prevalence of both pad and bilateral ear lobe creases increases over time , age matching was performed to normalize these risks between the groups . the anklebrachial index was chosen as the method to select controls owing to its high specificity ( 99% ) and sensitivity ( 89% ) for identification of pad.11 moreover , the accidental inclusion of cases among controls would have diluted the association of ear creases and atherosclerosis , rather than strengthened it . the noninvasive identification of external findings , including dlc and atc , digital clubbing , cyanosis , and hair loss on extremities provides information additional to epidemiological aspects such as smoking , hypertension , diabetes and dyslipidemia in raising a suspicion of atherosclerosis and risk of cardiovascular events.12 further investigations should be performed with larger samples , heterogeneously distributed according to age , gender , ethnicity , and pad severity , to provide a stratified risk analysis and a longterm follow up evaluation of prognostic factors in pad . bilateral dlc and atc are semiological findings which are quick and easy to evaluate and were independently associated with pad in this population .

introduction : peripheral arterial disease is a severe manifestation of atherosclerosis that can lead to critical ischemia of the lower limbs and is also associated with high cardiovascular risk . diagonal lobular and anterior tragal ear creases have been associated with coronary artery disease , but they have not yet been investigated in patients with peripheral arterial disease.objectives:to evaluate the prevalence of ear creases among patients with peripheral arterial disease of the lower limbs , compared with patients without documented atherosclerotic disease.methods:crosssectional study including 60 male patients with peripheral arterial disease of the lower limbs and 60 dermatologic outpatients matched for age and gender . the associations were adjusted for other risk factors by conditional logistic regression.results:the prevalence of diagonal and anterior tragal ear creases was higher among cases ( 73% vs. 25% and 80% vs. 43% , respectively ) than controls ; these associations remained significant even when adjusting for other known risk factors of atherosclerosis ( odds ratio = 8.1 and 4.1 , respectively).conclusions : ear creases are independently associated with peripheral arterial disease and may be an external marker for risk identification .

in modern cellular systems , rnas frequently associate with proteins to form ribonucleoprotein ( rnp ) complexes [ 15 ] . in these rnp complexes , biological functions ascribed to the main component ( rna or protein ) are regulated by the accessory component ( protein or rna ) . during molecular evolution , both main and accessory components often evolved from single- to multi - molecular systems to elaborate and diversify their functions . a typical example can be seen in ribonuclease p ( rnasep ) , which plays a pivotal role in trna processing [ 68 ] . most rnasep enzymes act as ribonucleoproteins ( rnps ) , which have been proposed to have evolved from a catalytic rna . while prokaryotic rnasep is composed of a single rna subunit and single protein subunit , eukaryotic rnasep has 9 - 10 protein subunits that decorate one rna subunit [ 68 ] . a different type of multicomponent system is seen in the evolutionary fragmentation of rna components in caulobacter and euglena , in which the tmrna and large ribosomal rna are reconstituted by assembling 2 and 14 fragments , respectively . for example , it is well known that some protein subunits of eukaryotic rnase p are shared with rnase mrp , which processes non - trna substrates [ 11 , 12 ] . more recently , the core proteins of telomerase rnp complex were reported to play novel roles by associating with nontelomerase rna [ 13 , 14 ] . these features of multicomponent rnp complexes are not only advantageous in natural molecular evolution but also attractive from the viewpoint of artificial design of functional biomacromolecular systems . previously , we reported self - folding rna ( figure 1(a ) , left ) and self - assembling rna ( figure 1(a ) , middle ) as platforms to design rna templates for assisting in chemical peptide ligation [ 1517 ] . previous molecular design for template rnas had limitations due to the use of the bacteriophage boxb motifs as a dual module for rna - rna interaction in the template rnas and also recognition of the substrate n - peptides ( prm1 in figure 1(a ) , left and middle ) . , we designed a new template system based on a two - piece derivative of the tetrahymena group i intron rna ( figure 1(a ) , right ) . the tetrahymena group i intron rna is a unimolecular rna with modular architecture that exhibits self - splicing activity . its structural modules can be classified into conserved core modules responsible for catalysis and nonconserved peripheral modules . peripheral modules , which are not directly involved in the mechanism of catalysis , stabilize the conserved core by organizing a tertiary interaction network that wraps and fixes the core modules ( figures 1(b ) and 1(c ) ) [ 1922 ] . the p5abc domain is a self - folding peripheral element playing crucial roles in stabilization of the core modules . these interactions are so strong that the function of p5abc is preserved in the two - piece format in which the p5abc domain rna ( p5 rna ) and the rest of the intron ( p5 rna ) are physically separated ( figures 1(b ) , 1(c ) , and 2(a ) ) . the resulting two rnas stably form a p5 : p5 complex ( figures 1(b ) , 1(c ) , and 2(a ) ) that is capable of conducting in vitro ribozyme functions . we employed this two - piece ( p5 : p5 ) complex as a platform to arrange two peptide recognition modules ( figure 1(a ) , right ) [ 24 , 25 ] . although the system designed in this study is still primitive , the rna design based on the p5 : p5 platform is potentially attractive for development of versatile rna - based template for chemical peptide ligation . molecular models were constructed from the coordinates of the crystal structure of tetrahymena group i intron ( protein date bank i d 1x8w ) and the nmr structures of the complex of n - peptide and boxb rna ( 1qfq ) , the complex of rev - peptide and rre rna ( 1etf ) , and the complex of p22n - peptide and p22 boxb rna ( 1a4 t ) . molecular modeling was performed by using discovery studio ( accelrys software inc . ) and the pymol molecular graphics system ( schrdinger , llc ) . protected fmoc aminoacids , n , n - diisopropylethylamine ( diea ) , hbtu , hobt , piperidine , ho - ch2-pam resin , and fmoc - ala - alko - peg resin were purchased from watanabe chemical ( hiroshima , japan ) . other reagents and solvents were purchased from nacalai tesque ( kyoto , japan ) , tci ( tokyo , japan ) , kishida chemical ( osaka , japan ) , or kanto chemical ( tokyo , japan ) . synthesis ( 0.1 mmol scale ) was manually carried out using ho - ch2-pam resin ( 1.0 mmol / g ) . fmoc - gly - oh ( 1.2 mmol ) and hobt ( 1.2 mmol ) were dissolved in dimethylacetamide ( dma ) ( 4 ml ) . then n , n-dicyclohexylcarbodiimide ( dcc ) ( 1.2 mmol ) was added to the solution with cooling on the ice for 10 min . to the resulting solution , ho - ch2-pam resin ( 0.3 mmol ) the resin was then washed with dmf ( 3 ml ) and ch2cl2/ch3ch2oh ( 1/1 ) ( 3 ml ) three times , respectively . the resin was further washed with ch2cl2 ( 3 ml ) and dmf ( 3 ml ) three times , respectively . the resin was added to benzoic anhydride ( 1.5 mmol ) dissolved in pyridine / dmf ( 3/1 ) ( 3 ml ) and agitated for 1 hour . finally , the resin was washed with dmf ( 3 ml ) and ch2cl2 ( 3 ml ) four times . after the introduction of gly , the second aminoacid was introduced by using hobt ( 3 equiv . ) , hbtu ( 3 equiv . ) , and diea ( 6 equiv . ) . repetitive removal of the fmoc groups followed by introduction of protected fmoc aminoacids was performed according to the standard procedure to give the protected peptide resin . protected peptide resin ( 0.033 mmol ) in 1.0 ml of dry ch2cl2 was stirred for 15 min under argon in a 20 ml flask . in a second flask , 0.67 ml of ( ch3)3al ( 2 m hexane solution , 0.67 mmol ) diluted with 2.33 ml of dry ch2cl2 was cooled to 0c under argon [ 26 , 27 ] . to this solution , 0.15 ml of ethanethiol ( 124 mg , 2.0 mmol ) was added dropwise , and the resulting mixture was allowed to stir for 15 min at 0c . this solution was added at once to the suspension of peptide resin in ch2cl2 . after stirring for 5 hr at room temperature , protected peptide thioester was treated with tfa ( 2.7 ml ) in the presence of m - cresol ( 60 l ) , thioanisole ( 60 l ) , ethanedithiol ( 60 l ) , phenol ( 60 g ) , and water ( 60 l ) for 2 hr at room temperature . the resulting crude peptide was repeatedly purified by hplc with cosmosil 5c18-ar - ii ( 20 25 mm and 4.6 25 mm ) under gradient conditions for ch3cn in 0.1% aqueous tfa . concentrations of peptide were determined and adjusted by using absorption of amide bonds at 220 nm as follows : h2n - mdaqtrrrerraekqaqwkaaaaggg - cosch2ch3 ( n - peptide1 ) ; maldi - tof - ms : m / z = 2944.282 [ m ] ( calcd . mw = 2944.38 ) ; h2n - aaaatrqarrnrrrrwrerqrg - cosch2ch3 ( rev - peptide 1 ) ; maldi - tof - ms : m / z = 2824.679 [ m ] ( calcd . mw = 2825.29 ) ; h2n - naktrrherrrklaierdtaaggg - cosch2ch3 ( p22n - peptide 1 ) ; maldi - tof - ms : m / z = 2766.51 ( calcd . h2n - mdaqtrrrerraekqaqwkaaaaggg - cosch2ch3 ( n - peptide1 ) ; maldi - tof - ms : m / z = 2944.282 [ m ] ( calcd . mw = 2944.38 ) ; h2n - aaaatrqarrnrrrrwrerqrg - cosch2ch3 ( rev - peptide 1 ) ; maldi - tof - ms : m / z = 2824.679 [ m ] ( calcd . mw = 2825.29 ) ; h2n - naktrrherrrklaierdtaaggg - cosch2ch3 ( p22n - peptide 1 ) ; maldi - tof - ms : m / z = 2766.51 ( calcd . mw = 2764.17 ) . solid - phase peptide synthesis ( 0.1 mmol scale ) was manually carried out using fmoc - ala - alko - peg resin ( 0.25 mmol / g ) . the resin was treated with 20% piperidine in dmf to remove fmoc group at the amino - terminus . then , protected fmoc aminoacid was introduced to the resin by using with hobt ( 3 equiv . ) , hbtu ( 3 equiv . ) , and diea ( 6 equiv . ) . repetitive removal of the fmoc groups followed by introduction of protected fmoc aminoacids was performed according to the standard procedure to give the protected peptide resin . after the deprotection of fmoc group of cysteine , the resin was dried under high vacuum . then , the protected peptide resin was treated with tfa ( 2.6 ml ) in the presence of m - cresol ( 150 l ) , thioanisole ( 150 l ) , and ethanedithiol ( 75 l ) for 1 the resulting crude peptide was purified by hplc with cosmosil 5c18-ar - ii ( 20 25 mm and 4.6 25 mm ) under gradient conditions for ch3cn in 0.1% aqueous tfa . concentrations of peptide were determined and adjusted by using absorption of amide bonds at 220 nm as follows : h2n - cggaaamdaqtrrrerraekqaqwka - cooh ( n - peptide2 ) ; maldi - tof - ms : m / z = 2949.034 [ m ] ( calcd . mw = 2950.38 ) ; h2n - cgaaatrqarrnrrrrwrerqraaaa - cooh ( rev - peptide2 ) ; maldi - tof - ms : m / z = 3096.007 [ m ] ( calcd . mw = 3095.54 ) . h2n - cggaaamdaqtrrrerraekqaqwka - cooh ( n - peptide2 ) ; maldi - tof - ms : m / z = 2949.034 [ m ] ( calcd . mw = 2950.38 ) ; h2n - cgaaatrqarrnrrrrwrerqraaaa - cooh ( rev - peptide2 ) ; maldi - tof - ms : m / z = 3096.007 [ m ] ( calcd . plasmid encoding the tetrahymena group i intron ( ptz - ivsu ) was used as a template for pcr amplification of dna fragments for transcription of the p5 , ez-5half , ez-3half , and tet l-30 rna . plasmid encoding the p5abc mutant intron ( pl21-p5abc ) was used as a template for pcr amplification of a dna fragment for transcription of the p5 rna . peptide recognition modules ( prm-1 and -2 ) were attached to the template dnas for the p5- and ez-5half rna , respectively by pcr with primers containing the sequence of prm . transcription reactions with t7 rna polymerase were performed according to the published protocol , and transcripts were purified on 6% denaturing polyacrylamide gels . the concentrations of rnas were determined from the intensities of uv absorption at 260 nm . chemical ligation of peptides in the presence of rna molecules was carried out as follows . the p5 rna , ez-5half rna , and ez-3half rna were separately denatured in water for 2.5 min at 80c and then mixed together . to this rna solution , concentrated reaction buffer ( final compositions of the reaction mixture were 40 mm hepes , ph 8.0 , 80 mm kcl , and 10 mm mgcl2 ) was added , and the mixture was warmed for 5 min at 37c . reaction was started by adding aqueous solution of substrate peptides ( 7.5 m ) and 2-mercaptoethanesulfonate ( mesna , final concentration was 1% ( w / v ) ) at 37c . samples ( 20 l ) were quenched with 2 l of 40% aqueous tfa , and the reaction mixtures were analyzed by hplc with cosmosil 5c18-ar - ii ( 4.6 25 mm ) . gradient conditions for ch3cn in 0.1% aqueous tfa were as follows : 5%32.5% in 22.5 min ; flow rate : 1 ml / min . the fractions corresponding to the ligated products were collected and their molecular weights were confirmed by maldi - tof - mass spectrometry . the mean values are shown in the figures in which error bars indicate the minimal and maximal values . for samples forming complexes , aqueous solutions of the p5 rna ( 5 pmol , final concentration 0.5 m ) , the 3 ends of which were labeled with bodipy fluorophore , nonlabeled ez-5half and ez-3half rna ( 10 pmol , final concentration 1.0 m ) were heated separately at 80c for 2.5 min . , a 10 concentrated folding buffer was added to adjust the mixture to 50 mm tris - oac ( ph 7.5 ) and 10 mm mg(oac)2 . the resulting mixture was incubated at 37c for 30 min . after adding 6 concentrated loading buffer consisting of glycerol and xc ( 0.1% ) , the samples were loaded onto a 5% nondenaturing polyacrylamide gel ( 29 : 1 acrylamide : bisacrylamide ) containing 50 mm tris - oac ( ph 7.5 ) and 10 mm mg(oac)2 . electrophoresis was carried out at room temperature with 200 v for the initial 5 min and then 75 v for 5 h. the resulting gels were analyzed with a fluoroimager pharos fx ( biorad , hercules , ca , usa ) . to modify the tetrahymena intron rna as a self - assembling template assisting chemical peptide ligation , a three - step redesign was carried out ( figures 1(b ) , 1(c ) , and 2(a ) ) . the first step involves conversion of the parent unimolecular rna ( tet l-30 ) to a two - piece derivative consisting of the p5 rna and the rest of the intron ( p5 rna ) as described in the introduction ( figures 1(b ) , 1(c ) , and 2(a ) ) . the second step involves the introduction of a break in the p5 region of the p5 intron rna ( figures 1(b ) , 1(c ) , and 2(a ) ) . the resulting two fragments ( ez-5half and ez-3half ) the reconstitution of p5 intron rna ( termed ez in this study ) from the two rna fragments is convenient for installation of the peptide - recognition motif ( prm ) . in the template dna for in vitro transcription , the introduction of prm to the ez-5half fragment of the bimolecular version of the p5 rna ( ez rna ) can be achieved simply by modifying the pcr primers . on the other hand , for the introduction of a prm sequence to the corresponding position ( the 92nd nucleotide from the 5 end ) of the unimolecular p5 rna ( 309 nucleotides ) , template dnas need to be prepared using either a reliable but laborious method ( plasmid construction using standard recombination techniques ) or a convenient but less reliable method ( preparation of a double - stranded dna using multistep pcr to assemble a set of dna oligonucleotides ) . gel mobility shift assay was carried out to confirm reconstitution of the tetrahymena group i intron ( tet l-30 ) by the three rnas ( p5 , ez-5half , and ez-3half ) ( figure 3(a ) ) . in the presence of the three rnas , mobility of the main band was similar to those of the parent unimolecular tet l-30 rna and the bimolecular complex ( p5 : p5 ) , indicating that the two - piece ( p5 and ez ) rna consisting of three rna fragments ( p5 , ez-5half , and ez-3half ) can be used as a platform rna ( termed the p5 : ez platform ) for chemical peptide ligation . the third step involves the introduction of two peptide - recognition motifs ( prms ) to the complex ( figures 1(b ) , 1(c ) , and 2(a ) ) . the one prm ( prm1 ) was introduced to the p5 rna and the other prm ( prm2 ) was introduced to the ez-5half fragment of the ez rna . to determine the versatility of the p5 : ez platform in chemical peptide ligation , we prepared six template rna complexes designed for peptide chemical ligations with six combinations of substrate peptides ( figure 2(a ) ) . to confirm that the introduction of prm1 and prm2 does not interfere with the complex formation between p5 and ez , we performed gel mobility shift assay with six template rna complexes , none of which showed significant reduction of the complex formation ( figure 3(b ) ) . since the binding between the p5 rna and the p5 rna was reported to be highly sensitive to misfolding , these results also indicate that neither the folding of p5 and ez nor their complex formation was disturbed severely by the introduction of prm1 and prm2 . structure - based design of the template rnas was carried out by installing two prms ( prm1 and prm2 ) on the platform p5 : ez complex ( figure 1 ) . each prm1 captures its target substrate ( peptide1 ) bearing the reactive thioester group in its c - terminus . each prm2 captures its target substrate ( peptide2 ) bearing cysteine in its n - terminus . to employ three rna - binding peptides ( n - peptide1 , p22n - peptide1 , and rev - peptide1 , figure 2(b ) ) as the substrate peptide-1 , we designed three p5 rna derivatives with prm1s for substrate peptides1 ( boxb , p22boxb , and rre ) . as the substrate peptides2 recognized by the ez rna , we used two peptides ( n - peptide2 and rev - peptide2 , figure 2(b ) ) . their target boxb and rre motifs were installed to the ez rna as prm2s ( figure 2(a ) ) . basal activities of six combinations of the substrate peptide-1 and -2 were investigated ( figure 4 ) . in the absence of template rnas , products yields in 7 h reaction of chemical ligations with six combinations of substrates ( 7.5 m each ) were around 1020% ( figure 4(b ) ) . to evaluate the ability of template rna derived from the platform complex ( p5 : ez ) , we designed and examined six combinations of peptide ligation reactions based on the model 3d structures of the substrate - template complexes ( figure 4(a ) ) . in the presence of stoichiometric amounts of the cognate template rna complex ( 7.5 m ) , product yields of the four reactions were improved to around 40% ( figure 4(b ) ) . the reaction between p22n - peptide1 and rev - peptide2 exhibited remarkable improvement of the product yield that reached 73% . on the other hand , the reaction between n - peptide1 and n - peptide2 was not promoted by the template rna ( figure 4(b ) ) . to determine whether the improvement of the reaction is due to the complex formation modeled in figure 4(a ) , control reactions were carried out using the parent p5 and ez rnas lacking prm1 and prm2 . in the reactions between rev - peptide1 and rev - peptide2 and between n - peptide1 and n - peptide2 ( figure 4(b ) ) , prms gave no positive effect ( figure 4(b ) ) . these results are predictable because these templates capture the two substrates in a productive manner ( peptide1/prm1 + peptide2/prm2 ) and inhibitive manners ( peptide2/prm1 + peptide1/prm2 , peptide1/prm1 + peptide1/prm2 , and peptide2/prm1 + peptide2/prm2 ) . since the yield of the reaction between p22n - peptide1 and rev - peptide2 ( 58% ) was close to the yield in the presence of the cognate template rna ( 73% ) , the template effect for this reaction was compared in the early phase of the reaction ( 1-h reaction , figure 4(c ) ) . the product yield of the cognate template rna was twofold higher than the yield with the control rna . on the other hand , the effect of the control rna on the reaction between p22n - peptide1 and rev - peptide2 was more efficient than the effect of the cognate template rna on the reaction of n - peptide1 and rev - peptide2 ( figure 4(c ) ) . among the six combinations of chemical peptide ligations , the reaction between n - peptide1 and rev - peptide2 was chosen for further investigation of the template ability of the self - assembling rna derived from the p5 : ez complex . reaction time courses indicated that the ligated product was produced through ( i ) intrinsic reactivity of the two peptides , ( ii ) assistance of rna in a prm - independent manner , or ( iii ) assistance of rna in a prm - dependent manner ( figure 5(a ) ) . to evaluate the contributions of ( ii ) and ( iii ) , we systematically prepared mutant complexes and investigated their effects . to determine the importance of two prms being a pair in the prm - dependent promotion of the ligation , we examined the ligation reaction in the presence of only the p5-boxb rna or the ez - rre rna ( figure 5(b ) ) . the product yields of the two reactions ( 15% and 13% ) were lower than the yield with cognate complex ( 44% ) and close to the yield with the platform p5 : ez complex ( 17% ) . to further investigate the effect of prm complex formation between p5 and ez was not affected by the number of prm in the p5 : ez complex ( figure 5(c ) ) . the resulting two complexes lacking either prm1 or prm2 served as templates much less effectively than the complex with two prms , and their effects were comparable to that of the platform complex ( p5 : ez ) ( figure 5(d ) ) . these results indicated that the prm - dependent enhancement was achieved by cooperative effects of prm1 ( boxb motif ) and prm2 ( rre motif ) . the product yields in the presence of the mutant complexes possessing one prm ( p5-boxb : ez and p5 : ez - rre in figure 5(d ) ) were slightly higher than those in the presence of p5-boxb or ez - rre alone ( figure 5(b ) ) . this difference may reflect the prm - independent enhancement contributed by the ez and p5 components in the mutant p5-boxb : ez and p5 : ez - rre complexes , respectively . in this study , we employed the two - piece derivative of the tetrahymena intron rna ( p5 : p5 complex ) as a self - assembling platform for a template rna to assist peptide ligation . the structural organization of the p5 : p5 platform ensures structural independence between the modules for rna - rna assembly and the modules for recognition of substrate peptides ( prm ) , suggesting the versatility and flexibility of the p5 : p5 platform in molecular design . this study confirmed the versatility of the p5 : p5 platform for template design because six types of template rna complex were designed and their template capabilities were tested experimentally . on the other hand , this study also revealed the current limitation of the p5 : p5 platform . in the case of the reaction between n - peptide1 and rev - peptide2 , the prm - dependent facilitation by the p5 : ez complexes possessing two prms was similar to the facilitation by the self - folding p4p6 rna with two prms . the prm - independent facilitation by the parent p5 : ez platform , however , was more efficient than those by the previous platforms based on the self - folding rnas . the negatively charged surface of 3d - rnas electrostatically accumulates positively charged substrate peptides [ 33 , 34 ] . this effect may be stronger in the p5 : p5 platform than in previous platforms because of the considerably larger surface area of the p5 : p5 platform . this property is undesirable if the p5 : p5-based rna is applied as a template to conduct prm - dependent ligation of the cognate substrate pair in the presence of non - cognate substrates . therefore , reduction of the surface size of 3d - rna may be a strategy to suppress prm - independent facilitation of peptide ligation . in the p5 : p5 platform , however , most of the core and peripheral modules contribute to self - assembly of p5 and p5 and also maintenance of its 3d structure . addition of cosolute molecule to the buffer solution may be an alternative approach [ 35 , 36 ] . positively charged small molecules or polymers may competitively block the substrate peptide to assemble with the platform region of the template rna , thus competitively suppressing the prm - independent reaction promotion . on the other hand , an appropriate amount of cationic cosolute may not inhibit specific binding between peptides and their target prms , resulting in preservation of prm - dependent reaction . an unexpected finding of the present study was the exceptionally efficient rna assistance of ligations between p22n - peptide1 and rev - peptide2 and between rev - peptide1 and rev - peptide2 without prms ( figure 4(b ) ) . in contrast to the two reactions , rna templates gave no positive effect on the reaction between n - peptide1 and n - peptide2 ( figure 4(b ) ) . no prm - independent facilitation was observed in the reaction between p22n - peptide1 and n - peptide2 ( figure 4(b ) ) . these results suggest that the prm - independent reaction promotion by the p5 : p5 platform depends on the combination of rna - binding peptides . rev - peptide and p22n - peptide seem more congenial to the rna - promoted chemical ligation than n - peptide ( figure 4 ) . although the molecular basis underlying these results remains to be elucidated , it is important to see whether apparently different results originate from the physical property of each peptide . n - peptide is less hydrophilic than p22n- and rev - peptides ( figure 2(b ) ) , and p22n - peptide has markedly higher affinity to cognate and noncognate rna motifs than n- and rev - peptides . thus , it is also important to determine whether the possible approaches to suppress prm - independent reaction promotion are effective on the reaction between p22n - peptide1 and rev - peptide2 .

multicomponent rna - peptide complexes are attractive from the viewpoint of artificial design of functional biomacromolecular systems . we have developed self - folding and self - assembling rnas that serve as templates to assist chemical ligation between two reactive peptides with rna - binding capabilities . the design principle of previous templates , however , can be applied only to limited classes of rna - binding peptides . in this study , we employed a two - piece derivative of a group i intron rna from the tetrahymena large subunit ribosomal rna ( lsu rrna ) as a platform for new template rnas . in this group i intron - based self - assembling platform , modules for the recognition of substrate peptides can be installed independently from modules holding the platform structure . the new self - assembling platform allows us to expand the repertoire of substrate peptides in template rna design .

traumatic hemothorax is the most common thoracic emergencies and patients are usually in critical conditions because of other combined serious complications such as hemorrhagic shock , which leads to a high mortality . retrospective analysis of 19 cases of traumatic hemothorax with hemorrhagic shock who received thoracoscopic surgery together with blood washing and continuous autologous transfusion was conducted . there were 16 males and 3 females , aged 2147 years , mean ( 28.76 5.24 ) years . the injury type included rib fracture combined with intercostal artery rupture bleeding in 8 cases , lung laceration following thoracic sharp injury in 6 cases , and intercostal vascular rupture & pulmonary laceration caused by thoracic sharp injury in 5 cases . the systolic blood pressure was ( 68 5 ) mmhg and diastolic blood pressure ( 53 6 ) mmhg . the mean blood loss was ( 1100.27 230.51 ) ml : 10001200 ml in 12 patients , 12001500 ml in 5 cases , and > 1500 ml in 2 cases . the diagnosis of hemorrhagic shock strictly followed the principle of patients with an estimated blood loss > 800 ml ( or more than 20% of the total ) . under combined anesthesia through double - lumen tracheal intubation , the patients underwent thoracoscopic surgery to achieve full detection and hemostasis . intraoperatively aseptic operation was strictly controlled . at the same time , type 3000p blood recycling machine ( beijing jingjing medical equipment co. , china ) was used to perform blood washing and transfusion . after filtration , centrifugation and washing , the anticoagulant , part of platelets , red blood cell ( rbc ) debris , tiny particles , free plasma protein and plasma components were removed ; the clean concentrated rbcs were pumped and transfused back to the patient . the recycled amount of rbcs among the 19 patients reached maximumly 1680 ml , minimally 490 ml , mean ( 662.41 269.15 ) ml . the two patients with a blood loss over 1500 ml had a poor recycle rate with many blood clots . on postoperative day 1 , totally they were infused with 14 u stored rbc suspension . for the whole group , the mean transfusion volume of rbc was 2.8 u. none of the 19 cases had postoperative transfusion reactions , secondary hemorrhagic disease or sepsis . patients combined with hemorrhagic shock and other serious complications often need thoracotomy . in recent years , the development of video - assisted thoracoscopic surgery is popular because it is a minimally invasive technique with less intraoperative blood loss and rapid postoperative recovery . it proves to be safe , effective and appearance - beneficial , and thus has been widely used . all our 19 patients of traumatic hemothorax & hemorrhagic shock received thoracoscopic surgery rather than traditional thoracotomy , even those with the injury types of rib fractures accompanying intercostal artery hemorrhage and sharp instrument - induced injury to the intercostal vessels or / and the lungs . the reasons are : for all the abovementioned combined injuries , thoracoscopic exploration can well define the patient 's injury conditions in a very short time ; as for the intercostal bleeding and pulmonary laceration bleeding , hemostasis can be easily done under thoracoscopy , which can greatly shorten the operation time and reduce surgical trauma as well as the chance of chest contamination . therefore thoracoscopic surgery has the advantages of minimally invasive , rapid recovery , and few complications . moreover the surgeons need to do a comprehensive , prompt and accurate judgment on the patient 's condition . the main indications of video - assisted thoracoscopic surgery in treating traumatic hemothorax are : ( 1 ) open or closed thoracic trauma associated with persistent hemothorax ; and ( 2 ) thoracic penetrating injury with primary drainage amount greater than 800 ml . whereas the contraindications include : ( 1 ) severe shock shortly after injury or drainage of a large amount of coagulated bloody fluid , which is often suspected or determined to have major vessels and heart damage ; ( 2 ) severe pulmonary laceration or tracheal / bronchial injury ; ( 3 ) combined esophageal rupture ; ( 4 ) severe combined injury and unstable hemodynamics ; ( 5 ) patient can not tolerate one - lung ventilation ; and ( 6 ) serious pleural adhesions on the operation side due to previous surguries . as long as the abovementioned surgical indications are strictly followed , the surgery can obtain satisfactory results . however , if intraoperative exploration shows the need for traditional thoracotomy , surgical strategies should be immediately changed . for patients with chest trauma - induced hemorrhagic shock , surgery and blood transfusion are both indispensable . some critical patients need emergent blood transfusion . but due to the blood preparation time or shortage of matched blood , surgery combined with autologous blood transfusion is very important . autologous transfusion of the blood free of contamination in the chest cavity was conducted , which won the valuable time for anti - shock treatment and played a very important role in patient 's rehabilitation . , blood transfusion is a key factor determining the rescue success . in the emergent condition of acute massive bleeding , intraoperative autologous blood transfusion can increase the success rate and win valuable time for the patient , and moreover avoid as high as 60%70% of allogeneic blood transfusion . for all our 19 patients , no one had blood transfusion reactions . in the emergent surgery of traumatic thoracic injury - induced bleeding , autologous blood transfusion is more time - saving , more convenient , safer and more effective than the stored blood transfusion , and the patient 's intraoperative blood loss will not be wasted . moreover the former is fresh autologous rbcs with a better cell viability and higher oxygen carrying capacity , which can rapidly supply blood volume and maintain an effective cycle without adverse transfusion reactions . but the indications and contraindications for autologous blood transfusion should be well known . blood that flows out of the vessels for more than 6 h or was suspected with contamination or contains cancer cells must not be re - transfused . the preservation technology of salvaged blood for intraoperative autologous transfusion has obtained more and more attention . combined with thoracoscopic surgery , the treatment in some patients with trauma - induced progressive hemothorax can make a great progress from conservative observation into more active surgical treatment , which greatly improves the rescue success rate and treatment safety .

from january 2013 to january 2015 , 19 patients of traumatic hemothorax with hemorrhagic shock were treated in our department by thoracoscopic surgery combined with autologous blood transfusion . this study retrospectively analyzed the therapeutic effect and shared our experience . the average amount of blood transfused back was 662.41 ml 269.15 ml . none of the patients developed transfusion reaction and were all discharged uneventfully . thoracoscopic surgery combined with autologous blood transfusion is effective in the rescue of patients with progressive hemothorax and hemorrhagic shock . when corresponding indications are well managed , treatment for these patients is quicker , safer , and more effective .

af and left ventricular diastolic dysfunction often co - exist. it was proven in previous studies , that diastolic dysfunction promotes the initiation and recurrence of af . , on the other hand , patients with af have a 3-fold higher risk of developing heart failure . thus , early recognition and appropriate therapy of diastolic dysfunction is worthwhile to prevent progression to diastolic heart failure and subsequent death in af patients . among the echocardiographic parameters of diastolic dysfunction , the ratio of early diastolic mitral inflow velocity ( e ) to early diastolic mitral annular velocity ( e ' ) has been reported to be a useful parameter for estimating left ventricular filling pressure ( lvfp ) . in the study by li , et al . e / e ' > 9 has a sensitivity of 72.7% and a specificity of 70.4% for identification of elevated lvfp ( > 15 mmhg ) in af patients . previous studies have shown that e / e ' at rest correlates with exercise capacity in af patients . , however , in many cases it is only under conditions of cardiovascular stress that lvfp increases and exercise - limiting symptoms develop . it has been reported that exercise stress echocardiography could assess lvfp during or after exercise , and is a non - invasive diagnostic test for early diastolic dysfunction . however , whether e / e ' after exercise is correlated with exercise capacity in af patients was unknown . so we hypothesized that exercise induced elevation of lvfp estimated by e / e ' ratio is associated with reduced exercise capacity in af patients . the study population consisted of 145 consecutive patients with persistent non - valvular af and normal left ventricular systolic function [ left ventricular ejection fraction ( lvef ) 50% ] , who were screened from january 2012 to december 2014 at peking university third hospital . other exclusion criteria were : significant valvular heart disease or congenital heart disease ; lvef < 50% ; nyha class iv ; coronary heart disease ; ecg or echocardiographic evidence of exercise - induced ischemia ; uncontrolled baseline heart rate ( > 100 beats / min ) ; moderate or severe respiratory disease , or functional disability . this study was approved by the ethics review boards of peking university health science center . all patients underwent a symptom - limited cardiopulmonary exercise test ( cpet ) with a treadmill using the bruce protocol in accordance with the american thoracic society / american college of chest physicians ( ats/ accp ) . peak oxygen uptake ( vo2 peak ) , anaerobic threshold ( at ) , carbon - dioxide production , and minute ventilation were measured during the test . vo2 peak and at were computed as weighted terms ( ml / min per kilogram ) . patients were not asked to discontinue -blockers , calcium channel blockers ( ccbs ) or digoxin before the test . the primary reason for discontinuing the exercise test included limiting dyspnea , chest pain , peripheral muscle fatigue , severe st - segment depression , or severe ventricular arrhythmia . comprehensive transthoracic echocardiography was performed using a vivid s6 ( ge ) machine and a 3.5-mhz transducer . standard m - mode , 2-dimensional , and color doppler imaging were performed in parasternal and apical views with patients in the left lateral decubitus position before exercise . e was measured using the pulsed wave doppler method , by placing the sample volume at the level of the mitral valve leaflet tips . the e ' was measured from the lateral corner of the mitral annulus in the apical 4-chamber view . lvfp was estimated by e / e ' ratio , and elevated lvfp was defined as e / e ' > 9 . af patients with normal lvfp ( e / e ' 9 ) at rest were classified into two groups according to lvfp after exercise : patients with elevated lvfp after exercise ( e / e ' > 9 after exercise ) and the ones with normal lvfp ( e / e ' 9 after exercise ) . continuous variables are expressed as mean sd , and compared by student 's unpaired t - tests between two groups . percentage was calculated for categorical variables , and comparisons between groups were performed by chi - squared tests . predictors of exercise capacity were identified by univariate analyses , and then all univariate predictors were entered in a stepwise manner into a multivariate linear regression model with the entry and retention set at a significance level of less than 0.10 . values for n - terminal pro - b - type natriuretic peptide ( nt - probnp ) were normalized by logarithmic transformation ( lnnt - probnp ) . all analyses were performed with spss for windows version 15.0 ( spss , chicago , il ) . the study population consisted of 145 consecutive patients with persistent non - valvular af and normal left ventricular systolic function [ left ventricular ejection fraction ( lvef ) 50% ] , who were screened from january 2012 to december 2014 at peking university third hospital . other exclusion criteria were : significant valvular heart disease or congenital heart disease ; lvef < 50% ; nyha class iv ; coronary heart disease ; ecg or echocardiographic evidence of exercise - induced ischemia ; uncontrolled baseline heart rate ( > 100 beats / min ) ; moderate or severe respiratory disease , or functional disability . this study was approved by the ethics review boards of peking university health science center . all patients underwent a symptom - limited cardiopulmonary exercise test ( cpet ) with a treadmill using the bruce protocol in accordance with the american thoracic society / american college of chest physicians ( ats/ accp ) . peak oxygen uptake ( vo2 peak ) , anaerobic threshold ( at ) , carbon - dioxide production , and minute ventilation were measured during the test . vo2 peak and at were computed as weighted terms ( ml / min per kilogram ) . patients were not asked to discontinue -blockers , calcium channel blockers ( ccbs ) or digoxin before the test . the primary reason for discontinuing the exercise test included limiting dyspnea , chest pain , peripheral muscle fatigue , severe st - segment depression , or severe ventricular arrhythmia . comprehensive transthoracic echocardiography was performed using a vivid s6 ( ge ) machine and a 3.5-mhz transducer . standard m - mode , 2-dimensional , and color doppler imaging were performed in parasternal and apical views with patients in the left lateral decubitus position before exercise . e was measured using the pulsed wave doppler method , by placing the sample volume at the level of the mitral valve leaflet tips . the e ' was measured from the lateral corner of the mitral annulus in the apical 4-chamber view . lvfp was estimated by e / e ' ratio , and elevated lvfp was defined as e / e ' > 9 . af patients with normal lvfp ( e / e ' 9 ) at rest were classified into two groups according to lvfp after exercise : patients with elevated lvfp after exercise ( e / e ' > 9 after exercise ) and the ones with normal lvfp ( e / e ' 9 after exercise ) . continuous variables are expressed as mean sd , and compared by student 's unpaired t - tests between two groups . percentage was calculated for categorical variables , and comparisons between groups were performed by chi - squared tests . predictors of exercise capacity were identified by univariate analyses , and then all univariate predictors were entered in a stepwise manner into a multivariate linear regression model with the entry and retention set at a significance level of less than 0.10 . values for n - terminal pro - b - type natriuretic peptide ( nt - probnp ) were normalized by logarithmic transformation ( lnnt - probnp ) . all analyses were performed with spss for windows version 15.0 ( spss , chicago , il ) . a total of 145 af patients ( 81 men and 64 women ) were included in this study . in all , 101 patients ( 69.7% ) had a history of hypertension , 35 ( 24.1% ) diabetes and 48 patients ( 33.1% ) were current smokers . patients were classified into two groups according to lvfp estimated by e / e ' after exercise : 39 ( 26.9% ) with elevated lvfp after exercise and 106 ( 73.1% ) with normal lvfp . there were no significant differences in age , gender , body mass index ( bmi ) , concomitant illnesses , medications , heart rate , systolic blood pressure , or lnnt - probnp between the two groups ( all p > 0.05 , table 1 ) . ace : angiotensin converting enzyme ; arb : angiotensin receptor blocker ; bmi : body mass index ; bp : blood pressure ; ccb : calcium channel blocker ; lvfp : left ventricular filling pressure ; nt - probnp : n - terminal pro - b - type natriuretic peptide . the average e / e ' ratio of patients with elevated lvfp and normal lvfp were 10.2 1.0 and 7.9 0.7 separately . echocardiographic parameters including left ventricular end diastolic diameter ( lvedd ) , left ventricular mass index ( lvmi ) , lvef and left atrial area ( laa ) were not significantly different between the two groups ( all p > 0.05 ) . as compared with the patients with normal lvfp , the ones with elevated lvfp after exercise had significantly lower e ' ( p = 0.005 ) and higher e / e ' at rest ( p < 0.001 ) , and higher e velocity ( p = 0.001 ) and lower e ' ( p < 0.001 ) after exercise ( p < 0.001 ) . however , e velocity at rest was not significantly different between the two groups ( p = 0.920 ) . vo2 peak ( 21.7 2.3 vs. 26.4 3.8 ml / min per kilogram , p < 0.001 ) , at ( 19.9 2.5 vs. 26.0 4.0 ml / min per kilogram , p < 0.001 ) and exercise time duration ( 6.2 0.8 vs. 7.0 1.3 min , p < 0.001 ) were significantly lower in patients with elevated lvfp after exercise than the ones with normal lvfp . univariate regression analyses showed that age , gender , e ' and e / e ' at rest , e ' and e / e ' after exercise , and lnnt - probnp were associated with vo2 peak . multivariate analysis identified 3 significant variables that were predicative of vo2 peak : age ( r = 0.351 , p < 0.001 ) , gender ( 26.4 4.4 and 23.6 3.0 ml / min per kilogram for male and female separately , p < 0.001 ) and e /e ' after exercise ( r = 0.632 , p data are represented as mean sd . at : anaerobic threshold ; e : early diastolic mitral inflow velocity ; e ' : early diastolic mitral annular velocity ; laa : left atrial area ; lvedd : left ventricular end diastolic diameter ; lvef : left ventricular ejection fraction ; lvfp : left ventricular filling pressure ; lvmi : left ventricular mass index ; nt - probnp : n - terminal pro - b - type natriuretic peptide ; vo2 peak : peak oxygen uptake . bmi : body mass index ; e : early diastolic mitral inflow velocity ; e ' : early diastolic mitral annular velocity ; laa : left atrial area ; lvef : left ventricular ejection fraction ; lvmi : left ventricular mass index ; nt - probnp : n - terminal pro - b - type natriuretic peptide ; vo2 peak : peak oxygen uptake . e : early diastolic mitral inflow velocity ; e ' : early diastolic mitral annular velocity ; vo2 peak : peak oxygen uptake . a total of 145 af patients ( 81 men and 64 women ) were included in this study . in all , 101 patients ( 69.7% ) had a history of hypertension , 35 ( 24.1% ) diabetes and 48 patients ( 33.1% ) were current smokers . patients were classified into two groups according to lvfp estimated by e / e ' after exercise : 39 ( 26.9% ) with elevated lvfp after exercise and 106 ( 73.1% ) with normal lvfp . there were no significant differences in age , gender , body mass index ( bmi ) , concomitant illnesses , medications , heart rate , systolic blood pressure , or lnnt - probnp between the two groups ( all p > 0.05 , table 1 ) . ace : angiotensin converting enzyme ; arb : angiotensin receptor blocker ; bmi : body mass index ; bp : blood pressure ; ccb : calcium channel blocker ; lvfp : left ventricular filling pressure ; nt - probnp : n - terminal pro - b - type natriuretic peptide . the average e / e ' ratio of patients with elevated lvfp and normal lvfp were 10.2 1.0 and 7.9 0.7 separately . echocardiographic parameters including left ventricular end diastolic diameter ( lvedd ) , left ventricular mass index ( lvmi ) , lvef and left atrial area ( laa ) were not significantly different between the two groups ( all p > 0.05 ) . as compared with the patients with normal lvfp , the ones with elevated lvfp after exercise had significantly lower e ' ( p = 0.005 ) and higher e / e ' at rest ( p < 0.001 ) , and higher e velocity ( p = 0.001 ) and lower e ' ( p < 0.001 ) after exercise ( p < 0.001 ) . however , e velocity at rest was not significantly different between the two groups ( p = 0.920 ) . vo2 peak ( 21.7 2.3 vs. 26.4 3.8 ml / min per kilogram , p < 0.001 ) , at ( 19.9 2.5 vs. 26.0 4.0 ml / min per kilogram , p < 0.001 ) and exercise time duration ( 6.2 0.8 vs. 7.0 1.3 min , p < 0.001 ) were significantly lower in patients with elevated lvfp after exercise than the ones with normal lvfp . univariate regression analyses showed that age , gender , e ' and e / e ' at rest , e ' and e / e ' after exercise , and lnnt - probnp were associated with vo2 peak . multivariate analysis identified 3 significant variables that were predicative of vo2 peak : age ( r = 0.351 , p < 0.001 ) , gender ( 26.4 4.4 and 23.6 3.0 ml / min per kilogram for male and female separately , p < 0.001 ) and e /e ' after exercise ( r = 0.632 , p data are represented as mean sd . at : anaerobic threshold ; e : early diastolic mitral inflow velocity ; e ' : early diastolic mitral annular velocity ; laa : left atrial area ; lvedd : left ventricular end diastolic diameter ; lvef : left ventricular ejection fraction ; lvfp : left ventricular filling pressure ; lvmi : left ventricular mass index ; nt - probnp : n - terminal pro - b - type natriuretic peptide ; vo2 peak : peak oxygen uptake . bmi : body mass index ; e : early diastolic mitral inflow velocity ; e ' : early diastolic mitral annular velocity ; laa : left atrial area ; lvef : left ventricular ejection fraction ; lvmi : left ventricular mass index ; nt - probnp : n - terminal pro - b - type natriuretic peptide ; vo2 peak : peak oxygen uptake . e : early diastolic mitral inflow velocity ; e ' : early diastolic mitral annular velocity ; vo2 peak : peak oxygen uptake . in this study , our data showed that elevated lvfp after exercise was present in 26.9% of af patients with normal lvfp at rest . multivariate regression analysis showed age , gender and e / e ' after exercise were independently associated with vo2 peak . af impairs cardiac function by several mechanisms , such as the loss of atrioventricular synchrony and atrial contraction , the reduction of the diastolic filling and the induction of a tachycardia - induced cardiomyopathy . the study by kosiuk , et al . showed that diastolic dysfunction was present in 37% of patients referred for af catheter ablation . park , et al . reported that e / e ' ratio was a useful independent prognostic parameter for predicting mortality in patients with af whose left ventricular systolic function was preserved . in these studies , echocardiography was all done at rest . however , resting estimation of lvfp gives incomplete information . during exercise , to maintain adequate left ventricular filling and stroke volume , the filling pressures raise provoking symptoms to patients with diastolic dysfunction . , previous studies confirmed that in subjects with normal myocardial relaxation , e / e ' remained unchanged during exercise because of a proportionally increase in both e and e ' velocities . in patients with impaired myocardial relaxation , however , the increase in e ' with exercise was lower than that of e velocity , so that the e / e ' ratio increased . takagi , et al . reported that elevated e / e ' ratio after exercise was valuable in predicting new - onset of af in non - ischemic elderly patients . this study found that elevated e / e ' after exercise was present in 26.9% of af patients with normal lvep at rest , indicating a presence of early diastolic dysfunction in these patients . the study by kosiuk , et al . showed that diastolic dysfunction was correlated with symptom severity in af patients . lee , et al . reported a negative correlation between e / e ' at rest and exercise capacity in af patients . because many patients may have normal lvfp in the resting state and cardiac symptoms may be precipitated only by exertion , it may be important to assess lvfp during exercise . studies had shown lvfp during or after exercise was associated reduced exercise capacity in patients with heart failure as well as non - obstructive hypertrophic cardiomyopathy . however , the value of e / e ' during exercise in af patients were uncertain . as far as we know , this study was the first to show that af patients with elevated e / e ' after exercise had lower exercise capacity than patients with normal e / e ' . so we concluded that early diastolic dysfunction detected by exercise stress echocardiography was associated with reduced exercise capacity . in this study , both e / e ' at rest and after exercise were correlated with vo2 peak in univariate analysis . however , multivariate regression analysis showed that e /e ' after exercise , but not e / e ' at rest , was an independent predictor of vo2 peak . this study also showed age and gender were independently associated with vo2 peak in af patents . it had been reported that elderly women were predominantly observed among patients with heart failure with preserved ejection fraction . previous study also showed that age and female gender were associated with heart failure in af patients . in this study , it has been proved that lenient heart rate control is as effective as strict rate control in terms of major clinical events . however , previous studies had controversial results about the relationship between heart rate control and exercise capacity . heart rate at rest was an independent predictor of vo2 peak , while cooper , et al . nt - probnp , an established biomarker for heart failure , could predict exercise capacity in patients with heart failure . in this study , patients with elevated e / e ' after exercise did not have elevated lnnt - probnp than patients with normal e / e ' . thus , nt - probnp may be not as sensitive as e / e ' in identifying early diastolic dysfunction in af patients . in univariate regression analysis , lnnt - probnp was associated with vo2 peak , however , lnnt - probnp was not an independent predictor of vo2 peak in multivariate regression analysis . the accuracy and reproducibility of e / e ' has been debated in af patients because of the beat - to - beat variations . demonstrating a good correlation between lvfp and e / e ' in af patients . in this study , five consecutive measurements of e and e ' were taken and averaged in order to reduce the effect of variance from beat to beat . elevated lvfp estimated by e / e ' ratio after exercise was independently associated with reduced exercise capacity . exercise stress echocardiography could be a useful diagnostic test for early diastolic dysfunction in af patients . af impairs cardiac function by several mechanisms , such as the loss of atrioventricular synchrony and atrial contraction , the reduction of the diastolic filling and the induction of a tachycardia - induced cardiomyopathy . the study by kosiuk , et al . showed that diastolic dysfunction was present in 37% of patients referred for af catheter ablation . park , et al . reported that e / e ' ratio was a useful independent prognostic parameter for predicting mortality in patients with af whose left ventricular systolic function was preserved . in these studies , echocardiography was all done at rest . however , resting estimation of lvfp gives incomplete information . during exercise , to maintain adequate left ventricular filling and stroke volume , the filling pressures raise provoking symptoms to patients with diastolic dysfunction . , previous studies confirmed that in subjects with normal myocardial relaxation , e / e ' remained unchanged during exercise because of a proportionally increase in both e and e ' velocities . in patients with impaired myocardial relaxation , however , the increase in e ' with exercise was lower than that of e velocity , so that the e / e ' ratio increased . takagi , et al . reported that elevated e / e ' ratio after exercise was valuable in predicting new - onset of af in non - ischemic elderly patients . this study found that elevated e / e ' after exercise was present in 26.9% of af patients with normal lvep at rest , indicating a presence of early diastolic dysfunction in these patients . the study by kosiuk , et al . showed that diastolic dysfunction was correlated with symptom severity in af patients . lee , et al . reported a negative correlation between e / e ' at rest and exercise capacity in af patients . because many patients may have normal lvfp in the resting state and cardiac symptoms may be precipitated only by exertion , it may be important to assess lvfp during exercise . studies had shown lvfp during or after exercise was associated reduced exercise capacity in patients with heart failure as well as non - obstructive hypertrophic cardiomyopathy . however , the value of e / e ' during exercise in af patients were uncertain . as far as we know , this study was the first to show that af patients with elevated e / e ' after exercise had lower exercise capacity than patients with normal e / e ' . so we concluded that early diastolic dysfunction detected by exercise stress echocardiography was associated with reduced exercise capacity . in this study , both e / e ' at rest and after exercise were correlated with vo2 peak in univariate analysis . however , multivariate regression analysis showed that e /e ' after exercise , but not e / e ' at rest , was an independent predictor of vo2 peak . this study also showed age and gender were independently associated with vo2 peak in af patents . it had been reported that elderly women were predominantly observed among patients with heart failure with preserved ejection fraction . previous study also showed that age and female gender were associated with heart failure in af patients . in this study , it has been proved that lenient heart rate control is as effective as strict rate control in terms of major clinical events . however , previous studies had controversial results about the relationship between heart rate control and exercise capacity . , in the study by lee , et al . heart rate at rest was an independent predictor of vo2 peak , while cooper , et al . nt - probnp , an established biomarker for heart failure , could predict exercise capacity in patients with heart failure . in this study , patients with elevated e / e ' after exercise did not have elevated lnnt - probnp than patients with normal e / e ' . thus , nt - probnp may be not as sensitive as e / e ' in identifying early diastolic dysfunction in af patients . in univariate regression analysis , lnnt - probnp was associated with vo2 peak , however , lnnt - probnp was not an independent predictor of vo2 peak in multivariate regression analysis . the accuracy and reproducibility of e / e ' has been debated in af patients because of the beat - to - beat variations . however , li , et al . demonstrating a good correlation between lvfp and e / e ' in af patients . in this study , five consecutive measurements of e and e ' were taken and averaged in order to reduce the effect of variance from beat to beat . elevated lvfp estimated by e / e ' ratio after exercise was independently associated with reduced exercise capacity . exercise stress echocardiography could be a useful diagnostic test for early diastolic dysfunction in af patients .

backgroundelevated left ventricular filling pressure ( lvfp ) is an important cause of exercise intolerance in patients with atrial fibrillation ( af ) . exercise stress echocardiography could assess lvfp during exercise . the objective of this study was to investigate the relationship between exercise induced elevation of lvfp and exercise capacity in patients with af.methodsthis study included 145 consecutive patients ( 81 men and 64 women ; mean age 65.5 8.0 years ) with persistent non - valvular af and normal left ventricular systolic function ( left ventricular ejection fraction 50% ) . all patients underwent a symptom - limited cardiopulmonary exercise test ( cpet ) . doppler echocardiography was performed both at rest and immediately after exercise . five consecutive measurements of early diastolic mitral inflow velocity ( e ) and early diastolic mitral annular velocity ( e ' ) were taken and averaged . e / e ' ratio was calculated . elevated lvfp was defined as e / e ' > 9 , and patients with elevated lvfp at rest were excluded.resultspatients were classified into two groups according to lvfp estimated by e / e ' ratio after exercise : 39 ( 26.9% ) with elevated lvfp after exercise and 106 ( 73.1% ) with normal lvfp . as compared with patients with normal lvfp , the ones with elevated lvfp after exercise had significantly lower peak oxygen uptake ( vo2 peak ) ( 21.7 2.3 vs. 26.4 3.8 ml / min per kilogram , p < 0.001 ) , lower anaerobic threshold ( 19.9 2.5 vs. 26.0 4.0 ml / min per kilogram , p < 0.001 ) , and shorter exercise time duration ( 6.2 0.8 vs. 7.0 1.3 min , p < 0.001 ) . multivariate analysis showed that age , gender and e / e ' after exercise were significantly correlated with vo2 peak.conclusionelevated lvfp estimated by e / e ' ratio after exercise is independently associated with reduced exercise capacity in af patients .

in addition to exogenous factors , it is believed that genetic variation also plays an important role in susceptibility to sepsis [ 46 ] . caspase-12 is an inflammatory caspase , in which a loss - of - function genetic variant has been fixed in some populations by still undefined evolutionary pressures [ 79 ] . this loss - of - function is due to the presence of a t / c single nucleotide polymorphism ( rs497116 ) on nucleotide position 125 in the caspase-12 gene . although the ancestral variant is still present in african and african - american populations , of which 2030% express the active variant of caspase-12 , it is absent in asian and caucasian populations [ 9 , 10 ] . functional studies have suggested that functional caspase-12 is a negative regulator of caspase-1 activation , which might result in less cytokine production in response to recognition through pattern recognition receptors . thus , based on the proposed inhibitory effect on caspase-1 and , consequently , lower il-1 and il-18 production , functional caspase-12 may increase the susceptibility to severe sepsis and/or the clinical outcome of sepsis patients . therefore , it is compelling to assess whether genetic variation in caspase-12 plays a role in the susceptibility to candida sepsis . the aim of this study was to assess whether genetic variants of caspase-12 influence the incidence , severity , and mortality of candida sepsis in a cohort of african - american patients . subjects were enrolled between january 2003 and january 2009 after informed consent ( or waiver , as approved by the institutional review board ) at the duke university hospital ( dumc , durham , nc , usa ) . non - infected controls were recruited from the same hospital wards as infected patients , with no history or evidence of candida sepsis / invasive candidiasis or any invasive fungal infection . circulating cytokine concentrations of il-6 , il-8 , and ifn in infected patients were measured by multiplex fluorescent bead immunoassays ( xmap technology , bio - rad , veenendaal , the netherlands ) , from day 0 up to day 5 after the initial positive blood culture . statistical comparisons of frequencies were made between infected versus non - infected subjects using chi - square tests . overall , a p - value < 0.05 was considered to be statistically significant . a total of 93 african - american patients and 88 non - infected african - american controls had genetic and clinical data available for the analysis . table 1baseline patient characteristics of african - american patients with candida systemic infection or uninfected controls recruited at the duke university hospital ( dumc , durham , nc , usa ) ( n = 181)variableinfected cohort ( n = 93 ) , % control cohort ( n = 88 ) , % mean age ( years)5252gendermale51.648.9family48.451.1immunocompromised state54.848.9hsct00solid organ transplant7.53.5active malignancy*22.613.8 solid tumor148.0 leukemia5.43.5 lymphoma3.22.3chemotherapy within past 3 months12.95.6neutropenia ( anc < 500 cells / mm)4.31.2hiv - infected5.40surgery within past 30 days34.430.7receipt of total parenteral nutrition19.45.75dialysis - dependent15.18.0acute renal failure36.633.0liver failure25.81.2intensive care unit admission within the past 14 days39.831.8median baseline serum creatinine ( mg / dl)2.271.9median baseline wbc count ( cells / mm)13.010.87candida spp . * * albicans44.2 glabrata23.7 parapsilosis17.2 tropicalis10.8 krusei3.2 other candida spp.0.9*subjects could have more than one active malignancy**sixteen subjects had > 1 species isolated baseline patient characteristics of african - american patients with candida systemic infection or uninfected controls recruited at the duke university hospital ( dumc , durham , nc , usa ) ( n = 181 ) * subjects could have more than one active malignancy * * sixteen subjects had > 1 species isolated no significant differences in the distribution of caspase-12 genotypes were seen when comparing infected patients ( cc 3.9% , ct 25.3% , tt 72.4% ) and non - infected controls ( cc 2.9% , ct 30.0% , tt 66.1% ) ( p > 0.05 ) . no associations between the caspase-12 genotypes and disseminated disease , persistent fungemia , or 30-day mortality were observed ( data not shown ) . serum samples collected from infected patients during the first 5 days after the initial positive blood culture were measured for concentrations of il-6 , il-8 , and ifn. also , measurements of il-1 and il-18 were performed in these samples . however , the concentrations of these cytokines were too low to detect ( data not shown ) . no differences in cytokine concentrations were apparent between individuals bearing different caspase-12 genotypes ( fig . 1 ) . 1il-6 , il-8 , and ifn circulating concentrations in infected patients from day 0 up to day 5 after initial positive blood culture , in relation to the caspase-12 genotype . the data are presented as mean standard error of the mean ( sem ) il-6 , il-8 , and ifn circulating concentrations in infected patients from day 0 up to day 5 after initial positive blood culture , in relation to the caspase-12 genotype . the data are presented as mean standard error of the mean ( sem ) caspase-12 has been suggested to inhibit caspase-1 processing of proil-1 and proil-18 into the active cytokines . genetic variation of caspase-12 in populations of african descent has been previously associated with susceptibility to bacterial sepsis . the present study was performed in order to assess the role of caspase-12 in sepsis caused by candida spp . the results indicate that the caspase-12 genotype has no significant effect on the susceptibility and severity of systemic infections with candida . candida is one of the leading pathogens causing sepsis [ 2 , 11 , 12 ] . pro - inflammatory cytokines such as il-1 and il-18 are a crucial factor in eliciting an effective immune response to eradicate the infection . a modulatory step in the production of these cytokines is exerted at the level of caspase-1 , a protease that cleaves the pro - form of these cytokines into shorter bioactive proteins [ 13 , 14 ] . it has previously been reported that caspase-12 knockout mice were better capable of clearing both local and systemic bacterial infections compared to wild - type mice , through an improved inflammatory response . the same authors described a similar effect of caspase-12 in patients with bacterial sepsis , with individuals bearing functional caspase-12 being more susceptible to this condition . however , the role of caspase-12 genetic variants in fungal sepsis has not been addressed so far . firstly , the comparison of caspase-12 genotype frequencies in african - american patients with non - infected controls revealed no statistically significant differences . secondly , no effects of the caspase-12 genotype was observed in relation to the clinical outcome of infection , assessed as disseminated disease , persistent fungemia , and 30-day mortality . furthermore , serum cytokine concentrations during the first 5 days of infection were shown to be unaffected by the caspase-12 genotype . our findings on the lack of influence of the caspase-12 genotype on fungal sepsis contrast with those of saleh et al . [ 10 , 15 ] , who suggested an important role of this genetic variant in bacterial sepsis . moreover , circulating cytokine concentrations in infected patients were also not influenced by the caspase-12 genotype . it should be emphasized that this is , in particular , true for il-6 and ifn , cytokines that are induced by il-1 and il-18 , respectively [ 1618 ] . this provides indirect evidence that functional caspase-12 has no clear effect on the production of il-1 and il-18 in the context of candida sepsis . one possible explanation for the discrepancy between this study and that of saleh et al . is represented by the different cause of sepsis in the two studies , fungal and bacterial , respectively . however , one has to concede that the pro - inflammatory cytokines , of which production is reportedly regulated by the caspase-12 genotype , exert similar protective effects in bacterial and fungal sepsis [ 1821 ] . in this respect , a recent study has also failed to reproduce the inhibitory effects of the caspase-12 genotype of lipopolysaccharide and gram - negative bacteria - induced cytokine production , bringing into question the biological activity of caspase-12 . in conclusion , although an effect of the caspase-12 genotype on the susceptibility to bacterial sepsis has been previously reported in a small cohort of african - american patients , this could not be confirmed in our larger cohort of fungal sepsis patients . furthermore , clinical outcome and in vivo cytokine responses were not influence by the caspase-12 genotype .

candida is one of the leading causes of sepsis , and an effective host immune response to candida critically depends on the cytokines il-1 and il-18 , which need caspase-1 cleavage to become bioactive . caspase-12 has been suggested to inhibit caspase-1 activation and has been implicated as a susceptibility factor for bacterial sepsis . in populations of african descent , caspase-12 is either functional or non - functional . here , we have assessed the frequencies of both caspase-12 alleles in an african - american candida sepsis patients cohort compared to uninfected patients with similar predisposing factors . african - american candida sepsis patients ( n = 93 ) and non - infected african - american patients ( n = 88 ) were genotyped for the caspase-12 genotype . serum cytokine concentrations of il-6 , il-8 , and ifn were measured in the serum of infected patients . statistical comparisons were performed in order to assess the effect of the caspase-12 genotype on susceptibility to candidemia and on serum cytokine concentrations . our findings demonstrate that caspase-12 does not influence the susceptibility to candida sepsis , nor has any effect on the serum cytokine concentrations in candida sepsis patients during the course of infection . although the functional caspase-12 allele has been suggested to increase susceptibility to bacterial sepsis , this could not be confirmed in our larger cohort of fungal sepsis patients .

alzheimer s disease ( ad ) is a pathophysiological process leading to , but starting long before dementia emerges . due to aging of the human population in developed and developing societies , it is expected that this number will double by the year 2030 and more than triple by 2050 . in the us alone , the cost of caring for individuals with ad is expected to rise from 200 billion dollars in 2012 to 1.1 trillion in 2050 . thus , ad and related forms of dementia have become one of the most severe socioeconomic and medical burdens impacting modern society . even small advances in therapeutic strategies could lead to a delay in the onset and progression of ad and would significantly reduce this burden . alterations in brain insulin metabolism have been suggested as one pathophysiological factor underlying this neurodegenerative disorder [ 68 ] . in line with this hypothesis , ad patients show reduced brain insulin receptor sensitivity [ 9 , 10 ] , hypophosphorylation of the insulin receptor and downstream second messengers such as the insulin receptor substrate-1 [ 10 , 11 ] and attenuated insulin and insulin - like growth factor receptor expression . furthermore , reduced cerebrospinal fluid ( csf ) insulin levels have been observed in moderate and severe cases of ad ; however , this research is not conclusive . other studies have demonstrated either normal or increased csf insulin levels in ad patients . moreover , in the only existing study measuring csf insulin levels in ad brains that explicitly used age - matched healthy control brains , normal levels of the hormone were detected . similar findings have been reported for insulin and insulin - like growth factor receptor expression evidence for attenuated levels , but also for normal or increased levels exists [ 10 , 15 ] . figure 1 provides an overview on the putative role of insulin in ad pathology.fig . 1overview of the role of insulin and insulin sensitivity in the pathology of alzheimer s disease overview of the role of insulin and insulin sensitivity in the pathology of alzheimer s disease as enhanced brain insulin signaling improves memory processes in cognitively healthy humans [ 1618 ] and possesses neuroprotective properties [ 19 , 20 ] , increasing brain insulin concentrations in ad patients could be a promising approach to prevent or slow the progression of this devastating disease . thus , it is not surprising that intranasal administration of insulin in ad patients enabling insulin to directly access the brain [ 21 , 22 ] was recently selected by the national institute of health ( nih ) as one of two therapeutic strategies receiving substantial funding as part of the national alzheimer s plan in the us . this plan is a federal initiative to find an effective way to prevent or treat ad by 2025 . in this review we will explore the underlying neural network and possible molecular mechanisms that mediate the protective effects of insulin in the human brain . also , we aim to provide an overview of the concept and current findings regarding the use of intranasal insulin to improve memory function in healthy and cognitively impaired humans . in healthy participants , intranasal insulin administration over the course of 8 weeks improved performance on a declarative memory task ( delayed recall ) based on a word list that had to be remembered acutely and 1 week later . this enhancing effect on declarative memory , a hippocampus - dependent function , was amplified when insulin aspart , a fast - acting insulin analog , was used . more recent studies have provided evidence that even a single acute dose of insulin ( 160 iu ) enhances hippocampus - dependent memory processes , as measured by both spatial and working memory tasks [ 18 , 26 ] . furthermore , verbal working memory , a capacity that relies on activation of the frontal cortex , is improved after a single intranasal application of insulin . a single dose of intranasal insulin acutely improved memory in memory - impaired older adults with ad or mild cognitive impairment ( mci ) and also improved memory and cognitive function with multiple treatments of patients with ad or mci . insulin was effective in improving performance on a verbal memory test in a group of ad and mci patients ; however , within this group subjects carrying the apoe4 allele , a strong genetic predictor for ad , showed poorer recall following insulin administration compared to patients that do not posses this allele and healthy controls . response curve to intranasally applied insulin , such that word and story recall was enhanced by a relatively low dose of insulin in patients that do not posses the apoe4 allele while apoe4 patients demonstrated a relative decline in memory . additional research indicated that attention and functional status ( e.g. , orientation , judgment , social interactions , home activities , personal care , speech / language ) of this patient group was enhanced by insulin . in the first pilot clinical trial on the use of intranasal insulin therapy in 64 mci and 40 ad patients , participants received either placebo , 20 iu of insulin , or 40 iu of insulin administered with a nasal spray over the course of 4 months . the main outcome measures were delayed story recall , dementia severity rating scale , alzheimer disease s assessment scale - cognitive subscale and alzheimer s disease cooperative study - activities of daily living scale . subsets of participants further underwent lumbar puncture and a positron emission tomography ( pet ) study before and after treatment . the between - subject comparison showed that intranasal insulin improved delayed memory and cognitive function , and , importantly , also preserved the functional ability of the patients as estimated by caregiver ratings , suggesting that the improvements were clinically relevant . based on pet findings , the authors also provided direct evidence for a higher f fluorodeoxyglucose uptake in the parietotemporal , frontal , precuneus and cuneus regions of the cns following intranasal insulin compared to placebo administration , linking the enhancement in functioning to processes in those brain areas . , this clinical trial employed a relatively low sample size , the treatment period was comparatively short , and , in absolute terms , the improvements were moderate . nevertheless , the improvements in episodic memory were still present 2 months after cessation of treatment . a meta - analysis investigating structural brain changes in patients with mci revealed convergent gray matter atrophy in the bilateral amygdala and hippocampus , extending to the left medial temporal pole , thalamus and bilateral precuneus . significant reduction of gray matter volume in perirhinal and hippocampal regions and a functional decline in inferior parietal lobules and precuneus has also been established in patients during the conversion from amnestic mci to ad , as well as patients with early ad [ 34 , 35 ] . taken together , these imaging studies reveal brain atrophy in the hippocampal region , which seems to be the most consistent structural marker of the associated cognitive deterioration and a strong predictor of ad . in conclusion , the present research is promising because small but consistent improvements of cognitive memory processes induced by insulin have been discovered ; however , further clinical trials are needed to assess the clinical relevance of intranasal insulin in the treatment of cognitive disorders . impaired insulin sensitivity of the brain and in the periphery of the human body , so - called insulin resistance , is associated with cognitive decline and smaller total brain size . further , in the insulin - resistant state reduced spontaneous ( task - independent ) cortical activity has been observed as reflected by decreased theta band activity , a low - frequency eeg component of which is positively related to memory performance . supporting this view , biochemical analysis of brains of ad patients revealed central nervous system ( cns ) insulin desensitization , which was correlated with the magnitude of cognitive impairments [ 10 , 39 ] . disturbances in glucose and energy metabolism and brain insulin signaling are therefore considered a pathological hallmark of ad [ 7 , 40 ] . in a recent f pet study , insulin resistance was also associated with a pattern of reduced cerebral glucose metabolism in frontal , temporo - parietal and cingulate regions in cognitively intact adults with prediabetes or type 2 diabetes . bearing in mind that such patterns of hypometabolism have likewise been observed in patients with mci and ad [ 42 , 43 ] and that diabetes is a risk factor for ad , screening for insulin resistance may provide a relatively low - cost , non - invasive means for identifying adults at risk to develop the disease . in a voxel - based morphometric ( vbm ) study in a large population of healthy elderly subjects ( at the age of 75 years ) , benedict and colleagues were able to show that the homa - ir score , a measure of peripheral insulin resistance , was negatively correlated with verbal fluency performance , total brain size and regional gray matter volume in bilateral areas of the middle and superior temporal gyri , i.e. , typical speech - processing areas . willette et al . confirmed this finding for middle - aged healthy subjects ( at the age of 58 years ) . further results from a structural imaging study in healthy participants point toward an inverse correlation of peripheral insulin resistance with total cerebral volume , but also with executive functions , verbal and visuospatial memory . the homa - ir score of patients at risk for ad was negatively associated with right and total hippocampal volume as well as overall cognitive performance , and verbal and non - verbal memory tests ; however , unlike in the previously mentioned study involving mci patients , this correlation was not influenced by the presence of the apoe4 allele . in conclusion , these studies shed light on anatomical correlates of healthy and cognitively impaired individuals and provide evidence for diminished total brain and hippocampal volume with higher insulin resistance rates . using resting - state imaging methods the activity of the brain in the absence of an external stimulus or task is assessed . here , spontaneous fluctuations that are useful to explore the brain s functional organization are of interest . the most important resting - state network is the default - mode network ( dmn ) , which is activated when we are awake and at rest and is deactivated during goal - oriented behavior . the dmn is an interconnected system of medial prefrontal , medial temporal , posterior cingulate , medial , lateral and inferior parietal cortical areas as well as the ventral precuneus . the resting - state activity and also the degree of structural and functional connectivity of the resting - state network is a biomarker for cognitive function and aging . in women at risk for ad , elevated plasma insulin levels have been associated with a decrease of connectivity between the dmn areas prefrontal cortex and hippocampus , pointing toward an involvement of the connectivity of these brain areas in ad pathology . it has further been revealed in magnetoencephalography ( meg ) studies in cognitively healthy subjects that intranasal insulin modifies the brain network during the resting state by changing the characteristic path length in the theta band . the theta band or theta rhythm is a frequency component that tends to appear during rest . the authors considered this phenomenon an increase in communication efficiency , particularly between brain areas involved in satiation and homeostatic control of eating behavior caused by the rise in csf insulin level . in addition , insulin modulated intrinsic cortical activity in hypothalamic and orbitofrontal cortex areas , two brain areas involved in the modification of reward during food consumption . whereas intranasally applied insulin had no effects on basal cerebral blood flow ( cbf ) or task - induced cbf after visual stimulation , a decrease of the blood oxygen level - dependent ( bold ) response after visual stimulation with food pictures was found in several areas of the fronto - temporal network . the authors of the latter studies primarily attribute their findings to the importance of insulin signaling during processes related to food consumption and the location of food sources . however , these findings do not allow conclusions regarding the effect of intranasal insulin on the connectivity strength of brain components affected in the early stages of ad , such as the hippocampus . since the homeostatic system heavily depends upon the sense of smell and , interestingly , the brain regions first affected by ad are those involved in the sense of smell ( e.g. , olfactory bulb , entorhinal cortex , hippocampus ) , it seems reasonable that distortions in olfactory performance are experienced very early in the progression of the pathology . this hypothesis is further supported by research in the rat model proving that ablation of the olfactory bulbs leads to a deficiency in spatial memory and higher levels of beta amyloid in the hippocampal areas . olfactory performance scores are hence considered behavioral markers for an early diagnosis of ad or other neurodegenerative disorders . as olfactory information enters the human cortical areas without a thalamic relay , they have direct access to brain areas responsible for emotional processing ( amygdala ) and memory formation ( hippocampus ) . since the brain regions involved in olfactory and memory processes both contain high amounts of insulin receptors [ 57 , 58 ] , future studies should elaborate whether the memory - enhancing effects of insulin could be potentiated by presenting associated odor cues . although defective brain insulin signaling is increasingly considered an important feature of ad pathology , the biological mechanisms of brain insulin resistance in ad have just recently started to be unraveled . initial molecular clues came from studies demonstrating that amyloid beta oligomers ( aos ) , toxins that accumulate in brains of ad patients and instigate synapse damage , bind to hippocampal neurons and trigger the removal of insulin receptors from the plasma membrane [ 20 , 60 , 61 ] . buttressing the clinical relevance of these findings , moloney and co - workers verified this phenomenon . aos also alter neuronal insulin receptor function , and this effect seems to be an important aspect of the overall synaptic / neuronal pathology induced by aos [ 39 , 60 , 61 ] . more recently , evidence linking pathogenic mechanisms triggered by aos in the ad brain to mechanisms present in metabolic diseases has emerged [ 39 , 63 , 64 ] . in type 2 diabetes and obesity - related insulin resistance , activation of c - jun n - terminal kinase ( jnk ) leads to irs-1 serine phosphorylation ( irs-1pser ) , resulting in peripheral insulin resistance [ 65 , 66 ] . similarly , aos induce activation of the jnk pathway and irs-1 inhibition in cultured hippocampal neurons [ 39 , 63 ] as well as within the brain of mice and monkeys . likewise , post - mortem brains of ad patients show abnormal distribution of irs , increased serine phosphorylation of irs-1 [ 10 , 61 ] and reduced cytosolic and/or membrane levels of pi3k and pi3k - dependent kinase 1 ( pdk1 ) [ 62 , 67 ] . ad brains further display elevated levels of phosphorylated jnk , which is known to counteract peripheral insulin signaling [ 39 , 68 ] . in conjunction , these studies suggest that the relationship between cns insulin impairment and ao accumulation in the brain is rather complex and should be evaluated in future studies . the discovery of a molecular parallel between defective brain insulin signaling in ad and peripheral insulin signaling dysfunction in diabetes [ 10 , 39 ] provides a rationale for using anti - diabetes agents as novel therapeutics in ad . supporting this view , a treatment with the glucagon - like peptide 1 ( glp-1 ) analog liraglutide ( commonly used in the treatment of diabetes ) leads to an amelioration of insulin resistances in the brains of ad patients and a mouse model of ad and positive effects on neuropathology and cognition in the mouse model . the next important step is to understand if and how stimulation of insulin signaling in the brain might facilitate neuroprotection , thereby preserving normal brain functioning . as touched on previously , insulin s actions seem to be important for proper hippocampal function , a region equipped with insulin receptors that is profoundly involved in the acquisition , consolidation and recollection of memories . in cell culture experiments using highly differentiated cultures of rat hippocampal neurons , the dendritic distribution of insulin receptors shows a punctate pattern , consistent with localization to synapses [ 20 , 60 ] . , insulin receptor signaling contributes to long - term memory consolidation and improves spatial learning [ 7375 ] . insulin has also been proposed to regulate neuronal survival and to act as a growth factor , possibly by activating insulin - like growth factor ( igf ) receptors . an additional possible mechanism of the protective action of insulin could be the decrease in stress - induced hypothalamic - pituitary - adrenal ( hpa ) axis activity responsiveness . insulin has been found to block both ao - induced reductions in insulin receptors on dendritic surfaces and irs-1pser induced by aos . remarkably , insulin protects synapses against aos , and the mechanism of protection entails a decrease of ao binding sites . as a consequence , insulin prevents the overall negative impact of aos on neurons [ 20 , 79 ] . collectively , these studies provide a rational basis for the use of insulin as an effective therapeutic agent in ad . insulin has been revealed not only to control whole - body energy and glucose homeostasis in the periphery of the human body [ 8184 ] , but also to exert specific effects in the cns . insulin receptors have been identified in a variety of brain areas , especially in the olfactory bulb , hippocampus and hypothalamus [ 57 , 58 ] . one possible method for studying the cerebral actions of insulin is via intravenous ( iv ) administration . in humans , it has been shown that iv infusions of insulin are followed by increases in csf levels of the hormone , indicating that plasma insulin accesses the brain . although iv insulin has been linked to improved memory functions in humans , this route of administration possesses little therapeutic potential for the treatment of cns disorders . intravenous application of insulin is a highly invasive technique that leads to hypoglycemia , which itself has detrimental effects on brain function and thus has to be accompanied by iv glucose infusion in order to maintain stable blood glucose levels constant in the experimental setting . the approach of intranasal delivery of neuropeptides , however , has provided us with an alternative for the effective and rapid delivery of insulin exclusively to the brain and thereby provides the means of therapeutic use [ 22 , 87 ] . applying this method , following intranasal application , insulin enters the nasal mucosa and is transported extracellularly along the axon bundles of the olfactory receptor cells in the roof of the nasal cavity leading through the foramina of the lamina cribrosa to the olfactory bulb , hippocampus and other regions of the brain and upper spinal cord [ 21 , 22 , 88 ] . the trigeminal neural pathway has also been shown to provide a pathway from the nasal mucosa through the lamina cribrosa and the pons to the cns [ 21 , 89 ] . hence , using this intranasal delivery method , insulin directly enters the brain , bypassing the blood - brain barrier ( bbb ) , and can be detected in biologically relevant concentrations in the csf 3040 min following intranasal application . furthermore , these effects can be achieved with doses that do not produce changes in peripheral blood levels of insulin and glucose . thus , this method selectively increases cns insulin levels and is therefore suitable for the investigation of both the short- and long - term effects of insulin on the human brain without the potential confound of peripheral side effects . however , at this point it is important to state that an intranasal dose of 40 iu insulin as applied in many previous studies results in short - term elevations of csf insulin to 25 pm levels , which is 410 times lower than the insulin levels ( 0.11 nm ) found to trigger brain insulin signaling in postmortem brains . thus , future studies are needed to show that after intranasal insulin uptake , brain insulin levels are sufficiently raised to enhance brain insulin signaling under in vivo conditions . nevertheless , many intranasal insulin studies have yielded effects on cns functions in humans that resembled those found in animals after intracerebroventricular insulin administration ( e.g. , reduced food intake , improved spatial memory functions ) [ 16 , 18 ] . that said , we believe that the current evidence is strong enough to support the view that intranasal insulin represents a promising pharmacological strategy to support insulin - sensitive cns functions in humans . according to the existing literature , intranasal insulin administration generally neither causes nasal irritation nor destroys the olfactory epithelium or glomerular projections [ 93 , 94 ] , and it therefore constitutes a clinically safe application method for the dissociation of central and peripheral insulin effects . collectively , the presented studies present evidence for the hypothesis that improving central nervous insulin signaling via intranasal insulin or insulin sensitizers , which cross the blood - brain barrier , could represent an effective way to prevent or treat ad . however , chronic insulin treatment can cause desensitization of insulin signaling pathways in peripheral tissues , raising concerns about the long - term efficacy of this approach . another aspect that requires critical consideration is that earlier studies have not shown a beneficial effect of acute intranasal insulin administration in ad and mci patients carrying at least one copy of the apoe4 allele [ 28 , 96 ] . in the most recent 4-month clinical trial , however , positive effects of intranasally applied insulin on ad and mci patients carrying one or two copies of the apoe4 allele have been shown , whereas the optimal insulin dose seems to vary between both groups . considering that the presence of one 4 allele increases the risk of developing ad two- to threefold , while having two 4 alleles increases the risk by about 15-fold , larger studies of longer duration are needed to fully evaluate intranasal insulin s therapeutic potential in the treatment of ad . an additional important next step for this research path is determining the mechanism of insulin s effects on cognition . this entails answering some basic questions in rodents and primates , e.g. , which levels of extracellular brain insulin ( as opposed to csf insulin ) are actually achieved by intranasal insulin doses that exert optimal effects on cognition ? is an increase above baseline of extracellular brain insulin levels sufficient to enhance brain insulin signaling or to normalize central nervous insulin signaling in the case of brain insulin resistance ? does intranasal insulin act equally with regard to neuroprotection and synaptic function ? how fundamental is brain insulin resistance to the development of both familial and sporadic forms of ad ? while there are many open questions regarding longitudinal efficacy and the mechanisms underlying its effect on cognition , recent clinical trials support the hypothesis that intranasal insulin may be a promising option to slow the progress of ad . with this in mind , it is not surprising that intranasal administration of insulin in ad patients was recently selected by the nih to receive substantial funding as part of the national alzheimer s plan in the us . in this multicenter study ( recruiting has not started yet ) , the effects of intranasally administered insulin on cognition , entorhinal cortex , hippocampal atrophy and csf biomarkers in amnestic mci or mild ad will be examined in a sample of 240 people ( clinicaltrials.gov identifier : nct01767909 ) . it is hypothesized that after 12 months of treatment with intranasal insulin subjects will demonstrate improved performance on a global measure of cognition , on a memory composite and in daily functioning . in addition to the examination of csf biomarkers and hippocampal and entorhinal atrophy , the study aims to examine whether the baseline ad biomarker profile , gender or apoe-4 allele carriage predicts treatment response . taken together , the findings reviewed in this article provide a strong rationale for hypothesizing that intranasal delivery of insulin or insulin sensitizers ( e.g. , liraglutide ) represents a promising pharmacological strategy to support insulin - sensitive cns functions in humans , including hippocampus - dependent memory formation and neuroprotection against ad and mci .

research in animals and humans has associated alzheimer s disease ( ad ) with decreased cerebrospinal fluid levels of insulin in combination with decreased insulin sensitivity ( insulin resistance ) in the brain . this phenomenon is accompanied by attenuated receptor expression of insulin and insulin - like growth factor , enhanced serine phosphorylation of insulin receptor substrate-1 , and impaired transport of insulin across the blood - brain barrier . moreover , clinical trials have demonstrated that intranasal insulin improves both memory performance and metabolic integrity of the brain in patients suffering from ad or its prodrome , mild cognitive impairment . these results , in conjunction with the finding that insulin mitigates hippocampal synapse vulnerability to beta amyloid , a peptide thought to be causative in the development of ad , provide a strong rationale for hypothesizing that pharmacological strategies bolstering brain insulin signaling , such as intranasal administration of insulin , could have significant potential in the treatment and prevention of ad . with this view in mind , the review at hand will present molecular mechanisms potentially underlying the memory - enhancing and neuroprotective effects of intranasal insulin . then , we will discuss the results of intranasal insulin studies that have demonstrated that enhancing brain insulin signaling improves memory and learning processes in both cognitively healthy and impaired humans . finally , we will provide an overview of neuroimaging studies indicating that disturbances in insulin metabolism such as insulin resistance in obesity , type 2 diabetes and ad and altered brain responses to insulin are linked to decreased cerebral volume and especially to hippocampal atrophy .

functional obstruction and bowel dilatation proximal to the affected segment , first described by harald hirschsprung in 1888 , is a complex disorder resulting from absence of ganglion cells in the bowel wall leading to functional obstruction and bowel dilatation proximal to the affected segment [ 1 , 2 , 3 ] . it is usually diagnosed in infants but hirschsprung 's disease ( hd ) may be diagnosed in the older children presenting with chronic constipation , which is unresponsive to conven - tional treatment . as constipation is a common problem among children and only small minority of patients have an organic cause for their constipation , it is necessary to distinguish between these two conditions . constipation is defined in neonates as failure to pass meconium within the first 48 hours of life and in the older children as the infrequent passage of stool of increased consistency . a number of constipated children are referred for rectal biopsy to exclude a possible hd . the diagnosis of hd is based on a combination of symptoms , radiological study , rectal manometry , and histological features of rectal biopsy . the histological features of hd include the absence of ganglion cells of the myenteric ( auerbach 's ) and submucosal ( meissner 's ) plexuses , and increased number of hypertrophic nerves [ 2 , 3 ] . rectal biopsy with histopathologic examination can reliably exclude hd and it is the gold standard for the definitive diagnosis of aganglionosis . it is said that 12% to 17% of children undergoing rectal biopsy are found to have hd [ 46 ] and so about 80% of patients receive an unnecessary surgical procedure . lewis et al hypothesized that key features in the history , physical examination , and radiographic evaluation would allow us to avoid unnecessary rectal biopsies . in a retrospective study they found that a history of delayed passage of meconium , abdominal distension , vomiting or the results of a contrast enema identified all patients with hd and excluded hd in approximately 36% of patients with idiopathic constipation ; in a child presenting only with constipation and none of the above features , it is not necessary to perform a rectal biopsy . ghosh and griffiths in a retrospective study in 186 children concluded that if the age at onset of constipation is after the neonatal period , rectal biopsy is unnecessary and it is unlikely that the child has hd . the aim of our study was to evaluate rectal biopsies from constipated children of different age groups to see in which age hd is more likely in order to avoid unnecessary rectal biopsy and on the other hand in which age group we need to pay more attention to avoid missing hd . the names and record numbers of children with chronic unrelenting constipation undergoing a rectal biopsy to exclude hd were obtained from the histopathology department of children 's medical center . cases were divided into 4 groups according to their age : a ) neonatal period : the first 4 weeks of life , b ) between 5 to 12 weeks old , c ) 13 weeks to 1 year old , and d ) above 1 year old . a detailed retrospective demographic review , including age of beginning of signs and symptoms , gender , family history of disease , signs and symptoms , paraclinical data and congenital anomalies , was made in all cases . rectal biopsies are preformed at least 1 cm above the pectinate line and types of biopsies were submucosal biopsies and full thickness muscle coat stained with hematoxilin and eosin . in aganglionic biopsies at least 30 - 50 sections were examined to make the diagnosis of hd . aganglionic cases less than 0.5 cm in diameter , presence of squamous epithelium or striated muscle fibers or submucosal biopsies in which the amount of submucosa was less than mucosa were excluded as the signs of inadequacy or inappropriateness . no extra sampling was imposed to individuals and no extra the parents were not charged . with exclusion of inadequate biopsies ( 6% of all biopsies ) , 172 biopsies were available from 168 children with constipation , 127 ( 75% ) had hd and 41 cases had normal biopsies with normal ganglion cells in appearance and distribution . the mean age of constipated patients at the time of biopsy was 39 months and the mean age of patients with proven hd was 18 months . table 1 shows frequency of hd among patients in different age groups based on the time of diagnosis . frequency of hd among patients in different age groups ( at the time of biopsy ) in 93 children with hd , the parents could remember the beginning time of constipation , in 85 ( 91% ) cases it was in neonatal period ( p<0.005 ) although the time of taking biopsy was later in many . in 8 cases the beginning of clinical manifestation was beyond neonatal period . in the remaining children ( 28 cases ) the parents could not remember the time they noticed constipation ( at or beyond neonatal period ) . from 27 cases without hd , 5 cases had constipation from neonatal period . in 22 cases the beginning of clinical manifestation was beyond neonatal period and in the remainder ( 14 children ) the beginning of clinical manifestation was not clear . frequencies of different clinical manifestations in patients with hd are listed in table 2 . frequency of different clinical manifestations of patients with hd congenital anomalies associated with hd were found in 9.6% and included 4 cases with cns malformation , 3 cases with congenital heart disease , 2 cases with anatomical gastrointestinal malformation , 2 cases with genitourinary malformation , 1 case with congenital dislocation of hip , 1 case with polydactyly and one case with down syndrome . barium enema was done in 103 children and there were false positive and false negative results in 7 and 3 cases , respectively , the diagnostic sensitivity was 91.3% . constipation accounts for nearly 3% of visits to a general pediatric office and 25% of visits to a pediatric gastroenterologist[7 , 1 ] . 95% of children with chronic constipation have functionalconstipation while only 5% have an organic cause for their symptoms , and a rectal biopsy , the goldstandard for the diagnosis of hd , is necessary to exclude this condition[1 , 8 ] . some authors are of opinion that if the onset of constipation is beyond neonatal period , the child is unlikely to have hd . the indications for rectal biopsy in children before 6 months of age : ( a ) delayed meconium passage ; ( b ) low intestinal obstruction of unknown cause ; ( c ) severe constipation ; ( d ) chronic abdominal distention and ( e ) failure to thrive [ 9 , 10 , 11 ] . our results show that males were affected 4 times more than females and it is parallel to those of other studies like results reported by amiel and halevy . in a study in korea the most frequent sign of disease in neonates in our study was abdominal distension and delay in meconium passage which is the same as in other studies [ 1 , 2 ] . most cases with hd were diagnosed in neonatal period ( 1.8% of patients without and 30% of patients with hd were neonates , p<0.004 ) . in 85 ( 91% ) cases the beginning of constipation was in neonatal period and in five non - hd cases the constipation was present in newborn infant ( p<0.008 ) . our data supports griffith 's results suggesting that if the onset of constipation occurs after the neonatal period , it is unlikely that the child has hd and therefore a rectal biopsy is unnecessary . also nicola in showed that in sixty percent of patients with hd and 15% of patients without hd the onset of symptoms was in the first week of life . in our study , a large number of suspected children had hd . this may be because our hospital as a referral one is usually attended by cases with prolonged or complicated diseases ; also the reason may be the good selection of patients for taking biopsy since in suspicious cases anorectal manometry and/or a barium enema is undertaken as screening methods before undergoing rectal biopsy . in 9.6% of our cases this is lower than in australian and boston studies which show 16% ( australia ) and 22% ( boston ) of children with associated anomalies [ 13 , 14 ] . these findings plus the fact that 9% of these children had also down 's syndrome , is evidence for the assumption that the etiology of hd may be partially genetic . the diagnostic sensitivity of barium enema in our study was about 91.3% and was close to amsterdam study in 2005 . according to our data and those of griffith and also nicola studies it can be concluded that delay in meconium passage is the most important clinical sign of hd and it should be emphasized in the patients history if noticed . furthermore , radiologic findings in older cases that are unresponsive to medical therapy are other means to select correct patients for biopsy .

objectivehirschsprung 's disease ( hd ) is a complex disorder resulting from absence of ganglion cells in the bowel wall leading to functional obstruction and bowel dilatation proximal to the affected segment . the aim of our study was to evaluate rectal biopsies from constipated children in different age groups to see in which age it is more likely to encounter hd to avoid unnecessary rectal biopsy.methodsrecords of all children with chronic constipation undergoing a rectal biopsy to exclude hd were obtained from the files of children 's medical center in tehran , iran . a detailed retrospective demographic review , including age of beginning of signs and symptoms was made of all cases.findingstotally , 172 biopsies were taken from 168 children in a five year period , of which 127 cases ( 75% ) had hd . the mean age of constipated patients at biopsy was 39 months and the mean age of patients with proven hd was 18 months . males were affected more than females . congenital anomalies associated with hd were found in 9.6% . in 85 ( 91% ) cases constipation had begun in neonatal period.conclusionour data supports previous studies that if constipation begins after the neonatal period , the child is unlikely to have hd . in neonates delay in meconium passage is the most important clinical sign of hd .

this study was conducted using formalin - fixed , paraffin - embedded tissue samples obtained from 170 ptc patients who underwent thyroid surgery at kangdong sacred heart hospital between january 2006 and december 2007 . among the 170 patients , 84 had available data including the preoperative serum thyroid autoantibodies and pathology reports for ht . ht was diagnosed on the basis of histological findings of diffuse lymphoplasmacytic infiltration with germinal centers , parenchymal atrophy with oncocytic change , and variable amounts of stromal fibrosis throughout the thyroid gland . lymphovascular invasion was assessed with light microscopy and defined as tumor cells that were present within a vascular space with identification of endothelial lining.15 after histological review , ptc cases were categorized into two groups , ptc with ht ( n=84 ) or ptc without ht ( n=86 ) . clinical information including age , sex , treatment modality , and survival or recurrence was adapted from medical records and radiologic findings and then analyzed . all glass slides from 170 patients with ptc were reviewed by two pathologists for diagnosis confirmation and selection of a representative section for immunohistochemical study . diagnosis and histologic differentiation were evaluated according to the world health organization classification , and tumor staging was based on the american joint committee on cancer updated tumor - node - metastasis cancer staging system . this study was approved by institutional ethics committee of kangdong sacred heart hospital seoul , korea . after a case review for diagnostic confirmation a circle was drawn on the slide around the most representative area . using the slide as a guide , core samples were obtained from each paraffin - embedded block using a tissue microarray tool ( quick - ray , unitma , seoul , korea ) . a punch size nine cores were embedded in each block in 33 arrangements . in total , 19 tissue microarray blocks were produced from the 170 tumor samples . cc1 standard ( ph 8.4 buffer contained tris / borate / ethylenediaminetetraacetic acid ) was used for antigen retrieval . dab inhibitor ( 3% h2o2 endogenous peroxidase ) was blocked for 4 minutes at 37. slides were incubated with anti - p2x7r antibodies ( 1:300 , goat igg , abcam , cambridge , uk ) for 40 minutes at 37 , and then incubated with a secondary antibody ( universal hrp multimer , ventana medical systems , melbourne , vic , australia ) for 8 minutes at 37. after incubation , slides were stained with the dab h2o2 substrate for 8 minutes , followed by hematoxylin and bluing reagent counterstaining at 37. a reaction buffer ( ph 7.6 , tris buffer ) was used as a washing solution . both the intensity of immunohistochemical staining and the proportion of stained tumor cells were semi - quantitatively evaluated . the staining intensity was scored as follows : 0 , negative ; 1 , weak ; 2 , moderate ; and 3 , strong . staining proportion was rated according to the percentage of positive cells and scored as follows : 0 , less than 10% ; 1 , 11% to 25% ; 2 , 26% to 75% ; 3 , more than 75% . the scores of staining intensity and proportion were multiplied to produce a weighted immunoreactive score ( 0 - 6 ) . cases with a score 3 were considered high expression and those with a score 2 were defined as low expression . two pathologists blinded to the patients ' clinical data interpreted all immunostained slides , and cases with discrepant scores were reevaluated to achieve a consensus score . comparisons between groups were performed using the student 's t - tests for continuous data . univariate and multivariate analyses were used to estimate the influence of p2x7r expression on clinicopathological parameters . chicago , il , usa ) was used for all statistical analyses , and a p<.05 was considered statistically significant . this study was conducted using formalin - fixed , paraffin - embedded tissue samples obtained from 170 ptc patients who underwent thyroid surgery at kangdong sacred heart hospital between january 2006 and december 2007 . among the 170 patients , 84 had available data including the preoperative serum thyroid autoantibodies and pathology reports for ht . ht was diagnosed on the basis of histological findings of diffuse lymphoplasmacytic infiltration with germinal centers , parenchymal atrophy with oncocytic change , and variable amounts of stromal fibrosis throughout the thyroid gland . lymphovascular invasion was assessed with light microscopy and defined as tumor cells that were present within a vascular space with identification of endothelial lining.15 after histological review , ptc cases were categorized into two groups , ptc with ht ( n=84 ) or ptc without ht ( n=86 ) . clinical information including age , sex , treatment modality , and survival or recurrence was adapted from medical records and radiologic findings and then analyzed . all glass slides from 170 patients with ptc were reviewed by two pathologists for diagnosis confirmation and selection of a representative section for immunohistochemical study . diagnosis and histologic differentiation were evaluated according to the world health organization classification , and tumor staging was based on the american joint committee on cancer updated tumor - node - metastasis cancer staging system . this study was approved by institutional ethics committee of kangdong sacred heart hospital seoul , korea . a circle was drawn on the slide around the most representative area . using the slide as a guide , core samples were obtained from each paraffin - embedded block using a tissue microarray tool ( quick - ray , unitma , seoul , korea ) . a punch size nine cores were embedded in each block in 33 arrangements . in total , 19 tissue microarray blocks were produced from the 170 tumor samples . the 4-m thick tissue sections were deparaffinized using ez prep solution . cc1 standard ( ph 8.4 buffer contained tris / borate / ethylenediaminetetraacetic acid ) was used for antigen retrieval . dab inhibitor ( 3% h2o2 endogenous peroxidase ) was blocked for 4 minutes at 37. slides were incubated with anti - p2x7r antibodies ( 1:300 , goat igg , abcam , cambridge , uk ) for 40 minutes at 37 , and then incubated with a secondary antibody ( universal hrp multimer , ventana medical systems , melbourne , vic , australia ) for 8 minutes at 37. after incubation , slides were stained with the dab h2o2 substrate for 8 minutes , followed by hematoxylin and bluing reagent counterstaining at 37. a reaction buffer ( ph 7.6 , tris buffer ) was used as a washing solution . both the intensity of immunohistochemical staining and the proportion of stained tumor cells were semi - quantitatively evaluated . the staining intensity was scored as follows : 0 , negative ; 1 , weak ; 2 , moderate ; and 3 , strong . staining proportion was rated according to the percentage of positive cells and scored as follows : 0 , less than 10% ; 1 , 11% to 25% ; 2 , 26% to 75% ; 3 , more than 75% . the scores of staining intensity and proportion were multiplied to produce a weighted immunoreactive score ( 0 - 6 ) . cases with a score 3 were considered high expression and those with a score 2 were defined as low expression . two pathologists blinded to the patients ' clinical data interpreted all immunostained slides , and cases with discrepant scores were reevaluated to achieve a consensus score . comparisons between groups were performed using the student 's t - tests for continuous data . univariate and multivariate analyses were used to estimate the influence of p2x7r expression on clinicopathological parameters . chicago , il , usa ) was used for all statistical analyses , and a p<.05 was considered statistically significant . patients consisted of 18 men and 152 women with an age range of 26 to 76 years ( mean , 47.511.7 years ) . the 170 cases consisted of 150 conventional ptc ( 88.2% ) and 20 variants ( 11.8% ) , including follicular variants ( n=16 ) , oncocytic variants ( n=3 ) , and a diffuse sclerosing variant ( n=1 ) . extrathyroid extension and lymphovascular invasion were identified in 45.9% ( 78/170 ) and 48.8% ( 83/170 ) of cases , respectively . the clinical and pathological characteristics according to the presence of ht are summarized in table 1 . ptc patients with ht were more likely to be women ( p=.01 ) , with less lymphovascular invasion ( p<.001 ) and extrathyroid extension ( p<.001 ) . less lymph node metastasis ( p<.001 ) was evident among the ptc with ht group compared to the ptc without ht group . there were no statistical differences in terms of age , histological variant , tumor size , or tumor multifocality between the ptc with ht and ptc without ht groups . of the 84 patients with ht , only one patient ( 1.2% ) had recurrence during a mean follow - up of 64.311.1 months , whereas six ( 7% ) patients without ht had recurrence during a mean follow - up of 64.88.6 months . lower recurrence ( p=.026 ) was noted in the ptc with ht group , however , this observation is not mentioned in the table because of the low number of patients . two patients expired during the follow - up period , but their cause of death was not clear ; hence , they were excluded . the 170 patients consisted of 90 having high p2x7r expression and 80 having low p2x7r expression . of the 90 patients with high p2x7r expression , 66 patients had ptc only ( 73.3% ) and 24 patients had ptc with ht ( 26.7% ) . high p2x7r expression was significantly associated with lymphovascular invasion ( p<.001 ) , extrathyroid extension ( p<.001 ) , and lymph node metastasis ( p<.001 ) . however , tumor size , tumor multifocality , and histological variant were not statistically associated with p2x7r expression ( table 2 ) . table 3 shows results of the multivariate logistic analysis investigating factors that potentially affect p2x7r expression . no significant association between p2x7r expression and age ( odds ratio [ or ] , 1.42 ; 95% confidence interval [ ci ] , 0.63 to 3.21 ; p=.40 ) , gender ( or , 0.75 ; 95% ci , 0.2 to 2.8 ; p=.67 ) , tumor multifocality ( or , 1.15 ; 95% ci , 0.31 to 3.99 ; p=.87 ) , lymphovascular invasion ( or , 1.31 ; 95% ci , 0.48 to 3.61 ; p=.59 ) , or lymph node metastasis ( or , 1.81 ; 95% ci , 0.71 to 4.6 ; p=.21 ) was found . however , there was a significant association between high p2x7r expression and extrathyroid extension ( or , 3.16 ; 95% ci , 1.31 to 7.6 ; p=.01 ) . high p2x7r expression was significantly associated with an absence of ht ( or , 5.43 ; 95% ci , 2.45 to 12 ; p<.001 ) . in the group with coexisting ht , p2x7r expression was significantly higher in patients with tumor multifocality ( p=.05 ) , lymphovascular invasion ( p<.001 ) , and extrathyroid extension ( p<.001 ) ( table 4 ) . for the ptc without ht group , p2x7r expression was significantly higher in women ( p<.001 ) and the tumor multifocality group ( p<.001 ) ( table 5 ) . patients consisted of 18 men and 152 women with an age range of 26 to 76 years ( mean , 47.511.7 years ) . the 170 cases consisted of 150 conventional ptc ( 88.2% ) and 20 variants ( 11.8% ) , including follicular variants ( n=16 ) , oncocytic variants ( n=3 ) , and a diffuse sclerosing variant ( n=1 ) . extrathyroid extension and lymphovascular invasion were identified in 45.9% ( 78/170 ) and 48.8% ( 83/170 ) of cases , respectively . the clinical and pathological characteristics according to the presence of ht are summarized in table 1 . ptc patients with ht were more likely to be women ( p=.01 ) , with less lymphovascular invasion ( p<.001 ) and extrathyroid extension ( p<.001 ) . less lymph node metastasis ( p<.001 ) was evident among the ptc with ht group compared to the ptc without ht group . there were no statistical differences in terms of age , histological variant , tumor size , or tumor multifocality between the ptc with ht and ptc without ht groups . of the 84 patients with ht , only one patient ( 1.2% ) had recurrence during a mean follow - up of 64.311.1 months , whereas six ( 7% ) patients without ht had recurrence during a mean follow - up of 64.88.6 months . lower recurrence ( p=.026 ) was noted in the ptc with ht group , however , this observation is not mentioned in the table because of the low number of patients . two patients expired during the follow - up period , but their cause of death was not clear ; hence , they were excluded . the 170 patients consisted of 90 having high p2x7r expression and 80 having low p2x7r expression . of the 90 patients with high p2x7r expression , 66 patients had ptc only ( 73.3% ) and 24 patients had ptc with ht ( 26.7% ) . high p2x7r expression was significantly associated with lymphovascular invasion ( p<.001 ) , extrathyroid extension ( p<.001 ) , and lymph node metastasis ( p<.001 ) . however , tumor size , tumor multifocality , and histological variant were not statistically associated with p2x7r expression ( table 2 ) . table 3 shows results of the multivariate logistic analysis investigating factors that potentially affect p2x7r expression . no significant association between p2x7r expression and age ( odds ratio [ or ] , 1.42 ; 95% confidence interval [ ci ] , 0.63 to 3.21 ; p=.40 ) , gender ( or , 0.75 ; 95% ci , 0.2 to 2.8 ; p=.67 ) , tumor multifocality ( or , 1.15 ; 95% ci , 0.31 to 3.99 ; p=.87 ) , lymphovascular invasion ( or , 1.31 ; 95% ci , 0.48 to 3.61 ; p=.59 ) , or lymph node metastasis ( or , 1.81 ; 95% ci , 0.71 to 4.6 ; p=.21 ) was found . however , there was a significant association between high p2x7r expression and extrathyroid extension ( or , 3.16 ; 95% ci , 1.31 to 7.6 ; p=.01 ) . high p2x7r expression was significantly associated with an absence of ht ( or , 5.43 ; 95% ci , 2.45 to 12 ; p<.001 ) . in the group with coexisting ht , p2x7r expression was significantly higher in patients with tumor multifocality ( p=.05 ) , lymphovascular invasion ( p<.001 ) , and extrathyroid extension ( p<.001 ) ( table 4 ) . for the ptc without ht group , p2x7r expression was significantly higher in women ( p<.001 ) and the tumor multifocality group ( p<.001 ) ( table 5 ) . in the present study , we found that ptc with ht correlated with more favorable biological characteristics than ptc without ht . in ptc , the absence of ht was associated with high frequencies of female patients , extrathyroid extension , lymph node metastasis , lymphovascular invasion , and frequent recurrences . similarly , the presence of autoimmune thyroiditis in thyroid cancer has been correlated with good prognosis . recent meta - analysis demonstrated that ptcs with coexisting ht are strongly associated with female patients , tumor multifocality , the absence of extrathyroidal extension , absence of lymph node metastasis , and high recurrence - free survival rates.16 kim et al.7 also suggested that ptc coexisting with ht may protect against central lymph node metastasis . huang et al.17 reported that coexisting ht with either ptc or follicular thyroid carcinoma is linked with improved clinical stage and favorable prognosis . lymphocytic infiltrates in thyroid cancer contain cytotoxic t lymphocytes , and fas - mediated apoptosis is the major mechanism by which cytotoxic t lymphocytes cause target cell lysis.3 in addition , interleukin-1 , secreted by infiltrating lymphocytes , inhibits human thyroid carcinoma cell growth.18 only three articles have been published on the possible link between thyroid cancer and p2x7r expression.13,14,19 in vitro study has shown that thyroid papillary carcinoma cell lines express high levels of p2x7r.13 gu et al.14 suggested that p2x7r expression is associated with lymph node metastasis in ptcs . in their logistic regression analysis , p2x7r expression , tumor size , and capsular invasion are predictors for lymph node metastasis , suggesting that p2x7r expression may predict the aggressiveness of ptc.14 however , these studies have not demonstrated the association between p2x7r expression and ptc with ht . in the present study , ptc with high p2x7r expression showed significantly higher frequencies of lymphovascular invasion , extrathyroid extension , lymph node metastasis , and absence of ht . in the multivariate analysis , high p2x7r expression was independently associated with the absence of ht and the presence of extrathyroid extension . our results suggested that p2x7r expression in ptc correlates with poor prognostic factors . in the ptc with ht group , the expression of p2x7r was significantly higher in tumor multifocality , lymphovascular invasion , and extrathyroid extension . as for the ptc without ht group , the expression of p2x7r was significantly higher in females and those with tumor multifocality . these results may imply that a different mechanism of p2x7r expression may be involved according to coexistence of ht . recently , beynon et al.20 reported that activated memory t - cells primed by interferon- suppress the activation of monocytes by inhibiting p2x7r - mediated signaling , indicating that p2x7r expression in ht may be associated with activated t lymphocytes of ht . in conclusion , the occurrence of ptc in ht individuals may predict a favorable tumor behavior such as less tumor multifocality , lymphovascular invasion , and extrathyroid extension , compared to those having ptc without ht . p2x7r expression in ptc was correlated with poor prognostic factors and the absence of ht .

backgroundthis study was aimed at investigating the relation of p2x7 receptor ( p2x7r ) expression with the clinicopathological features of papillary thyroid carcinoma ( ptc ) coexisting with hashimoto 's thyroiditis ( ht).methodswe examined 170 patients ( 84 , ptc with ht ; 86 , ptc without ht ) . p2x7r expression was examined by immunohistochemical methods . the staining intensity and patterns were evaluated and scored using a semi - quantitative method.resultsthe ptc with ht group was more likely to contain women and had less extrathyroid extension , lymph node ( ln ) metastasis , lymphovascular invasion , and recurrence than the ptc without ht group . patients positive for p2x7r had significantly higher frequencies of lymphovascular invasion , extrathyroid extension , ln metastasis , and absence of ht . as shown by multivariate analysis , the expression of p2x7r was significantly higher if ht was absent and extrathyroid extension was present . in the ptc with ht group , the expression of p2x7r was significantly higher in patients with tumor multifocality , lymphovascular invasion , and extrathyroid extension . in the ptc without ht group , the expression of p2x7r was significantly higher in women and those having tumor multifocality.conclusionscoexistence of ptc with ht is associated with good prognostic factors , and p2x7r expression in ptc was correlated with poor prognostic factors and the absence of ht .

the centers for disease control and prevention recommend screening all pregnant women for chlamydia trachomatis infection and syphilis at the first prenatal visit . repeated screening for these infections during the third trimester is recommended for those women at increased risk for contracting these infections . screening for gonorrhea once or twice during pregnancy is recommended for those women at risk . most states have laws requiring that women undergo screening for syphilis at least once during pregnancy . nine states , including florida , have statutory requirements that pregnant women undergo screening for syphilis both at the first visit and again in the third trimester . at least in florida , there is no similar law requiring prenatal screening either for gonorrhea or chlamydia trachomatis infection . screening for sexually transmitted diseases ( std ) during pregnancy has been advocated as being cost - effective as long as the prevalence exceeds 1% . the reported prevalence of gonorrhea during pregnancy varies widely , with rates varying between 0 and 10% , depending on the risk status of patients [ 46 ] . the prevalence of chlamydia trachomatis infection during pregnancy is higher than that of gonorrhea [ 7 , 8 ] . syphilis is the least common of the sexually transmitted infections that are endemic to the united states . in 2003 , cases of primary and secondary syphilis occurred at a rate of 2.5 cases per 100 000 population . since reaching a nadir in 1999 , the number of cases has increased slightly over the past few years . however , this increase has been confined to men who have sex with men . we sought to evaluate the prevalence of syphilis in the obstetric population delivering at shands hospital at the university of florida . by doing so , we wanted to evaluate the utility of repeating screening tests for syphilis during the third trimester . we performed a historical cohort analysis of all women delivering at shands hospital at the university of florida from 1 july 2003 to 30 june 2004 . for consideration of screening for syphilis at the first prenatal visit , all women who attended at least one prenatal visit were included . for consideration of the utility of repeating the screening in the third trimester , women were included in the cohort if they began prenatal care prior to 27-weeks gestation and delivered after 32 weeks , so as to allow a minimum of 6 weeks between first - visit testing and delivery . screening for syphilis utilized nontreponemal tests of serum samples confirmed with treponemal tests . screening for gonorrhea and chlamydia trachomatis infection the exact types of tests and manufacturers varied , depending on the payer - specified laboratory for specific patients . subjects meeting criteria for inclusion in the cohort were identified from the delivery log maintained in the labor and delivery unit at shands hospital at the university of florida and from the database maintained by the division of maternal - fetal medicine at the university of florida . medical record charts of these women were abstracted , and data were entered into a relational database ( access 2000 , microsoft corporation , redmond , wash ) . sas version 9.0 ( sas institute , cary , nc ) was utilized for statistical analysis . rates of screening for syphilis at the first prenatal visit and again in the third trimester were calculated . the prevalence of infection with 95% confidence intervals ( cis ) ( modified wald method ) was calculated . we anticipated that the inclusion of one year of delivered patients would yield a cohort of over 1000 patients undergoing repeated third - trimester screening , allowing 95% confidence intervals of less than 3% . categorical data were analyzed with the uncorrected chi - square and fisher 's exact tests as appropriate . all tests of statistical significance were two - tailed and utilized an alpha of 0.05 . during the study interval , 2244 women delivered at our hospital , and 2085 charts were available for review . of the 1962 women who had at least one prenatal care visit , the mean age was 25.8 ( standard deviation ( sd ) = 6.3 ) years . fifty seven percent of women were white , 25% were black , and 18% were of other ethnicities . sixty two percent of women were parous , 64% were unmarried , 75% had either government - sponsored or self-payer status , and 18% reported a prior history of at least one sexually transmitted infection ( gonorrhea , chlamydia , syphilis , genital herpes , trichomoniasis , or hiv ) . the first prenatal visit for these patients occurred at a mean gestational age of 13.7 ( sd = 6.9 ) weeks . of the 1962 women who had at least one prenatal care visit , 1724 ( 87.9% ) were screened for gonorrhea and chlamydia trachomatis infection at the first prenatal care visit . of these women , 19 ( 1.1% ) had gonorrhea and 107 ( 6.2% ) had a chlamydia trachomatis infection . of the 1962 women who had at least one prenatal care visit , 1940 ( 98.9% ) were screened for syphilis at the first prenatal care visit . no cases of syphilis were identified with this initial screening ( 95% confidence interval ( ci ) = 0,0.24% ) . of the 1627 women beginning prenatal care prior to 27 weeks and delivering after 32 weeks similar to the results of screening at the first prenatal visit , no cases of syphilis were identified with third trimester screening ( 95% ci = 0,0.34% ) . our study is limited by its retrospective nature . also , these data are from a single center during a single year , and they may not be generalizable to other obstetric populations . however , the prevalence rates of gonorrhea and chlamydia trachomatis infection in our population are similar to those in other high - risk clinic populations . therefore , we believe that it is unlikely that the prevalence of syphilis in our population is significantly lower than it would be in the majority of practice settings in the united states . in this study , compliance with our state 's legal requirement for screening for syphilis at the first visit was excellent ( 98.9% ) . others have reported similarly high first - visit screening rates for syphilis and for other tests that are included in some sort of prenatal battery . compliance with the legal requirement for repeating screening in the third trimester was lower ( 84.6% ) . reasons for this lower rate of screening than at the first visit likely include patient refusal and provider apathy due to the rarity of the infection . the primary goal of screening pregnant women for syphilis is to prevent cases of congenital syphilis . currently , the total number of cases in the united states each year is approximately 400 . as with the rate of syphilis in women , the incidence of syphilis in newborns also is decreasing . most counties in our state and others rarely report a case of primary or secondary syphilis in adults , much less a case of congenital syphilis . clearly , because of the untoward consequences of congenital syphilis , screening women once during pregnancy for this infection is reasonable and well justified . however , we believe that the statutory requirement , as currently exists in florida and 8 other states , to screen all women twice during pregnancy for an infection that is as rare as syphilis currently should be reevaluated .

objectives . our aim is evaluating the need for repeating tests for syphilis on pregnant women in the third trimester . study design . a single - center retrospective cohort study was performed on all women delivering 7/036/04 . results . during the study interval , 2244 women delivered at our hospital . of those women having available records and attending at least one prenatal visit , 1940 ( 98.9% ) were screened for syphilis at the first prenatal visit . of the 1627 women beginning prenatal care prior to 27 weeks and delivering after 32 weeks , 1377 ( 84.6% ) were rescreened in the third trimester . no cases of syphilis were identified with either the initial ( upper limit of 95% ci 0.24% ) or repeat ( upper limit of 95% ci 0.34% ) screening . conclusions . in our obstetric population , syphilis is so uncommon that mandated prenatal screening on more than one occasion seems unjustified and laws requiring repeated screening should be reevaluated .

fibrous dysplasia of bone is characterized by the replacement of medullary bone with fibro - osseous tissue . this results in the distortion and overgrowth of bone , leading to characteristic deformities . based on the bony involvement , fibrous dysplasia is divided into three types : monostotic , polyostotic , and mccune albright syndrome ( mas ) . mas is characterized by a triad of monostotic / polyostotic fibrous dysplasia , caf-au - lait macules ( calms ) , and endocrine hyperfunction . the disease is congenital but not hereditary and is due to an activating mutation in g protein - coupled receptor . it manifests usually in childhood , with recurrent fractures , distorted bones , precocious puberty , acromegaly , or hyperthyroidism . untreated disease leads to permanent disfigurement , and the disease is classified as paucibacillary or multibacillary depending on the proliferation of the bacilli . the risk factors for the disease are living in endemic areas with poor hygiene conditions and defects in cell - mediated immunity . we recently encountered a patient with mas who presented with cutaneous features of hansen 's disease . we report the same for the unusual association and to highlight the relevant review of the literature . a 30-year - old man presented with hypoesthetic , depigmented patches over the skin of the chest and back of 1 year duration . he gave history of recurrent fractures since the age of 4 years with shortening of right leg . the parents gave history of hyperpigmented macules over the trunk since birth and denied similar features in family members . the patient attained puberty at the age of 14 years and denies history suggestive of acral enlargement , thyrotoxicosis , cushing 's syndrome , and swellings in any part of the body . examination revealed ( height 152 cm ) asymmetry of the face with expansion of right maxilla and lower jaw and short right leg ( 82 vs 86 cm ) [ figures 1 and 2 ] . cutaneous examination revealed a large calm over the back , extending across the midline and multiple anesthetic patches over scapula and lower back [ figure 3 ] . there was no evidence of goiter , acromegaloid features , and thickened nerves , and the rest of the examination was normal . facial photograph showing craniofacial dysplasia legs showing short right leg ( arrow ) with expansile lesion of tibia ( arrow head ) skin over the back showing a large calm crossing midline ( long arrows ) with depigmented hypoesthetic patches ( short arrow ) hormonal profile revealed normal thyroid adrenal and gonadal axes evaluation . skeletal survey revealed expansile osteolytic lesion over right maxilla , right hip , and shoulder . skin biopsy specimens were obtained from ear lobes , legs , and depigmented lesions over the back , avoiding areas of calm . specimens revealed the presence of acid - fast bacilli along with noncaseating granulomas consistent with the diagnosis of tuberculoid leprosy . the diagnosis of fibrous dysplasia is made by the presence of bone pains , recurrent fractures , deformity of bone , and radiological evidence of fibro - osseous tissue showing expansile lytic lesion . biopsy and tc - mdp scan of the bone were not considered necessary to establish the diagnosis in this classical case . other laboratory workup showed elevated alkaline phosphatase ( 388 u / l ) with normal calcium , phosphorus , parathyroid hormone , and 25-hydroxy vitamin d levels . he was treated with triple drug regimen for leprosy and also given oral calcium carbonate 1.5 g daily along with the first dose of zoledronic acid 4 mg as an intravenous infusion . mas is characterized by the triad of polyostotic fibrous dysplasia , calm , and endocrinopathy . the diagnosis of fibrous dysplasia is based on classical radiological findings supported by histopathology and bone scan . hyperfunctioning of endocrine glands in mas presents with precocious puberty , hyperthyroidism , acromegaly , or cushing 's syndrome . our patient had only two features of the triad and did not have any evidence of endocrine hyperfunction syndromes . the calm is unusual in our patient in that it crossed the midline and was seen on both sides of the body . the calms in mas do not cross the midline and follow the developmental lines of blashko , thus differentiating from those in neurofibromatosis . our patient had an exceptionally large calm that crossed the midline interspersed with anesthetic depigmented macular lesions of hansen 's disease . hansen 's disease is caused by mycobacterium leprae , and the disease spreads via respiratory droplets between close personal contacts over a prolonged duration . immunocompromised situations are the predominant risk factors for the disease , and defects in cell - mediated immunity are implicated in persons with genetic predisposition . the mode of acquisition of hansen 's disease in our patient is possibly due to frequent stay in the hospitals due to multiple fractures and contact with a patient . screening of the persons involved in personal care of the patient did not reveal features of hansen 's disease . the association of hansen 's disease with mas appears coincidental as there is no evidence that mas predisposes to the infectious diseases or leads to immunosuppressed state . extensive literature search did not reveal any previous reports of association between hansen 's disease and mas . they are given as intravenous or oral preparation and act by suppressing the osteoclast activation . treatment of mas involves bisphosphonate therapy to reduce fracture rate apart from treating any associated endocrinopathy . to conclude , we present an interesting case of mas without associated endocrinopathy who had coexisting hansen 's disease . this unusual combination was not reported earlier in world literature .

mccune albright syndrome ( mas ) comprises a triad of fibrous dysplasia of bone , caf - au - lait macule , and endocrinopathy . the disease is due to activating mutation of g protein - coupled receptor leading to hyperfunction of glands . hansen 's disease is caused by infection with mycobacterium leprae and is seen with underlying immunosuppressed conditions in genetically predisposed individuals . we recently encountered a patient with hansen 's disease along with underlying mas and report the same in this report .

the growth of gene and genomic databases provides motivation for developing tools to extract information about the function of a protein from sequence data , with the ultimate goal of understanding the collection of functions represented in an organism 's genome . work on molecular evolution over 30 years has shown that such questions must be phrased carefully , and always with cognizance of the darwinian paradigm that insists that the only way of obtaining functional behavior in living systems is through natural selection superimposed on random variation in structure . a behavior is functional if the organism would be less able to survive and reproduce if that behavior were different . an amino - acid residue is functional if , upon mutation , the organism is less able to survive and reproduce . a long literature has sought to interpret the evolutionary behavior of protein sequences , in the hope of drawing inferences about the relationship between fitness and sequence . what has emerged is the recognition that a family of orthologous proteins displays a diversity of structure and a corresponding diversity in behavior , where some of the behavioral differences have a strong impact on fitness ( are functional ) , and others are neutral ( or nearly so ) . without resolving , in a general way , questions regarding the relationship ( neutrality versus selection ) between fitness and protein sequence , we can build interpretive tools that capture information from patterns of evolution of genomic sequences that is informative about function - in particular , events that are characterized by the biological scientist as a change in function . for a protein to change its function , it must change its behavior ; this in turn requires that it change its amino - acid sequence . a protein being recruited for a different function over a very short time ( geologically speaking ) frequently experiences an episode of rapid sequence evolution , an episode where the number of amino - acid substitutions per unit time is large . therefore , molecular evolutionists have long been interested in the rates at which substitutions accumulate in protein sequences . these rates are known to vary widely in different protein families . calculating rates in the units of substitutions / time requires knowledge of the geological dates of divergence of protein sequences . because geological times are frequently not known ( and almost never known precisely ) , alternative approaches for identifying episodes of rapid sequence evolution have been sought . the number of nucleotide substitutions that change the sequence of the encoded protein ( nonsynonymous substitution ) by the number of nucleotide substitutions that do not change the sequence of the encoded protein ( synonymous substitution ) , and then normalizes these for the number of nonsynonymous and synonymous sites . this is the ka / ks rate ratio [ 3 , 4 , 5 ] . high ka / ks rate ratios for reconstructed ancestral episodes of sequence evolution are hypothesized to be signatures of positive adaptation , and have been associated with significant change in function [ 6 , 7 ] . in general , ka / ks values are low . for example , the average ka / ks value in proteins between rodents and primates is 0.2 . this is taken to indicate that most of these proteins , selected over millions of years , attained an optimum function prior to the divergence of rodents and primates . this implies that subsequent evolution was conservative ; most nonsynonymous mutations were detrimental to the fitness of the organism . functional change can be defined as mutation that alters organismal fitness and is subject to selective pressure . for an example of intraspecific variation , for example , the hemoglobin in the bar - headed goose has undergone adaptive change relative to the hemoglobin from the closely related greylag goose in response to a reduced partial pressure of oxygen at high altitudes . adaptive evolution is also believed to be displayed by paralogous mammalian mhc class i genes and relate to a birth - and - death model of gene duplication . traditionally , positive selection is defined by a ka / ks rate ratio significantly greater than unity . while 0.6 < ka / ks < 1 can occur by relaxation of functional constraint , the theoretical cut - off of 1 is well known to miss significant functional changes in proteins for several reasons . long branches can dilute an episode of positive adaptation ( with ka / ks > 1 ) with episodes of conservative evolution . ka / ks values can miss positive selective pressures on individual amino acids because they average events over the entire protein sequence . behavior in a protein can change significantly if only a few amino acids change while the remainder of the sequence is conserved in order to retain core behaviors of the old and new functions ( for example , the protein fold ) . these adaptive events will only be detected on sufficiently short branches which pinpoint the adaptive change . alternative ways of identifying ka / ks values below unity that are suggestive of adaptive evolution involve comparison of these values for an individual branch of a tree with those values for branches in the tree generally . if one branch has a ka / ks value far outside of the norm for the family ( but still below 1 ) , we can guess that this branch represents an episode of positive selection . this will work for gene families that generally display conservative evolution ( such as the sh2 ( src homology 2 ) domains ) , but not for others . for example , many immune - system genes show a much more continuous distribution of values , which may indicate that they are perpetually under different amounts of positive selective pressure . in this case , the designation of a cut - off value of ka / ks , below which two homologous genes have the same function , and above which they have different functions , is arbitrary . ultimately , this level should be determined by benchmarking adaptivity with specific functions and specific protein folds . ka / ks rate ratios are well known to be useful starting points for generating stories about the interaction between protein sequences and the darwinian processes that shape these sequences . these stories help us understand how these sequences contribute to the fitness of the host . this means that biologists would find useful a comprehensive database of examples where ka / ks values are high . most useful would be a database that presents families where ka / ks is greater than 1 , and a separate family where ka / ks is greater than some arbitrary cut - off less than 1 , but still relatively high compared to the average value in the average protein . taed is designed to provide , in raw form , evolutionary episodes in specific chordate and embryophyte ( flowering plants , conifers , ferns , mosses and liverworts ) protein families that might be candidates for adaptive evolution . taed contains a collection of protein families where at least one branch in the reconstructed molecular record has a ka / ks value greater than unity , or greater than 0.6 . the second cut - off is arbitrary , chosen to be high relative to the average ka / ks value for the average episode of evolution in a protein family . empirically , the lower cut - off seems to admit additional examples of gene families that might have undergone adaptive evolution . taed should be used as a raw list of potentially adaptively evolving genes for experimentalists seeking gene families to study in further detail , and for bioinformaticists interested in studying large datasets of examples of genes with high ka / ks rate ratios . the master catalog is a database of 26,843 families of protein modules generated from an all - against - all search of genbank release 113 . a protein is broken into independently evolving modules on the criterion of the presence of a subsection of a gene as a complete open reading frame in another species . pairs that were within 180 pam ( point accepted mutation ) units with a minimum length requirement were grouped into the same family . this database was the starting point for the exhaustive calculation of ka / ks rate ratios . the master catalog is different , both in concept and execution , from other resources ( for example hovergen pfam , and cogs ) that offer databases of protein families . the master catalog incorporates reconstructed ancestral states within its data structure , in addition to multiple sequence alignments ( msas ) and evolutionary trees . having these reconstructed ancestral states provides a value to the database , especially for functional interpretation , that is not offered by databases that contain only trees , or only multiple sequence alignments , or only trees and multiple sequence alignments . further , because the master catalog is explicitly developed as a tool for doing functional genomics relying on reconstructed intermediates , and as the information about function is extracted from analysis of patterns of variation and conservation in genes and proteins within a family , it emphasizes the generation of high - quality trees , msas , and reconstructed ancestral states . for this reason , instead , it constructs nuclear families , where the trees , msas , and ancestral states are not compromised by poor gap placement , a common problem in clustal - based multiple sequence alignments of sets of highly divergent protein sequences . whereas maximum likelihood methodologies perform better in some situations , they are too computationally intensive to apply exhaustively . further , they are based upon an explicit model of evolution that may not be appropriate along all branches analyzed , a situation where maximum parsimony may outperform maximum likelihood on some branches . therefore , to generate the initial version of this database , first , we were concerned that silent positions would be ' saturated ' with substitutions , rendering the ks measurements meaningless . whereas reconstruction back to the last common ancestor of chordates or embryophytes with no intermediates frequently bears the signature of synonymous position equilibration , synonymous position saturation can be avoided if individual branches are shorter than the period required for saturation to occur ( t1/2 to saturation of approximately 120 million years ) . saturation was measured through the examination of the extent to which twofold redundant codon systems had reached equilibration . branches that showed equilibration greater than five half - lives towards saturation were excluded from taed on the basis of differences between reconstructed ancestral sequences at the beginning of branches and sequences at the end . a second significant problem is that of short branches bearing fractional mutations . these are known to generate ka / ks values with large errors . to prevent these errors from biasing the database , a new simple robustness test was implemented to ensure that an ' interesting ' ka / ks value ( one above the cut - off ) was not recorded in the database if it became ' uninteresting ' ( below the cut - off ) through the shift of a single mutation reconstructed in the branch . the test modified the ka / ks calculation in a simple way , as described below : modified ka / ks = kamod / ksmod kamod = ( number of nonsynonymous - 1)/total nonsynonymous sites ksmod = ( number of synonymous + 1)/total synonymous sites in general , the smaller the difference between ka / ks and kamod / ksmod , the more significant or robust the branch . to exclude short branches with fractional mutations ( arising through ambiguous ancestral sequence reconstruction ) without excluding other short branches , branches with kamod / ksmod values below 0.5 were excluded from taed . of 5,305 families of modules containing chordate proteins , 280 contained at least one branch with a ka / ks value greater than 1 , representing 643 branches emanating from 63 different nodes of the tree of life . some 778 families had at least one branch with a ka / ks value greater than 0.6 , totaling 2,232 branches emanating from 92 nodes of the tree of life . thus 15% of all families of chordate modules are likely to have modified their function at least once during the course of evolution . of 3,385 families of modules representing embryophyte proteins , 123 have at least one branch with a ka / ks value greater than 1 , representing 228 families emanating from 25 nodes . some 407 families had at least one branch with a ka / ks value greater than 0.6 , totaling 1,105 branches from 43 nodes . here , perhaps 12% of all embryophyte families have modified their function along at least one branch . this result based on ancestral sequence reconstruction contrasts greatly with the result of endo , ikeo and gojobori , where the search for gene families undergoing adaptive evolution yielded only two families . they compared extant sequences rather than reconstructed evolutionary intermediates , counted families only where a majority of the pairs were at high ka / ks values , and used a smaller database . a list of candidate protein module families that have undergone modification of function is available at . the version described here is designated taed 2.1 and will remain available at this site . as more sophisticated methods are developed and applied , as correlations with functional and structural databases are pursued , and as data from other types of evolution beyond coding sequence evolution is added , links to these datasets will be provided . taed 2.1 contains two image - mapped trees ( for chordates and embryophytes ) , where the node that an adaptive branch emanates from can be clicked on to obtain a list and master catalog reference number . multiple sequence alignments and phylogenetic trees corresponding to these entries can be obtained from eragen biosciences . this study represents the first comprehensive analysis of ka / ks rate ratios throughout the chordata and the embryophyta . although the methods utilized were rough and designed to give a quick snapshot into a global picture of evolution , the taed , as a raw resource , should be valuable in the analysis of much of chordate evolution . functional genomics analyses of many of the protein families that have suffered recruitment and functional change within the past 500 million years will soon emerge . many of the episodes of functional change recorded in taed can be correlated with events in the geological or paleontological record , in response to changing environments , evolving paleoecology or the development of new physiology . gene families may display evolutionary episodes with high ka / ks values , and therefore appear within taed , for several possible reasons . for example , branches resulting from gene duplication events that give rise to paralogs with very different behaviors will presumably have high ka / ks values , as will orthologous pairs from species that place very different demands on their function . this search was done without distinguishing paralogs from orthologs , and the user of taed should be careful in the analysis of specific families in recognition of this fact . because there is no reliable true set of protein families ' known ' to have suffered functional adaptation , it is important to remember that a darwinian definition of function differs from the functional annotation of genomes , and it is possible for a protein to alter or change its function while retaining the same annotation . to examine this dataset , specific proteins must be examined individually . many protein families already believed to be candidates for functional recruitment appear on the list . these include plasminogen activator in vampire bats which is expressed in saliva and involved in blood clotting , phospholipase a2 in snakes which is expressed in venom and involved in tissue damage and mhc proteins in mammals , which are involved in the immune system as part of the host - parasite arms race , all having obvious explanations of why they may have suffered functional change . several families are newly identified as being candidates for functional change , such as the previously proposed obesity protein leptin in primates . a third category of discovery in taed is in the detection of episodes of adaptive change at new points in the divergent evolution of proteins , for example myostatin in the bovidae . these are the candidate genes that were identified as showing rapid sequence evolution emanating from this node in the tree of life . they potentially include orthologs between two species of bovids , paralogs , alternatively spliced transcripts and intraspecific evolution . the genes on the list have roles in the immune system , body musculature and reproduction , traits frequently under selective pressure . these examples and many others are candidates for further experimental study through cloning from additional species and through functional study in laboratories expert in the particular protein . a sample listing from taed indicating candidate adaptively evolving genes detected that emanated from the bovidae node these examples potentially include orthologs between different species of bovidae , paralogs , alternatively spliced cdnas with potentially different functional effects and intraspecific modifications . from a phylogenetic perspective , the knowledge of candidate genes evolving at the same time in the same organism can allow one to begin to ask if entire pathways or phenotypic functions are under selective pressure at particular points in evolutionary history . where tertiary structures for the proteins exist , mutations along branches can be mapped onto three - dimensional structures first to evaluate the validity of specific examples , and second , to understand the nature of adaptive evolution at a structural level . one analysis of taed indicates that among branches with ka / ks rate ratios > 1 , only 3% of synonymous sites had mutated compared with 10% on the average branch in the database . this is consistent with the notion that episodes of adaptive evolution can be lost in long branches , as these are combined with prior and/or subsequent episodes characterized by lower ka / ks rate ratios characteristic of functional constancy . as more genes are sequenced from more species , the greater articulation of trees will not only increase the accuracy of sequence reconstructions , but will also allow us to detect new examples of functional change that are buried in long branches . at a biological level , the dataset generated here can be mined to provide global pictures of how evolution has occurred . correlation of data in this database with that in other functional databases will enable a leap from genotype to organismal phenotype . the high ka / ks rate ratio in leptin in a branch connecting primates with rodents may have been a useful predictor of change of function for pharmaceutical companies interested in the mouse model of leptin for human obesity . for the experimentalist , mutations occurring along putatively adaptive branches can be assayed for functional importance in systems of interest . finally , this database represents a growing framework for the study of adaptive evolution . as datasets become available , changes in gene expression , alternative splicing patterns , imprinting patterns , recombination events and other molecular mechanisms of adaptation will be added to this database in a phylogenetic perspective . the ultimate goal is a dynamic resource depicting candidate molecular events that are responsible for phenotypic differences between closely related species . starting with the master catalog ( version 1.1 derived from genbank release 113 ; free of charge for academic users through ) , ka / ks rate ratios were reconstructed database - wide for each ancestral branch in every evolutionary tree containing genes from the chordata and the embryophyta . this analysis was restricted to these organisms because there is less evidence for codon and gc - content biases which complicate the accurate calculation of ks . the master catalog uses multiple sequence alignments generated from clustal w and neighbor - joining trees , both derived from protein sequences . because the master catalog is based on an analysis of nuclear families , rather than extended families , these inexpensive tools generate acceptable multiple sequence alignments . ka / ks values were calculated for branches on an evolutionary tree between nodes using the method of li and pamilo and bianchi [ 3 , 4 , 5 ] modified to allow full treatment of probabilistic ancestral sequences . while reconstructed ancestral sequences contain ambiguities , using probabilistic ancestral sequences takes this into account ( by weighting ambiguous positions according to their probabilities ) and allows us to construct a model of evolutionary history that is robust . two cut - offs were used to identify ' interesting ' values for the ka / ks rate ratio , 1 and 0.6 . we are indebted to the national institutes of health ( grants hg 01729 and mh 55479 ) for partial support of this work .

backgroundthe master catalog is a collection of evolutionary families , including multiple sequence alignments , phylogenetic trees and reconstructed ancestral sequences , for all protein - sequence modules encoded by genes in genbank . it can therefore support large - scale genomic surveys , of which we present here the adaptive evolution database ( taed ) . in taed , potential examples of positive adaptation are identified by high values for the normalized ratio of nonsynonymous to synonymous nucleotide substitution rates ( ka / ks values ) on branches of an evolutionary tree between nodes representing reconstructed ancestral sequences.resultsevolutionary trees and reconstructed ancestral sequences were extracted from the master catalog for every subtree containing proteins from the chordata only or the embryophyta only . branches with high ka / ks values were identified . these represent candidate episodes in the history of the protein family when the protein may have undergone positive selection , where the mutant form conferred more fitness than the ancestral form . such episodes are frequently associated with change in function . an unexpectedly large number of families ( between 10% and 20% of those families examined ) were found to have at least one branch with high ka / ks values above arbitrarily chosen cut - offs ( 1 and 0.6 ) . most of these survived a robustness test and were collected into taed.conclusionstaed is a raw resource for bioinformaticists interested in data mining and for experimental evolutionists seeking candidate examples of adaptive evolution for further experimental study . it can be expanded to include other evolutionary information ( for example changes in gene regulation or splicing ) placed in a phylogenetic perspective .

niche partitioning in ecological communities may entail differential responses to a large number of environmental axes ( hutchinson 1957 ) . this partitioning is often considered in spatial terms , but the partitioning of resources through time can also facilitate the coexistence of ecologically similar species ( townsend et al . evidence of temporal partitioning may be found at various scales , from differing activity times within the diel cycle ( kronfeldschor and dayan 2003 ) , to interspecific variation in reproductive effort between years ( silvertown 2004 ) . however , differences in phenology are the most familiar forms of temporal partitioning within ecological communities . here , examples include sympatric mantids whose annual life cycles are staggered to allow coexistence ( hurd and eisenberg 1989 ) and temporal segregation of larval development in odonate communities to reduce competitive and predatory interactions ( crowley and johnson 1982 ) . in communities of amphibians , interspecific variation in reproductive phenology and its implications for coexistence has received some attention , particularly for species whose reproductive activity can be tracked by male advertisement calls ( wells 2007 ) . advertisement calling is exhibited by most species of frogs and toads . within species , calls convey information about the location and fitness of males to prospective partners and competitors ; among species , calls evolve distinct acoustics that act as an important premating isolation mechanism ( wells 2007 ) . however , phenological differences also represent a key source of interspecific variation in advertisement calling in anuran communities , signaling considerable variation in the timing of reproduction . in tropical environments , calling occurs primarily during the rainy season , but there may be considerable heterogeneity in the timing of calling linked to the wide variety of reproductive modes displayed . for example , among 31 species of frogs studied by gottsberger and gruber ( 2004 ) in french guiana , species could be characterized as obligate early season callers ( species that lay foam nests or display direct development ) , mid or late season callers ( species with embryonic development over water ) , sporadic and explosive callers ( species that lay eggs directly into water ) , or generalists that call right through the rainy season ( species with parental care of larvae ) . a comparable diversity of strategies has been reported for other tropical anuran communities , including species that call exclusively during the dry season ( aichinger 1987 ; donnelly and guyer 1994 ; bertoluci 1998 ; bertoluci and rodrigues 2002 ) . temperate anurans may also vary substantially in calling seasonality . among 13 species in eastern texas , ( 2006 ) documented calling seasons ranging from 2 to 12 months . in ontario , de solla et al . ( 2006 ) monitored a community of eight species and found little or no overlap in the calling seasons of several taxa . sympatric anurans can also vary markedly in their meteorological cues for calling . even among aquaticbreeding amphibians from the same community , responses to rainfall can vary considerably , from explosive initiation of calling in response to rain , to dampening or even cessation of calling in response to rain ( oseen and wassersug 2002 ; gottsberger and gruber 2004 ; saenz et al . positive and negative relationships between environmental temperature and calling activity are also known in anuran communities , with some species favoring warm conditions and others cooler conditions ( gottsberger and gruber 2004 ; steelman and dorcas 2010 ) . similarly , while the fundamental effects of relative humidity and wind strength on water balance and thermoregulation in anurans ( tracy 1976 ) suggest that the former should be positively correlated with calling activity and the latter negatively , the relationship between these variables and calling may be idiosyncratic ( oseen and wassersug 2002 ) . while it is clear that the phenology of calling can vary widely within anuran communities , it is also true that the available data on the subject are overwhelmingly derived from anuran communities in the americas ( wells 2007 ) . few , if any , studies are available for large parts of africa and eurasia . the same is true for australia . of the 241 frogs so far described in australia ( anstis 2013 ) , only nine species ( 4% ) have been the focus of dedicated phenological studies on calling and reproduction to our knowledge ( macnally 1984 ; williamson and bull 1992 ; driscoll 1998 ; brooke et al . likewise , we are aware of only seven studies of the phenology of calling across an australian frog community ( humphries 1979 ; gillespie 2001 ; morrison 2001 ; lemckert and mahony 2008 ; lemckert and grigg 2010 ; dostine et al . , we describe the phenology of advertisement calling in an anuran community from the temperate climes of southern australia , focusing on interspecific variation in both the seasonality of calling and the meteorological cues for calling . we combined data collected over 11 years to develop models of the nightly probability of calling by the focal species . as has been documented for tropical and temperate anuran communities elsewhere , our data reveal considerable interspecific variation in the seasonality of calling and the weather conditions that favor calling , which may be indicative of temporal partitioning of reproductive activity in this community . we conducted 1432 nocturnal surveys for frogs between 2001 and 2012 at 253 sites across the volcanic plains north and west of melbourne , victoria , australia . sites were distributed in the catchments of the darebin , merri , moonee ponds , and kororoit creeks . the landscape is undulating , rising to a maximum elevation of ~300 m asl at the study sites . upper catchments are dominated by grazing land , graduating to industrial and urban estates in the lower catchments . the climate is temperate , with cool winters ( july mean daily temperature range = 5.413.1c ) and warm summers ( february mean daily temperature range = 14.126.6c ) ( bom 2014 ) . rainfall averages 538 mm , with the highest monthly rainfall occurring in late winter and spring ( august sites included slow flowing pools along the main channel and tributaries of the streams listed above , as well as lentic wetlands such as farm dams , flooded quarries , swamps , and water treatment ponds . site selection was haphazard , including both randomized protocols ( mostly for selecting pools along streams ) and nonrandomized protocols that aimed to maximize geographic coverage while accounting for both access and logistical constraints ( for further details , see canessa et al . surveys were conducted between september and april ( inclusive ) in each year , encompassing the austral spring , summer , and autumn . survey timing sought to optimize detection rates of litoria raniformis in most cases ; this endangered species having been studied intensively in this region since 2001 ( heard et al . 2012a , b , 2013 , 2015 ) . however , aural surveys during the spring and early summer months optimize detection rates across the frog community in this region ( canessa and parris 2013 ) . surveys commenced at least 30 min after dark and began with a period of 510 min listening quietly for male advertisement calls . surveys extended up to 265 min , with up to 10 repeat surveys at a site in any given year . surveys were completed under permits 10001816 , 10003005 , and 10005649 issued by the victorian department of environment , land , water and planning . to supplement the above survey dataset , we collated data from 263 nocturnal surveys for frogs completed at the same study sites in the same period by private consultants , other researchers , and local naturalists . all surveys were conducted during the austral spring , summer , or autumn , and followed surveys protocols analogous to our own . survey data were gathered from the relevant research report or thesis , or through personal communications . air temperature ( dry bulb , c ) and relative humidity ( % ) were recorded during most surveys with the aid of a compact whirling hygrometer ( brannan p / l , cumbria , uk ) . wind strength was recorded subjectively on an ordinal scale between 0 ( still ) and 3 ( strong , regular gusts ) . rainfall ( mm ) was derived from the australian bureau of meteorology ( bom 2014 ) , taking measurements from the melbourne airport weather station , located roughly in the center of the study area ( 37.67 s , 144.83 e ) . two measures of rainfall were used : rainfall on the day of the survey and cumulative rainfall over the preceding 7 days ( lagged rainfall ) . we used bayesian logistic regression to model the nightly probability of calling for those species with a sufficient number of detections . for each of these species , we began by filtering the nondetection data to exclude records from sites in years when no detections of calling were made . hence , for each species , only detection and nondetection data from sites that were known to be occupied by reproductively active males in a given year were included . we also removed any surveys when the focal wetland was dry ( which precludes calling among the focal species ) and randomly filtered the remaining dataset for each species such that only one site was included for any given survey night . repeat surveys of the same site in a given year were assumed to be independent . hence , for each species , the detection ( 1 ) or nondetection ( 0 ) of calling on survey night i at a site known to be occupied by reproductively active males was treated as a bernoulli variable with probability p i. covariate effects on p i were modeled using a logistic equation and linear link function : ( 1)logpi1pi=+xxi , where is the intercept , and x is the regression coefficient for covariate x. the underlying seasonal variation in p i was modeled using a fourier series approach following burnham and anderson ( 2002 , p. 188 ) . the approach treats phenomena as displaying a regular pattern that may be described using cosine functions of increasing complexity . the change in p i with days since the start of the austral spring ( september 1 , daysi ) was modeled as : ( 2)logpi1pi=+1cos2daysi365+2sin2daysi365 . with the exception of wind strength ( which did not show any obvious seasonality ) , raw meteorological data for each survey were first adjusted to represent the anomaly from the seasonal mean . ( 2012 ) , we fitted up to fourth degree polynomial regressions between each meteorological variable and survey date using maximum likelihood in r version 3.0.3 ( r core team 2014 ) . the bestfitting model for each meteorological variable was selected using akaike 's information criterion ( burnham and anderson 2002 ) , and the residuals from this model used as inputs for the logistic models . fourteen models of p i were fitted to the detection data for each species . each model contained one of the two rainfall variables , plus one or more of temperature , relative humidity , and wind strength . models were fitted twice for each species ; once with temperature represented by air temperature and once with temperature represented by water temperature . correlations between predictors were weak in the model set ( pearson 's r correlations ranged from 0.13 to 0.25 ) , except for a moderate negative correlation between air temperature and humidity ( pearson 's r of 0.46 ) . an effect of survey effort ( survey duration multiplied by the number of observers ) was included in all models to account for the fact that the probability of detecting males that are calling sporadically increases with survey duration , and multiple surveyors have a higher cumulative chance of hearing sporadic or weakly calling individuals . a random year models were fitted to the data using markov chain monte carlo ( mcmc ) sampling in openbugs version 3.2.3 ( thomas et al . 2006 ) , called from r. uninformative priors were used for each parameter . with the exception of survey date , parameter estimates and their 95% credible intervals ( 95% ci ) were drawn from 40,000 mcmc samples after a burnin of 60,000 samples . was used to predict their nightly probability of calling across 366 days , beginning and ending on september 1 . in turn , these predictions were used to estimate the seasonal peak in the probability of calling for each species . we conducted 1432 nocturnal surveys for frogs between 2001 and 2012 at 253 sites across the volcanic plains north and west of melbourne , victoria , australia . sites were distributed in the catchments of the darebin , merri , moonee ponds , and kororoit creeks . the landscape is undulating , rising to a maximum elevation of ~300 m asl at the study sites . upper catchments are dominated by grazing land , graduating to industrial and urban estates in the lower catchments . the climate is temperate , with cool winters ( july mean daily temperature range = 5.413.1c ) and warm summers ( february mean daily temperature range = 14.126.6c ) ( bom 2014 ) . rainfall averages 538 mm , with the highest monthly rainfall occurring in late winter and spring ( august sites included slow flowing pools along the main channel and tributaries of the streams listed above , as well as lentic wetlands such as farm dams , flooded quarries , swamps , and water treatment ponds . site selection was haphazard , including both randomized protocols ( mostly for selecting pools along streams ) and nonrandomized protocols that aimed to maximize geographic coverage while accounting for both access and logistical constraints ( for further details , see canessa et al . surveys were conducted between september and april ( inclusive ) in each year , encompassing the austral spring , summer , and autumn . survey timing sought to optimize detection rates of litoria raniformis in most cases ; this endangered species having been studied intensively in this region since 2001 ( heard et al . 2012a , b , 2013 , 2015 ) . however , aural surveys during the spring and early summer months optimize detection rates across the frog community in this region ( canessa and parris 2013 ) . surveys commenced at least 30 min after dark and began with a period of 510 min listening quietly for male advertisement calls . surveys extended up to 265 min , with up to 10 repeat surveys at a site in any given year . surveys were completed under permits 10001816 , 10003005 , and 10005649 issued by the victorian department of environment , land , water and planning . to supplement the above survey dataset , we collated data from 263 nocturnal surveys for frogs completed at the same study sites in the same period by private consultants , other researchers , and local naturalists . all surveys were conducted during the austral spring , summer , or autumn , and followed surveys protocols analogous to our own . survey data were gathered from the relevant research report or thesis , or through personal communications . six meteorological variables were considered as potential determinants of calling activity . air temperature ( dry bulb , c ) and relative humidity ( % ) were recorded during most surveys with the aid of a compact whirling hygrometer ( brannan p / l , cumbria , uk ) . wind strength was recorded subjectively on an ordinal scale between 0 ( still ) and 3 ( strong , regular gusts ) . rainfall ( mm ) was derived from the australian bureau of meteorology ( bom 2014 ) , taking measurements from the melbourne airport weather station , located roughly in the center of the study area ( 37.67 s , 144.83 e ) . two measures of rainfall were used : rainfall on the day of the survey and cumulative rainfall over the preceding 7 days ( lagged rainfall ) . we used bayesian logistic regression to model the nightly probability of calling for those species with a sufficient number of detections . for each of these species , we began by filtering the nondetection data to exclude records from sites in years when no detections of calling were made . hence , for each species , only detection and nondetection data from sites that were known to be occupied by reproductively active males in a given year were included . we also removed any surveys when the focal wetland was dry ( which precludes calling among the focal species ) and randomly filtered the remaining dataset for each species such that only one site was included for any given survey night . repeat surveys of the same site in a given year were assumed to be independent . hence , for each species , the detection ( 1 ) or nondetection ( 0 ) of calling on survey night i at a site known to be occupied by reproductively active males was treated as a bernoulli variable with probability p i. covariate effects on p i were modeled using a logistic equation and linear link function : ( 1)logpi1pi=+xxi , where is the intercept , and x is the regression coefficient for covariate x. the underlying seasonal variation in p i was modeled using a fourier series approach following burnham and anderson ( 2002 , p. 188 ) . the approach treats phenomena as displaying a regular pattern that may be described using cosine functions of increasing complexity . the change in p i with days since the start of the austral spring ( september 1 , daysi ) was modeled as : ( 2)logpi1pi=+1cos2daysi365+2sin2daysi365 . with the exception of wind strength ( which did not show any obvious seasonality ) , raw meteorological data for each survey were first adjusted to represent the anomaly from the seasonal mean . ( 2012 ) , we fitted up to fourth degree polynomial regressions between each meteorological variable and survey date using maximum likelihood in r version 3.0.3 ( r core team 2014 ) . the bestfitting model for each meteorological variable was selected using akaike 's information criterion ( burnham and anderson 2002 ) , and the residuals from this model used as inputs for the logistic models . fourteen models of p i were fitted to the detection data for each species . each model contained one of the two rainfall variables , plus one or more of temperature , relative humidity , and wind strength . models were fitted twice for each species ; once with temperature represented by air temperature and once with temperature represented by water temperature . correlations between predictors were weak in the model set ( pearson 's r correlations ranged from 0.13 to 0.25 ) , except for a moderate negative correlation between air temperature and humidity ( pearson 's r of 0.46 ) . an effect of survey effort ( survey duration multiplied by the number of observers ) was included in all models to account for the fact that the probability of detecting males that are calling sporadically increases with survey duration , and multiple surveyors have a higher cumulative chance of hearing sporadic or weakly calling individuals . a random year models were fitted to the data using markov chain monte carlo ( mcmc ) sampling in openbugs version 3.2.3 ( thomas et al . 2006 ) , called from r. uninformative priors were used for each parameter . with the exception of survey date , parameter estimates and their 95% credible intervals ( 95% ci ) were drawn from 40,000 mcmc samples after a burnin of 60,000 samples . 2002 ) . the bestfitting model for each species was used to predict their nightly probability of calling across 366 days , beginning and ending on september 1 . in turn , these predictions were used to estimate the seasonal peak in the probability of calling for each species . eight frog species were detected during this study : crinia signifera , crinia parinsignifera , limnodynastes dumerilii , limnodynastes tasmaniensis , litoria ewingii , litoria peronii , litoria raniformis , and litoria verreauxii . six species were commonly detected : c. signifera ( 396 detections total , 177 detections on independent nights ) , lim . although there was considerable temporal overlap in calling activity ( fig . 1 ) , the seasonality and predicted peak of calling varied among species ( figs . 2 , 3 ) . of 15 possible speciestospecies comparisons of the predicted peak in the nightly probability of calling , the 95% credible intervals overlapped in only four cases ( c. signifera & lim . the proportion of surveys in each month during which calling was recorded for the six commonly detected species . only surveys at sites that were known to be occupied in a given year are included for each species ( calling was detected at least once at those sites in those years ) . the estimated nightly probability of calling over 366 days for the six commonly detected species , beginning and ending on the september 1 . estimates are taken from the top model for each species and assume that all other variables influencing the detection of calls ( meteorological variables and survey effort ) are at their mean values . models were fitted to survey data from sites that were known to be occupied in a given year ( calling was detected at least once at those sites in those years ) . the estimated seasonal peak of the nightly probability of calling for the six commonly detected species . estimates are taken from the top model for each species and assume that all other variables influencing the detection of calls ( meteorological variables and survey effort ) are at their mean values . ewingii were recorded calling in all survey months , but most frequently in early spring ( fig . 3 ) , with a steep decline in the nightly probability of calling from november to february , followed by a rapid recovery during autumn ( fig . 2 ) . limnodynastes tasmaniensis displayed a similar ( although weaker ) pattern , with observed calling in all survey months ( fig . 1 ) , but an estimated peak in the nightly probability of calling in september and trough in march ( figs . 2 , 3 ) . , the proportion of surveys in which calling was detected declined in a roughly linear fashion after september ( fig . 1 ) ; however , the estimated seasonal peak in the probability of calling was in october ( fig . 3 ) . thereafter , the nightly probability of calling declined sharply , reaching near zero in midautumn ( fig . 2 ) . the nightly probability of calling was estimated to peak in early december for both species ( fig . 3 ) , before declining to zero or slightly thereabouts during autumn and winter ( fig . 2 ) . interspecific variation was also apparent in relationships between the nightly probability of calling and the meteorological variables considered ( tables 1 , 2 ) . relatively high rainfall in the preceding 7 days increased the probability of calling by c. signifera , lim . however , there was a clear negative effect of lagged rainfall on the probability of calling by lit . raniformis ( mean estimate = 0.60 , 95% ci = 1.10 , 0.11 ; table 2 ) . likewise , while the probability of calling by both lim . raniformis increased as the temperature anomaly increased , the former responded more strongly to air temperature , while the later responded to water temperature ( tables 1 , 2 ) . ewingii : mean estimate = 1.15 , 95% ci = 0.39 , 1.96 ; lit . verreauxii : mean estimate = 1.1 , 95% ci = 0.06 , 2.22 ; table 2 ) . the five topranked models of the nightly probability of calling for the six commonly detected species . all models include cosine effects of days since september 1 and a linear effect of survey effort ( to account for the fact that the probability of detecting calls increases with effort ) . they are as follows : air or water temperature anomaly ( temp ) , humidity anomaly ( hum ) , wind strength ( wind ) , rainfall anomaly ( mm ) either for the day of survey ( rain ) or for the preceding 7 days ( rain lag ) . model selection statistics are the deviance information criterion ( dic ) , distance from the top model ( dic ) , and model weight ( w ) temp is air temperature for these species . variables are as follows : days since september 1 ( days ) , air or water temperature anomaly ( temp ) , humidity anomaly ( hum ) , wind strength ( wind ) , rainfall anomaly ( mm ) either for the day of survey ( rain ) or for the preceding 7 days ( rain lag ) . coefficients for the effect of survey effort are not shown temp is air temperature for these species . there are two obvious mechanisms through which temporal partitioning of reproductive effort may facilitate coexistence in anuran communities . intraspecifically , male anurans compete acoustically for mates in various ways , including extrinsic factors such as caller location ( littlejohn 1977 ; parris 2002 ) and intrinsic factors such as call duration and acoustic frequency ( littlejohn 1977 ; wagner 1992 ; welch et al . 1998 ) . for example , littlejohn and martin ( 1969 ) demonstrated inhibition of calling by pseudophryne semimarmorata in direct response to acoustic interference from sympatric geocrinia victoriana in southern australia . likewise , schwartz and wells ( 1984 ) reported reductions in the attractiveness of calls of hyla ebraccata in panama when the primary notes overlapped with calls of the sympatric h. microcephala . in an extensive study of 39 species across three anuran communities in brazil , duellman and pyles ( 1983 ) demonstrated considerable overlap in the acoustic properties of calls . they concluded that temporal partitioning of calling , both in terms of the seasonal timing and environmental conditions under which calling takes place , was an important mechanism by which acoustic interference is mitigated ( and coexistence facilitated ) in these communities . the second mechanism by which partitioning of reproductive phenology may facilitate coexistence in anuran communities is mitigation of larval competition . competitive interactions between larval anurans are a key determinant of larval survival and metamorphic success ( alford 1999 ) . resource competition among larvae also has important carry over effects on the fitness and survival of postmetamorphic anurans ( chelgren et al . hence , larval competition represents a key determinant of anuran population dynamics and community structure ( wells 2007 ) . the early work of alford and wilbur on the subject of larval competition in anuran communities produced evidence both for and against temporal partitioning as a mechanism for coexistence ( alford and wilbur 1985 ; wilbur and alford 1985 ; alford 1989a , b ) . temporal partitioning was important for the outcomes of some interactions ( alford and wilbur 1985 ; wilbur and alford 1985 ; alford 1989a ) , but not others ( alford 1989b ) . later work confirmed that the strength of larval competition between species varies intrinsically , and mediates the importance of temporal partitioning for larval fitness ( gascon 1992 ) . it also showed that the temporal order in which interactions take place ( socalled priority effects ) may be an important determinant of the strength of competitive interactions among anuran larvae ( lawler and morin 1993 ) . we can not demonstrate that the interspecific variation in calling phenology documented here is adaptive with regard to coexistence , but there are several interesting points to be made in this regard . in the focal community , two species pairs could be expected to benefit most from partitioning of reproductive phenology : lit . these frogs are closely related , have analogous larval ecologies and similar advertisement calls ( loftushills 1973 ; watson et al . 1985 ; anstis 2013 ) . however , there was considerable uncertainty surrounding our estimates of calling seasonality for lit . verreauxii , and we note that an earlier , winter peak in calling activity has been recorded elsewhere for this species ( humphries 1979 ; smith et al . 2003 ; lemckert and grigg 2010 ) . verreauxii suggested by our study could be indicative of temporal partitioning when in sympatry with lit . our data are very limited for c. parinsignifera ; however , they suggest that the peak calling period of c. parinsignifera occurs later than that of c. signifera , and this agrees with observations elsewhere ( humphries 1979 ; macnally 1979 ) . indeed , macnally ( 1979 ) reported that c. signifera truncates its spring calling period to minimize overlap with c. parinsignifera when the two species occur in sympatry . it is also notable that the species pairs with the closest seasonal patterns of calling activity c. signifera & lit . obs . ) . as such , resource competition between larval c. signifera and lit . 2009 ) , the former calls from concealed positions under vegetation or crevices , while lit . elevated perches can significantly increase the effective distance of calls ( parris 2002 ) ; hence , acoustic interference of lit . dumerilli calls from hidden positions under banks and rocks , or in emergent vegetation ( anstis 2013 ; pers . . these two species also favor slightly different meteorological conditions for calling , and there calls differ acoustically ( dominant frequency of 680 hz for lim . our data revealed both expected and unexpected relationships with regard to the meteorological correlates of calling in this community . the probability of calling increased with increasing rainfall over the preceding 7 days for c. signifera , lim . hence , the influence of rainfall on calling activity is most likely mediated by its effect on wetland water levels , as is often the case for aquaticbreeding anurans ( wells 2007 ) . although somewhat counterintuitive , this relationship has been reported for ecologically analogous ranid frogs from north america . ( 2006 ) demonstrated a negative relationship between calling activity and rainfall for both lith . clamitans are all largely aquatic , summer breeding frogs that occupy permanent wetlands ( heard et al . they also all favor nights with warmer water temperatures for calling ( oseen and wassersug 2002 ; saenz et al . 2006 ; this study ) . while it may be supposed that water temperature is lower during rainy periods , and this could account for a negative effect of rainfall on calling activity by lit . raniformis , lagged rainfall and water temperature were unrelated in our dataset ( pearson 's r = 0.04 ) . ( 2006 ) hypothesized that acoustic interference from storms or rainfall itself may be the underlying mechanism . raniformis reduce calling activity following rainfall to reduce acoustic interference with other species in the community , with calling by four species being positively related to lagged rainfall ( as above ) . raniformis may stem from the sensitivity of this species to rising water levels in lotic situations , where increased flows represent a threat to eggs and weakly swimming earlystage tadpoles . of the frogs detected in our study area , lit . raniformis was the most frequently observed in lotic habitats . in line with lemckert and grigg ( 2010 ) , our data suggest that calling by lim . dumerilii is a terrestrial species that can range up to 1 km from wetlands during the nonbreeding season ( humphries 1979 ; anstis 2013 ) . dumerilii to call on nights with comparatively warm air temperatures relates to the suitability of such nights for female migration to breeding sites . finally , the positive relationship between the probability of calling and relative humidity demonstrated here for lit . verreauxii may be readily explained by their propensity to call from elevated perches or on land , rather than in the water ( anstis 2013 ; pers . higher relative humidity facilitates hydroregulation in these circumstances ( tracy 1976 ) , allowing prolonged calling . other ecological mechanisms could account for the variation in reproductive phenology observed during this study . for example , the focal species may vary in their susceptibility to terrestrial or aquatic predators , and time reproduction to minimize exposure to particular predators ( e.g. , lips 2001 ) . invertebrates and fish are important larval predators for some of the frogs studied here ; however , the available evidence suggests that they respond spatially rather than temporally to these predators ( hamer and parris 2010 , 2013 ) . we encourage further research on the reproductive phenology of amphibians and the mechanisms underlying interspecific variation in this trait within communities . as well as insights into niche partitioning and community dynamics , research on reproductive phenology can inform assessments of the effects of anthropogenic disturbances on amphibian communities , including disruptions from climate change ( todd et al . 2011 ) . in particular , we encourage research on the reproductive phenology of amphibians from australia , africa , and eurasia , for which data are scarce .

abstractspatial and temporal partitioning of resources underlies the coexistence of species with similar niches . in communities of frogs and toads , the phenology of advertisement calling provides insights into temporal partitioning of reproductive effort and its implications for community dynamics . this study assessed the phenology of advertisement calling in an anuran community from melbourne , in southern australia . we collated data from 1432 surveys of 253 sites and used logistic regression to quantify seasonality in the nightly probability of calling and the influence of meteorological variables on this probability for six species of frogs . we found limited overlap in the predicted seasonal peaks of calling among these species . those shown to have overlapping calling peaks are unlikely to be in direct competition , due to differences in larval ecology ( crinia signifera and litoria ewingii ) or differences in calling behavior and acoustics ( limnodynastes dumerilii and litoria raniformis ) . in contrast , closely related and ecologically similar species ( crinia signfera and crinia parinsignifera;litoria ewingii and litoria verreauxii ) appear to have staggered seasonal peaks of calling . in combination with interspecific variation in the meteorological correlates of calling , these results may be indicative of temporal partitioning of reproductive activity to facilitate coexistence , as has been reported for tropical and temperate anurans from other parts of the globe .

thrombotic microangiopathy ( tma ) results from red cell fragmentation and platelet trapping with subsequent microangiopathic hemolytic anemia and thrombocytopenia and represents a final common pathway of a multitude of clinical syndromes . in this paper , we attempt to summarize the salient features of many of these clinical entities and provide suggestions to help guide the efforts to differentiate between them . this is by no means meant to be an exhaustive discussion of these disorders , but rather strives to be a useful summary of some of the important causes of tma . kasabach - merritt phenomenon ( kmp ) typically describes thrombocytopenia and consumptive coagulopathy in the presence of a vascular tumor . however , further research has shown that kmp is most commonly associated with the rare benign vascular tumors kaposiform hemangioendothelioma ( khe ) and tufted angioma ( ta ) [ 2 , 3 ] . more than 50% of the cases of khe and ta occur in the first year of life . khe and ta tend to occur on the extremities , neck or trunk , but can sometimes involve the retroperitoneum . for this reason , thrombocytopenia in a newborn unexplained by other routine testing ( such as for sepsis ) should prompt imaging for occult vascular lesions . the pathogenesis of this condition has not been fully elucidated , but to the best of our understanding , the abnormal endothelium of these tumors results in platelet trapping , fibrin deposition with subsequent red blood cell damage , and a consumptive coagulopathy . while microangiopathic features are often apparent , anemia is not a common presenting sign unless significant bleeding has occurred . bleeding into the lesion leads to rapid enlargement of the tumor , continuing the cycle of platelet trapping and activation with consumption of clotting factors . kmp has been reported as having 3040% mortality , usually due to uncontrolled bleeding , but also secondary to cardiac failure or invasion of vital structures by the lesion . historically , interferon alpha has been used successfully as treatment for kmp , but the side effect of irreversible spastic diplegia , particularly in children under 1 year of age , has led to this drug falling out of favor , though it is still used in some cases . first - line treatment now typically relies upon corticosteroids and vincristine , though no prospective randomized trials have been completed comparing this regimen to an alternate treatment [ 5 , 6 ] . use of other chemotherapies such as cyclophosphamide , actinomycin - d , and methotrexate and of antiplatelet treatments such as ticlodipine has been reported [ 7 , 8 ] . there are studies currently underway investigating the benefit of novel therapies such as rapamycin . surgical resection , radiation , and embolization of the lesions thrombotic thrombocytopenic purpura ( ttp ) has classically been defined as the pentad of microangiopathic hemolytic anemia , thrombocytopenia , fever , and neurological and renal compromise . however , the diagnosis requires only microangiopathic anemia and thrombocytopenia , with or without ischemic organ damage , and not attributable to an alternate recognizable cause . untreated , the mortality of ttp was historically as high as 8090% . with the use of plasma exchange treatment , the overall mortality has been reduced to 2030% . the term ttp ( or secondary ttp ) has also been used to refer to microangiopathic hemolytic anemia associated with vasculitis , infection , pregnancy , medication , or which develops post - stem - cell transplant . for the purposes of this section of this paper , we will focus on congenital and idiopathic acquired ttp . recent research has illuminated some of the underlying pathophysiology associated with this disorder , though many questions still remain . ttp is the result of thrombi in the microcirculation , leading to thrombocytopenia and microangiopathic hemolytic anemia due to red cell damage within the microthrombi . one of the most exciting developments in the field of ttp has been the identification of the von willebrand factor - cleaving protease , adamts13 ( a disintegrin and metalloprotease with thrombospondin type 1 repeats ) . this enzyme is responsible for the cleavage of extra large multimers of von willebrand factor ( vwf ) into smaller moieties . the lack of this enzyme , either constitutionally or acquired , has been linked to the development of ttp [ 15 , 16 ] . this phenomenon does not account for all of the cases of ttp , however , and further research is required to explain the complete link between this enzyme and the clinical variability of the disease . idiopathic ttp typically occurs due to the development of autoantibodies to adamts13 , usually type g immunoglobulins . patients ' risk of relapse is somewhat predicted by very low adamts13 levels at diagnosis . however , variations in adamts13 activity and inhibitor levels after treatment has been completed have not been consistently predictive of pending relapse , putting the utility of following these levels into question [ 12 , 19 , 20 ] . plasma infusion or exchange is the mainstay of treatment for acquired ttp [ 12 , 21 ] . additional treatment modalities such as steroids , vincristine , cyclophosphamide , antiplatelet agents , and rituximab ( anti - cd20 antibody ) have also been used to varying success . inherited ttp , also known as upshaw - schulman syndrome , results from genetic mutations in the adamts13 gene and represents approximately 10% of those patients with ttp . this is an extremely rare disorder , estimated to have an incidence of only 1 : 1,000,000 . it is inherited in an autosomal recessive pattern , sometimes homozygously , but more often due to compound heterozygosity , and penetrance can be variable within the same family . most patients present in infancy , but there are patients who have presented during the second or third decades of life , and there are individuals who are known to carry mutations who show no symptoms . this has led to the hypothesis that , in some individuals carrying 2 mutations , an additional triggering event in conjunction with low adamts13 levels is required to develop ttp [ 11 , 14 ] . in addition , a novel aptamer which blocks platelet activation in ttp is currently under investigation and has completed phase i studies [ 24 , 25 ] . gene therapy has also been suggested as a treatment for this disorder , though research is still in the preclinical stages . hemolytic uremic syndrome ( hus ) is characterized by microangiopathic hemolytic anemia and thrombocytopenia with accompanying renal impairment . it can be divided into 3 subtypes : diarrhea - associated hus ( d+hus ) , pneumococcal ( also known as atypical or diarrhea negative ) hus , and congenital hus ( which has also been referred to as atypical ) . while the three entities share clinical features , the underlying epidemiology , pathogenesis , and treatment are markedly different . like ttp , the term hus has been used to refer to a spectrum of thrombotic microangiopathy caused by medications or associated with pregnancy or other clinical conditions [ 13 , 28 ] . we will only address the three entities listed above in this section of the paper . it usually occurs in children and is most commonly caused by infection with shiga - toxin producing escherichia coli o157 ( stec ) . the mean incidence of hus in children under 18 years of age ranges from 0.28 to 0.71 cases per 100,000 persons . the disease is more common in those < 5 years of age , in whom the mean incidence has been reported between 0.85 and 1.87 cases per 100,000 children [ 29 , 30 ] . d+hus is also the most common cause of acute renal failure in children in the usa . mortality ranges from 1 to 33% with the highest mortality in those > 60 years of age . the pathogenesis of d+hus results from the production of shiga - toxins ( stx ) by the infective e. coli . while the most common type of e. coli associated with this illness is the o157 : h7 strain , it is well known that other strains of e. coli may also produce stx . stec lead to a food- or water - borne diarrheal illness that can be bloody or nonbloody . the disease progresses to hus due to the release of stx in the intestine , which then are translocated across the gut epithelium into the circulation . the primary locations of gb3 in the human body are vascular endothelial cells , particularly in the glomeruli . exposure to stx damages the endothelium , resulting in the release of tissue factor and exposure of the platelets to extra large von willebrand factor multimers and collagen . this then triggers activation of the platelets and the coagulation cascade and leads to thrombus formation . the impact of the actions of stx in these locations results in microangiopathic hemolytic anemia and thrombocytopenia . in addition , stx has also been shown to bind to and directly activate platelets via the gb3 receptor . there have been recent studies regarding synsorb pk , an agent composed of silicone dioxide particles linked to the pk trisaccharide that is responsible for stx binding to the endothelial cell surface . monoclonal antibodies to stx are also under investigation . in experimental studies in mice these antibodies have been shown to neutralize stx . hus associated with pneumococcal infection ( d - hus or p - hus ) is one of the atypical forms of hus . it is much less common that d+hus accounting for only approximately 510% of hus cases overall , though it may be responsible for as many as half of d - hus cases . it has been associated with a higher morbidity and mortality than d+hus , though increased awareness and improved treatment has improved this finding . p - hus tends to occur in very young children , with the average age typically under 1 year . the overall mortality rate is currently approximately 10% , which is much improved from previous reports of mortality from 2950% [ 35 , 36 ] . streptococcus pneumoniae release neuraminidase , an enzyme that results in desialylation of red blood cell membrane proteins and exposure of the thomsen - friedenreich cryptantigen ( t - antigen ) . the t - antigen is a ubiquitous antigen on human blood cells to which all people have igm antibodies . it is not clear if these symptoms are antibody mediated or are a result of another mechanism . in addition , supportive care with dialysis may be required for those patients in kidney failure . many patients require blood product transfusion , though exposure to plasma should be limited because of the possibility of worsening the hemolysis due to infusion of igm anti - t antibody . vaccination against pneumococcus would reasonably be expected to reduce the prevalence of p - hus , since the most common serotypes associated with hus are included in the vaccine . however , it has recently been shown that serotype 19a may have emerged as an important invasive pathogen leading to p - hus . it is thought that up to one - third to one - half of patients with ahus have a mutation in one of the complement regulatory proteins . the deficiency of factor h , a regulatory glycoprotein in the alternative complement pathway , leads to a form of congenital hus . the absence leads to overactivity of the alternative complement pathway and subsequent features of hus . mutations causing deficiencies in factor i , c3 , and membrane cofactor protein cd46 have also been reported [ 31 , 37 , 38 ] . gain - of - function mutations in complement factor b and c3 , have been identified in families with ahus and persistent activation of the alternative complement pathway . families carrying mutations show incomplete penetrance of disease and may be modulated by the presence or absence of other complement gene variants [ 3840 ] . autoantibodies to factor h which develop in individuals with mutations in factor h related proteins 1 and 3 have also been implicated in the pathogenesis of the disease . the prognosis for ahus is grim , with mortality approaching 25% in the acute phase and as many as 50% of patients developing end stage renal disease . if transplantation is attempted , 60100% of patients will experience a relapse , which almost inevitably results in loss of the renal graft . treatment relies on infusion of plasma which contains the missing factor . in the case of those patients with autoantibodies , recent studies regarding the use of the recombinant complement inhibitor eculizumab have been promising , particularly in those patients who have failed plasma - replacement therapy . microangiopathic hemolytic anemia ( maha ) , also called posttransplant thrombotic microangiopathy ( ta - tma ) has been described as a complication of bone marrow transplantation , both autologous and allogeneic with variable incidence rates ranging from 0 to 74% depending on the diagnostic criteria used to make the diagnosis [ 42 , 43 ] . it is also a well - known complication following solid organ transplants , such as kidney transplants , with a variable time from transplant to diagnosis and could be due to viral infections , arise de novo , or be related to the use of calcineurin inhibitors , mainly the microemulsion form of cyclosporine [ 37 , 44 ] . tma was observed in 26 of 188 patients with renal transplants with an incidence of 14% with the majority of these patients having no systemic manifestation of tma . it has also been reported following liver transplantation ; five of 100 patients who received living donor liver transplant ( ldlt ) developed tma with an incidence of 5% and at a median interval of 18 days following transplant . another major factor in the development of tma is drugs used for graft versus host disease ( gvhd ) , mainly fk506 and cyclosporine or with other gvhd prophylaxis . factors that can contribute to this condition include high - dose chemotherapy for autologous stem cell transplant ( asct ) , conditioning regimen for allogeneic stem cell transplant , total body irradiation , and use of immune suppressive therapy [ 38 , 47 , 48 ] . the role of gvhd and renal involvement in the pathogenesis of ta - tma has also been investigated . reviewed 314 renal autopsies on patients who died after their first hematopoietic stem cell transplant over a 7-year period . twenty percent of autopsies reviewed showed tma , with an additional 15% with evidence of thrombus formation . the risk of tma in that patient population increased 4-fold with incidence of acute gvhd , independent of the use of cyclosporine . the presence of acute gvhd was an independent risk factor in the development of ta - tma in the multivariate analysis in addition to sex mismatch , higher doses of total body irradiation ( tbi ) , and viral infections . in addition to the significant renal involvement with acute gvhd , ta - tma has also been found to involve the gut tissue . in a study of 16 patients with ta - tma , all were found to have acute gvhd . seven patients were retrospectively diagnosed with ta - tma , while 9 were specifically investigated for it . all patients that were retrospectively diagnosed died as a result of increasing the immune suppression intensity due to the impression that it was purely gut gvhd , while 8 of the other nine patients improved following reduction of immune suppression . infections have also been shown to be associated with maha after transplant ; bacterial infection with helicobacter pylori was found to be associated with six patients among 74 consecutive transplants through involvement of elevated cytokines . viral infections such as parvob-19 , cmv , and hhv-6 have also been implicated as a cause of ta - tma . typically postinfectious cases are due to reactivation of these viruses within the first 36 months after transplant . viral infections should be considered in recipients of transplant who develop refractory cases of ta - tma [ 39 , 52 , 53 ] . pregnancy - associated thrombotic microangiopathy is a rare but serious disorder that is associated with significant maternal and perinatal morbidity and mortality , due to the deposition of fibrin and platelet thrombi in the microcirculation of the placenta . it has been reported to occur in 1 : 25,0001 : 100,000 pregnancies [ 54 , 55 ] . pregnancy can precipitate the disease for the first time or can exacerbate the recurrence of an existent condition [ 56 , 57 ] . pregnancy - associated microangiopathy typically occur during late pregnancy ; however , it has also been reported in the first trimester of pregnancy , in which case the patient was managed very intensely with continuous plasma therapy throughout the pregnancy . both mother and fetus survived without complications . in reviewing the oklahoma ttp - hus registry in 335 patients , terrell et al . did not find any gender or race predilection that was associated with pregnancy - associated microangiopathy , as well as with hematopoietic stem cell transplant or drugs . recently published data on the largest collection of pregnancy - associated microangiopathy encompassing over 4 decades . one hundred and sixty - six patients with pregnancy - associated thrombotic thrombocytopenic purpra were reported on . patients either had their first episode ( n = 106 ) or recurrent episode ( n = 32 ) . median time presentation was 23.7 9 weeks and 22 10 weeks , respectively . the only significant laboratory differences between both groups were hemoglobin and hematocrits ( 6.8 versus 11.4 g / dl and 19% versus 25% , resp . ) . perinatal and maternal mortalities were high in both groups ( 32% versus 44% and 26% versus 10.7% ) ; however , both mortality rates were significantly lower in the years after 1980 compared to before that year . one of the confusing clinical pictures was the association of pregnancy - associated ttp and hellp syndrome ( hemolysis , elevated liver enzymes , low platelets ) , which had both preeclampsia / eclampsia and ttp concurrently . this was reported in 29 of the 166 patients in the series and significantly increased maternal mortality from 21.8% to 44.4% . pregnancy - associated microangiopathy can be confused with other medical and obstetrical conditions such as severe preeclampsia / eclampsia , hellp syndrome , sepsis , and pregnancy - induced fatty liver changes [ 57 , 61 ] . the confusion in making the diagnosis usually happens due to the non - specific clinical and laboratory criteria needed to make the diagnosis . this is more of a problem when the initial management is done by physicians who are not accustomed to taking care of these patients . stella et al . reported on 14 cases of pregnancy - associated microangiopathy in 12 pregnancies by reviewing the charts of these patients . 4/12 cases were evaluated in the er initially and were given the diagnoses of domestic violence , gastroenteritis , itp , and panic attack . 8/12 patients were seen by obstetric services ; however , all were given the wrong diagnosis , mainly hellp syndrome , despite of being normotensive and having no proteinuria . half of the patients in the series were diagnosed before 24 week gestation but only 3 had term live births . over the last 2 decades , there has been increasing evidence that adamts13 is significantly involved in the pathogenesis of pregnancy - associated microangiopathy . this could be due to congenital deficiency of the enzyme activity which is usually severe or due to the presence of inhibitors with less sever deficiency [ 63 , 64 ] . however , some patients with pregnancy - associated microangiopathy were found to have a normal enzyme activity while other patients with ttp due to other clinical syndromes had low enzyme activity . the congenital deficiency of the adamts13 activity is also known as upshaw - schulman syndrome ( uss ) . in their research paper , fujimura and nine females with a total of 15 pregnancies developed significant thrombocytopenia during the second half of their pregnancies leading to stillbirth in 8 out of total 16 babies . eight of the nine women had heterozygous mutations and only one had a homozygous abnormality . although adamts13 enzyme deficiency ( both congenital and acquired ) is the main underlying mechanism in pregnancy - induced microangiopathy , dysregulation of alternative c3 convertase enzyme , due to mutations in the gene coding for the protein has also been shown to cause an atypical form of hus during pregnancy ( mainly with renal involvement and not due to shiga toxin ) . in a retrospective analysis of 100 women with this rare and ill - defined type of hus , it was found to be associated with pregnancy in 21 patients ( 21% incidence ) . the disease occurred during the first pregnancy in 38% and occurred mainly during postpartum period ( 79% ) . the unusual feature of this syndrome was the disproportionate severe renal involvement compared to the thrombocytopenia ; 81% of patients required hemodialysis during the acute phase while 61% eventually reached end - stage renal disease in less than a month from the beginning of the episode . a variety of the therapeutic interventions have been tried with pregnancy - induced microangiopathy ( and other forms of ttp ) with limited success . therapies such as steroids , antiplatelet agents , ivig , and even splenectomy have been tried . not until plasma infusion and plasma exchange were introduced that the survival rate increased from 10% up to 80% currently [ 59 , 64 ] . plasma exchange and/or plasma infusion need to be initiated whenever there is a good suspicion of microangiopathy . in general this therapy will have to be continued to the end of delivery or throughout the postpartum period [ 56 , 59 , 61 , 64 ] . one of the clinical dilemmas when it comes to systemic vasculitis is the overlap of manifestations of both disorders . one of the earliest reports has been by ross et al . in 1996 when they reported the incidence of 2 cases of maha that was associated with a case of wagner 's granulomatosis with positive antineutrophil antibodies ( ancas ) and another case of guillain - barre syndrome with positive nerve biopsy for anca with specificity to myeloperoxidase . a more common association of systemic autoimmune disorders and maha is usually with systemic lupus erythematosus ( sle ) , with large number of case reports and small case series [ 62 , 6668 ] . in a single - institution large series of patients with sle and maha , kwok et al . twenty - six patients were found to have maha in addition to their sle diagnosis-87 patients without maha matched for age and sex were also included as controls . the study showed that sle is an independent risk factor for the development of microangiopathy and that this risk was associated with higher sle disease activity index ( sledai ) . the study also showed that infection was the only independent risk factor for mortality for those ttp patients . maha has been also reported in a large variety of connective tissue disorders other than sle , including systemic sclerosis , rheumatoid arthritis , dermatomyositis , and still 's disease [ 70 , 71 ] . similar to other conditions with microangiopathy and maha , adamts 13 enzyme has been linked to the pathophysiology of maha in autoimmune disorders . severe reduction in the enzyme activity as well as the presence of inhibitors has been consistently found in patients with sle and other similar disorders . since adamts 13 deficiency is considered very rare in sle and similar disorders with maha , testing for this enzyme is considered a good method to distinguish between the two [ 65 , 67 , 70 ] . maha is a rare disorder that has the hallmark of fragmented red blood cells and thrombocytopenia that is nonimmune in nature . maha is not a single disease or even a single group of disorders ; rather it is a spectrum of disorders that usually present as an hus / ttp picture . ttp / hus is caused by a vast array of different agents ( drugs , toxins , infections , pregnancy , and autoimmunity ) that damage the endothelium via multiple and varied mechanisms . functionally altered endothelium provokes intravascular platelet aggregation , resulting in the variable clinical manifestations of the syndrome . because of the rarity of these disorders and the nonspecific clinical and laboratory features , this diagnosis ( and its underlying causes ) can be easily overlooked or missed leading to the high morbidity and mortality that can be seen with this problem . therefore , when a patient presents with unexplained thrombocytopenia and a coombs - test - negative hemolytic anemia , a presumptive diagnosis of maha should be considered . one of the newer advancements in the management of this disorder is the discovery of association of the adamts13 enzyme 's level and activity with ttp that has led to earlier and improved detection rate which was translated to better survival . no specific therapy can be claimed to cure this group of disorders , however , few interventions have been proven to be helpful ( and even life saving ) in some disorders such as the use of plasma infusion and plasma exchange in the management of ttp .

thrombotic microangiopathy results from thrombotic occlusion of the microvasculature leading to fragmentation of red blood cells , profound thrombocytopenia , and a microangiopathic hemolytic anemia with elevation of lactate dehydrogenase and negative direct coomb 's test . this constellation of clinical and laboratory findings is not due to one disease entity ; rather , it represents a variety of underlying diagnoses . among the major disease entities are ttp / hus , which can be congenital or acquired , bacterial infections , medications , vascular or endothelial pathology like kasabach - merritt phenomenon , and stem cell transplantation . in this paper , we offer a review of some of the major causes of thrombotic microangiopathy .

wound healing requires a concerted effort of remodeling of various components of the connective tissue in the presence of appropriate cytokines and growth factors . wound healing is a complex process involving a cascade of events , orchestrated by interactions between many cell types , soluble factors and matrix components . various steps like microbial control , subsidence of inflammation , regeneration of connective tissue , angiogenesis and epithelialisation should take place in a time - bound sequence . any acute wound initially goes through the phase of haemostasis and inflammation . immediately after the injury , the cell membranes release potent vasoconstrictors like prostaglandin 2 alpha and thromboxane a2 . the collagen in the wound sets off the clotting cascade through both the intrinsic and the extrinsic pathways . neutrophils undergo chemotaxis into the wound in the presence of interleukin-1 , tumour necrosis factor ( tnf)-alpha , transforming growth factor ( tgf)-beta , platelet factor-4 and bacterial products . the wound then enters into a proliferative phase in the vital presence of the activated macrophage . collagenases secreted by the activated macrophage debride the wound . interleukins and tnfs stimulate fibroblasts and tgf stimulates keratinocytes . epithelialisation , angiogenesis , granulation tissue and collagen deposition are the cardinal events that take place . in the proliferative phase , the fibrin clot is replaced by a template of fibrocytes that lay down collagen . platelet - derived growth factor ( pdgf ) and epidermal growth factor secreted , respectively , by platelets and macrophages lead to fibroblast activation . in response to tgf - beta 1 secreted by the macrophages , fibroblasts situated inside the wound stimulated by pdgf , fibroblasts lay down a provisional matrix composed of type iii collagen , glycosaminoglycans and fibronectin . an organised deposition of a matrix of collagen is the hallmark of the third phase of remodeling or maturation . this phase usually begins 1 week after the injury , and extends up to 1 year [ figure 1 ] . in the early part of the third phase ( 45 weeks ) , a lot of collagen deposition takes place and the tissue is indurated . if there are problems with matrix deposition , the strength of the wound is considerably impaired . the collagen in granulation tissue is fundamentally different from the collagen in the normal dermis . it has thinner fibres , probably due to greater hydroxylation and glycosylation of the lysine residues . the strength of the wound reaches a maximum of 80% at around 3 months after injury . these wounds develop whenever there is prolongation of one of the phases of normal healing . these fail to heal when other wounds of similar size would have otherwise healed . according to leaper and durani , any wound that that has not shown a 2040% reduction in area after 24 weeks of optimal treatment should be labeled as chronic . a chronicity may be considered when there is no complete healing after 6 weeks or if there is poor response to a treatment change . wound healing is said to be impaired when such sequential progression of events for normal healing does not take place and , instead , local injury and chronic inflammation persist.[157 ] wound healing is a complex process that is susceptible to abnormalities at many levels . low oxygen tension in the wound results in endothelial cell apoptosis , decreased neutrophil and fibroblast activity . oedema in the wound increases the distance between capillaries at the tissue level and hence local perfusion of oxygen is decreased . indeed , hyperoxia recruits bone marrow - derived progenitor cells into diabetic and ischaemic wounds . the resident fibroblasts in chronic wounds may be phenotypically senescent and show decreased responsiveness to tgf and pdgf . the presence of bacteria in the wound increases pro - inflammatory mediators and decreases growth factor levels . the neutrophils and macrophages release proteases and oxidants , which degrade cytokines and extracellular matrix . combined , tissue hypoxia and bacterial proliferation are two major local obstacles in the healing of any wound . the components of tissue engineering are ( 1 ) gene therapy , ( 2 ) cytokines and other growth factors , ( 3 ) scaffold and ( 4 ) cells . a stem cell is a cell that can self - replicate and give rise to more than one type of mature daughter cell . thus , stem cell incorporates a broad range of cells with different capacities for proliferation and differentiation . totipotent stem cells are capable of giving rise to an intact organism , including germinal tissues . pleuripotent stem cells can give rise to cells derived from all three germ layers . multipotent or organ - specific stem cells are capable of giving rise to the cells that comprise a single organ system or tissue . therefore , a stem cell is characterized by ( 1 ) unlimited self - renewal capacity , ( 2 ) long - term viability , ( 3 ) multi - lineage potential , ( 4 ) participation in tissue repair and ( 5 ) preservation of somatic homeostasis . a classical stem cell must be capable of asymmetric cell division , i.e. it must produce as progeny , one exact multipotent replica cell and a transit amplifying ( ta ) cell that performs a more specialized function . thus , replication of a single cell can potentially result in whole populations of cells . this will lead on to produce tissue regeneration along with retention of a population of identical stem cells . embryonic stem cells are formed from the inner cell mass of a pre - implantation blastocyst . these have the potential to differentiate into any type of cell ( multipotent ) . with suitable media and in the presence of growth factors , these can give rise to keratinocytes . however , the stability and oncogenicity of embryonic stem cells have to be further defined . cells have also been harvested from the peripheral blood , umbilical cord blood , amniotic membrane , adipose tissue , etc . organ systems also harbour indigenous holoclones of cells , with high - renewal capacity and low levels of differentiation . these are called resident stem cells . in the skin , these are the dermal sheath cells of the hair follicle . the uncommitted mesenchymal stem cells ( mscs ) , residing in the bone marrow stroma , are fibroblast - like cells that optimize the microenvironment of the haematopoietic cells . the same kinds of cells are also found in connective tissues across various organs in the body . these mscs are capable of differentiating into tissues like bone , cartilage , adipose tissue and endothelium , depending on their microenvironment ( niche ) . the adult bone marrow aspirate can provide 30% more multipotent stem cells as compared with umbilical cord blood . mscs can be expanded ( multiplied ) due to their property of adhering to tissue culture surfaces . infusions of mscs have been found to upregulate the anti - inflammatory cytokines such as il-10 and il-12 while decreasing the concentrations of pro - inflammatory cytokines like interferon - gamma , il-1 , il-6 and macrophage inflammatory protein-1 alpha . in murine studies , mscs have been found to have anti - apoptotic effects , upregulation of bcl-2 and suppression of caspase enzymes in adjacent cells . in mice pre - treated with endotoxin , intrapulmonary infusions of mscs resulted in reduction in pulmonary vascular congestion , decreased alveolar wall thickening and increased survival . ex vivo expanded mscs are known to interact with a broad range of immunocytes , including t lymphocytes , b lymphocytes , nk cells and dendritic cells . these mscs then home in to the sites of injury and seed the wound . inside the microvasculature of the developing granulation tissue , endothelial progenitor cells , again from the marrow , augment new vessel formation ( vasculogenesis ) . integrating themselves into the wound , the mscs are postulated to directly participate in its repair . transplanted mscs are thought to impact locally through five major paths ( 1 ) increased angiogenesis , ( 2 ) decreased local inflammation , ( 3 ) anti - apoptotic and chemotactic signaling , ( 4 ) normalization of extracellular matrix and ( 5 ) stimulation of nearby resident stem cells . they have been reported to secrete vascular endothelial growth factor ( vegf ) , fibroblast growth factor ( fgf ) , transforming growth factor ( tgf beta ) , hepatocyte growth factor , interleukins 10 and 13 and other cytokines and growth factors ( paracrine signaling ) , which help in remodeling of the extracellular matrix and in neovascularisation . even in an environment with low oxygen tension , up to 20% of fibroblasts in the wound are postulated to arise from bone marrow precursors . again , mscs have the potential to stimulate and differentiate the resident progenitor cells and promote recovery of injured local cells . the immunosuppressive properties of mscs also help in prolonged wound coverage by delay of allograft rejection . only around 5% of the stem cells that are injected into the wound engraftment levels of locally delivered stem cells were observed to be too low to significantly regenerate functional tissue , especially in cardiac myocytes and smooth muscle tissue . the injected human mscs have not been described to transform into keratinocytes and dermal appendage cells . the immuno - attenuative response afforded by the local presence of stem cells can be replicated by cell - free conditioned media derived from stem cells . all these evidence point to the theory that stem cells benefit through paracrine pathways , rather than by replacing functional tissue . thus , in contrast to any specific drug that affects a single pathway for its action , mscs exhibit their therapeutic power through diverse systemic and local interrelated routes . nearly half a century has elapsed since friedenstein and co - workers first isolated mscs from rat bone marrow . these cells have been experimented in diseases as diverse as myocardial infarction , segmental bone defect and lung fibrosis . cultured different clones of keratinocytes in vitro and introduced the concept of stem cells in wound care . another approach is the local application of stem cells for optimizing the regenerative capability of wounds . badiavas and falanga in 2003 introduced the use of bone marrow - derived cells in chronic wounds . three patients with recalcitrant wounds present for more than 1 year and resistant to standard treatment were chosen for the study . the authors reported healing of two of the wounds with three sittings of cell treatment alone . ichioka and colleagues used a collagen matrix impregnated with bone marrow aspirate on a chronic leg ulcer that had not healed for more than 1 year . humpert et al . examined the nature of wound healing in a type 2 diabetic patient after the use of topical mononuclear bone marrow cells . an in vitro study revealed that the levels of bfgf , vegf and collagen synthesis were much higher in bone marrow stromal cells than in dermal fibroblasts . the question is whether these stem cells differentiate into elements of connective tissue in the microarchitecture of the wound . the consensus postulated is that the wounds change their character from a chronic non - healing one to an acute regenerating one . currently , this change of character requires surgical debridement . by removing diseased tissue , debridement recruits fresh fibroblasts , burd reports the use of autologous bone marrow on chronic non - healing leg ulcers . document using this procedure in a patient who sustained a 40% total burn surface area tbsa burn . they applied allogenic fibroblast - like mscs on the burn wounds prior to skin grafting . they reported that there was a decrease in serous discharge from the wound after the application of stem cells . the patient had less pain after the procedure . on follow - up , they noted neo - angiogenesis , increased epithelialisation and increased graft uptake in the wound . they hypothesize that healing would accelerate if inflammatory cells are brought to the wound from the bone marrow . they describe the topical application of autologous bone marrow on two patients with chronic non - healing ulcers . a small residual raw area persisted that healed totally with a split - thickness skin graft . falanga and colleagues expanded autologous bm - mscs ex vivo and combined them with fibrin spray for topical application . they studied the efficacy of this system in healing of four acute and six chronic wounds . the authors reported healing of these wounds within 8 weeks with the topical application alone . the chronic wounds that were chosen by the authors were refractory wounds present for more than 1 year . all of those healed within 1620 weeks , with up to three sittings of the fibrin msc delivery system . lataillade et al . reported the use of mscs in a patient who sustained a severe radiation burn injury to his left buttock . the patient underwent multiple sittings of debridement followed by split - skin grafting and local stem cell injection . these cells were cultured from bone marrow aspirate and underwent a two - step expansion process ex vivo , before local injection . the wounds were of diverse origin and included post - burn , diabetic and decubitus ulcers . the mscs were cultured and expanded and impregnated into a collagen sponge that served as a dermal replacement . adipose - derived stem cells ( ascs ) are similar to mscs in morphology , differentiation and paracrine secretion under hypoxic conditions . plastic surgeons , lately adipose - tissue engineers , take advantage of their abundance in liposuction specimens for cell - assisted lipotransfer . ascs were used along with suitable scaffolds to regenerate bone in human calvarial and maxillectomy defects . a stem cell is a cell that can self - replicate and give rise to more than one type of mature daughter cell . thus , stem cell incorporates a broad range of cells with different capacities for proliferation and differentiation . totipotent stem cells are capable of giving rise to an intact organism , including germinal tissues . pleuripotent stem cells can give rise to cells derived from all three germ layers . multipotent or organ - specific stem cells are capable of giving rise to the cells that comprise a single organ system or tissue . therefore , a stem cell is characterized by ( 1 ) unlimited self - renewal capacity , ( 2 ) long - term viability , ( 3 ) multi - lineage potential , ( 4 ) participation in tissue repair and ( 5 ) preservation of somatic homeostasis . a classical stem cell must be capable of asymmetric cell division , i.e. it must produce as progeny , one exact multipotent replica cell and a transit amplifying ( ta ) cell that performs a more specialized function . thus , replication of a single cell can potentially result in whole populations of cells . this will lead on to produce tissue regeneration along with retention of a population of identical stem cells . embryonic stem cells are formed from the inner cell mass of a pre - implantation blastocyst . these have the potential to differentiate into any type of cell ( multipotent ) . with suitable media and in the presence of growth factors , these can give rise to keratinocytes . however , the stability and oncogenicity of embryonic stem cells have to be further defined . on the contrary , cells have also been harvested from the peripheral blood , umbilical cord blood , amniotic membrane , adipose tissue , etc . organ systems also harbour indigenous holoclones of cells , with high - renewal capacity and low levels of differentiation . these are called resident stem cells . in the skin , these are the dermal sheath cells of the hair follicle . the uncommitted mesenchymal stem cells ( mscs ) , residing in the bone marrow stroma , are fibroblast - like cells that optimize the microenvironment of the haematopoietic cells the same kinds of cells are also found in connective tissues across various organs in the body . these mscs are capable of differentiating into tissues like bone , cartilage , adipose tissue and endothelium , depending on their microenvironment ( niche ) . the adult bone marrow aspirate can provide 30% more multipotent stem cells as compared with umbilical cord blood . mscs can be expanded ( multiplied ) due to their property of adhering to tissue culture surfaces . infusions of mscs have been found to upregulate the anti - inflammatory cytokines such as il-10 and il-12 while decreasing the concentrations of pro - inflammatory cytokines like interferon - gamma , il-1 , il-6 and macrophage inflammatory protein-1 alpha . in murine studies , mscs have been found to have anti - apoptotic effects , upregulation of bcl-2 and suppression of caspase enzymes in adjacent cells . in mice pre - treated with endotoxin , intrapulmonary infusions of mscs resulted in reduction in pulmonary vascular congestion , decreased alveolar wall thickening and increased survival . ex vivo expanded mscs are known to interact with a broad range of immunocytes , including t lymphocytes , b lymphocytes , nk cells and dendritic cells . these mscs then home in to the sites of injury and seed the wound . inside the microvasculature of the developing granulation tissue , they differentiate into dermal fibroblasts , myofibroblasts , lymphoid tissue and antigen presenting cells . endothelial progenitor cells , again from the marrow , augment new vessel formation ( vasculogenesis ) . integrating themselves into the wound , transplanted mscs are thought to impact locally through five major paths ( 1 ) increased angiogenesis , ( 2 ) decreased local inflammation , ( 3 ) anti - apoptotic and chemotactic signaling , ( 4 ) normalization of extracellular matrix and ( 5 ) stimulation of nearby resident stem cells . they have been reported to secrete vascular endothelial growth factor ( vegf ) , fibroblast growth factor ( fgf ) , transforming growth factor ( tgf beta ) , hepatocyte growth factor , interleukins 10 and 13 and other cytokines and growth factors ( paracrine signaling ) , which help in remodeling of the extracellular matrix and in neovascularisation . even in an environment with low oxygen tension , up to 20% of fibroblasts in the wound are postulated to arise from bone marrow precursors . again , mscs have the potential to stimulate and differentiate the resident progenitor cells and promote recovery of injured local cells . the immunosuppressive properties of mscs also help in prolonged wound coverage by delay of allograft rejection . only around 5% of the stem cells that are injected into the wound engraftment levels of locally delivered stem cells were observed to be too low to significantly regenerate functional tissue , especially in cardiac myocytes and smooth muscle tissue . the injected human mscs have not been described to transform into keratinocytes and dermal appendage cells . the immuno - attenuative response afforded by the local presence of stem cells can be replicated by cell - free conditioned media derived from stem cells . all these evidence point to the theory that stem cells benefit through paracrine pathways , rather than by replacing functional tissue . thus , in contrast to any specific drug that affects a single pathway for its action , mscs exhibit their therapeutic power through diverse systemic and local interrelated routes . nearly half a century has elapsed since friedenstein and co - workers first isolated mscs from rat bone marrow . these cells have been experimented in diseases as diverse as myocardial infarction , segmental bone defect and lung fibrosis . cultured different clones of keratinocytes in vitro and introduced the concept of stem cells in wound care . another approach is the local application of stem cells for optimizing the regenerative capability of wounds . badiavas and falanga in 2003 introduced the use of bone marrow - derived cells in chronic wounds . three patients with recalcitrant wounds present for more than 1 year and resistant to standard treatment were chosen for the study . the authors reported healing of two of the wounds with three sittings of cell treatment alone . ichioka and colleagues used a collagen matrix impregnated with bone marrow aspirate on a chronic leg ulcer that had not healed for more than 1 year . examined the nature of wound healing in a type 2 diabetic patient after the use of topical mononuclear bone marrow cells . an in vitro study revealed that the levels of bfgf , vegf and collagen synthesis were much higher in bone marrow stromal cells than in dermal fibroblasts . the question is whether these stem cells differentiate into elements of connective tissue in the microarchitecture of the wound . the consensus postulated is that the wounds change their character from a chronic non - healing one to an acute regenerating one . currently , this change of character requires surgical debridement . by removing diseased tissue , debridement recruits fresh fibroblasts , burd reports the use of autologous bone marrow on chronic non - healing leg ulcers . document using this procedure in a patient who sustained a 40% total burn surface area tbsa burn . they applied allogenic fibroblast - like mscs on the burn wounds prior to skin grafting . they reported that there was a decrease in serous discharge from the wound after the application of stem cells . the patient had less pain after the procedure . on follow - up , they noted neo - angiogenesis , increased epithelialisation and increased graft uptake in the wound . they hypothesize that healing would accelerate if inflammatory cells are brought to the wound from the bone marrow . they describe the topical application of autologous bone marrow on two patients with chronic non - healing ulcers . a small residual raw area persisted that healed totally with a split - thickness skin graft . falanga and colleagues expanded autologous bm - mscs ex vivo and combined them with fibrin spray for topical application . they studied the efficacy of this system in healing of four acute and six chronic wounds . the authors reported healing of these wounds within 8 weeks with the topical application alone . the chronic wounds that were chosen by the authors were refractory wounds present for more than 1 year . all of those healed within 1620 weeks , with up to three sittings of the fibrin msc delivery system . lataillade et al . reported the use of mscs in a patient who sustained a severe radiation burn injury to his left buttock . the patient underwent multiple sittings of debridement followed by split - skin grafting and local stem cell injection . these cells were cultured from bone marrow aspirate and underwent a two - step expansion process ex vivo , before local injection . the wounds were of diverse origin and included post - burn , diabetic and decubitus ulcers . the mscs were cultured and expanded and impregnated into a collagen sponge that served as a dermal replacement . adipose - derived stem cells ( ascs ) are similar to mscs in morphology , differentiation and paracrine secretion under hypoxic conditions . plastic surgeons , lately adipose - tissue engineers , take advantage of their abundance in liposuction specimens for cell - assisted lipotransfer . ascs were used along with suitable scaffolds to regenerate bone in human calvarial and maxillectomy defects . a pilot study was conducted in our hospital on burn patients using bone marrow mscs expanded in vitro ( publication pending ) . two wounds of similar size were compared . both wounds underwent closure by split - skin grafting . the test wound was injected with up to 23 million mscs at the time of grafting . while a majority of the test wounds healed faster than their counterparts , we did encounter some areas of concern for clinical application of stem cells . falanga refined the local delivery of stem cells by incorporating them in fibrin as a spray . he postulated that incorporating into fibrin might be ideal for transporting cells and soluble mediators into sites of injury . envisaged that 3d constructs of mscs , with enhanced anti - inflammatory properties , could be mass produced as off - the - shelf medication . future technology could involve the use of biogels , biodegradable matrices and microparticles that integrate into the wound bed without the risk of foreign body reaction or infection . dermal matrix carrier has been found to improve the therapeutic effects of bone marrow and ascs in murine models . composite skin with xenogenic dermis seeded with epidermal stem cells and dermal papilla cells has also favoured wound healing . impregnating the carrier with growth factors that induce the commitment of resident stem cells into keratinocytes is also another possibility . genetic modulation and transfection of cutaneous stem cells for induction into keratinocyte lineage has also been postulated . msc mobilization strategies with factors such as granulocyte macrophage colony stimulating factor gm - csf are envisaged . development of smart biomaterials that would augment the transit of circulating stem cells is also sought after . this can potentially avoid the application of exogenous cells , using the patient 's own cells for wound healing . the amount of stem cells required for optimisation of the wound is also not standardised . the number and location of stem cells within skin may contribute to rapid healing seen in the foetus . however , cells migrate to distant organs like spleen , when therapeutically administered numbers exceed 1 10 . adequate culture , expansion and characterisation of stem cells take 3 weeks to 1 month in the laboratory the current status of stem cells in chronic wounds would be as an additional tool to the existing therapeutic armamentarium . future goals of stem cell therapy for chronic wounds should focus on rapid expansion of cells in vitro . another option would be to maximise the number of precursor cells resident in the wound . effective delivery and optimal engraftment of these cells are also of paramount importance . a bioengineered dermal epidermal composite enriched with expanded autologous stem cells that can immune - modulate the wound it covers remains a challenge for reconstructive surgery .

chronic wounds continue to be a major challenge for the medical profession , and plastic surgeons are frequently called in to help in the management of such wounds . apart from the obvious morbidity to the patient , these problem wounds can be a major drain on the already scarce hospital resources . sometimes , these chronic wounds can be more taxing than the underlying disease itself . although many newer methods are available to handle such situations , the role of stem cells in the management of such wounds is an exciting area that needs to be explored further . a review of literature has been done regarding the role of stem cells in the management of chronic wounds . the abnormal pathology in such wounds is discussed and the possible role of stem cells for optimal healing in such cases would be detailed .

juvderm is produced by a proprietary manufacturing process referred to as hylacross technology , which refers to the fact that juvderm is not sized in contrast to the other ha fillers ( prevelle silk , restylane , perlane ) which use sizing technology . sizing is the process by which crosslinked ha is pushed through a specially sized screen and broken into pieces . the medium size pieces of ha are made into restylane while the larger ones are made into perlane . it is unknown what effect the sizing technology or the hylacross technology have on a filler s performance , or if they offer any benefit in the efficacy of the product . for example , smith ( 2007 ) noted a difference of the homologous juvderm gel fillers compared to fillers with gel particle suspensions , mentioned in his publication on the practical use of juvderm . he claimed that after injection , the juvderm filler remained in the area where it was injected because of its cohesive nature and high viscosity , and did not flow away from the injection point . he stated that gel - particle fillers in contrast seem to flow away from the injection point , causing filling of unintended areas and waste of product . it is important to realize that this is one person s opinion and has not been substantiated by scientific research . to the authors knowledge , no studies have scientifically characterized the diffusion and spread of the various ha fillers . the amount of ha in a product may contribute to its stiffness and longevity . theoretically , the higher the amount of ha in the product , the stiffer it is and the longer it will last . however , not all of the ha in the product is crosslinked so one must take into account the overall percentage of cross - linking ( how much of the ha is crosslinked ) and the degree of crosslinking ( is the ha molecule completely or partially crosslinked ) . often , uncrosslinked ha is added to a filler product to increase its ease of injection as it functions as a lubricant . juvderm ultra is 9% crosslinked while juvderm ultra plus is 11% crosslinked ( table 2 ) . other fillers are cross - linked with different crosslinking agents , such as divinyl sulphone ( dvs ) for prevelle , captique , and hylaform . puragen is cross - linked with 2 , 7 , 8-diepoxyoctane ( deo ) , which forms both ether and ester crosslinks . crosslinking quality has to be in the right balance to maintain both duration and the biocompatibility of the ha filler . the stiffness or g ( pronounced g prime ) of a product is one of the most important considerations . the measurement of resistance to deformation is known as the elastic modulus or the g. together with the cohesivity of the product , g values could be used to determine the appropriate placement of an ha dermal filler . for example more robust products ( higher g values and higher cohesivities ) such as juvderm ultra plus and perlane , should be used in deeper lines , such as nasolabial folds and marionette lines , as well as to lift the lateral brow , to correct the nasal bridge , to give the ear lobe youthful volume , to evert the nipples , and to raise the nasal tip . more fluid products such as juvderm ultra and restylane are more suited to be used over large areas such as the cheekbones and cheeks . low g products such as hylaform and prevelle silk are necessary in areas that require a softer agent , such as the body of the lip or the tear trough . as new products reach the market , knowing the g will help practitioners match fillers with indications . in contrast to juvderm ultra , juvderm ultra plus has a higher proportion ( 11% ) of crosslinked ha , which makes ultra plus more viscous . consequently , ultra plus is more suitable for adding volume and correcting the deeper facial grooves and furrows , whereas juvderm ultra is best suited for contouring and volumizing facial wrinkles and folds ( figures 2 and 3 ) ( fda 2006 ) . juvderm ultra and ultra plus can be grouped in the medium range of product stiffness , which makes them suitable for the use in any wrinkles , moderate or deep , as well as scar correction . unmodified ha is included as a lubricant to help decrease extrusion force and make injection easier . juvderm ultra is injected into the mid - dermis via 30-gauge needle while juvderm ultra plus is implanted deeper via a 27-gauge needle . it is important to tightly attach the needles to the luer - lock syringe to prevent detachment during injections . as juvderm is not completely hydrated with water in the syringe , and ha is well known to be able to bind 1,000 times its weight in water , juvderm will absorb water after injections and thus slightly expand within 24 hours after correction . the patients can thus be informed , that the effect will be even better 24 hours after the injection . however , it is important to consider this feature in clinical practice , especially when injecting the body of the lips , therefore one should always undercorrect to allow for expansion . restylane and puragen are also not completely hydrated in the syringe , whereas captique , hylaform , and prevelle are completely hydrated and will not expand after injection . the longevity of juvderm ultra is about 69 months and ultra plus may last up to 12 months , which is similar to restylane and perlane . captique and prevelle silk are thought to last 46 months and the duration of puragen is unknown at the time of publication of this article . both juvderm products are packaged in 0.8-ml syringes as a clear gel and are stored at room temperature with a shelf - life of 24 months . the adverse - event profile of juvderm is mild and transient . as with all ha products , as juvderm ultra and ultra plus lack an anesthetic , patients do feel pain during injection . therefore , a topical anesthetic or a nerve block can be used to minimize discomfort . both juvderm ultra and ultra plus have been approved for use in the nasolabial folds . in the authors experience , both products may also be used off - label for lip augmentation , for the correction of marionette folds , prejugal sulci , and as volume fillers for atrophy and acne scars . furthermore , juvderm can be placed in the tear trough area , but extra care is necessary , due to the proximity to the eye with the risk of the needle popping off , thus one should inject very slowly with only moderate extrusion force . the needle is more likely to pop off when the syringe is almost empty so inject the tear trough area with a new syringe and save the last part of the syringe for less dangerous areas such as the nasolabial folds . juvderm ultra can easily be placed in the vermillion border or the body of the lip . again , one should be cautious as not to over - inject the vermillion border due to postponed expansion of the product . as juvderm has been on the us marked only since late 2006 , only a few publications have assessed the various characteristics of the juvderm products . in the pivotal trial that led to fda approval of juvderm , baumann et al ( 2007 ) compared the safety and effectiveness of 3 types of smooth - gel ha dermal fillers vs cross - linked collagen in the treatment of nlf in 439 subjects in a multicenter , double - masked , randomized , within - subject study . the subjects randomly received one of three types of smooth - gel ha dermal filler in one nlf and cross - linked bovine collagen in the other . the three different smooth - gel has used were j30 ( juvderm 30 ) , 24 hv ( juvderm ultra ) , or 30 hv ( juvderm ultra plus ) , of which only the latter two are currently marketed in the us . the cross - linked bovine collagen filler used was zyplast ( allergan , formerly inamed ) . the nlfs were to be filled to full correction ( 100% of the defect ) , and not overcorrected , and a maximum of 3 treatments first treatment and up to 2 touch - ups at roughly 2-week intervals were allowed to achieve optimal correction . nlf severity was assessed using the5-pointwrinkle assessment scale ( was ) , with 0 = none ( no wrinkle ) ; 1 = mild ( shallow , just perceptible wrinkle ) ; 2 = moderate ( moderately deep wrinkle ) ; 3 = severe ( deep wrinkle , well - defined edges but not overlapping ) ; 4 = extreme ( very deep wrinkle , redundant fold with overlapping skin ) . the results showed that all three dermal fillers proved longer - lasting clinical corrections than bovine collagen . twenty - four weeks after the last treatment , 90% of subjects treated with 30 hv ( juvderm ultra plus ) dermal filler retained a clinically significant improvement , 88% treated with 24 hv ( juvderm ultra ) and 81% with j30 dermal filler . the bovine collagen treated nlfs showed clearly shorter longevity with lasting improvement after 24 weeks ranging from 36% to 45% . in addition to its superior longevity , the injection volume for ha dermal fillers proved to be lower ( median , 1.6 ml ) compared with bovine collagen ( median , 2.0 ml ) , representing an additional important advantage for the patient in treatment costs and comfort . the only treatment - related adverse events observed were localized site reactions in the area of injection , which were mild to moderate in severity and did not differ between any filler type . in decreasing percentage those were injection site induration , erythema , edema , pain , nodule formation , bruising , discoloration , and pruritus ; they lasted no more than 7 days . the preferred filler by the patients used was 24 hv ( juvderm ultra ) with 88% , followed by 84% for 30 hv ( juvderm ultra plus ) and 78% for j30 ; the majority of subjects preferred ha fillers to the collagen fillers . in an almost identical study design ( pinsky et al 2007 ) , the safety and effectiveness of juvderm dermal fillers compared to zyplast bovine collagen for the correction of nasolabial folds ( nlfs ) was assessed in a multicenter , double - blind , randomized , within - subject controlled trial . 292 subjects were randomly treated with juvderm ultra or juvderm ultra plus in one nlf and zyplast bovine collagen in the other nlf . the treating investigators were instructed to fill each nlf to full correction ( 100% of the defect ) , but not to overcorrect . a maximum of 3 treatments first treatment and up to 2 touch - ups at roughly 2-week intervals were allowed to achieve optimal correction . an average injection volume of 1.5 ml ( 2 syringes ) of juvderm dermal filler was used for initial treatment and 0.7 ml ( 1 syringe ) for repeat treatment . nlf severity was assessed using the 5-point wrinkle assessment scale ( was ) , and a validated photographic guide . after 6 month subjects showed a clinically significant mean level of improvement for the nlfs treated with juvderm ultra or juvderm ultra plus , but not for nlfs treated with zyplast , supporting the above stated findings by showing a longer longevity for juvderm ultra and ultra plus than for zyplast . the mean level of improvement was still clinically significant for subjects who returned for a follow - up treatment beyond 9 months , with the proportion of nlfs still showing clinically significant improvement in 75% with juvderm ultra and 81% with juvderm ultra plus . again , the frequency and severity of treatment site reactions ( eg , erythema , induration , pain , edema , nodule formation , bruising , pruritus , and discoloration ) were mild or moderate and were similar for all fillers . the authors concluded , that due to its superior longevity , individuals treated with these juvderm dermal fillers may require to repeat treatments less frequently than those treated with bovine collagen fillers , and that less product will be needed at repeat treatments . the results of the above mentioned studies are supported by a recent study by lupo et al which compared juvderm ultra plus ha filler with zyplast bovine collagen in a multicenter , double - blind , randomized , within - subject , controlled study ( lupo et al 2008 ) . in a split face mode , severe nlfs of 87 subjects were treated , one side with juvderm ultra plus and the other side with zyplast . in the study population all fitzpatrick skin types were represented , 36% having darker skin types ( fitzpatrick types iv through vi ) . up effectiveness was assessed using the validated , static , 5-point wrinkle assessment scale ( was ) with a photographic guide . the juvderm ultra plus filler showed significantly better nlf severity scores compared to zyplast at each follow - up time point from 4 to 24 weeks . at 24-week follow - up clinically significant correction of nlf treated with juvderm the clinical correction with juvderm ultra plus remained high , whereas the scores for zyplast nearly returned to baseline over the period of 24 weeks . at 24 weeks , the mean improvement was still 1.7 with the juvderm ultra plus product but only 0.5 with bovine collagen . longevity was shown by maintenance of the clinical correction for 1 year or more in 81% of nlfs treated with juvderm . the median volume of juvderm required was 0.7 ml ( one syringe ) , significantly less than for zyplast ( 1.6 ml ) . for the initial treatment , the median volume of juvderm ultra plus injected was 2 syringes ( 1.6 ml ) , and less than 1 syringe ( 0.7 ml ) for the retreatment at after 69 months . treatment site reactions were similar for juvderm ultra plus and zyplast and were similar to those in the above - mentioned trials . as for patient satisfaction , most subjects preferred juvderm ultra plus ( 85% ) versus collagen ( 10% ) ; 5% showed no preference . in summary , all three studies above show superior longevity of the ha juvderm fillers compared to bovine collagen fillers . initial treatments required roughly two syringes of juvderm , and retreatments required only one syringe . treatment site reactions and side effects were similar for all fillers , ha and collagen , and were always short in duration ( less than 7 days ) and mild in severity . juvderm ultra plus was compared with radiesse in an european study by moers - capri et al ( 2007 ) that compared the hydroxylapatite filler with two ha fillers for the treatment of the nasolabial folds . the objective of this multicenter , blinded , randomized trial was to compare patient satisfaction , efficacy and durability of the various fillers . a total of 205 patients were randomized into 3 arms , receiving radiesse ( caha gel ) , juvderm ultra , or perlane . after the first treatment a touch - up was performed 4 months later and patients were followed up at 8 , and 12 months , without any additional touch - ups . the injections were performed with a 27-gauge needle into the mid to deep dermis . at 8 months follow - up , nlfs treated with caha gel were significantly more improved , than with any ha , as assessed by global aesthetic improvement scale ( gais ) ( narins et al 2003 ) . moreover , the volumes used for caha gel were lower than for the has . in the patient satisfaction there are many factors to be understood , in order to know which ha filler to use ( table 3 ) . because no peer - reviewed scientific publications have reviewed the above - mentioned properties , it is impossible at this point to know how important these various characteristics are in choosing a filler . more data need to be collected to properly understand if , for example , sizing technology makes a difference or if ester bonds last longer than ether bonds . these distinctions will become clearer and more important as more ha fillers reach the market and more data are collected .

soft tissue augmentation with temporary dermal fillers is a continuously growing field , supported by the ongoing development and advances in technology and biocompatibility of the products marketed . the longer lasting , less immunogenic and thus more convenient hyaluronic acid ( ha ) fillers are encompassing by far the biggest share of the temporary dermal filler market . since the approval of the first ha filler , restylane , there are at least 10 ha fillers that have been approved by the fda . not all of the approved ha fillers are available on the market , and many more are coming . the juvderm product line ( allergan , irvine , ca ) , consisting of juvderm plus and juvderm ultra plus , was approved by the fda in 2006 . juvderm is a bacterium - derived nonanimal stabilized ha . juvderm ultra and ultra plus are smooth , malleable gels with a homologous consistency that use a new technology called hylacross technology . they have a high concentration of cross - linked has , which accounts for its longevity . juvderm ultra plus is used for volumizing and correcting deeper folds , whereas juvderm ultra is best for contouring and volumizing medium depth facial wrinkles and lip augmentation . various studies have shown the superiority of the ha filler products compared with collagen fillers for duration , volume needed , and patient satisfaction . restylane , perlane , and juvderm are currently the most popular dermal fillers used in the united states .

granular cell tumor ( gct ) is a benign tumor believed to originate from neural cells ; it may occur at many sites , although it occasionally affects the oral cavity , skin , soft tissue , breast , and female genital organs.1,2 the gastrointestinal tract is an uncommon location of gcts , and the most common site is the esophagus followed by the large intestine and stomach.1,3 most of the colorectal gcts were located in the proximal colon , cecum , appendix , and rectum , and they usually appeared as a solitary growth and followed a benign course.4 although gcts are usually benign , malignant transformation or de novo primary malignant tumors have been described , particularly for large lesions.5,6 to date , only a few cases of benign gct in the sigmoid colon have been reported , and to our knowledge , there is only one case report in the english literature of malignant gct in the colon incidentally detected by positron emission tomography / computed tomography.3,7,8,9 here , we report a second case , involving a 43-year - old woman with histologically benign or atypical but clinically malignant gct with invasion of the lymph nodes and vessels in the sigmoid colon , and treated by segmental colon resection with lymph node dissection . a 43-year - old female patient visited sam medical center for further evaluation of a submucosal tumor that was seen on prior colonoscopy in another clinic . the laboratory results showed a white blood cell count of 4,100/mm , hemoglobin of 7.7 g / dl , platelet count of 340,000/mm , albumin of 4.4 g / dl , aspartate aminotransferase of 13 iu / l , and alanine aminotransferase of 7i iu / l . colonoscopy showed a yellowish , round submucosal tumor with intact mucosa , approximately 1 cm in diameter in the sigmoid colon ( fig . endoscopic ultrasonography ( eus ) revealed an approximately 11 cm hypoechoic heterogeneous mass with a smooth margin originating from the third echo layer ( fig . because the diagnosis was benign gct or carcinoid tumor , and the tumor was relatively small , we decided to follow the patient after 1 year , after she was given sufficient explanation about the malignant potential of the lesion . at the follow - up colonoscopy 1 year later , the size of the tumor increased to approximately 3 cm . we performed segmental colon resection with lymph node dissection , and the pathologic gross finding showed the tumor to be well circumscribed and located at the submucosal and muscular layers with central infiltration of the serosa . histologic examination revealed proliferation of large polygonal cells with abundant eosinophilic granular cytoplasm , which was positive for s-100 and invasion of local lymph nodes and vessels ( fig . two of the histologically malignant criteria of gct - presence of vesicular nuclei with large nucleoli and pleomorphism - were met , and the tumor was diagnosed as a histologically benign or atypical but clinically malignant gct occurring in the sigmoid colon . gct , which was first described occurring in the tongue by abrikossoff in 1926 , is a rare , benign tumor that can occur anywhere in the body ; however , only approximately 8% of gcts are found in the gastrointestinal tract.10 in endoscopic findings , gastrointestinal gcts are typically yellow - white , sessile , small ( < 20 mm ) , and covered by a normal - appearing mucosa.3 on endosonography , gcts are usually hypoechoic , homogeneous lesions in the submucosa , and these features are helpful for determining the depth of invasion and planning the surveillance and therapeutic strategy.3,7 in our patient , eus showed a hypoechoic , heterogeneous submucosal tumor originating from the third echo layer , and the lesion was initially considered as a benign gct or carcinoid tumor . the origin of gcts has not yet been definitely determined , and the recent use of immunohistochemical stains , including s-100 protein or neuron - specific enolase ( nse ) , suggests its origin to be the schwann cells as well as the possibility of a neuroendocrine origin.2 histologically , gcts are composed of large polygonal cells containing numerous eosinophilic granules and small , uniform nuclei and neural markers , including s-100 protein or nse.7 although a gct usually appears as a solitary small nodular growth and follows a benign course , a malignant potential has been described in a few cases , particularly for large lesions > 4 cm in diameter.7 comparisons between malignant transformation and de novo primary malignant tumor are based on primary histologic findings and the clinical course of the patient , especially in recurrent cases during the follow - up period . malignant gct is usually difficult to diagnose because of its rarity , and it was first described by ravich in 1945.11 these tumors have been classified into two categories : histologically and clinically malignant type , and histologically benign but clinically malignant type . although metastasis , among the worse prognostic features including a larger tumor size , advanced patient age , and local tumor recurrence , remains the most important criteria for defining malignancy , not all malignant gcts actually metastasize . therefore , attempts have been made to identify specific malignant histologic features to predict malignant behavior . histologically malignant features include spindling of tumor cells , the presence of vesicular nuclei with large nucleoli , increased mitotic rate , a high nuclearto - cytoplasmic ratio , pleomorphism , and necrosis , and malignant gct is histologically diagnosed when three or more of those six criteria are fulfilled . tumors that meet one or two criteria are histologically diagnosed as atypical , and those that exhibit only focal pleomorphism are diagnosed as benign.5 because malignancy can be definitely proven only by clinical findings , especially metastasis , clinical features such as metastasis , large size , rapid growth , and invasion into adjacent tissue are reported to be more important criteria of malignancy than the histologic features of a tumor with histologically undetermined malignant potential.7,12 in our patient , two microscopic criteria - the presence of vesicular nuclei with large nucleoli and pleomorphism - were observed in the resected tumor , and the lesion was considered as histologically benign or atypical ; however , the lesion manifested clinically malignant features such as rapid growth and invasion of lymph node and vessel . therefore , this case can be classified as a histologically benign or atypical but clinically malignant type of gct . some similar cases of histologically benign or atypical gct , which were diagnosed using the classic histological criteria , were reported previously as a malignant gct owing to their clinical course.5,12 in addition , malignant transformation from a benign tumor rather than being a de novo primary malignant tumor was suggested in our patient because microscopic histology showed atypical or benign findings according to the above six histologic criteria but other clinical findings showed relatively rapid tumor growth and lymphatic and vascular invasion at 1 year follow - up . in the treatment of gastrointestinal gcts , benign lesions < 2 cm in diameter and separated from the muscularis propria can be successfully removed by endoscopic resection ; however , for large lesions ( > 2 cm ) infiltrating the muscularis propria , surgery is the best therapeutic option for complete excision especially if the lesion was suspicious for malignancy.4,5,10 the prognosis of malignant gct is considered poor ; 30% to 40% of local recurrence and 50% to 60% of metastasis were reported in previous reviews ; and the role of chemotherapy and radiation therapy is not yet verified.5,12 in summary , this case describes a submucosal tumor showing continuity to the third echo layer on eus , and a rapid growth up to 3 cm was observed during the 1-year follow - up . the tumor showed histologically benign or atypical features but was diagnosed as a clinically malignant gct with invasion of lymph nodes and vessels after segmental colon resection and lymph node dissection .

granular cell tumor ( gct ) is an uncommon , usually benign neoplasm ; however , a malignant potential has been described . malignant gct is an extremely rare neoplasm showing rapid growth and invasion into adjacent muscles , lymph nodes , or vessels , or even distant metastasis . we recently experienced a case of a histologically benign or atypical but clinically malignant gct , with invasion of the lymph nodes and vessels in the sigmoid colon , diagnosed by segmental colon resection with lymph node dissection . we also performed a review of relevant medical literature .

s. globisoprus wild - type and mutant strains were grown on isp4 agar medium or cultured in a9 liquid medium as described.15 escherichia coli dh5 and et1256716 were cultured in luria - bertani ( lb ) broth.17 micrococcus luteus atcc 9431 was grown on lb agar.17 cosmid pbs10064 and plasmid pwhm125018 were described previously . general dna manipulations were performed according to standard procedures.17 pcr reactions were performed using pfx dna polymerase according to the manufacturer 's instructions ( invitrogen , carlsbad , ca ) . the pcr fragment obtained using pfx was treated with taq dna polymerase from invitrogen ( carlsbad , ca ) for 10 min at 72 c to add the terminal streptomyces protoplasts preparation and transformation were performed as described previously.19 homologous sequence database searching was executed with blast . cosmid pbs1006 was used as a template to clone the three regulators for c-1027 production by pcr . the sgcr1 gene was amplified by pcr using primers 5-cgaggatccgcggatggattacacg-3 ( sgcr1 upstream , bamhi ) and 5-cctctagaagtaggccgtcaacggc-3 ( sgcr1 downstream , xbai ) and inserted into pgem - t easy after adding terminal the sgcr1 gene was excised as a 1.1-kb bamhi / xbai fragment and inserted into the same sites of pwhm1250 to generate pbs1107 , in which the expression of sgcr1 is under the control of the constitutive erme * promoter . similarly , the sgcr2 gene was amplified by pcr using primers 5-cttctagagggagttacggtgaccacg-3 ( sgcr2 upstream , xbai ) and 5-ttctgcagggccctggtcaccc-3 ( sgcr2 downstream , psti ) and inserted into pgem - t easy after adding terminal plasmid pbs1097 with sgcr2 under the control of erme * was then constructed by inserting the 0.9-kb psti / xbai sgcr2 fragment into the same sites of pwhm1250 . for sgcr3 overexpression , the sgcr3 gene was amplified by pcr using primers 5-cgaaagcttgcccaggagaagggaaac-3 ( sgcr3 upstream , hindiii ) and 5-gggacgcgtcgctcagctcc-3 ( sgcr3 downstream , mlui ) , inserted into pgem - t easy after adding terminal after removal of the terminator between the erme * promoter and the hindiii site in pwhm1250 as a 0.5-kb sphi fragment , the sgcr3 gene was cloned as a 1.2-kb hindiii / mlui fragment into the same sites of the modified pwhm1250 to afford pbs1108 , in which the expression of sgcr3 is under the control of erme*. finally , pbs1107 , pbs1097 , and pbs1108 were introduced into the wild - type s. globisporus strain by protoplast transformation to afford recombinant s. globisporus strains sb1014 , sb1015 , and sb1016 that overexpress sgcr1 ( sb1014 ) , sgcr2 ( sb1015 ) , and sgcr3 ( sb1016 ) , respectively.4 plasmid pbs1109 , in which sgcr2 and sgcr3 are expressed in tandem under the control of erme * , was constructed by inserting the 1.2-kb hindiii / mlui fragment of sgcr3 into the same sites of pbs1097 . similarly , insertion of the 1.1-kb bamhi / xbai fragment of sgcr1 into the same sites of pbs1109 yielded pbs1110 , in which erme * controlled the expression of all three genes sgcr1 , sgcr2 , and sgcr3 . introduction of pbs1109 and pbs1110 into the wild - type s. globisporus strain by protoplast transformation finally afforded the recombinant strains sb1017 ( sgcr2r3 overexpressing ) and sb1018 ( sgcr1r2r3 overexpressing ) , respectively , that overexpress the selected regulatory genes in tandem.4 s. globisporus wild - type and recombinant strains were cultured in a9 medium using a two - step fermentation for c-1027 and heptaene production as described4,15 and then treated to obtain three fractions as follows : ( fraction 1 ) the cells were pelleted by centrifugation ; ( fraction 2 ) the supernatant was adjusted to ph 4.0 and centrifuged to collect the precipitate ; and ( fraction 3 ) the remaining supernatant was saturated to 70% with ammonium sulfate , incubated at 4c for 3 hours , and the precipitated proteins were collected by centrifugation . the c-1027 chromophore was isolated from fraction 3 by acetone extraction as described previously.15 fractions 1 and 2 were combined and extracted with acetone to isolate the heptaene metabolite.11 the acetone extracts were detected directly by hplc after being concentrated.5,11 c-1027 production was assayed against m. luteus atcc 9431 as described previously.4,15 for the liquid a9 medium cultures , the ammonium sulfate - precipitated fraction 3 containing the c-1027 chromoprotein complex was dissolved in the same volume of water and 5 ul of the resultant solution was used for each bioassay . for the solid isp4 plates ( 25 ml isp4 in a 9 cm petri dish ) growing s. globisporus strains , a round agar block of 0.5 cm diameter was placed directly onto an m. luteus - seeded lb agar plate for antibacterial assay . hplc analysis was performed with a c18 column ( 5 m , 250 mm 4.6 mm , alltech , lexington , ky ) on a varian hplc system with an in - line prostar 330 pda detector ( woburn , ma ) . hplc detection of the c-1027 chromophore was the same as described previously.15 for hplc analysis of the heptaene , the column was eluted at a flow rate of 1 ml / min with a gradient of acetonitrile with 0.1% trifluoroacetic acid from 50% to 100% for 15 min and maintained 100% for a further 10 min . s. globisoprus wild - type and mutant strains were grown on isp4 agar medium or cultured in a9 liquid medium as described.15 escherichia coli dh5 and et1256716 were cultured in luria - bertani ( lb ) broth.17 micrococcus luteus atcc 9431 was grown on lb agar.17 cosmid pbs10064 and plasmid pwhm125018 were described previously . general dna manipulations were performed according to standard procedures.17 pcr reactions were performed using pfx dna polymerase according to the manufacturer 's instructions ( invitrogen , carlsbad , ca ) . the pcr fragment obtained using pfx was treated with taq dna polymerase from invitrogen ( carlsbad , ca ) for 10 min at 72 c to add the terminal streptomyces protoplasts preparation and transformation were performed as described previously.19 homologous sequence database searching was executed with blast . cosmid pbs1006 was used as a template to clone the three regulators for c-1027 production by pcr . the sgcr1 gene was amplified by pcr using primers 5-cgaggatccgcggatggattacacg-3 ( sgcr1 upstream , bamhi ) and 5-cctctagaagtaggccgtcaacggc-3 ( sgcr1 downstream , xbai ) and inserted into pgem - t easy after adding terminal the sgcr1 gene was excised as a 1.1-kb bamhi / xbai fragment and inserted into the same sites of pwhm1250 to generate pbs1107 , in which the expression of sgcr1 is under the control of the constitutive erme * promoter . similarly , the sgcr2 gene was amplified by pcr using primers 5-cttctagagggagttacggtgaccacg-3 ( sgcr2 upstream , xbai ) and 5-ttctgcagggccctggtcaccc-3 ( sgcr2 downstream , psti ) and inserted into pgem - t easy after adding terminal plasmid pbs1097 with sgcr2 under the control of erme * was then constructed by inserting the 0.9-kb psti / xbai sgcr2 fragment into the same sites of pwhm1250 . for sgcr3 overexpression , the sgcr3 gene was amplified by pcr using primers 5-cgaaagcttgcccaggagaagggaaac-3 ( sgcr3 upstream , hindiii ) and 5-gggacgcgtcgctcagctcc-3 ( sgcr3 downstream , mlui ) , inserted into pgem - t easy after adding terminal after removal of the terminator between the erme * promoter and the hindiii site in pwhm1250 as a 0.5-kb sphi fragment , the sgcr3 gene was cloned as a 1.2-kb hindiii / mlui fragment into the same sites of the modified pwhm1250 to afford pbs1108 , in which the expression of sgcr3 is under the control of erme*. finally , pbs1107 , pbs1097 , and pbs1108 were introduced into the wild - type s. globisporus strain by protoplast transformation to afford recombinant s. globisporus strains sb1014 , sb1015 , and sb1016 that overexpress sgcr1 ( sb1014 ) , sgcr2 ( sb1015 ) , and sgcr3 ( sb1016 ) , respectively.4 plasmid pbs1109 , in which sgcr2 and sgcr3 are expressed in tandem under the control of erme * , was constructed by inserting the 1.2-kb hindiii / mlui fragment of sgcr3 into the same sites of pbs1097 . similarly , insertion of the 1.1-kb bamhi / xbai fragment of sgcr1 into the same sites of pbs1109 yielded pbs1110 , in which erme * controlled the expression of all three genes sgcr1 , sgcr2 , and sgcr3 . introduction of pbs1109 and pbs1110 into the wild - type s. globisporus strain by protoplast transformation finally afforded the recombinant strains sb1017 ( sgcr2r3 overexpressing ) and sb1018 ( sgcr1r2r3 overexpressing ) , respectively , that overexpress the selected regulatory genes in tandem.4 s. globisporus wild - type and recombinant strains were cultured in a9 medium using a two - step fermentation for c-1027 and heptaene production as described4,15 and then treated to obtain three fractions as follows : ( fraction 1 ) the cells were pelleted by centrifugation ; ( fraction 2 ) the supernatant was adjusted to ph 4.0 and centrifuged to collect the precipitate ; and ( fraction 3 ) the remaining supernatant was saturated to 70% with ammonium sulfate , incubated at 4c for 3 hours , and the precipitated proteins were collected by centrifugation . the c-1027 chromophore was isolated from fraction 3 by acetone extraction as described previously.15 fractions 1 and 2 were combined and extracted with acetone to isolate the heptaene metabolite.11 the acetone extracts were detected directly by hplc after being concentrated.5,11 c-1027 production was assayed against m. luteus atcc 9431 as described previously.4,15 for the liquid a9 medium cultures , the ammonium sulfate - precipitated fraction 3 containing the c-1027 chromoprotein complex was dissolved in the same volume of water and 5 ul of the resultant solution was used for each bioassay . for the solid isp4 plates ( 25 ml isp4 in a 9 cm petri dish ) growing s. globisporus strains , a round agar block of 0.5 cm diameter was placed directly onto an m. luteus - seeded lb agar plate for antibacterial assay . hplc analysis was performed with a c18 column ( 5 m , 250 mm 4.6 mm , alltech , lexington , ky ) on a varian hplc system with an in - line prostar 330 pda detector ( woburn , ma ) . hplc detection of the c-1027 chromophore was the same as described previously.15 for hplc analysis of the heptaene , the column was eluted at a flow rate of 1 ml / min with a gradient of acetonitrile with 0.1% trifluoroacetic acid from 50% to 100% for 15 min and maintained 100% for a further 10 min . manipulation of pathway regulation is an efficient strategy to increase specific secondary metabolite production.12,13 among the three regulators indentified within the c-1027 biosynthetic gene cluster , sgcr3 has been characterized as a tylr - like activator,14 and sgcr1 and sgcr2 have been proposed to be positive regulators based on bioinformatic analyses . sgcr2 belongs to the arac / xyls activator family,20 while sgcr1 is a strr - like protein ; the only two studied strr - like regulators , strr ( 36.7% identity)21 and novg ( 35.1% identity),22 are both activators . based on the premise that all three c-1027 regulators are potential activators , the three regulatory genes were overexpressed individually under the control of the constitutive erme * promoter on the high - copy - number plasmid pwhm1250 in s. globisporus c-1027 to generate strains sb1014 ( sgcr1 overexpressing ) , sb1015 ( sgcr2 overexpressing ) and sb1016 ( sgcr3 overexpressing ) . evaluation of the metabolic profiles of each of the recombinant strains revealed that sgcr1 was a particularly effective activator for the production of a heptaene , an early metabolite of the enediyne biosynthetic machineries.11 the sgcr1-overexpressing strain s. globisporus sb1014 produced 46 4 mg / l of the heptaene at day 2 , which was about 9-fold higher than in wild - type ( 5 0.4 mg / l ) , and by day 5 yielded 66 5 mg / l , which is about 5-fold higher than the 14 3 mg / l observed in wild - type . mg / l ) were also higher than wild - type at day 5 ( figure 2 ) , supporting a positive regulatory role for all three regulators . the production of c-1027 in each strain was detected using a bioassay against m. luteus atcc 9431 . in the case of a9 liquid medium , all three regulator - overexpressing recombinant strains produced c-1027 earlier than did the wild - type control ( figure 3 ) . the s. globisporus wild - type or recombinant strains achieved highest c-1027 production at day 4 ( sb1016 ) or day 5 ( wild - type , sb1014 and sb1015 ) , and all three recombinant strains exhibited larger inhibition zones than observed with wild - type at their highest titer ( figure 3b ) , confirming the up - regulating characteristics of sgcr1 , sgcr2 and sgcr3 . the sgcr1-overexpressing mutant sb1014 produced the highest titer of c-1027 at day 5 , which was 2 to 3 times the c-1027 titer in wild - type when analyzed by hplc ( figure 3c ) . in the case of isp4 agar medium , sb1015 and sb1016 also produced c-1027 earlier , but their largest inhibition zones were comparable in size to those of the s. globisporus wild - type ( figure 3a ) . in summary , overexpression of each individual regulator induced similar titer increases for both c-1027 and heptaene in a9 medium , indicating a common regulatory system controlling the biosyntheses of both compounds . moreover , sgcr1 overexpression was more effective at titer improvement than overexpression of either sgcr2 or sgcr3 , implying that one or more genes controlled by sgcr1 are bottlenecks in c-1027 production . given that sgcr1 , sgcr2 and sgcr3 behave as activators , we reasoned that their co - overexpression in the s. globisporus wild - type strain could further improve the production of c-1027 and related compounds . two pwhm1250-derived constructs , containing sgcr2r3 ( pbs1109 ) or sgcr1r2r3 ( pbs1110 ) in tandem under the control of the erme * promoter , were transformed into s. globisporus wild - type to afford sb1017 and sb1018 , respectively . / l ) at day 5 than sb1015 and sb1016 , but less than sb1014 . to our surprise , sb1018 produced heptaene with an efficiency comparable to that of wild - type ( 12 1 mg / l ) ( figure 2 ) . for c-1027 titers in a9 liquid media , sb1017 reached optimum yield at day 4 , and the inhibition zone was larger than those of c-1027 wild - type . in contrast , the c-1027 titer in sb1018 was not improved ( figure 3 ) . in the case of isp4 agar media , however , the largest inhibition zones were comparable in size to those found for c-1027 wild - type . the counterintuitive observation that overexpression of all three activators together in s. globisporus sb1018 did not further improve c-1027 or heptaene production implies the multifaceted regulation of c-1027 biosynthesis . due to their low titers and inherent reactivity , hplc analysis of enediyne production is laborious , often requiring a length process of sample preparation , in particularly for the 9-membered enediynes produced as chromoprotein complexes . although antibacterial bioassays provide a convenient detection method , they are inevitably limited by a lack of specificity as other antibiotics may also inhibit the tested bacterial growth . moreover , some newly discovered enediyne analogs like the sporolides from salinispora tropica have no detectable antibacterial activity,23 which limits the utility of antibacterial bioassays for discovering novel enediynes . the heptaene metabolite was previously shown to be produced by all 9- or 10-membered pkses and their cognate thioesterases thus far tested using in vivo and in vitro approaches.9 - 11 subsequent discovery of this heptaene in fermentations with all enediyne producers inspired its use as a unique and convenient phenotypic indicator for enediyne production.11 since both the heptaene and the final enediyne natural products are biosynthesized by pkses , production of the heptaene indicates that the pkse is functionally expressed and capable of synthesizing the polyketide precursor of the enediyne core . previous work on s. globisporus showed that production of both c-1027 and heptaene was abolished in a sgce pkse null mutant , but could be restored by complementation with a functional copy of the sgce gene in trans.11 here we demonstrate that in all five s. globisporus recombinant strains , c-1027 and hepatene production was similarly affected . these data complement previously reported genotypic screenings for new enediyne producers.24,25 more importantly , the use of heptaene production as an indicator of enediyne production presents novel opportunities for fermentation optimization efforts and the identification of cryptic , and otherwise undetectable , enediyne producers . in conclusion , we have demonstrated that , in addition to sgcr3 , the other two regulators , sgcr1 and sgcr2 , identified in the c-1027 biosynthetic gene cluster also play positive regulatory roles in c-1027 production . the titers of both c-1027 and the heptaene metabolite were significantly increased in the sgcr1-overexpressing s. globisporus strain sb1014 . moreover , data presented here support the application of heptaene production as a convenient phenotypic indicator for enediyne production .

manipulation of pathway regulation is an efficient strategy to increase specific secondary metabolite production . here we successfully improved production of both the enediyne antitumor antibiotic c-1027 and a heptaebe , an early metabolite of the c-1027 pathway , by manipulating the three regulatory genes , sgcr1 , sgcr2 , and sgcr3 , within the c-1027 biosynthetic gene cluster . sgcr3 has previously been established as an activator , and we now propose that sgcr1 and sgcr2 are also positive regulators based on their up - regulation effects on titer and/or timing of heptaene and c-1027 production in streptomyces globisporus . specifically , overexpression of sgcr1 significantly improved production of the heptaene ( about 5-fold ) and c-1027 ( 2- to 3-fold ) compared to the wild - type strain . however , the titers of heptaene and c-1027 were not increased by overexpressing all three activators together , underscoring the complexity of c-1027 biosynthetic pathway regulation . the possibility of exploiting the heptaene as a readily identifiable and unique indicator for rapidly detecting enediyne production was also assessed .

geneexpression profiling approaches divided all into several subcategories , in which prognoses and frequencies according to age differ significantly.12 herein , we introduce one of the new all categories , phlike all , which is related to highrisk all . philadelphia chromosome positivity signifies the result of a translocation that gives rise to the bcrabl1 oncogene and is one of the most detrimental hallmarks observed in all patients . two groups independently proposed that multiple patients with phnegative blineage all had geneexpression profiles similar to those of patients with phpositive all.13 , 14 such phnegative all cases were categorized as philadelphia chromosomelike all ( phlike all ) . philadelphia chromosomelike all comprises 10% and 13% of standard and highrisk childhood blineage all , respectively.15 the frequency of phlike all increases with age , accounting for > 25% of young adult cases , whose eventfree and overall survival expectation is extremely poor , similar to that of phpositive cases.15 given that the definition of phlike all was based only on the similarity of geneexpression profiles to phpositive all , the genetic abnormalities associated with phlike all cases were unlikely to be homogeneous . therefore , transcriptome and wholegenome sequencing was carried out to understand genetic alterations underlying phlike all.15 , 16 among 1725 blineage all cases , 154 patients were determined as phlike all and underwent genomic analyses . these approaches subcategorized phlike all into seven groups : type i , ablclass fusions ( abl1 , abl2 , csf1r , pdgrb ) ; type ii , erythropoietinreceptor ( epor ) or jak2 rearrangements ; type iii , cytokine receptorlike factor 2 ( crlf2 ) rearrangements ( often accompanied by jak2 mutations and jakstat signal activation ) ; type iv , other mutations activating jakstat signaling ( il7r , flt3 , sh2b3 , tyk2 , il2rb ) ; type v , uncommon miscellaneous kinase mutations ( ntrk3 , dgkh ) ; type vi , raspathway mutations ( kras , nras , ptpn11 , nf1 ) ; and type vii , no mutations in kinase genes ( fig . 2 ) . categorization of philadelphia chromosomelike acute lymphoblastic leukemia ( phlike all ) according to genetic abnormalities . genomic analyses have shown that phlike all is heterogeneous , but involves a high frequency of kinase gene alterations . the data summarized in this figure are from roberts et al.15 the type i category with ablclass fusions accounted for 22% of phlike all cases and was independent from the abnormalities associated with the jakstat signaling pathway . the type ii category accounted for 18% of phlike all , while type iii cases showed the most frequently observed genetic abnormality ( approximately 20% ) in phlike all cases . interestingly , more than half of the crlf2 rearrangements associated with type iii cases also harbored missense or multiple mutations in jak2 , which activate jakstat signaling . the type iv category was related with other jakstatactivating abnormalities , in which il7r and/or flt3 mutations were common . cases involving the type v and type vii categories were infrequent and difficult to characterize , and type vi occurred in a minority of phlike all cases , exhibiting genomic abnormalities activating ras signaling . importantly , the high frequency of such kinaseactivating mutations in phlike all suggested that the treatment outcomes of phlike all might improve with specific inhibitors ( fig . , the mullighan group reported that the addition of imatinib induced remission in a refractory phlike all patient who had an activating rearrangement of pdgfb.17 kinase gene alterations and their inhibitors in philadelphia chromosomelike acute lymphoblastic leukemia ( phlike all ) . the tyrosine kinase inhibitors for each kinase mutation in phlike all are indicated as a possible therapy . deletions , amplifications , mutations , and structural rearrangements in key transcription factors promoting early lymphoid differentiation ( e.g. , tcf3/e2a , ebf , lef1 , ikzf1 , ikzf3 , pax5 , and blnk ) were detected in 40% of blineage all cases.18 among these , mutations in the ikzf1 gene encoding the ikaros transcription factor were frequently observed and more highly correlated with poor prognosis associated with all than were mutations in genes encoding other transcription factors . notably , many phlike all cases , regardless of the subcategories mentioned above , revealed mutations in the ikzf1 gene , which is also a common finding in phpositive all . ikzf1 encodes the transcription factor ikaros , which is indispensable for the induction of blineage differentiation in hscs.19 , 20 its mutations are also strongly associated with lymphoid blast crisis of cml.21 therefore , here , we introduce accumulating data pertaining to ikzf1 mutations associated with highrisk all ( table 1 ) . features of ikzf1mutated acute lymphoblastic leukemia ( all ) aberrant ikzf1 mutations are likely some of the most detrimental driver mutations , accounting for > 80% of phpositive all . ikzf1 deletions were not detectable in chronicphase cml , but emerged simultaneously when cml transformed to lymphoid blast crisis.21 ikzf1 alterations were also common in phlike all cases , regardless of the type of kinase gene mutation described above,15 and suggested significantly lower 5year eventfree survival rates of phlike all patients compared to those without an ikzf1 alteration . with respect to tlineage all , ikzf1 mutations were also observed more frequently in etpall , the phenotype of which is characterized as tlineage markernegative and hsc / myeloid markerpositive , than in other tlineage all cases.22 homozygous germline ikzf1null mice lacked t , b , and nk lymphocytes and their early progenitors , and heterozygous dominantnegative ikzf1mutated mice rapidly acquired tlineage all.19 , 23 , 24 dominantnegative ikzf1 mutations are more deleterious and oncogenic , likely due to crossinterference with other ikaros family members . most ikzf1 deletions identified in phpositive all were monoallelic and lacked exons 36 of the ikzf1 gene , which encode the nterminal zinc finger dnabinding domain.21 this deletion results in dominantnegative isoforms that inhibit both wildtype ikaros and other family members.25 interestingly , genomic breakpoints in the ikzf1 gene are located in the vicinity of cryptic heptamerrecombinationsignal sequences , which are recognized by the rag enzyme complex.21 these observations suggested that ikzf1 deletions likely occurred with rag expression , which marked the specification of hematopoietic stem / progenitor cells toward the lymphoid lineage.26 , 27 , 28 recent studies have indicated that histone h3 , trimethylated on lysine at position 4 , in proximity to cryptic recombinationsignal sequences , might contribute to misleading the rag complex into producing aberrant recombination and promoting oncogenes.29 thus , epigenetic instability in phpositive hscs or their proximate progenitors could underlie the incidence of ikzf1 mutations . although ikzf1 alterations strongly correlate with refractory all , the underlying mechanism of which remains unknown , recent efforts to investigate the biological features of ikzf1mutated cells offered clues to overcome this intractable disease . the georgopoulos group showed that induction of dominantnegative ikaros isoforms in early preb cells arrested their differentiation at the proliferative large preb cell stage and culminated in oligoclonal expansion with the occurrence of blineage all in transplanted recipients.30 , 31 these ikarosdeleted preleukemic and leukemic cells expressed higher levels of integrins than those in normal counterpart cells , and were more dependent on bm stromal cells through integrinmediated adhesion for their growth and survival . notably , inhibitors for focal adhesion kinase , which transduces integrin signaling into cells , significantly abrogated adhesion and induced apoptosis in ikarosdeleted leukemic cells.30 the mullighan group also reported that ikzf1 alterations induced adhesive potential and hscrelated characteristics in phpositive all cells , while reducing their responsiveness to tyrosine kinase inhibitors.32 ikzf1altered all cells infiltrated bm and interacted with perivascular mesenchymal cells and arterial endothelial cells , which are thought to comprise the hsc niche.33 it is noteworthy that treatment with retinoidreceptor agonists enhanced ikzf1 expression , reversed hsclike features of ikzf1altered phpositive all cells , and recovered their sensitivity to tyrosine kinase inhibitors.32 while retinoids have long been known to affect the integrity of hscs and the differentiation of lymphohematopoietic progenitors by directly or indirectly regulating a number of transcription factors,34 , 35 , 36 numerous nuclear receptors also potentially regulate transcription factors in hscs.37 thus , it is worth examining the types of nuclearreceptor signals that upregulate ikzf1 expression . intragenic deletions of erg , which encodes an ets family member transcription factor , were recently identified in a subset of childhood blineage all.38 intriguingly , although dominantnegative ikzf1 deletions were frequently associated with ergdeleted all cases , the response to treatment and survival of these patients were unexpectedly positive . in fact , within blineage all cases involving ikzf1 deletion , 8year eventfree survival improved to > 85% in cases involving erg deletions , whereas the rate was only 51% without the deletions.38 an independent group also reported similar results , suggesting that erg deletions might mitigate the negative impact of ikzf1 deletion in all prognoses.39 future studies on the molecular mechanisms underlying such exceptional cases might discover more sophisticated strategies to overcome ikzf1 alterations in all cells . historically , germline variations inherited from ancestors were rarely considered to influence all incidence or features . until a decade ago , all pathogenesis was thought to be mostly attributed to acquired mutations in hscs or lymphoid progenitors . however , recent studies have identified multiple genomic variants that increase all susceptibility and affect prognosis . genomewide association studies for childhood all cases identified multiple germline polymorphisms showing high association with all incidence and characteristics.11 some of the inheritable variants were found in lymphoid neoplasmrelated genes , including ikzf1 , arid5b , and cdkn2.40 , 41 , 42 , 43 , 44 ikzf1 encodes the lymphoidlineage transcription factor ikaros mutation , which is intimately associated with all as previously discussed . notably , this protein is also an integral component combining transcription factors with the chromatinremodeling network.45 arid5b is a member of the atrich dnainteraction domain family,46 and although the arid5b function in lymphohematopoiesis has not been well studied , it may be involved in epigenetic regulation of gene expression in hscs and early lymphoid progenitors , similar to other atrich dnabinding proteins.47 , 48 , 49 , 50 interestingly , arid5b polymorphisms are associated with blineage hyperdiploid all and racial differences associated with all incidence.40 , 51 cdkn2 encodes ink4a / arf , which regulates hsc selfrenewal and differentiation under the control of the transcriptional repressor and polycomb group protein bmi1.52 , 53 therefore , hereditary predisposition to all may be related to the epigenetic instability of hscs and lymphoid progenitors . a hyperdiploid karyotype is more common in childhood all compared to adult all ; however , the associated mechanisms remain unknown . moriyama and colleagues studied familial all and identified a nonsense variant of etv6 , a member of the erythroblast transformationspecific family , which is involved in hematopoiesis and oncogenesis , with high prevalence among familial all cases.54 , 55 , 56 they undertook a broad survey for etv6 mutations in > 4405 all children and identified 31 somatic etv6 variants potentially related to all susceptibility . interestingly , all children with etv6 variants were significantly older than those without such variants when diagnosed , and more often had a hyperdiploid karyotype . holmfeldt and colleagues used genomic profiling on 124 childhood all cases with a hypodiploid karyotype57 and identified inherited alterations in tp53 , as well as ras , dnarepair , and receptor tyrosine kinase signalingrelated genes . the higher frequency of all in children relative to adults suggests that the influence of genetic predisposition to the disease might be more profound at younger ages . however , a recent study showed that inherited gata3 variants strongly enhanced susceptibility to all in adolescents and young adults.58 the detected gata3 polymorphisms were also detectable in childhood phlike all,59 and the frequency of all patients with those gata3 variations was positively correlated with patient age at diagnosis . furthermore , gata3 variations were also associated with poor treatment response and high risk of relapse . these findings suggested that agerelated differences in all biology might reflect , at least in part , the genetic variations resident in hscs . in the last decade , advances in geneexpression profiling and genomewide sequencing have revolutionized our understanding of all pathogenesis . as described in this short review , many pathogenic all mutations have been identified in hscrelated genes , which are often associated with all treatment failure and early relapse . as some of the mutations are also associated with activation of certain kinase pathways , the invention of simple , convenient , and costeffective sequencing technologies would enable earlier and more sophisticated therapeutic intervention with specific inhibitors . on the other hand , we must stress that leukemiainitiating mutations are still undetectable in > 10% of childhood all patients , and that the genetic information of older adult all patients has not been catalogued at this stage . nonetheless , we believe that studies in the coming decade will completely describe the genomic landscape of all across all generations and refine the therapeutic algorithm to be more targeted and individualized . the authors have no conflicts of interest relating to the topic of this article . abbreviationsallacute lymphoblastic leukemiaamlacute myeloid leukemiabmbone marrowcmlchronic myeloid leukemiaetpearly tlineage progenitorflt3fmslike tyrosine kinase3hschematopoietic stem cellil7rinterleukin7 receptornknatural killerphlikephiladelphia chromosomelikeragrecombinationactivating genestatsignal transducer and activator of transcription acute lymphoblastic leukemia acute myeloid leukemia chronic myeloid leukemia early tlineage progenitor fmslike tyrosine kinase3 hematopoietic stem cell interleukin7 receptor philadelphia chromosomelike recombinationactivating gene signal transducer and activator of transcription

acute lymphoblastic leukemia ( all ) occurs with high frequency in childhood and is associated with high mortality in adults . recent technical advances in nextgeneration sequencing have shed light on genetic abnormalities in hematopoietic stem / progenitor cells as the precursor to all pathogenesis . based on these genetic abnormalities , all is now being reclassified into newly identified subtypes . philadelphia chromosomelike blineage all is one of the new highrisk subtypes characterized by genetic alterations that activate various signaling pathways , including those involving cytokine receptors , tyrosine kinases , and epigenetic modifiers . philadelphia chromosomelike all is essentially heterogeneous ; however , deletion mutations in the ikzf1 gene encoding the transcription factor ikaros underlie many cases as a key factor inducing aggressive phenotypes and poor treatment responses . wholegenome sequencing studies of all patients and ethnically matched controls also identified inherited genetic variations in lymphoid neoplasmrelated genes , which are likely to increase all susceptibility . these findings are directly relevant to clinical hematology , and further studies on this aspect could contribute to accurate diagnosis , effective monitoring of residual disease , and patientoriented therapies .

cesarean sections ( cs ) performed following a medical indication is necessary for life - saving . it is a surgical intervention to prevent maternal or perinatal complications and the appropriate rate of use should be one associated with the lowest rate of maternal and perinatal morbidity and mortality . however , women are increasingly undergoing cs without any medical indication , which may contribute to the world - wide higher rates of cs . there has been a debate about the appropriateness of cs performed due to maternal request or what physicians suggest to their patients . result of the world health organization ( who ) global survey in a large cross - sectional study demonstrated severe maternal morbidity in planned cesarean delivery are higher than planned vaginal delivery ( vd ) . who recommended 10 - 15% rate of cesarean section . this recommendation was based on the cesarean section rates of the countries with the lowest maternal and neonatal mortality rates in the world at that moment . many studies have shown that the actual rate of cs in numerous countries is far higher than the recommended range . the rate of cesarean section out of all deliveries in iran increased by six - fold over the past three decades . these results are in accordance with previous reports that confirm the growing rate of cs in iran . however , the debate on the best practices ( vd vs. cs ) to minimize postnatal morbidity still is a matter of controversy both from professionals perspectives and from women 's perceptions of the childbirth experience . the postpartum period are critical life events for women leading to physiological , emotional , and social changes . postpartum mothers experience certain physical health conditions that may affect their quality - of - life ( qol ) , future health , and health of their children . several studies confirm that socio - economic deficiencies and medical problem are risk factors for decreased qol and depressive symptoms in women during the postnatal period . mode of delivery and childbirth experience may have a long - term effect on self - rated health . several studies have investigated the association between type of delivery and health related quality - of - life ( hrqol ) , but there is conflicting , with some studies reporting decreased hrqol in cesarean delivery . whereas , other studies did not confirm any relations between cs and hrqol . in a study it was found that patients after vd had higher mean physical hrqol scores than after cs while mean mental hrqol were similar among vd and cs groups . in another study , however , careful assessment of the predictor variables of poorer physical and mental health after childbirth may improve the quality of postpartum care . the aim of this study was to explore the impact of delivery mode on women 's postpartum physical and mental health related qol . a prospective study was performed in the period august 2007 to october 2008 of pregnant women who admitted for prenatal care in the health center . a total of 10 health center were selected randomly in kashan city in iran . after applying inclusion and exclusion criteria , there were 365 consecutive women that agreed to take part in the study and all women gave their informed consent before entering the study . of whom 342 women entering the study after delivery consist of 175 ( % 51.2 ) mothers after vd and 167 ( % 48.8 ) mothers after cs ( elective or emergency cs ) , in follow - up 2 months after delivery ( time 1 assessment ) 162 women after vd and 159 after cs completed qol questionnaire . of whom 150 women in each type of delivery completed qol questionnaire 4 months after delivery again ( time 2 assessment ) in health centers . in these analyses , we have included all women with singleton , live born infant , term pregnancy , prenatal care started before 20 weeks gestation , having uncomplicated pregnancies , parity 1 - 3 and the study population was ethnically iranian , not having : others pre - existing diseases , history of infertility , and divorce . excluding criteria were instrumental delivery , birth weight less than 2,500 g , child or fetal dead , child abnormality , not breast feeding and medical problems in child and mother . all patients were interviewed by the trained midwives and a structured questionnaire was used to evaluate women 's socio - demographic characteristics and obstetric variables . also we used a generic hrqol instrument to measure qol ( categorized into physical and mental health ) . qol was assessed using the iranian version of short form-36 health survey social functioning ( sf-36 ) . sf-36 is a standard and well - known generic health related qol instrument and proved to be highly feasible , reliable and is a good choice to measure health related qol after delivery . it consists of 36 items , organized into eight scales : physical functioning ( pf ) , role limitation due to physical problems or role physical ( rp ) , bodily pain ( bp ) , general health ( gh ) , vitality ( vt ) , social functioning ( sf ) , role limitation due to emotional problems or role emotional ( re ) , and emotional well - being ( ew ) . the scores on each subscale range from 0 to 100 with higher scores indicating a better condition , the sf-36 furthermore provides physical health ( based on the pf , rp , bp , and gh scales ) and a mental health ( relating to vt , sf , re , and ew ) . we compared women 's socio - demographic characteristics and physical and mental health scores between women with vd and cs that completed qol questionnaire at 2 and 4 months after delivery ( 150 vd and 150 cs ) . furthermore score differences within each mode of delivery between time 2 and time 1assessment were evaluated . differences in means were analyzed using the student 's t - test and the mann the study protocol was approved by the local research committee in kashan university of medical sciences . there were no significant differences between vd and cesarean groups with regard to these variables . maternal age was between 20 and 40 and vd was performed up to % 80 with episiotomy or laceration . the physical and mental health parameters related qol at 2 months follow - up has shown in table 2 ; total physical health score in vd women was higher than cs group . mean scores was 60.52 13.28 and 57.37 12.20 ( p = 0.034 ) respectively . on performing a detailed analysis , statistically significant differences in 2 parameters ; pf ( p = 0.001 ) and rp ( p = 0.026 ) were found . total mental health score in cs women was generally higher than vd , but differences were not significant . comparing physical and mental health parameters at 4 months postpartum results showed that the vd group in compared cs group had a higher score in total physical health ( 61.37 13.05 and 58.36 14.09 p = 0.05 ) and total mental health was ( 64.99 12.44 and 61.83 13.58 p = 0.036 ) respectively . in comparing subscales ; differences statistically significant were found for 1 parameter in physical health ( physical function p = 0.01 ) and for 2 parameter in mental health ( sf p = 0.036 and ew p = 0.042 ) . to compare the findings within each group results demonstrated that the vd group showed more improvements on total mental hrqol from time 1 to time 2 assessment [ table 4 ] . the mean score differences ( 4 months scores minus 2 months scores ) in vd and cs groups were 4.70 16.29 and 0.3 13.81 ( p = 0.01 ) respectively . in comparing subscales differences statistically significant were found for 2 parameter ( sf p = 0.03 and ew p = 0.01 ) . general characteristics of patients according to mode of delivery distribution of postpartum physical and mental health according to mode of delivery at 2 months after delivery distribution of postpartum physical and mental health according to mode of delivery at 4 months after delivery the mean score differences within each mode of delivery ( 4 months scores 2 months scores ) * our study showed that physical hrqol at 2 and 4 months after delivery is better in women with vd . there were significant differences in the pf and rp parameters in time 1 and pf in time 2 assessments . also mental hrqol at 4 months after delivery and improvement on mental hrqol from time 1 to time 2 assessments was better in vd group ; differences were significant in sf and ew . mental hrqol at 2 months after delivery was better in cs group but differences were not significant in each of parameters ; a reason for this occurred is mothers after cs supported better due to surgical intervention . also , in our study , nearly % 80 of vd was performed with episiotomy or laceration . in a study , the median time to restart intercourse in the normal vd with episiotomy was 40 days and in the cs group was 10 days postpartum and there was significant decreases in the sexual functioning scores after vd when compared to cs group . concerning its effects on postpartum sexual functioning . sexual health is perceived as an integral part of gh as it can affect postpartum depression . there are many studies that assess morbidity resulting from vd and cs , but a few studies have focused on women 's postpartum hrqol . however , our results are consistent with several studies but differ from some of the previous studies ; in a prospective study 100 women were interviewed ( 50 with normal delivery and 50 with cs ) . qol was measured using the sf-36 at two points ( time 1 : 6 - 8 weeks after delivery and time 2 : 12 - 14 weeks after delivery ; result showed vd group had a better qol for almost all subscales in both assessment times , the differences were significant for vt , mental health and pf . another study was performed to investigated fatigue and hrqol in women after vd , elective cs and emergency cs . a total of 141 women ( 71 after vd , 36 after elective cs and 34 after emergency cs ) completed the hrqolsf-36 questionnaires ; result showed patients after vd had higher mean physical hrqol scores than after cs , but the mean mental hrqol scores of the study groups were similar . in contrast these results a cross - sectional study evaluated the effect of delivery mode on women 's postpartum qol in rural china . in this study , none of six dimensions and total score of qol displayed significant difference between women with normal delivery and cs . in this study , cs rate was 70.0% , and most of them ( 59.7% ) were selected by maternal request . the most important in this data being a high frequency of cs due to maternal request also other factors such as lower education level and male gender of infant were associated with poor qol and whereas , in our study , these factors were similar in two groups . however , socio - cultural determinants may contribute in influencing postnatal qol . safarinejad et al . in a cohort study showed the qol parameters by using sf-36 questionnaire in planned cs women were generally higher than vd . there were differences significant for two physical health domains ( gh and rp ) and all of the mental health domains . in this study planned cs compared to vd and all of the women were parity 1 and younger age . one might argue the findings were influenced by the fact that women with elective or emergency cesarean may experience rather different qol during the postnatal period . furthermore in primipara women other reasons are associated with poor qol . in our study , cs consist of elective or emergency and parity was 1 - 3 . in another study dunn and oherlihy compared satisfaction levels by early postnatal questionnaire among 140 women who had a vaginal birth after delivery ( vbac ) with women who had cesarean section after vaginal delivery . the vbac group experienced minimal pain after delivery and had felt better prepared for delivery . in our study , subscale pain at 2 month after delivery was similar in two groups ; one reason was high frequency of episiotomy in vd group , it is important to pay attention to this risk factor to be able to offer good care and treatment to women during postpartum where necessary . in a prospective study in mothers who requested cesarean section in the absence of medical indication , their reason for the request , self - estimated health and experience of delivery were investigated . results showed women requesting cesarean section experienced their health as less good and were more often planning for one child only . they more often reported anxiety for lack of support during the labor , for loss of control and concern for fetal injury or death . after planned cesarean section women in this group reported a better birth experience compared to women planning a vaginal birth . there were no differences in signs of postpartum depression between the groups 3 months after birth . however intended caesarean delivery was found to be an independent risk factor for complications and is associated with an increased risk of maternal rehospitalization . furthermore mean duration of sickness and mean days required for returning to normal activities were also higher in case of caesarian section also it is expensive than normal vaginal delivery . our research was a prospective study , data were obtained from the participants by trained questionnaires and study population was ethnically uniform . unfortunately , we only used a general instrument and this might be considered as a limitation . another limitation of the study was that the type of cesarean section was not considered , women with elective or emergency cesarean may experience rather different qol during postpartum . the findings suggest that vd lead to a better physical health at 2 months after delivery and physical and mental health at 4 months after delivery . furthermore , we recommend the future studies include both general and specific questionnaire in assessing postpartum qol among women with vd , elective cs and emergency cs , in addition other studies needed for recognizing social and cultural factors that are related to physical and mental hrqol after delivery .

background : the postpartum period is a critical life event for women leading physical , emotional , and social changes . postpartum quality - of - life may be affected by delivery mode . the purpose of this study was to determine the association between postpartum health related quality - of - life ( hrqol ) and mode of delivery.materials and methods : in a prospective study , 300 women consisting of 150 vaginal deliveries ( vd ) and 150 cesarean sections ( cs ) were recruited between august 2007 and october 2008 from health centers . stratified random sampling was performed to select 10 health centers in kashan city , iran . physical and mental hrqol was measured using the sf-36 questionnaire and compared between vd and cs groups 2 and 4 months after delivery . data were analyzed by using the student 's t - test , mann whitney u - test , and chi - square test.results:results showed physical hrqol at 2 months after vd was better than cs significantly ; there were significant differences in the physical functioning and role physical subscales . furthermore mental hrqol at 4 months after vd was better than cs significantly ; there were significant difference in the social function and emotional health subscales.conclusion:the findings demonstrated that vd leads to a better physical health at 2 months after delivery and mental health at 4 months after delivery . efforts should be made to reduce cs .

cervical spine surgery for multiple pathologies including persistent radiculopathy and spondylotic myelopathy has been associated with significant improvements in health - related quality - of - life ( hrqol ) outcomes . however , there are significant risks associated with undergoing cervical spine surgery . some complications , such as mild dysphagia after an anterior cervical discectomy and fusion , are common , but rarely result in any long - term sequelae . conversely , rare complications such as a symptomatic epidural hematoma , esophageal injury , or vertebral artery injury can lead to significant morbidity and even death . asymptomatic postoperative epidural hematomas after spine surgery are extremely common ; in a study that looked at patients who had any type of spine surgery , mirzai et al reporting that magnetic resonance imaging can detect an epidural hematoma in up to 89% of patients postoperatively . fortunately , these hematomas are only symptomatic in 0.10% to 0.24% of all spine cases . when this complication is recognized quickly and the hematoma is evacuated , many patients can make a full neurologic recovery , but a delay in diagnosis and treatment can lead to an irreversible neurological injury . for surgeons to be able to accurately inform their patients about the risks and benefits of cervical spine surgery , it is important to establish the actual incidence of rare but potentially devastating complications . to date , almost all of the large studies published on this complication are single - institution studies that report the incidence of postoperative symptomatic epidural hematomas in all types of spine surgery . the published studies that are dedicated to postoperative epidural hematomas in the cervical spine are relatively small cases series . the purpose of the current study is to determine the incidence of symptomatic postoperative epidural hematomas following cervical spine surgery across multiple centers , and assess the impact of this complication on clinical outcomes . the study is a large retrospective multicenter case series study involving 21 high - volume surgical centers from the aospine north america clinical research network . centers were included if they are members of the aospine north america clinical trial research network . those centers that had interest in participating were prescreened concerning their ability to provide data per study protocol . altogether medical records for 17 625 patients who received cervical spine surgery , anterior or posterior ( levels from c2 to c7 ) , between january 1 , 2005 , and december 31 , 2011 , inclusive , were reviewed to identify occurrence of 21 predefined treatment complications . the complications included reintubation requiring evacuation , esophageal perforation , epidural hematoma , c5 palsy , recurrent laryngeal nerve palsy , superior laryngeal nerve palsy , hypoglossal or glossopharyngeal nerve palsy , dural tear , brachial plexopathy , blindness , graft extrusion , misplaced screws requiring reoperation , anterior cervical infection , carotid artery injury or cerebrovascular accident , vertebral artery injuries , horner s syndrome , thoracic duct injury , tetraplegia , intraoperative death , revision of arthroplasty and , pseudomeningocele . trained research staff at each site abstracted the data from medical records , surgical charts , radiology imaging , narratives , and other source documents for the patients who experienced one or more of the complications from the list . copies of case report forms were transferred to the aospine north america clinical research network methodological core for processing , cleaning , and data entry . test was used to analyze changes in clinical outcomes at follow - up compared to preoperative status . of the 21 involved sites , only 19 reported the incidence of postoperative epidural hematomas . a total of 16 582 cervical spine surgeries occurred between january 1 , 2005 , and december 31 , 2011 , at 19 different institutions , and 15 patients developed a postoperative epidural hematoma , for a total incidence of 0.090% . a total of 8887 anterior procedures and 7695 posterior procedures were evaluated . while rate of epidural hematoma was less in anterior procedures ( 5.63 per 10 000 anterior cases ) than posterior procedures ( 13.00 per 10 000 posterior cases ) substantial variation between institutions was noted , with 11 sites reporting no epidural hematomas , and 1 site reporting an incidence of 0.76% ( table 1 ) . the average time to presentation of symptoms was 4.67 7.90 days after surgery , and the average length of stay for patients with the complication was 9.36 9.35 days . all patients initially presented with a new neurologic deficit from the hematoma , but 9 ( 60% ) patients had complete resolution of the neurologic deficit after hematoma evacuation . table 3 reports the specific surgery , the time of symptom onset , the new symptoms , if there was a delay in diagnosis , and if there were residual neurologic symptoms for each of the 15 cases . importantly , 2 of the 3 patients ( 66% ) who had a delay in the diagnosis of the epidural hematoma had residual neurologic deficits compared to only 4 of the 12 patients ( 33% ) who had no delay in the diagnosis or treatment ( p = .53 ) . all 6 of the patients with persistent neurologic deficits had motor weakness , with 2 of the 6 having an asia ( american spinal injury association ) c spinal cord injury . importantly , among the patients who were diagnosed with a symptomatic postoperative epidural hematoma , there was no significant improvement in hrqol metrics between the preoperative evaluation and the final follow - up evaluation ( table 4 ) . the time of symptom onset , if there was a delay in diagnosis , and if there were residual neurologic symptoms for each of the 15 cases . abbreviations : acdf , anterior cervical discectomy and fusion ; asia , american spinal injury association ; mri , magnetic resonance imaging ; ct , computed tomography ; emg / ncs , electromyogram / nerve conduction study . the health - related quality - of - life metrics for patients who developed a postoperative epidural hematoma . abbreviations : ndi , neck disability index ; mjoa , modified japanese orthopaedic association scale ; sf-36 , short form-36 health survey ; phy - sf-36 , physical component of the sf-36 ; ment - sf-36 , mental component of the sf-36 . this study finds that symptomatic postoperative epidural hematomas in the cervical spine are a rare event , occurring in approximately 1 in 1000 cervical spine cases . the results of this study are consistent with other large studies that identify the rate of symptomatic postoperative epidural hematomas throughout the entire spine . in a review of 14 932 spine cases that were performed at a single institution over 18 years , awad et al identified 32 symptomatic postoperative epidural hematomas for an incidence of 0.20% . while the methodology of the current study did not allow for the identification of risk factors for this complication , awad et al reported 3 preoperative risk factors , including the use of nonsteroidal anti - inflammatory medication , rh - positive blood , and patients greater than 60 years of age . additionally , surgeries involving 6 or more levels , greater than 1 l of blood loss , and a hemoglobin of less than 10 g / dl were identified as intraoperative risk factors ; last , if the international normalized ratio rose above 2.0 in the first 48 hours , patients were also at an increased risk of a symptomatic epidural hematoma . in a similar , large , single - institution retrospective study of 12 000 spine surgeries , kou et al reported that the incidence of a symptomatic postoperative epidural hematoma was 0.10% , and they identified preoperative coagulopathy as well as multilevel surgery as risk factors . both the incidence rate reported in the current study and the fact that 11/15 cases were multilevel fusions are consistent with the results of kou et al . there are 3 large single - institution studies that reported the incidence of a symptomatic epidural hematoma in patients undergoing cervical surgery . aono et al identified 1376 patients who underwent cervical spine surgery , and they reported only a single epidural hematoma in 466 anterior cervical spine procedures ( 0.21% ) , and 4 symptomatic epidural hematomas in 910 patients who underwent a cervical laminoplasty ( 0.44% ) . these results are similar to those reported by amiri et al , who reported an overall incidence of 0.22% in 4568 spine cases , and a rate of 0.15% in 1962 patients who underwent cervical surgery . last , goldstein et al reported on 529 patients undergoing posterior cervical surgery over 10 years at a single intuition , and they reported that 1.5% of patients developed a symptomatic postoperative epidural hematoma . this incidence by goldstein et al is substantially higher than that identified in the current study or previous reports , and goldstein et al postulate that this may be due to the fact that almost 50% of the patients in the study underwent a posterior cervical decompression and fusion ; however , this is contradicted by their results , as a stepwise regression analysis identified an increased charlson comorbidities index as well as the use of nonsteroidal anti - inflammatory medication in the postoperative period as independent risk factors for a hematoma , but not the surgical treatment ( laminoplasty , laminectomy , or the use of instrumentation ) . because the study by goldstein et al is a relatively small , single - institution study , it is possible that the substantial increase in the risk of an epidural hematoma may be due to institutional protocols or population - specific factors . specifically , the rate of asymptomatic epidural hematomas has been reported to be almost 90% , so if the surgeons at the institution were more inclined to order advanced postoperative imaging , they may have attributed a neurologic deficit , such as a c5 palsy to the hematoma . with 16582 cervical spine cases , this study is the largest study in the literature looking at individual patients who developed a symptomatic postoperative epidural hematoma in the cervical spine , and because patients from 19 different institutions were reviewed , individual surgeon and institution - based risks have been significantly mitigated . while this methodology has many benefits , including that a series of 15 patients with a postoperative cervical epidural hematoma is the largest reported in any publication to date , there are still significant limitations in the study that must be acknowledged . while a series of 15 patients is the largest published , it was still too small to definitively establish if there is a significant difference in the neurologic outcome of patients who had a delay in the diagnosis . additionally , a substantial increase in the rate of epidural hematomas occurred in posterior surgeries ; however , with only 15 patients in total with this complication , it did not reach statistical significance ( anterior procedures5.63 per 10 000 cases ; posterior procedures13.00 per 10 000 cases ; p = .188 ) . furthermore , each institution identified all patients with a postoperative symptomatic epidural hematoma , but the individual patient data for all of the unaffected patients were not available . because of this , it was not possible to identify specific risk factors for a hematoma , such as the use of anticoagulation , or the presence of specific comorbidities ; additionally , it is not possible to determine the institutional risk factors . the incidence rate ranged from 0.00% to 0.76% at different institutions ; however , without reviewing the entire cohort from all the hospitals , it is not possible to determine which factors affected the hospital s rate of epidural hematomas . conversely , this methodology allowed for a large sample size , and because individual patient data were used rather than icd-9 codes , it is not subject to flaws of large , administrative database studies . another limitation to this study is the heterogeneity in the data that were reported . because the study spanned 7 years and 19 institutions , the available data varied significantly . only 4 patients ( 26.7% ) had pre- and postoperative sf-36 ( short form-36 survey ) and ndi ( neck disability index ) data , while mjoa ( modified japanese orthopaedic association scale ) and nurick grades were available for 1 ( 6.7% ) and 7 ( 46.7% ) patients , respectively . in spite of the limited numbers and variable outcomes measures , the current study is the only study in the literature to report any hrqol outcome measures after this rare complication , and regardless of the outcome measure reported , patients with a postoperative epidural hematoma showed no significant change from preoperative scores . a final limitation is that the diagnosis of a symptomatic postoperative epidural hematoma was left up to the treating physician . undoubtedly , some surgeons have a lower threshold for obtaining advanced imaging postoperatively , and therefore they are more likely to identify and treat an epidural hematoma than surgeons who rarely obtain advanced imaging . the current study is the largest series to date to analyze the incidence of an epidural hematoma following cervical spine surgery . the results of this study suggest that an epidural hematoma is a very rare event , occurring in approximately 1 out of 1000 cervical spine surgeries , and it may be slightly more common in posterior surgeries . prompt diagnosis and treatment resulted in a complete neurologic recovery in the majority of patients , but even without a delay in the diagnosis , 33% of patients still had persistent neurologic deficits . additionally , the sequelae of this complication results in patients having no improvement in hrqol outcomes from their preoperative state .

study design : a multicentered retrospective case series.objective:to determine the incidence and circumstances surrounding the development of a symptomatic postoperative epidural hematoma in the cervical spine.methods:patients who underwent cervical spine surgery between january 1 , 2005 , and december 31 , 2011 , at 23 institutions were reviewed , and all patients who developed an epidural hematoma were identified.results:a total of 16 582 cervical spine surgeries were identified , and 15 patients developed a postoperative epidural hematoma , for a total incidence of 0.090% . substantial variation between institutions was noted , with 11 sites reporting no epidural hematomas , and 1 site reporting an incidence of 0.76% . all patients initially presented with a neurologic deficit . nine patients had complete resolution of the neurologic deficit after hematoma evacuation ; however 2 of the 3 patients ( 66% ) who had a delay in the diagnosis of the epidural hematoma had residual neurologic deficits compared to only 4 of the 12 patients ( 33% ) who had no delay in the diagnosis or treatment ( p = .53 ) . additionally , the patients who experienced a postoperative epidural hematoma did not experience any significant improvement in health - related quality - of - life metrics as a result of the index procedure at final follow - up evaluation.conclusion:this is the largest series to date to analyze the incidence of an epidural hematoma following cervical spine surgery , and this study suggest that an epidural hematoma occurs in approximately 1 out of 1000 cervical spine surgeries . prompt diagnosis and treatment may improve the chance of making a complete neurologic recovery , but patients who develop this complication do not show improvements in the health - related quality - of - life measurements .

cis - diamminedichloroplatinum ii or cisplatin ( cp ) , an important antineoplastic drug , is widely used for treatment of solid tumors including head and neck , lung , testis , ovary , and breast tumors ( 1 ) . unfortunately , it has some side effects such as ototoxicity , gastrotoxicity , myelosuppression , and allergic reactions . the major side effect of cp is nephrotoxicity ( 2 ) , which occurs in about 30% of patients treated with cp due to tubular toxicity , inflammation , oxidative stress , and change in the renal blood flow ( 3 - 6 ) . various agents such as vitamins c and e , losartan , l - arginine , magnesium , and erythropoietin have been proposed as nephroprotective agents against cp - induced nephrotoxicity ( 7 - 14 ) . previous studies showed that cp - induced nephrotoxicity is sex - related and intensity of kidney damage in males treated with cp is significantly greater than that in females ( 15 , 16 ) . at first sight , it seems that the sex differences in cp - induced nephrotoxicity must be related to sex hormones ; however , according to our previous study , estrogen did not have protective effect against cp - induced nephrotoxicity and might promote toxicity in female rats ( 17 ) . the role of testosterone ( ts ) against cp - induced nephrotoxicity it seems that ts affects cp effectiveness in proximal tubule with vasodilatory effect on renal afferent arterioles via expression of androgenic receptors and activation of nitric oxide synthase ( nos ) ( 18 ) . previously , it was reported that ts enhances kidney susceptibility to ischemia - reperfusion injury and on the other hand , it had protective role in ischemia - reperfusion - induced acute kidney injury ( 19 , 20 ) . this study aimed to examine the role of different doses of ts in response to cp - induced nephrotoxicity in castrated rats . the investigation was performed on 54 adult male wistar rats ( animal center , isfahan university of medical sciences , isfahan , iran ) with the mean weight of 182 2.7 g. the rats were housed at the temperature of 23 to 25 c with free access to water and chow . the rats were anesthetized with intraperitoneal injection of ketamine ( 75 mg / kg ) . the epididymis and testis were pulled out , vas deferens and spermatic blood vessels were ligated , and finally , testes were removed . groups 1 , 2 , and 3 received 10 , 50 , and 100 mg / kg / wk intramuscular ts enanthate ( aburaihan co. , tehran , iran ) , respectively , for four weeks and at the end of the third week , the animals received 2.5-mg / kg / d cp intraperitoneally for seven days . groups 5 , 6 , and 7 received the same treatment regimen as groups 1 through 3 , respectively , for four weeks ; however , they received saline instead of cp for the following seven days . group 8 ( negative control ) received sesame oil and saline instead of ts and cp , respectively . when cp injection was initiated , the animals were weighed daily . at the end of the study , blood samples were obtained and then the animals were sacrificed . left and right kidneys were removed and weighed immediately for histopathologic investigation and measurements . the serum creatinine ( cr ) and blood urea nitrogen ( bun ) levels were determined using quantitative diagnostic kits ( pars azmoon , iran ) . the serum and kidney levels of nitrite ( stable no metabolite ) were measured using a colorimetric elisa kit ( promega corporation , usa ) that involved the griess reaction . the serum and kidney levels of malondialdehyde ( mda ) were determined by 10% trichloroacetic acid ( tca ) and 0.67% thiobarbituric acid ( tba ) . the serum level of ts was measured using enzyme immunoassay elisa kit ( diagnostics biochem canada inc . , the left kidney was fixed in 10% neutral formalin solution and embedded in paraffin for hematoxylin and eosin staining to examine the tubular damage . a pathologist who was completely unaware of the study protocol and administered medications evaluated the damage . kidney tissue damage score ( ktds ) was graded from i to iv , based on the intensity of tubular lesions ( hyaline cast , debris , vacuolization , flattening and degeneration of tubular cells , and dilatation of tubular lumen ) , while zero was assigned to normal tubules without any damage . comparison of the groups with regard to the bodyweight ( bw ) loss , kidney weight ( kw ) ; and levels of bun , cr , mda , ts , and nitrite were performed by one - way anova followed by post hoc dunnett 's test when applicable . the histopathologic damage score of the groups was compared by the kruskal - wallis and mann - whitney u tests . the investigation was performed on 54 adult male wistar rats ( animal center , isfahan university of medical sciences , isfahan , iran ) with the mean weight of 182 2.7 g. the rats were housed at the temperature of 23 to 25 c with free access to water and chow . the rats were anesthetized with intraperitoneal injection of ketamine ( 75 mg / kg ) . the epididymis and testis were pulled out , vas deferens and spermatic blood vessels were ligated , and finally , testes were removed . groups 1 , 2 , and 3 received 10 , 50 , and 100 mg / kg / wk intramuscular ts enanthate ( aburaihan co. , tehran , iran ) , respectively , for four weeks and at the end of the third week , the animals received 2.5-mg / kg / d cp intraperitoneally for seven days . groups 5 , 6 , and 7 received the same treatment regimen as groups 1 through 3 , respectively , for four weeks ; however , they received saline instead of cp for the following seven days . group 8 ( negative control ) received sesame oil and saline instead of ts and cp , respectively . when cp injection was initiated , the animals were weighed daily . at the end of the study , blood samples were obtained and then the animals were sacrificed . left and right kidneys were removed and weighed immediately for histopathologic investigation and measurements . the serum creatinine ( cr ) and blood urea nitrogen ( bun ) levels were determined using quantitative diagnostic kits ( pars azmoon , iran ) . the serum and kidney levels of nitrite ( stable no metabolite ) were measured using a colorimetric elisa kit ( promega corporation , usa ) that involved the griess reaction . the serum and kidney levels of malondialdehyde ( mda ) were determined by 10% trichloroacetic acid ( tca ) and 0.67% thiobarbituric acid ( tba ) . the serum level of ts was measured using enzyme immunoassay elisa kit ( diagnostics biochem canada inc . , the left kidney was fixed in 10% neutral formalin solution and embedded in paraffin for hematoxylin and eosin staining to examine the tubular damage . a pathologist who was completely unaware of the study protocol and administered medications evaluated the damage . kidney tissue damage score ( ktds ) was graded from i to iv , based on the intensity of tubular lesions ( hyaline cast , debris , vacuolization , flattening and degeneration of tubular cells , and dilatation of tubular lumen ) , while zero was assigned to normal tubules without any damage . comparison of the groups with regard to the bodyweight ( bw ) loss , kidney weight ( kw ) ; and levels of bun , cr , mda , ts , and nitrite were performed by one - way anova followed by post hoc dunnett 's test when applicable . the histopathologic damage score of the groups was compared by the kruskal - wallis and mann - whitney u tests . comparison of the positive and negative control groups ( group 4 and group 8 , respectively ) demonstrated that in the positive control group , kw , ktds , and serum levels of bun , cr , and mda had increased while the serum ts level , serum and kidney tissue levels of nitrite , and bw had decreased significantly ( p < 0.05 ) . abbreviations : bun , blood urea nitrogen ; cr , creatinine ; sn , serum nitrite ; smda , serum malondialdehyde ; ts , testosterone ; kn , kidney tissue levels of nitrite ; kmda , kidney tissue levels of malondialdehyde ; kw , kidney weight ; bw , bodyweight changes ; and ktds , kidney tissue damage score . comparison between the case groups ( groups 1 , 2 , and 3 ) and the positive control ( group 4 ) indicated that the serum levels of bun and cr , and ktds had significantly decreased in group 1 ( group treated with low - dose ts plus cp ) in comparison with group 4 ( positive control group ) ( p < 0.05 ) ( figure 2 ) . according to these findings as expected , significant increase in serum ts level was detected in group 3 ( group treated with high - dose ts ) in comparison with the positive control group ( p < 0.05 ) ( figure 2 ) . although the serum levels of ts in groups 1 and 2 were higher than that in group 4 , these differences were not statistically significant ( figure 2 ) . no significant differences were observed among the groups with respect to other parameters ( table 1 ) . significant difference from group 4 . abbreviations : bun , blood urea nitrogen ; cr , creatinine ; ts , testosterone ; and ktds , kidney tissue damage score . abbreviations : cp , cisplatin ; ts , testosterone ; sn , serum nitrite ; kn , kidney nitrite ; smda , serum malondialdehyde ; kmda , kidney tissue malondialdehyde ; kw , kidney weight ; and bw , bodyweight changes . ts10 , ts50 , and ts100 stand for treatment with testosterone doses of 10 , 50 , and 100 mg / kg / wk , respectively . comparison between ts alone treated groups ( groups 5 , 6 , and 7 ) in one hand and negative control group ( group 8) on the other hand indicated no significant differences in serum levels of bun , cr , and nitrite as well as tissue levels of nitrite and mda ( table 2 ) . nevertheless , increase in serum mda , kw , and ktds were observed in high - dose ts ( 100 mg / kg / wk ) group ( p < 0.05 ) . the bw reduced in ts treated groups and this reduction was significant only in group 7 in comparison with group 8 ( p < 0.05 ) . the serum level of ts was significantly higher in groups 6 and 7 ( p < 0.05 ) . abbreviations : cp , cisplatin ; ts , testosterone ; bun , blood urea nitrogen ; cr , creatinine ; sn , serum nitrite ; kn , kidney tissue nitrite ; smda , serum malondialdehyde ; kmda , kidney tissue malondialdehyde ; kw , kidney weight ; ktds , kidney tissue damage score ; and bw , bodyweight changes . significant difference from group 8 ( p < 0.05 ) . ts10 , ts50 , and ts100 stand for treatment with testosterone doses of 10,50 , and 100 mg / kg / wk , respectively . comparison of the positive and negative control groups ( group 4 and group 8 , respectively ) demonstrated that in the positive control group , kw , ktds , and serum levels of bun , cr , and mda had increased while the serum ts level , serum and kidney tissue levels of nitrite , and bw had decreased significantly ( p < 0.05 ) . abbreviations : bun , blood urea nitrogen ; cr , creatinine ; sn , serum nitrite ; smda , serum malondialdehyde ; ts , testosterone ; kn , kidney tissue levels of nitrite ; kmda , kidney tissue levels of malondialdehyde ; kw , kidney weight ; bw , bodyweight changes ; and ktds , kidney tissue damage score . comparison between the case groups ( groups 1 , 2 , and 3 ) and the positive control ( group 4 ) indicated that the serum levels of bun and cr , and ktds had significantly decreased in group 1 ( group treated with low - dose ts plus cp ) in comparison with group 4 ( positive control group ) ( p < 0.05 ) ( figure 2 ) . according to these findings , low - dose ts can prevent cp - induced nephrotoxicity . as expected , significant increase in serum ts level was detected in group 3 ( group treated with high - dose ts ) in comparison with the positive control group ( p < 0.05 ) ( figure 2 ) . although the serum levels of ts in groups 1 and 2 were higher than that in group 4 , these differences were not statistically significant ( figure 2 ) . no significant differences were observed among the groups with respect to other parameters ( table 1 ) . significant difference from group 4 . abbreviations : bun , blood urea nitrogen ; cr , creatinine ; ts , testosterone ; and ktds , kidney tissue damage score . abbreviations : cp , cisplatin ; ts , testosterone ; sn , serum nitrite ; kn , kidney nitrite ; smda , serum malondialdehyde ; kmda , kidney tissue malondialdehyde ; kw , kidney weight ; and bw , bodyweight changes . ts10 , ts50 , and ts100 stand for treatment with testosterone doses of 10 , 50 , and 100 mg / kg / wk , respectively . comparison between ts alone treated groups ( groups 5 , 6 , and 7 ) in one hand and negative control group ( group 8) on the other hand indicated no significant differences in serum levels of bun , cr , and nitrite as well as tissue levels of nitrite and mda ( table 2 ) . nevertheless , increase in serum mda , kw , and ktds were observed in high - dose ts ( 100 mg / kg / wk ) group ( p < 0.05 ) . the bw reduced in ts treated groups and this reduction was significant only in group 7 in comparison with group 8 ( p < 0.05 ) . the serum level of ts was significantly higher in groups 6 and 7 ( p < 0.05 ) . abbreviations : cp , cisplatin ; ts , testosterone ; bun , blood urea nitrogen ; cr , creatinine ; sn , serum nitrite ; kn , kidney tissue nitrite ; smda , serum malondialdehyde ; kmda , kidney tissue malondialdehyde ; kw , kidney weight ; ktds , kidney tissue damage score ; and bw , bodyweight changes . ts10 , ts50 , and ts100 stand for treatment with testosterone doses of 10,50 , and 100 mg / kg / wk , respectively . the major finding of the present study was protective role of low - dose ts against cp - induced nephrotoxicity in castrated rats . previous studies identified the role of estrogen in cp - induced nephrotoxicity ( 17 ) , but the role of ts in cp - induced nephrotoxicity has not been well - documented . the effect of cp on serum levels of bun , cr , and mda , kidney damage , kw , and bw loss were reported ( 11 , 21 - 28 ) . in addition , decrease in the serum levels of nitrite and ts with cp has been demonstrated ( 29 - 31 ) . the present study confirmed these findings . despite orchiectomy and elimination of ts source , the serum level of remaining ts significantly decreased in group 4 in comparison with group 8 . this suggests that cp might cause a depression in serum ts level ( 31 ) . in our study , the serum levels of bun and cr and kidney damage were decreased in low dose of ts plus cp treated group ( group 1 ) in comparison with the positive control group , which revealed the protective role of ts . it seems that ts at high doses did not have any protective role against cp - induced nephrotoxicity and might acts as a toxic agent . in addition , we found that ts alone at high doses had raised serum level of mda , kw , and ktds while it had reduced bw significantly . some researches demonstrated that in ischemia - reperfusion model , the serum level of cr and proximal tubule injury might decrease in castrated rats that had received ts . therefore , ts might protect the kidney from ischemia - reperfusion - induced acute kidney injury due to fast vasodilation effect on renal afferent arterioles , which is mediated by androgen receptors and activation of nitric oxide synthase due to an acute increase in glomerular filtration rate ( 18 , 20 ) . in addition , chronic diseases like obesity , hypertension , cardiovascular diseases , and chronic kidney disease ( ckd ) are associated with deficiency of ts in males ( 32 - 36 ) . reduced ts levels in men with nondialysis ckd and in males on hemodialysis have been reported and ts deficiency aggravates cardiovascular events and increases arterial stiffness and mortality ( 37 , 38 ) . furthermore , hypogonadism is a cause of anemia in men with ckd and ts replacement therapy decreases the prevalence of anemia in these patients ( 39 ) . in the current study , the high dose of ts in ts alone treated rats ( group 7 ) increased mda level and ktds . according to some studies , ts increases plasma mda , as an oxidative stress parameter , and cr . ts elevates kidney susceptibility to ischemia - reperfusion injury through decreased activation of nos and increased oxidative stress . it also promotes susceptibility of proximal tubule cells to apoptotic damage ( 19 , 40 , 41 ) . on the other hand , high dose of ts enhances oxidative stress and high dose of ts decreased animal bw , which was similar to reported observation in human where ts replacement therapy in hypogonadal men led to loss of bw ( 43 ) . our results suggest that ts in low doses plays a protective role against cp - induced nephrotoxicity and in high dose has a promoting role in nephrotoxicity ; however , ts does not possess antioxidant properties at pharmacologic concentrations ( 44 ) . it can be concluded that cp therapy should be avoided when serum ts level is high because ts in high concentrations promotes cp - induced nephrotoxicity .

background : cisplatin ( cp ) is an important antitumor drug with serious side effects such as nephrotoxicity . estrogens can affect cp - induced nephrotoxicity ; however , the role of testosterone ( ts ) , the main male sex hormone , is not clear.objectives:this study aimed to investigate the effect of ts on cp - induced nephrotoxicity in castrated male rats.materials and methods : a total of 54 male wistar rats were castrated and allocated into eight groups . groups 1 through 3 respectively received 10 , 50 , and 100 mg / kg / wk of ts and group 4 received sesame oil for four weeks ; then all four groups received 2.5 mg / kg / d cp for one week . groups 5 through 8 received the same treatment regimen as groups 1 through 4 during first four weeks but instead of cp , they received saline for one week . then the animals were sacrificed for biochemical and histopathologic studies.results:cp increased the serum levels of blood urea nitrogen ( bun ) , creatinine ( cr ) , and malondialdehyde ( smda ) as well as kidney weight ( kw ) , bodyweight ( bw ) loss , and kidney tissue damage score ( ktds ) . it significantly decreased the serum and kidney levels of nitrite and serum level of ts in comparison with the control group ( p < 0.05 ) . however , coadministration of cp and low dose of ts significantly decreased the serum levels of bun as well as cr and ktds ( p < 0.05 ) . administration of high - dose ts alone increased the smda level , ktds , and kw while decreased the bw significantly ( p < 0.05).conclusions : it seems that testosterone in low dose , i.e. physiologic dose , protects kidneys against cp - induced nephrotoxicity ; however , special care is needed in cp therapy of patients with high levels of ts .

clinical information was collected retrospectively for january 1 through july 31 , 2009 , and prospectively from august 1 , 2009 through april 30 , 2011 . retrospectively , we identified patients who had been tested for influenza by use of a rapid test kit and extracted personal and clinical information from medical records . prospectively , we collected the same information from patients tested for influenza by rapid test kit and used nasopharyngeal swab extracts from rapid test kits for virus typing . typing was performed by reverse transcription nested - pcr ( rt - npcr ) : multiplex for seasonal influenza virus ( subtypes a / h1 , a / h3 , b- , and a / h5 ) and simplex for influenza a(h1n1)pdm09 virus ( technical appendix table 1 ) . by amplifying product exclusively for influenza a(h1n1)pdm09 and for traditional influenza ( h1n1 ) viruses and by producing a different length product for each subtype , # iincludes 1 case for which tamiflu and relenza were prescribed . for the retrospective period , we identified 1,066 suspected cases of influenza ; 20 patients were nonresidents of izu - oshima island ; the address for 1 patient was unknown . for the prospective period , we identified 2,348 patients with suspected influenza ; 3 were excluded because they did not consent to study participation . patients from the prospective period were tested with a rapid test . in total , 97.8% ( 2,293/2,345 ) of the samples the total number of patients with suspected influenza in the prospective study was 2,219 ( a patient with multiple visits within 7 days was counted as 1 patient ) , of which 78 were not residents of izu - oshima island ( technical appendix table 2 ) . * age during january 2009 ( for 200809 ) , 2010 ( for 200910 ) , and 2011 ( for 201011 ) . population recorded in the resident registry in january 2009 ( for 200809 ) , 2010 ( for 200910 ) , and 2011 ( for 201011 ) . the sum of influenza a(h1n1)pdm09 cases among residents in izu - oshima during the study divided by the population during january 2011 . the sensitivity of the rapid test kit compared with rt - npcr was 90% for type a but lower ( 80% ) for type b ( technical appendix table 3 ) . among patients with positive rapid test results , the same influenza subtypes circulated in izu - oshima as in other areas ( 2,3 ) . we assessed the period from when a novel virus was introduced through the postpandemic season . the introduction of a(h1n1)pdm09 virus in izu - oshima occurred 11 weeks after confirmation of the first case in japan ( 4 ) and 10 weeks after confirmation in tokyo ( 5 ) . after the first case of a(h1n1)pdm09 infection was identified in japan on may 16 ( week 20 ) ( 4 ) , no other influenza a cases were diagnosed by a rapid test in izu - oshima until august 1 ( week 31 ) , when a patient with influenza a ( unspecified ) was determined to have had contact with a person with confirmed a(h1n1)pdm09 infection on mainland japan . the first outbreaks of influenza a in izu - oshima ceased within 5 weeks ( figure 1 ) . after 3 weeks with no cases , starting at the end of september ( week 39 ) , clusters of influenza cases were observed in schools and families . although immediate school or class closures were implemented , the pandemic began in the middle of november and peaked during week 50 , which was 6 weeks later than the tokyo peak ( 5 ) . on the island , the incidence of a(h1n1)pdm09 infection during the pandemic season was higher than the incidence caused by other subtypes during the 20092011 seasons ( table 2 ) . however , the overall incidence on the island ( 5.0% ) during the pandemic season was one third of the estimated incidence for all of japan ( 16.2% ) ( 3 ) . cases of influenza and influenza - like illnesses on izu - oshima island , japan , from week 1 of 2009 through week 17 of 2011 . the number of influenza cases and influenza - like illnesses are plotted weekly from the disease onset . influenza cases were defined as illnesses diagnosed by a rapid test combined with a reverse transcription nested pcr ( rt - npcr ) or by a rapid diagnostic test ( rdt ) alone , during the retrospective period ( unspecified ) . influenza - like illnesses were defined as cases for which influenza was ruled out by negative rt - npcr or cases for which influenza was ruled out by rdt results and further tests were not performed . disease onset was defined by the date when the patient first reported fever or upper respiratory symptoms . the disease onset for the case that had no date in the clinical records was defined as the day before the first clinical visit according to the median day of visit from the available study data . b ( rdt / pcr ) , cases diagnosed by a rdt or rt - npcr . a(h1n1)pdm09 and h3 , co - infection cases with 2 virus subtypes confirmed by rt - npcr . influenza seasons were defined as follows : week 130 of 2009 was the 200809 prepandemic season , week 31 of 2009week 33 of 2010 was the 200910 pandemic season , and week 34 of 2010week 17 of 2011 was the 201011 postpandemic season . the introduction and dissemination of a(h1n1)pdm09 virus varied by age ( figure 2 ) . on the island , as on the mainland , introduction of the emerging virus preceded the outbreak among high school age children ( 4,6 ) . however , at the end of the season , the incidence among persons 514 years of age exceeded that among persons 1519 years of age ( table 2 ) . the cumulative incidence of a(h1n1)pdm09 infections for 200910 was calculated for the sum of a(h1n1)pdm09 virus cases among residents on izu - oshima island , japan , divided by the population at the end of december 2009 and plotted by week in the 200910 season . the numbers adjacent to the lines indicate the age groups , in years . during the postpandemic season , in addition to a(h1n1)pdm09 virus , epidemics of influenza a / h3 and b viruses occurred . no patient with confirmed a(h1n1)pdm09 infection during the postpandemic season had a history of influenza in the previous season ; but 28% ( 50/180 ) of patients with influenza b and 3% ( 2/58 ) with influenza a / h3 virus did . the cumulative incidence of a(h1n1)pdm09 infection 2 seasons after the pandemic was estimated at 7.3% and was highest ( 43.1% ) among persons 1014 years of age . this population - based surveillance study determined the incidence of the influenza virus subtypes circulating in pandemic and postpandemic seasons . the estimated incidence of symptomatic cases was accurate because of the easy access to health care on izu - oshima island . care was sought for almost all ( 97% ) children with upper respiratory symptoms and fever , although the proportion of adults who sought clinical care was not high ( 7 ) . most ( 83.6% ; 1,774/2,122 ) patients for whom disease onset was identified had visited a medical institute within 2 days of disease onset despite their symptoms being only mild to moderate . furthermore , all suspected cases of influenza , except 2 , were confirmed by a rapid test ; most were tested further and isolates were subtyped by rt - npcr . the cumulative incidence 2 seasons after the pandemic indicates that early introduction of a(h1n1)pdm09 virus to those 1519 years of age was not caused by differential sensitivity to the virus . rather , it was probably caused by more frequent exposure to the emerging virus , possibly because of higher mobility of persons in this age group . considering the conservative antigenic property of a(h1n1)pdm09 virus in the postpandemic season ( 8) , the absence of a(h1n1)pdm09 infection in this season among those who had experienced it in the pandemic season suggests that immune memory persisted in the postpandemic season . the cumulative incidence suggests that nearly half of the school - age children had immunity to a(h1n1)pdm09 virus by infection after 2 seasons . the remaining virus - naive elderly population should be considered for future preventive intervention , although they might have some immunity against a(h1n1)pdm09 virus ( 912 ) . the delayed introduction of a(h1n1)pdm09 virus might primarily be explained by the isolated environment of the island ; introduction would be mediated solely by visitors carrying the virus . the delayed start and peak of the epidemic and the low incidence could be attributed early case identification plus early and extensive therapy ( including prompt initiation of antiviral medication according to results of proactively performed rapid tests ) ; easy access to health care ; and public health interventions ( such as school closures ) . in addition , unique social features might also have contributed to the delayed pandemic and low disease incidence . the proportion of children < 15 years of age ( 12% ) is the same on izu - oshima island as in tokyo ; whereas , the proportion of those 65 years of age is 31% on the island and only 20% in tokyo . assuming that persons 65 years of age had preexisting immunity against a(h1n1)pdm09 virus , as suggested by other studies ( 912 ) , the community possibly had a larger number of nonsusceptible persons . limited public transport and low population density ( 96/km ) might have reduced disease spread . school closures might have more effectively reduced the chance of transmission in such settings than in other areas . this study provides a sound basis for modeling studies that consider social structures to help explain the effects of public health interventions for influenza spread in a community . reverse transcription nested pcr ( rt - npcr ) primers used for seasonal ( multiplex ) and pandemic influenza ( simplex ) detection and typing ( table 1 ) . diagnosis of influenza cases and influenza - like illnesses from 200809 to 20102011 influenza seasons ( table 2 ) . sensitivity and specificity of quicknavi - flu kit ( nordic biolabs ab , taby , sweden ) compared with rt - npcr ( table 3 ) .

a population - based influenza surveillance study ( using pcr virus subtyping ) on izu - oshima island , japan , found that the cumulative incidence of influenza a(h1n1)pdm09 virus infections 2 seasons after the pandemic was highest for those 1014 years of age ( 43.1% ) . no postpandemic a(h1n1)pdm09 case - patients had been infected with a(h1n1)pdm09 virus during the pandemic season .

we report a case of intravesical polyvinyl - coated electric wire in the urinary bladder . a 34-year - old man presented in our clinic with irritative lower urinary tract symptoms . three weeks earlier he had seen and felt a wire in his urethra of which he was unaware of the origin . patients are usually too ashamed to admit the cause of an intravesical foreign body , which is often sexual or erotic in origin . radiologic evaluation usually reveals the nature and size of the foreign body in the bladder of patients evaluated for recurrent urinary tract infection . most organs of the genitourinary system , except for the male external genitalia , are well protected by the surrounding structures.1 for instance , the bladder lies directly behind the pelvic bone and anterior to the rectum , which makes it relatively inaccessible . the bladder functions in the storage and intermittent evacuation of urine with a capacity of about 450750 ml . the urethra , through which the contents are evacuated , measures about 1620 cm in males , which makes the presence of a foreign body in the male urinary bladder an enigma.2 foreign bodies in the urinary bladder may occur by self - insertion or migration from the neighboring organs.3 they vary from tissue originating from adjacent organs4 to metallic objects such as electric wires.5 the reasons for their presence in the urinary bladder also vary from sexual and erotic manipulations6 to iatrogeny7 and complimentary and alternative medicine practice.8 a foreign body in the urinary bladder , according to jung et al4 is a rare occurrence . martinez - valls et al9 describe the condition as being exceptional and not a common emergency . from a medline search , most cases of foreign bodies in the urinary bladder in nigeria have been due to migration of intrauterine contraceptive devices and have been presented as case reports . we report the case of a 34-year - old man with polyvinyl chloride ( pvc)-coated electric wire in the lumen of the urinary bladder , followed by a review of the literature . a 34-year - old man presented in our clinic with a 2-week history of frequency , urgency , and dysuria . the diagnosis of urinary tract infection was made , and he was requested to do a urine microscopy culture and sensitivity and then placed on empirical antimicrobial therapy . he subsequently presented with the same symptoms in addition to severe suprapubic tenderness and fever . this time , he felt and saw the tip of a wire in his urethra . ultrasonography was carried out , which suggested an intravesical foreign body without a urethral extension . the radiologist suggested that a plain pelvic x - ray be obtained to establish the nature of the intravesical foreign body . the x - ray showed a radiopaque intravesical substance fully coiled in the bladder without a urethral extension . this suggested an electric wire and helped in estimating the size and length of the foreign body ( figure 1 ) . finger inspection confirmed that the foreign body was intravesical without a urethral extension . with minimal effort , the wire , which was coated with pvc , measured about 46 cm , and was knotted close to one end ( figure 2 ) , was extracted . foreign bodies localized to the urogenital tract represent a relatively rare pathology and , according to mischianu et al10 in many cases , have legal implications . this is particularly so for the urinary bladder , as the urethra and ureters may be catheterized for a reasonable period because they are only conduit pipes . their presence in the urinary tract predisposes to infection , calculus formation , and bladder outlet obstruction , foreign bodies in the urinary bladder may occur by self - insertion or migration from the neighboring organs.11 this patient did not volunteer a reasonable history to enable a fair assertion of the method of insertion . that is not unusual , as , according to aliabadi et al12 most patients are usually too ashamed to admit they had inserted an object for autoerotic , psychiatric , therapeutic , or any other reasons . the length of the male urethra makes it impossible to assume that a foreign body found its way into the bladder unknown to the patient , unlike in females in a primitive agricultural setting . according to van ophoven and dekernion13 the most common motive associated with the insertion of foreign bodies into the genitourinary tract is sexual or erotic in nature . in adults , they opine that it is commonly caused by insertion of objects used for masturbation , which is frequently associated with a mental health disorder.14 such patients are also known to be prone to genital self - emasculation injuries,15 so they need to be identified and have psychiatric assessment . the variety of foreign bodies inserted into , or attached externally to , the genitourinary tract defies imagination and includes all types of objects.6 they vary with societies , levels of development , the aim of insertion , and cause . osca et al16 reported eight cases of self - inserted foreign bodies in the lower urinary tract treated from 1976 to 1990 , of which five were intravesical . objects found include electric cable , tweezers , a hair pin , a drawing pin , pebbles , and a paper clip . kochakarn and pummanagura17 studied 78 patients , and objects inserted included cotton swabs , tampons , paper clips , and pen casing . according to kochakarn and pummanagura , as most of thailand is an agricultural environment , some small living organisms , such as leeches , rafique7 has identified the intravesical foreign bodies as copper wire , a carrot , a lead pencil , an intrauterine device , surgical gauze , pieces of foley catheter , and a teflon beak of resectoscope sheath . there is clearly a remarkable variation in objects in relation to intravesical foreign bodies and their causes in different societies . the presentation of intravesical foreign bodies from iatrogenic causes requires a high index of suspicion for diagnosis , as the foreign body may remain in the body for a long period before symptoms develop . this duration is the period of migration from the primary site of introduction to the bladder lumen . for instance , izumi et al8 reported a case of an acupuncture patient with a bladder stone of 2 cm with a needle core who had symptoms of benign prostatic hyperplasia for 10 years . another example is the case report by irisawa et al18 of a 66-year - old male who underwent a rectal operation 6 years earlier and subsequently presented with a surgical needle in the bladder . it is supposed that it took the needle about 6 years to migrate from the perirectal tissue to the bladder . far more common are case reports of the migration of intrauterine contraceptive devices.19,20 the need for a high index of suspicion is reinforced by the rarity of this condition . the symptoms of intravesical foreign body are remarkably constant and are usually as a result of bladder irritation and , when the foreign body is large , reduced bladder capacity . hematuria may occur from trauma due to self - manipulation or rough objects that injure the bladder wall . these symptoms are most commonly irritative17 but may also be obstructive if there is an extension of the foreign body into the urethra21 and this is usually accompanied by copious bloody discharge . a wide variety of complications may follow the presence of foreign bodies in the urinary bladder . foreign bodies in the urinary tract increase the risk of urinary tract infection.22 such infections are often recurrent as a result of bacteria persistence within or on the foreign body and sometimes as a result of poor drainage . the risk of endotoxemia should always be borne in mind in the process of extraction of the foreign body . complications such as calculus formation have been widely reported in association with migrated intrauterine contraceptive devices and surgical needles.2325 life - threatening complications of intravesical foreign bodies are rare but do occur . ito et al26 reported in 2009 a case of intravesical foreign body with a vesical rupture and peritoneal invasion . to the authors knowledge , there had been only 10 similar cases in japan at the time of their report . severe infection and obstruction may lead to renal insufficiency,27 whereas urethral injury during insertion or self - extraction may predispose to urethral stricture formation . radiologic images are next only to a high index of suspicion in the evaluation of patients with intravesical foreign bodies . the extent and modality required depends on the type of foreign body and the presence or otherwise of complications . radiologic evaluation is necessary to determine the exact size , number , and nature of the foreign body . ultrasonography is usually able to localize the foreign body to the bladder and determine the exact size and number but is unable to evaluate the exact nature ( figure 1 ) . plain abdominal x - ray is , however , able to classify the objects into radiopaque and radiolucent , and therefore to a large extent determine their nature , eg , radiopaque metals ( figure 2 ) . in the presence of bladder perforation , computed tomographic cystography is a reliable diagnostic tool where enhancement of the bladder contents is necessary.28 cystoscopy confirms the diagnosis , and some foreign bodies are successfully removed during the process.29 foreign bodies in the urinary bladder represent a urologic challenge that requires prompt management7 and should be treated as emergencies . the wide variety of techniques for removal of intravesical foreign bodies indicates the ingenuity with which urologists have approached the subject . broadly , these are classified into endoscopic and open surgical approaches , the method used usually depending on the nature and size of the object and the available expertise and equipment . 7 endoscopic and minimally invasive techniques should be encouraged . however , in many cases , management can not be endoscopic and it is necessary to use the classical method . the simplest endoscopic approach to extraction of an intravesical foreign body is cystoscopy and retrieval with biopsy forceps . this is suitable for small objects such as intrauterine contraceptive devices , as reported by olaore et al19 sharma et al11 and forde et al30 in their different works . a more complex cystoscopic approach has been described by reddy and daniel.31 the cystoscope acts as the optical device and a laparoscope port is introduced suprapubically under vision for extraction of complex foreign bodies while the bladder is insufflated with carbon dioxide . laparoscopic approach to removal has also been described.4 when there is encrustation or stone formation on the foreign body , internal lithotripsy or stone forceps may be used to crush the stone in order to facilitate extraction of the foreign body . sometimes , open surgery is required because the foreign body is too large or its nature makes this imperative , as in the case of our patient . the presence of a foreign body in the urinary bladder is rare and exceptional and therefore requires a high index of suspicion for diagnosis . it should be considered in young patients with recurrent urinary tract infection and poor response to antibiotic therapy .

aimwe report a case of intravesical polyvinyl - coated electric wire in the urinary bladder.case reporta 34-year - old man presented in our clinic with irritative lower urinary tract symptoms . three weeks earlier he had seen and felt a wire in his urethra of which he was unaware of the origin . radiologic evaluation revealed an electric wire completely coiled up in the urinary bladder . it was removed through a suprapubic cystotomy.discussionpatients are usually too ashamed to admit the cause of an intravesical foreign body , which is often sexual or erotic in origin . radiologic evaluation usually reveals the nature and size of the foreign body in the bladder of patients evaluated for recurrent urinary tract infection . open surgery or an endoscopic approach may be used for their extraction .

it is well known that the kidney size of the patient undergoing chronic hemodialysis ( hd ) gradually decreases . however , only a few reports are there on the study of the actual decrease rate [ 24 ] . on the other hand , scheduled ultrasonographic ( us ) examination of kidney after induction of hd is important to check the progression of acquired cystic disease of kidney ( acdk ) and renal cancer which may arise in acdk [ 5 , 6 ] . we examined the renal longest diameter of chronic hd patient at the scheduled us kidney checkup , calculated the alteration rate of the size in the same patients , and studied the difference of the alteration rate among the factors such as gender , age , and original diseases . of 229 outpatient chronic hemodialysis patients , 109 patients , who had neither hereditary polycystic disease nor severe acquired cystic disease of kidney , were selected ; we performed ultrasonography ( us ) twice or three times to measure their maximum renal diameter ( mean of both kidneys ) , and the yearly alteration rate of the diameter was calculated . the largest caliber of the imaged kidney was measured using the measurement tool loaded in the us machine ( aroca ssd-280 , toshiba nemio 30 , japan ) ( figure 1 ) . the us study of the kidney was mainly carried out intercostal method through ribs with 3.75 mhz linear probe . some of the patient 's data had been measured more than three times , and for those cases , the longest interval between the two data was used for calculation . the yearly alteration rate ( cm / year ) was calculated as follows : { ( mean of both kidney diameter ( kd1 kd2 cm))/measurement interval ( month ) } 12 ( months ) . the results were studied to find out the factors that may influence the alteration rate of kidney diameter : age , gender , and the original disease ( compared among three groups , chronic glomerulonephritis and iga nephropathy , diabetes , and others including hypertension ) . to evaluate whether the alteration rate was stable all through the on hd period or not , patients were divided to nine groups according to the interval from the induction of hd to the first measurement . and all patients agreed with signature that their clinical data or physical findings would be used for statistical study of the japanese society for dialysis therapy , and other clinical studies . this study did not violate ethical standards of the declaration of helsinki and its revisions , because the measurements were performed during the annual routine health check up for hd patients without any additional invasive procedures , and the purpose of the measurement was explained by the examiner and verbal allowance was obtained by the patient at the time of examination . statistical evaluations of various parameters were performed using stat view 4.5 for mac ( abacas concepts , inc . ca usa ) , and each analytical method was indicated in the table . a p value of < 0.05 was regarded as statistically significant . raw data obtained by all 109 patient 's measurements and connected in line between the same patient 's data plots is indicated as in figure 2 . the average interval from the induction of hd to the first measurement was 29.5 2.5 months ( from 0.6 to 114 months ) , and the average interval of the two measurements was 35.9 2.2 months ( from 5.8 to 86 months ) . the average of all measured renal largest diameter was 6.91 0.06 cm ( 4.3 to 11.1 cm ) . the average decrease rate of renal diameter was 4.34 0.4 mm / year , which was simply obtained from the mean of 109 patients ' results . among the three original groups , cgn + iga and diabetes groups were younger than ht + others group ( p < 0.05 ) , and diabetes group showed larger renal diameter at the first measurement ( p < 0.05 ) as indicated in table 1 . however , no difference was seen on the decrease rate in relation to gender , age , and original disease ( tables 1 and 2 ) . comparing the samples those were divided to 9 categories according to the time of the first measurement after induction of hd , the decrease rate was large when the hd vintage was small , suggesting that the alteration rate reduced according to the duration of hd ( 5.3 0.8 mm / year , n = 33 , first measurement not more than 10 months after induction of hd , 3.9 0.6 mm / year , n = 18 , ~20 months , 5.4 0.9 mm / year , n = 16 , ~30 months , 4.9 3.5 mm / year , n = 6 , ~40 months , 3.3 1.0 mm / year , n = 11 , ~50 months , 3.6 0.8 mm / year , n = 11 , ~60 months , 2.1 1.3 mm / year , n = 5 , ~70 months , 0.6 0.6 mm / year , n = 5 , ~80 months , 1.5 1.6 mm / year , n = 4 , and ~80 , p = 0.014 , kruskal - wallis ) as indicated in figure 3 . although gradual decrease of the kidney size is frequently observed in hd patient , to the best of our knowledge , no studies have been found in the english medical literature reporting the actual decrease rate of the kidney size by measuring the same patient consecutively . therefore , the decrease rate and the influence of original disease on the decrease speed , were poorly understood . our study indicated that kidney size decreased approximately 4 mm per year in an average , and the decrease rate slowed as the dialysis vintage increased . original disease , gender and age did not affect the decrease rate . in the japanese literature , uchida studied on hd patients concerning kidney size , degree of cysts formation and possibility of cancer formation from the autopsy and surgical specimens . they reported that kidney size decrease the first three years after the induction of hd ( kidney size ( cm ) = k = 9.13 0.33x ( years ) ) , then , increased due to acdk formation until 8 years ( k = 5.43 + 0.15x ) , and after 8 years , kidney size decreased again ( k = 7.62 0.58x ) . yamaguchi et al . reported from us observation , that kidney size decreased the first three years after the induction of hd , kept the same size until 8 years , and then start to increase size due to acdk formation . according to their study , the renal shrink rate of patients who had diabetes was smaller than those who had other diseases in the first 5 years from induction of hd . these studies were not suitable to evaluate the common decrease rate of renal size after induction of hd , that in they include both cystic and noncystic kidneys [ 13 ] . we excluded 125 acdk - formed patients out of 234 patients and evaluate 109 patients . our study did not consider the initial renal diameter at the induction of hd , as there was no data , and it would be better if the measurement interval had been regular to obtain more accurate data . the shrinkage of kidney is thought to not start at the induction of hd but at the histological change into end - stage kidney by original diseases . other previous study reported that the renal diameter of the patients having diabetes was larger than that of those having other diseases such as cgn . renal hypertrophy is thought to one of the signs of renal impairment of diabetic nephropathy [ 8 , 9 ] . the decrease rate of renal diameter in patients who have diabetes is reported to be faster than in those who have other diseases in the beginning of hd . in our study , the mean of the first measured renal diameter of the diabetic patients was also larger than that of other original diseases as indicated in the table 2 . however , according to our study , there were no difference in the decrease rate of renal size among the original diseases . some cases showed different types of renal cortical shrinkage such as the increase of parapelvic adipose tissue , not altering total renal diameter so much . there were no malignant tumors in our study including acdk case , though they were excluded from the calculation of renal size . although more study will be needed to find out the relation between tumor formation and alteration of renal appearance , our study suggested that macroscopic change of end - stage kidney would be almost the same among the original diseases that were responsible for inducing hd . chronological change of renal size on chronic hd was studied for 109 hd patients using us examination consecutively . renal diameter decreased approximately 4.3 mm each year , and the decrease rate slowed as the duration of hemodialysis increased . the decrease rate was not affected by the original disease , age , and gender .

we here present the results of ultrasonographic ( us ) evaluations on the alteration of renal diameter of chronic hd patients . of 109 outpatient hd patients who had neither severe acquired cystic disease of the kidney nor hereditary polycystic kidney disease , we performed us two or three times to measure their maximum renal diameter ( mean of both kidneys ) , and the yearly alteration rate was calculated . the average interval of the two measurements was 35.9 months , and the average hd duration from the hd induction to the first measurement was 29.5 months . the average decrease rate of renal diameter was 4.34 0.4 ( se ) mm / year . no statistical difference was seen on the decrease rate in relation to gender , age and original disease ( among three groups , glomerulonephritis and iga nephropathy , diabetes , and others including hypertension ) . however , the decrease rate was large when the first measurement was close to the induction of hemodialysis , suggesting that the alteration rate reduced according to the hemodialysis vintage ( 5.3 0.8 mm / year , first measurement not more than 10 months after induction of hd and 1.5 1.6 mm / year , first measurement more than 80 months after induction of hd ) . renal diameter decreased approximately 4.3 mm each year , and the decrease rate slowed as the length of time on dialysis increased .

snake bite is an established important cause of morbidity and mortality among the poor , rural tropical population . hilly terrain is a special environment , which provides natural setting to study the challenges in the management of snakebite cases . eco - rich vegetation due to a long rainy season , abundant flora and fauna and a scattered population using paths traversing rural and forest lands makes people in these areas particularly prone to snake bite . existence of venomous snakes like trimeresurus albolabris ( white lipped pit viper ) , gloydius himalayanus ( himalayan pit viper ) and naja oxiana ( black cobra ) has been documented in the state of himachal pradesh , india in addition to the common big 4- the indian cobra ( naja naja ) , the common krait ( bungarus caeruleus ) , the russell 's viper ( daboia russelii ) and the saw - scaled viper ( echis carinatus ) which are found throughout india . the spectrum of envenomation is broadly assumed to be the same throughout india , but the diversity of venomous species questions the effective use of polyvalent anti - snake venom ( asv ) raised and imported from one state to another . on - scene resource constraints , ignorance , use of local herbal treatment and other indigenous medicines and transportation difficulties in the hilly terrain prevent patients from reporting in time to centers where snake antivenom is available . in 2009 the purpose of this study is to describe the clinical profiles and manifestations of patients of snakebite admitted to our hospital located in shivalik and the lesser himalayan region of himachal pradesh over the last 2 years . no studies on snake bite are available from this region to the best of our knowledge . in a hospital record - based retrospective descriptive study , we evaluated snakebite cases admitted to the hospital under the department of medicine from july 2010 to june 2012 . data were collected from the medical records department of the 570 bedded , tertiary care hospital which caters to the rural hilly population of the physiogeographic zone of shivalik and lesser himalayas of the state of himachal pradesh , india . recorded information was entered in a pre - coded proforma and included details on demography , clinical profile , treatment and outcome . we evaluated the records of all snakebite cases where complete information was available as per the pre - coded proforma parameters . excluded were five snakebite patients who absconded or were discharged against medical advice and where records were incomplete . most of the patients were young with the mean age of 36.52 standard deviation ( sd ) 13.43 ( range : 13 - 74 years ) and the number of male patients was higher than female patients . not a single case was recorded from december to march as shown in table 2 . on analysis for trends table 3 shows diurnal variation in the timing of snake bite with majority of the accidents happening in the evening , though this difference was not statistically significant . analysis of time taken between bite and presentation to hospital is represented in table 4 . the most frequently bitten site was lower limbs and foot happened to be the most common site as shown in table 5 . neuroparalysis was the most common presentation and was seen in 46% ( 53/114 ) patients among symptomatic patients . detailed clinical presentation is as depicted in table 6 . among patients with neuroparalysis early morning syndrome occurred in 26.4% ( 14/53 ) patients . out of 53 patients with neuroparalytic features , acute kidney injury was observed in 22 ( 11% ) patients and they belonged to the hemotoxic or the hemotoxic with local symptoms and signs group . dose used was 292.69 196.27 ml ( range : 50 - 950 ml ) . allergic reactions in the form of early anaphylaxis were noted in 7% ( 8/114 ) patients . deaths were due to ventilator - associated pneumonia in 4 , refractory shock in 2 , massive gastrointestinal bleed in 2 and cardiorespiratory arrest in 2 . age wise distribution of snake bite patients ( n=200 ) month wise distribution of snake bite patients ( n=200 ) time distribution of snake bite patients time taken between bite and presentation site distribution of snake bite patients clinical presentation of patients the state of himachal pradesh in the northwestern himalayas extends between 3222- 3312n and 7545-7904e covering an area of 56,090 km . topography of the state is dominantly mountainous with the altitudinal range of 350 - 6975 m. the state has an estimated forest cover of 13,880 km which is 25% of the total geographic area of the state . the state has a total population of 68,56,509 and 90.2% people live in rural setup . reptilean fauna in this region is somewhat different from that of other parts of india . attempts to record reptilian fauna of himachal pradesh has revealed presence of venomous snakes like t. albolabris ( white lipped pit viper ) , g. himalayanus ( himalayan pit viper ) and n. aoxiana ( black cobra ) in addition to the common big 4 - the indian cobra ( n. naja ) , the common krait ( b. caeruleus ) , the russell 's viper ( d. russelii ) and the saw - scaled viper ( e. carinatus ) . it is important that snakes belonging to viperidae group have been abundantly found up to an altitude of 4850 m. antivenom is the only specific treatment for snake bite envenoming , but existing products cover only the this diversity of venomous species questions the effective coverage of indian polyvalent asv . deaths due to snake bite occur predominantly in rural areas ( 97% ) and majority of them happen outside areas with access to health care facilities . in an analysis of snake bites in india , the death rate in the state of himachal pradesh was not known . in the present study , the predominance of young male victims suggests that more ambulant population is at the highest risk for snake bite in this region and has been reported in studies from different places in india . female preponderance has been observed in a study done in another physiographic zone of lower shivalik himalayan region of the state . all the cases of snake bite occurred in the months of april to november , which corresponds with summer , monsoon and the harvesting season in this region , all ideal for snakebites . the maximum bites occurred during the month of august , in the rainy season , when vegetation is abundant and people are involved in intense agricultural activities . this distinct seasonal pattern with peaks in the warm and rainy months has been observed in the state of himachal pradesh , as in other parts of the country . this observation is important from the prevention aspect as this is the time when maximum vigil is required to guard against the snake bites . no definite pattern for the time of bite was observed in our cases as is evident from wide variability . the time of bite corresponds to the outdoor activities and relative abundance of diurnal and nocturnal snakes . it was observed that only a few ( 13.5% ) of the patients attended hospital within the 1 h of bite and the majority ( 64% ) attended between 1 and 6 h. 22 ( 11% ) patients arrived at this hospital after 12 h of bite . most of these patients were either referred from neighboring health centers or arrived late at the appearance of symptoms or complications in the form of acute renal failure . difficult topography , delayed transportation in addition to lack of awareness of the hazards of snakebite and initial preference for alternative systems of medicine are the main reasons for delay in arrival as reported in the past . the bite - to - treatment delay varies greatly in studies from different health care centers of india . lower limbs were the commonest site of bite in 55% ( 110/200 ) patients and foot was involved in 61% ( 68/110 ) cases . it was followed - up by upper limbs out of which hand and fingers accounted for 22.5% ( 45/200 ) . bites on the uncommon sites like head and trunk are mostly due to nocturnal species biting people who are asleep . majority of the snakebite cases in the hills of himachal pradesh are due to either non - venomous snakes or dry bites of venomous snakes . among the symptomatic patients common were neuroparalytic followed by hemotoxicity . local signs only in the form of ecchymosis , swelling and necrosis were observed in 21.9% ( 25/114 ) patients . out of 36 patients with hemotoxicity , local symptoms and signs were found in 21 patients ( typical syndrome of viperidae species bite ) and four patients with neurotoxicity had additional local features . the reasons for local signs in these patients are difficult to attribute to a single cause as 116 patients had applied tourniquets and indigenous herbal medicines . t. albolabris ( white lipped pit viper ) , g. himalayanus ( himalayan pit viper ) bites are known for predominant local signs in the form of pain , marked swelling , bruising and bleeding . fortunately none of the patients had resorted to the incision and drainage of the bite site in this region , a practice currently not recommended . out of the cases presenting with neuroparalytic syndrome 26.4% presented with early morning symptoms . the kraits often enter the houses in search of food especially during midnight to early morning . the person sleeping on the floor / open environment is particularly susceptible to their bite . a false reassurance is created by paucity or absence of local pain and swelling after krait bite . the absence of bite mark with no specific history of snake bite makes diagnosis and management complex . at our center , out of a total of the 200 patients admitted with history of snake although the dose of antivenom is not yet fixed , the indications are well - known . national snake bite management protocol 2008 ( india ) has recommended a maximum dose for hemotoxic and neurotoxic bites as 30 vials ( 300 ml ) and 20 vials ( 200 ml ) respectively . one of the observations was higher dose used to reverse the effects of persistent coagulopathy in our patients . we have to move toward symptom analysis and treatment relevant to a specific snake species is the ideal but not always possible . adverse reactions in the form of early anaphylactic reactions to antivenom were seen in 7% patients , but there were no deaths . the death rate in our study was 5% and compares well with the death rate from other studies across india . a retrospective analysis was one of the limitations of this study , since some of the important data were incomplete or insufficient and they may not reflect the exact statistics . in spite of the limitations , the data can be generalized to some extent as the study center happens to cater to two thirds of the state population . further with strong public health infrastructure utilization and absence of organized private sector in the state lends credibility to this generalization . a retrospective analysis was one of the limitations of this study , since some of the important data were incomplete or insufficient and they may not reflect the exact statistics . in spite of the limitations , the data can be generalized to some extent as the study center happens to cater to two thirds of the state population . further with strong public health infrastructure utilization and absence of organized private sector in the state lends credibility to this generalization . a retrospective analysis was one of the limitations of this study , since some of the important data were incomplete or insufficient and they may not reflect the exact statistics . in spite of the limitations , the data can be generalized to some extent as the study center happens to cater to two thirds of the state population . further with strong public health infrastructure utilization and absence of organized private sector in the state lends credibility to this generalization . snake bite is a neglected tropical disease affecting poor villagers in rural areas . the data and research from this region is primitive on the clinically important subject of bites due to reptilian fauna and their manifestations .

objective : the objective of the following study is to assess the clinical profiles and manifestations of snakebite patients in the rural hilly setting of shivalik and the lesser himalayan region of himachal pradesh.materials and methods : a hospital record - based retrospective descriptive study was carried out that included details on demography , clinical profile , treatment and outcome among 200 patients over a period of 2 years . the data was analyzed using chi - square test for comparison.results:142 ( 71% ) patients were young ( age group of 16 - 45 years ) and the number of male patients was 118 ( 59% ) and female patients were 82 ( 41% ) . all the cases recorded presented in the months of april to november . not a single case was recorded from december to march . the most frequently bitten sites were the lower limbs particularly the feet . 86 ( 43% ) of the patients presented without any features of envenomation . neuroparalysis was the commonest presentation in 53 ( 46% ) patients followed by hemotoxicity in 36 ( 31% ) among symptomatic patients . early morning neuroparalysis syndrome was the presentation in 26.4% patients . allergic reactions in the form of early anaphylaxis were noted in 7% patients.conclusion:snake bite is a neglected tropical disease affecting poor villagers in rural areas . future research focusing on understanding epidemiological determinants of snake bite is desired .

proteins are the main perpetrators of most biological processes that take place within and between cells . however , proteins are very social in nature and often perform their function as part of large molecular machines , whose action is coordinated through complex regulatory networks of transient protein interactions . it is thus the relationships between molecules , rather than their mere presence , what will ultimately determine the behavior of a biological system . consequently , after the completion of the first genome sequencing projects , much effort has been devoted to unveiling protein interrelationships in a high - throughput manner , and recent years have witnessed the consecution of the first interactome drafts for several model organisms , including human ( 1,2 ) , setting the bases for future systems biology initiatives ( 3 ) . however , high - throughput interaction discovery experiments indicate only that two proteins interact , but do not provide information about the molecular details or the mechanism of the interaction . currently , this atomic level of detail can come only from high - resolution 3d structures , where the residue contacts are resolved and the protein interaction interfaces characterized . as a result , several databases have been developed in the last years to capture and store interactions of known 3d structure ( 46 ) . the database of 3d interacting domains ( 3did ) is a collection of protein protein interactions for which a high - resolution 3d structure has been solved . by exploring all interactions of known structure as stored in the protein data bank ( pdb ) ( 7 ) , we could divide them into two main categories on the basis of their contact interfaces : domain domain and domain peptide interactions ( 3 ) . we also used the finding that homologous pairs of interacting proteins tend to interact in the same way ( i.e. all fgfs bind the same fgf receptor pocket ) to further cluster and classify protein interactions in interaction types ( 8) , according to their binding and interface topologies . domain domain interactions involve the binding of two globular domains , which creates a large contact interface of 2000 on average ( 9 ) . these are the type of interactions that usually occur in multimeric enzymes and large multiprotein complexes , and they can be either intra- or inter - molecular ( i.e. between domains in the same or different proteins , respectively ) . to identify all the cases of domain domain interactions of known 3d structure , we first assigned pfam ( 10 ) domains to each individual protein in the pdb . we then computed all the physical interactions between domains requiring at least five contacts ( hydrogen bonds , electrostatic or van der waals interactions ) , and removed those lacking a significant interface as described in refs ( 11,12 ) . this procedure has proven efficient at identifying and purging interaction artifacts from crystal packing ; however , it is likely that 3did still contains some nonbiological associates . currently , 3did contains 115 559 domain domain interactions of known 3d structure comprising 120 980 proteins . we have classified them in 4887 unique interaction types according to the pfam families mediating them . of these , 3535 interaction types always occur between domains placed in different proteins ( intermolecular ) , 738 are only seen between domains in the same polypeptide chain ( intramolecular ) and the remaining 614 occur both inter- and intra - molecular . when available , 3did also contains functional information about the interacting domains as annotated in the gene ontology ( go ) database ( 13 ) . domain peptide , or peptide - mediated , interactions are those where a globular domain in one protein recognizes and binds a short linear motif in another , creating a relatively small interface . such interactions are found predominantly in signaling and regulatory networks ( 14 ) and , due to their transient nature , are much more difficult to handle biochemically . linear motifs are short patterns of around 10 residues with a common function ( i.e. binding to a globular domain ) that occur in otherwise unrelated proteins . in isolation , these motifs bind their target proteins with sufficient strength to establish a functional interaction . they are frequently found in disordered or unstructured regions and adopt a well - defined structure only upon binding . an example of this type of interactions is the well - studied src - homology-3 ( sh3 ) domain , which binds slightly different variants of proline - rich peptides ( e.g. [ rky]xxpxxp or pxxpx[kr ] ) . most of what is currently known about peptide - mediated interactions is compiled in the eukaryotic linear motif ( elm ) database ( 15 ) , which provides a literature - curated collection of motifs and their interaction partners . our procedure to detect all cases of peptide - mediated protein interactions of known 3d structure was recently described in ref . ( 16 ) . in brief , we first parsed the pdb and identified all those entries containing two or more interacting proteins . we extracted all the information regarding the different 66 ligands involved in peptide - mediated interactions from the elm database and assigned pfam families to all the globular domains involved in the interactions via literature curation . we then assigned pfam families to all interactions of known 3d structure . whenever we identified a protein chain containing an elm - binding domain , we searched all contacting chains for occurrences of the linear consensus motif . when we found a motif match in close vicinity of the globular domain ( 10 ) we considered it a potential domain finally , we went manually through the 2200 potential hits , comparing the interacting structures to those described in the literature , and removing false positives where the interaction was not mediated by the consensus peptide . because of the visual inspection , we are confident that the interactions reported here are biologically relevant . at present , 3did contains data on 829 hand - curated peptide - mediated interactions of known 3d structure , from 611 protein pairs , involving 32 globular domains and 51 linear motifs . the whole concept of interaction types relies on the observation that homologous pairs of interacting proteins very often interact in the same way , this is , using the same binding interfaces ( 8) . however , there are exceptions to the norm where homologous protein pairs can interact in a completely different manner . for instance , the interaction between the signaling proteins chey and chea - p2 differs by a rotation of 90 in different bacterial species , despite being close homologs ( 17 ) . this is particularly relevant for those proteins that have evolved to interact with many different partners by only changing a few binding residues , such as antibodies , ankyrin repeats , etc . we have computed and classified all the interaction interfaces for each interaction type using a clustering procedure reminiscent of the one used by kim et al . we then computed a distance matrix for all the interfaces based on the number of shared contacts , and performed a complete linkage hierarchical clustering analysis to discover the different modes of interaction between the two given domains . the result is that , for each interaction type , we are able to identify how many different interfaces are used and how often these occur . we have termed the alternative interaction interfaces within the same interaction type interface topologies , and they are stored in 3did together with the frequency in which they occur . although the vast majority of known interaction types only display one or a few different topologies , it is also true that some families are able to interact with many partners using a large number of surface patches ( figure 1 ) . it is thus important , if one wants to model the structure of one interaction onto another , to make sure that , for this particular interaction type , only one interaction topology is possible or , at least , that there is one whose occurrence clearly stands over the rest . half of the interaction types in 3did always interact using the same topology , and most of the remaining ones show only a few different topologies . for a handful of interaction types , we find over 50 interface topologies ( 66 for ras : ras up to 199 for v - set : v - set ) . half of the interaction types in 3did always interact using the same topology , and most of the remaining ones show only a few different topologies . for a handful of interaction types , we find over 50 interface topologies ( 66 for ras : ras up to 199 for v - set : v - set ) . the standard way of accessing 3did is through the web - based tool by querying it with a particular domain or motif , although it can also be queried by pasting a protein sequence or directly indicating the pdb codes or go terms of interest . as in previous versions , 3did will then display all domains , or peptides , that do physically interact with our domain of interest and for which the 3d structure of the interaction is known . all interaction partners will also be displayed in an interactive network ( figure 2 ) , where the user can choose the depth and a color scheme based on molecular function , biological process or cellular compartment as described by go . the network also gives information on the type of interaction ( domain domain or peptide - mediated ) and whether these interactions are intra- or inter - molecular . the user can then select a particular interaction and retrieve the specific details stored in 3did . the output page for each domain domain interaction displays a table with information concerning all the known 3d structures where this interaction is found ( figure 3 ) . the table shows the exact location of the two domains in the 3d complex and gives empirical potential scores and z - scores , which provide a measure of the number of favorable interacting residue - pairs at the interface ( 11,12 ) . the z - score generally accounts for interaction specificity : the higher it is the more specific the interaction . finally , clicking on the rasmol ( 20 ) icon pops up a display of the 3d complex . the two interacting domains are colored and shown in ribbons representation with the residues participating in the interface ( i.e. making hydrogen bonds , salt bridges or van der waals contacts ) shown in ball - and - stick . the newest version of 3did also includes a graphical representation of the different interaction topologies for each interacting domain . this representation indicates which residues of a domain are used in a particular interaction , as well as their frequency ( figure 3 ) . the query results for lig_sh2_src show the linear motif pattern and source database ( elm ) , links to the binding domain sh2 and all 3d structures containing this motif , followed by all motifs binding sh2 along with their patterns ( if available ) , sh2 's interface residues and a link to the corresponding domain motif interaction page . the network below visualizes domains and motifs interacting with lig_sh2_src as well as their interactions among each other . domain interaction view shows all topologies observed in 3d structures of this interaction type along with their frequencies . rainbow color scheme is used to visualize where interface residues lie in the sequence , from n - terminus ( blue ) to c - terminus ( red ) . each topology has an identifier ( i d ) of the form x : y , where x is the interface i d in domain 1 ( pdz here ) and y is the interface i d of domain 2 ( trypsin here ) . the interaction details provide pdb i d , domain positions , score and z - score as well as the topology i d , linked to the topology visualization above , for each interaction between these two domains in a known 3d structure . the query results for lig_sh2_src show the linear motif pattern and source database ( elm ) , links to the binding domain sh2 and all 3d structures containing this motif , followed by all motifs binding sh2 along with their patterns ( if available ) , sh2 's interface residues and a link to the corresponding domain motif interaction page . the network below visualizes domains and motifs interacting with lig_sh2_src as well as their interactions among each other . domain interaction view shows all topologies observed in 3d structures of this interaction type along with their frequencies . rainbow color scheme is used to visualize where interface residues lie in the sequence , from n - terminus ( blue ) to c - terminus ( red ) . each topology has an identifier ( i d ) of the form x : y , where x is the interface i d in domain 1 ( pdz here ) and y is the interface i d of domain 2 ( trypsin here ) . the interaction details provide pdb i d , domain positions , score and z - score as well as the topology i d , linked to the topology visualization above , for each interaction between these two domains in a known 3d structure . mysql and flat files containing the entire database are also available through the website for independent studies . 3did is weekly updated with new 3d structures , and major updates are implemented whenever new versions of pfam or elm are released . spanish ministerio de educacin y ciencia ( pse-010000 - 2007 - 1 and bio2007 - 62426 ) partially ; 3d - repertoire from the european commission under fp6 contract lshg - ct-2005 - 512028 .

the database of 3d interacting domains ( 3did ) is a collection of protein interactions for which high - resolution 3d structures are known . 3did exploits structural information to provide the crucial molecular details necessary for understanding how protein interactions occur . besides interactions between globular domains , the new release of 3did also contains a hand - curated set of transient peptide - mediated interactions . the interactions are grouped in interaction types , based on the mode of binding , and the different binding interfaces used in each type are also identified and catalogued . a web - based tool to query 3did is available at http://3did.irbbarcelona.org .

cd63 is a member of the tetraspanin family of cell surface associated membrane proteins characterized by presence of four hydrophobic domains . originally found to be present on the cell surface of activated blood platelets in humans , cd63 was later shown to function as the activation mark itself . at the cell surface , cd63 is known to be endocytosed via a clathrin - dependent pathway . in addition to the plasma membrane , cd63 is highly expressed in the endosome and lysosome of immune cells including antigen presenting cells , t - lymphocytes , and neutrophils [ 57 ] . in the macrophages and dendritic cells , cd63 can chaperone mhc molecules through the endosomal pathway to present antigens and initiate immune response , whereas , in the t - lymphocytes , cd63 is linked to the proper trafficking of cell surface associated chemokine receptor ( cxcr ) and its mutation leads to mistargeting of cxcr to late endosomes / lysosomes [ 3 , 7 ] . additionally , cd63 has also been associated with a wide variety of cancer cells , where it plays a role in cell activation , motility , differentiation , and tumor invasion making this protein an important research topic in vertebrates . in invertebrates , hemocyte - mediated phagocytosis is a specific form of endocytosis which involves engulfment of pathogenic bacteria . this process plays a central role in efficiently eliminating the invading pathogens aiding in host defense . the internalized vacuole harboring the pathogen , called phagosomes , then undergoes maturation through lysosomal fusion resulting in acidification and thorough digestion of engulfed bacteria [ 11 , 12 ] . we believe that cd63 protein is involved in the process of hemocyte - mediated phagocytosis . interestingly , immunofluorescence studies have demonstrated that cd63 is highly expressed in the granular and vesicular structures of mollusk hemocytes , supporting our hypothesis about its possible role in phagocytosis . p. undulata is one of the most important economic marine mollusks in the south china sea and gulf of thailand . recently , it has been suffering from severe pathogenic infections resulting in collapsing natural clam populations inflicting great losses to the aquaculture industry [ 15 , 16 ] . therefore , understanding the molecular mechanism of innate immunity in the clam p. undulata is of paramount importance . in this study we have investigated the possible role of p. undulata cd63 protein in promoting hemocyte - mediated phagocytosis against pathogenic challenge . in the process we have cloned and characterized the full - length cdna of cd63 from the clam . healthy clams were obtained from xiamen , fujian province , china , and were maintained in tanks at 18c22c with recirculating seawater for two week , prior to the bacterial infection . the clams were challenged with injection of 100 l vibrio parahaemolyticus or vibrio alginolyticus at a concentration of 1 10 cells / ml . the controls were injected with equal volume of pbs instead . at 24 h after injection , hemolymph was withdrawn from the adductor muscle of clams with a 0.5 mm diameter ( 25 g ) disposable needle and immediately centrifuged ( 700 g for 10 min at 4c ) to separate the hemocytes cells from plasma . for each treatment , a set of gene specific primers ( table 1 ) were used to amplify 5 and 3 cdna ends using generacer kit ( life technologies , usa ) . the race products were cloned into the pcr - topo vector ( life technologies , usa ) and were sequenced with abi prism 3730 dna sequencer ( perkinelmer , wellesley , ma , usa ) . finally , the full - length cdna sequence of p. undulata cd63 was constructed by combining the 3- and 5-end sequences . the full - length cd63 sequence was analyzed using the ncbi blast algorithm ( http://www.ncbi.nlm.nih.gov/blast ) and the expert protein analysis system ( http://www.expasy.org/ ) . protein domain prediction was performed by smart ( http://smart.embl-heidelberg.de/ ) web tool and the presence of transmembrane structure was analyzed using tmhmm ( http://www.cbs.dtu.dk/services/tmhmm/ ) . multiple sequence alignments of cd63 amino acid sequences were performed using the clustalx version 1.81 and the phylogenetic trees were generated by the mega 5.0 package utilizing neighbor - joining method . total rna was extracted from hemocytes with trizol reagent ( invitrogen , usa ) according to the manufacturer 's directions . cdna was reverse - transcribed using the primescript rt reagent kit ver.2.0 ( takara , japan ) from 1 g of total rna . the expression profile of cd63 in bacteria challenged hemocytes was measured by quantitative real - time pcr ( qpcr ) . the qpcrs were carried out in a 20 l reaction system containing 10 l of 2x master mix ( roche , usa ) , 0.4 l of each of the forward and reverse primers ( 10 mm ) , 1 l of diluted cdna ( 1 : 10 ) , and 8.2 l of pcr - grade water . glyceraldehyde 3-phosphate dehydrogenase ( gapdh ) was used as the reference gene and additional negative controls were also used to test for genomic dna contaminations . the specificity of the pcr amplifications was accessed by analyzing the melting curves at the end of the reaction . all data were generated by the rotor - gene version 6 - 0 - 22 software and the fold changes were calculated using the 2-ct method . appropriate fragments of cd63 and control luciferase ( obtained from pgl3-vector ) were pcr - amplified with t7 promoter linked primers ( table 1 ) . the products were used as templates for dsrna synthesis by in vitro transcription using megascript t7 transcription kit ( life technologies , usa ) . the rna integrity was examined by gel electrophoresis and the concentration was quantified by spectrophotometers of nanodrop 2000c ( thermo scientific ) . in vivo gene knockdowns were performed by injecting 20 g respective dsrna into the adductor of the clam . the phagocytic activity of hemocytes was measured by flow cytometry ( bd facscalibur ) as described previously . briefly , hemolymph was collected from normal and cd63 knockdown clams and fluorescent ( fitc ) conjugated v. parahaemolyticus and v. alginolyticus were incubated with the hemolymph . after 30 minutes of incubation , cell - associated fluorescence was quantified using flow cytometry at 530 nm ( fl1 ) to assess phagocytic activity . hemocytes cell populations were defined based on their size ( fsc ) and granularity ( ssc ) properties , and a total of 10,000 events were acquired for each sample . the statistical differences between groups were analyzed by one - way anova , followed by dunnett test and turkey multiple comparison test using the spss software package . in this study , the homolog of cd63 was identified and characterized from marine mollusk , paphia undulata , henceforth designated as pu - cd63 . the complete cdna of pu - cd63 is 1,738 bp in length and includes an open reading frame ( orf ) of 849 bp , a 5-untranslated region ( utr ) of 168 bp , and a 3-utr of 721 bp with a canonical polyadenylation signal ( aataaa ) ( supplementary figure 1 , in supplementary material available online at http://dx.doi.org/10.1155/2016/7893490 ) . this previously uncharacterized pu - cd63 cdna sequence has been deposited in genbank under accession number kt596763 . bioinformatics analysis showed that the orf encodes a 282-amino - acid protein with a calculated molecular mass of 32 kda and a theoretical isoelectric point of 8.32 . tmhmm analysis revealed that pu - cd63 contains four putative hydrophobic transmembrane helixes ( figure 1 ) , with the characteristic large extracellular loop ( lel ) between the third and the fourth transmembrane domain [ 4 , 20 , 21 ] . cd63 proteins are majorly trafficked to either the plasma membrane in cell surface or the endosomes / lysosomes . two characteristics motifs of cd63 , yxx consensus motif and n - glycosylation sites , assist in this targeting . the yxx motif contains an essential tyrosine residue , 2 hydrophobic xx residues , and a bulky hydrophobic amino acid and plays a role in both endocytosis of cd63 from the plasma membrane and lysosomal targeting . the sequence 110-yvii-113 is located within the third transmembrane helix and meets the criterion for yxx motif , implicating a possible role in pu - cd63 trafficking . on the other hand , the other motif , n - linked glycosylation , was shown to be required exclusively for endosomal / lysosomal targeting of cd63 in mammals [ 23 , 24 ] . bioinformatic analysis also revealed that pu - cd63 contains one putative n - glycosylation site 155-ngt-157 within the lel region , suggesting its conserved function in endosomal / lysosomal trafficking . in addition to four transmembrane domains the pu - cd63 proteins feature several conserved amino acid residues including a highly conserved ccg motif , the two cysteine residues of which are required for essential disulphide bond formations in the second extracellular loop [ 25 , 26 ] . phylogenetic trees were constructed from sequence alignments of homologous cd63 proteins from different species by utilizing the neighbor - joining method ( supplementary figure 2 ) . in agreement with the traditional evolutionary groupings , pu - cd63 was clustered together with its homolog from another mollusk species crassostrea gigas into the mollusk clade . to explore the possible involvement of pu - cd63 in the immune response , its temporal expression profile was determined in the hemocytes after bacterial challenge , using qpcr . in comparison to the uninfected controls , pu - cd63 transcript sharply increased to about 6-fold and 3-fold at 3 h after challenge with v. parahaemolyticus and v. alginolyticus , respectively ( figure 2(a ) ) . its expression peaked at 12 h after v. parahaemolyticus and v. alginolyticus challenge , with respective 12- and 7-fold change . 2448 h after challenge , however , the transcript abundance of pu - cd63 dropped to basal levels . these results indicate a clear induction in the expression of cd63 in mollusk hemocytes after pathogenic challenge and strongly support our hypothesis about an immunogenic role of pu - cd63 . a similar induction of the cd63-like gene was observed in the hemocytes of crassostrea ariakensis 12 h after challenge with lipopolysaccharide ( lps ) and polyinosinic : polycytidylic acid ( poly i : c ) , supporting our finding . as the predominant cell - mediated defense mechanism for tackling the invading pathogens , phagocytosis plays a central role in the innate immune response of invertebrates , including mollusks . to examine the potential role of cd63 on the hemocyte - mediated phagocytosis in p. undulata , dsrna - mediate rnai knockdown of cd63 was performed in vivo by injection into the adductor muscle . the knockdown efficiency of pu - cd63 mrna was assessed in the hemocytes via qpcr at 72 h after injection . the level of the pu - cd63 mrna decreased to about 20% of that of the controls ( figure 2(b ) ) . with 70% inhibition set as the accepted threshold for efficient rnai , our injection successfully knocked down the pu - cd63 gene in clam hemocytes . the fluorescent ( fitc ) conjugated v. parahaemolyticus and v. alginolyticus were incubated with hemolymph to assess phagocytic activity of ipu - cd63 clams . a dramatic 78% and 33% reduction in hemocytes phagocytic activity was recorded in ipu - cd63 clams by flow cytometry , after incubation with v. parahaemolyticus and v. alginolyticus , respectively ( figures 2(c ) and 2(d ) ) . this result clearly demonstrated the role of cd63 proteins in hemocytes mediated phagocytosis in response to pathogenic challenge in the clam p. undulata . supporting our observation , there are reports that internalization of cd63 is accompanied with phagocytosis of saccharomyces cerevisiae in human dendritic cells . although the exact mechanism of how cd63 promotes phagocytosis remains unclear , previous studies have demonstrated that it can serve as an adaptor protein linking different other proteins via direct / indirect interaction and facilitating the formation of endocytic machinery . apart from various cell surface receptors , cd63 has also been shown to associate with integrin proteins [ 31 , 32 ] . interestingly , in mollusks , blockage of integrin activity could significantly reduce the phagocytic ability of the hemocytes . we believe that cd63 promotes hemocyte - mediated phagocytosis via altering the integrin recruitment in endocytic machinery , a hypothesis which needs to be tested in the future . in summary , we have cloned and characterized the cd63 sequence from clam p. undulata , followed by its expression profiling in pathogen challenged hemocytes . a drastic induction in the pu - cd63 mrna was noted in the hemocytes after pathogen challenge . moreover , using flow cytometry we showed a definite role of pu - cd63 in promoting hemocyte - mediated phagocytosis . our work has enhanced our understanding about innate immunity of economically important clam p. undulata .

as one of the surface membrane proteins of tetraspanin family , cd63 plays a crucial role in cellular trafficking and endocytosis , which also is associated with activation of a wide variety of immune cells . here , the homolog of cd63 was characterized from one marine mollusk , paphia undulata , which is designated as pu - cd63 . the complete cdna of pu - cd63 is 1,738 bp in length with an open reading frame ( orf ) of 849 bp , encoding a 282 amino acid protein with four putative hydrophobic transmembrane helixes . bioinformatic analysis revealed that pu - cd63 contains one putative yxx consensus motif of 110-yvii-113 and one n - glycosylation site 155-ngt-157 within the large extracellular loop ( lel ) region , supporting its conserved function in plasma membrane and endosomal / lysosomal trafficking . moreover , temporal expression profile analysis demonstrates a drastic induction in the expression of cd63 in hemocytes after pathogenic challenge with either v. parahaemolyticus or v. alginolyticus . by performing dsrna - mediate rnai knockdowns of cd63 , a dramatic reduction in hemocytes phagocytic activity to pathogenic vibrio is recorded by flow cytometry , revealing the definite role of pu - cd63 in promoting hemocyte - mediated phagocytosis . therefore , our work has greatly enhanced our understanding about primitive character of innate immunity in marine mollusk .

gastrointestinal autonomic nerve tumors ( gants ) were first described by herrera et al . in 1984 . they have recently been distinguished from each other , leiomyoma , leiomyosarcoma , leiomyoblastoma , schwannoma , and gastrointestinal stromal tumor ( gist ) , on the basis of the tumor cell differentiation revealed by immunohistochemical studies . the term gist , a subgroup of gants , has recently been applied to mesenchymal tumors with nonspecific ultrastructual findings . schwannomas are tumors derived from schwann cells in the auerbach plexus which form a neural sheath , accounting for 2 - 6 of all neoplasms of the gastrointestinal tract . it is well established that schwannomas appear more frequently in the stomach and the small bowel ; those with a location in the colon are rare [ 4 , 5 ] . we report an unusual case of an appendicular schwannoma arising in the tip of the appendix . a review of the previous literature is also presented . a 68-year - old man presenting with intermittent cough and sputum for 1 month without any abdominal symptoms was admitted to our hospital . the laboratory findings were within normal limits , including a negative sputum culture ( gram stain and afb culture ) . there was a consolidation with the opacity of ground glass in the left upper lobe lingular division on the chest computed tomography ( ct ) , suggesting pneumonia or a malignancy such as a bronchioloalveolar carcinoma ( bac ) or a lymphoma . further evaluations were done for cancer stage work - up , and the abdomino - pelvic ct showed about a 1.4 cm , well - enhanced nodule on the tip of the appendix , suggesting carcinoid tumor ( fig . 1 ) . positron emission tomography ( pet ) ct also showed a lesion in the appendix that could not be ruled out for metastasis ( fig . the patient underwent a left upper lobectomy of the lung and a laparoscopic appendectomy ( fig . a frozen biopsy was done that showed a neurogenic stromal tumor with a clear resection margin . the appendix tumor was 2.3 1.8 1.1 cm in size with a 2.3 cm resection margin , suggestive of an appendix stromal tumor ( favoring a neurogenic tumor ) ( fig . the immunohistochemical staining showed positive s-100 protein , consistent with the diagnosis of a schwannoma ( fig . the patient was followed for 6 months later , and no evidence of specific complications or recurrence on abdomen was noted , but cancer recurred in the other lung , so the patient underwent palliative chemotherapy . they are mainly benign neurogenic tumors arising from the sheath of the peripheral nerves , but they may undergo malignant changes if left untreated [ 6 , 7 ] . there is no difference in the rates of incidence in men and women , and a median age of presentation is 65 years of age . these tumors frequently occur in the head and neck , the spinal cord , and the extremities , but they are seldom found in the abdomen . in the gastrointestinal tract , schwannomas are mostly found in the stomach , but reports of their being present in the colon and rectum are rare , and most grow outside the lumen contralateral to the mesenteric attachment [ 3 , 4 ] . although neurogenic tumors usually grow very slowly and the symptoms are vague and nonspecific , common symptoms are abdominal pain , apalpable mass , and hemorrhage ; however , intestinal obstruction is rare [ 3 , 5 , 8 ] . in the small intestine , hemorrhage is the most frequent symptom because when the tumor grows , abundant blood vessels in the submucosal tumor become exposed on the surface of mucosa and may easily be injured . approximately 30 - 50% of appendicular tumors show clinical symptoms and signs similar to those of appendicitis , and this rate is higher for carcinoid tumors than it is for other tumors . however , the majority of patients , as our patient , have nonspecific symptoms , which makes preoperative diagnosis very difficult ; thus , the tumor is often found incidentally [ 10 - 12 ] . for the diagnosis of a schwannoma , conventional imaging modalities , including ct , ultrasonography , barium enema , magnetic resonance imaging , and endoscopy , are useful for tumor localization [ 4 - 7 ] . also , blood loss scintigraphy , which can detect minor hemorrhage , has been reported to enable the diagnosis of jejunal tumors . however , we could not distinguish benign from malignant stromal tumors by using radiologic images alone . in our case , we found the appendicular tumor suggesting a carcinoid tumor by using an abdomino - pelvic ct , but pet ct could not rule out the possibility of malignancy , so surgical exploration was performed . one study showed that patients treated for an appendicular neoplasm had a higher rate of associated cancer than the normal population , especially synchronous colon carcinomas . thus , the authors of that study recommended a colonoscopic examination in patients with incidentally - discovered appendicular tumors . immunohistological studies of gants play a key role in the differentiating a schwannoma from other stromal tumors which have high potential of malignancy . s-100 protein , a nervous tissue - specific protein , and non - specific enolase , a molecular marker of peripheral and central neuroendocrine cells , aree useful for indicating schwannoma cell line tumors [ 2 , 3 , 6 , 7 , 13 ] . our patient also had positive s-100 protein , but showed negative for cd 34 , which is usually up to 80% positive for gastrointestinal stromal tumors other than schwannomas . a complete surgical resection is the treatment of choice while chemotherapy or radiotherapy is not effective . the surgical approaches depend on the tumor size and on the histologic features that mainly determine the prognosis , so preoperative imaging tests are very important in the selection of patients for surgical resection . however , in the case of malignant neurinomas , the prognosis is poor because hematogenous metastasis to the lung and the liver frequently occur [ 2 , 3 , 6 , 7 , 13 ] . because of its low incidence , the optimal treatment for a malignant schwannoma has not been not fully established , but like other soft tissue sarcomas , only a complete surgical resection can provide a chance for cure . the surgical margin has been advocated to be the most important prognostic factor , so every effort should be made to achieve a tumor - free surgical margin without unnecessary sacrifice of nerves . the indications for a laparoscopic approach to the resection of tumors have not yet been established definitively . one report indicates that laparoscopy does not increase the risk of local recurrence or metastasis compared with the open surgery , but another shows an increased rate of peritoneal seeding and abdominal wall metastasis after laparoscopic resection . it has been generally accepted that appendicular carcinoids can be treated by using a simple appendectomy with a clear resection margin . however , if they are larger than 2 cm in size or if the base of the appendix or the mesoappendix is involved , general agreement exists that a right hemicolectomy should be performed . benign appendicular tumors , including mesenchymal tumors without malignant components , are traditionally treated with a complete surgical resection or a limited cecal resection if the resection margin is involved . in the case of malignant lesions , improved survival was seen in patients who underwent a right hemicolectomy compared with patients who underwent an appendectomy alone [ 11 , 12 , 15 , 16 ] . the tumor should be assessed intraoperatively , and the treatment should be tailored to each individual patient based on the frozen biopsy results . in our case , the patient received a laparoscopic appendectomy with a free resection margin , favoring a neurogenic stromal tumor . therefore , the laparoscopic approach could be technically challenging even if the tumor is preoperatively suggested to be malignant . complete surgical excision is thought to be the best approach and to determine the overall outcome . laparoscopic surgery , however , with a clear resection margin should be available for treatment of an appendicular schwannoma and should result in a favorable prognosis with recurrence being unusual ; thus , an unnecessary laparotomy can be avoided .

appendicular schwannoma is a rare tumor originating from schwann 's cells in the auerbach plexus . the preoperative diagnosis is difficult because the clinical features are nonspecific , and it is mostly found accidentally via a radiologic image as a tumor , mimicking malignancy . we report a case of an appendicular schwannoma coexisting with an adenocarcinoma in the lung . a laparoscopic appendectomy was done with a clear resection margin , and the immunohistochemical staining showed positive s-100 protein , which confirmed the schwannoma . the patient also underwent a left upper lobectomy of the lung . the patient has been free of recurrence for the 6 months since the operation . the laparoscopic approach could be available for treatment of an appendicular schwannoma , thus avoiding an unnecessary laparotomy .

history is like a screen in which the past lightens the present and the present brightens the future . as a fast developing country , india 's pride is in its rich history , traditions and philosophies with its ideologies , requirements and support system different from western counterparts . the traditional indian system of medicine ayurveda is not only a herbal tradition , but a science with a comprehensive approach to life along with fundamental principles of diagnosis and treatment . when explored , we recognize the richness , comprehensiveness and intense possibility of paradigms and wisdom contained in this ancient indian literature , and its potential to enrich our professional knowledge . indian psychiatrists are increasingly open to the incorporation of ayurvedic concepts in practice . as we move forward hallucinations have fascinated human beings down the ages , and were sometimes regarded as messages from god , or as spiritual or religious visitations . however , the ancient indian treatment system of ayurveda was surprisingly modern in its interpretation of this phenomenon . in ayurveda , a hallucination is something that is perceived . ayurveda regards life as the combination ( samyoga ) of body ( sharira ) , organs of conation and cognition ( indriya ) , mind ( satva ) , and soul ( atma ) . but for practical purposes , the mind ( manas ) and body ( sharira ) were separately defined and their entity and doshas separated . bodily doshas ( sharirika doshas - vata , pitta , kapha ) and mental doshas ( manasika doshas - rajas , tamas ) mutually affect each other . as per ayurveda , the mind has immense potential along with its attributes of conscious and creative energy . according to charaka the father of ayurveda the mind is inactive ( achetana ) by itself but gets activated ( chetana ) by the self or soul ( atma or atman ) . although beyond sensory perception ( it can not be seen or felt ) , it is a material substance ( dravya ) , since it has both quality ( guna ) and action ( karm ) coexistent within itself . manas links the soul ( atma ) with sense organs and their sensory objects such as sound , touch , shape , taste , and smell . the soul ( atma ) is the basis of all experience , while mind ( manas ) is only the instrument of experience . mind is the mediator between soul ( atma ) and sensory organs ( indriyas ) . our ancient indian system emphasized the theory of unity of body and soul and explained how to deal with mental health problems using a psychosomatic approach . the concept of sadhaka pitta ( one of the five sub type of pitta dosha ) appears to be psycho physiological . the vitiation of one of the bodily humors - vata dosha is said to cause delirium ( pralapa ) , insomnia ( nidra nasha ) , etc . , kapha vitiation caused excessive sleep ( ati nidra ) and dullness ( avasada ) . trigunas specifically indicate the psychological traits - satva , rajas and tamas . the ordinary classification of mental states such as thinking , feeling , and willing could be explained within the scope of triguna . satva is indicated by knowledge acquisition , analyzing , satva is the nature of harmony in various forms . means , rajas is the initiator of thought , which gets converted to an action by desire , effort and memory . darkness , ignorance ( moha ) and is indicated by an inability to perceive , laziness . charaka says that the mind is a diverse disposition of these trigunas which function in mutual combination . the predominance of any one of them leads to a particular psychological constitution or predisposition - the pure sattvik , passionate rajasik and the ignorant tamasik . a psychological balance of all three is essential for a healthy state of mind . when satva is predominant mental outlook is healthy . the predominance of one or other qualities in a person at any given time determines his mental state at that time . however , satva , comparable to the concept of superego is not a dosha . the cognitive functions pertaining to memory , conception , judgment and reasoning all belong to satva . rajas is prominently responsible for the state of feeling and egoistic emotional states of mind like fear , anger , hope , envy , forethought , self - importance , hate etc . feelings like hunger , thirst and involuntary primitive and acquired conations which include reflex , instinctive , casual and habitual functions belong to tamas . auditory , visual , tactile , olfactory and gustatory sensations also may be related to tamas . i.e. , it has the characteristic of veiling or covering or concealing ( i.e. , it is unconscious ) . due to tamasik characteristics ( visadatmaka - depressive ) , unfulfilled urges and wishes once present in our mind are forced into unconsciousness which may subsequently result in mental disorders . according to ayurveda , sadhaka pitta ( one of the five types of pitta dosha ) is deemed to be essentially responsible for higher mental faculties and emotional states as mentioned in sushruta samhita , vagbhata ( ashtanga hridaya ) and charaka samhita , another treatise kashyapa samhita is of the same opinion . this is located in hridaya ( seat of mind ) and is responsible for the feelings of fear ( bhaya ) , courage , bravery , valor ( shourya ) , anger ( krodha ) , happiness ( harsha ) , intelligence ( buddhi ) , medha , and self - esteem ( abhimana ) . sadhaka pitta performs its functions by dispelling the kapha ( one of tridosha , which is responsible for dullness ) and ignorance ( tamas ) of the hridaya and thus enables manas to perceive things clearly . ayurveda has differentiated our organs into organs of perception ( jnanendriya ) namely auditory ( shabda ) , tactile ( sparsha ) , vision ( roopa ) , taste ( rasa ) , olfactory ( gandha ) and organs of execution ( karmendriya ) namely speech ( vak ) , hands ( pani ) , foot ( pada ) , excretion ( payu ) , sexual ( upastha ) . mind has been given the special status as ubhayendriya , meaning organ of both perception and action . according to ayurveda , although the mind ( manas ) is responsible for sensory perception , it has specific functions of its own . they are thinking ( chintya ) , analysis ( vicharya ) , speculation ( uhya ) , distinct thought ( dhyeya ) and decision ( sankalpa ) . the upanishads provide descriptions of theories of perception , thought , consciousness , and memory . prakriti is described , which can be considered as equivalent to personality in modern psychiatry . ancient indian psychology emphasized the significance of concentration and considered it means for perception of truth . the yoga system of philosophy dealt in depth with both theory and practice related to mental health . as per philosophy of yoga , the mind or manas performs two types of functions namely observable ( paridrishta ) and inferable ( aparidrishta ) . they are comprehension ( pramana ) , to oppose ( viparyaya ) , thinking of options / thinking by examples ( vikalpa ) , sleep ( nidra ) and memory ( smriti ) . ( avidya ) , unhappiness ( asmita ) , aggression ( raga ) , hatred ( dvesha ) and obsession ( abhinivesha ) . a mentally healthy individual has strong smriti ( memory ) and pramana ( comprehension ) . inferable functions ( aparidrishta ) consist of seven subfunctions namely self - discipline ( nirodha ) , righteousness ( dharma ) , culture ( sanskara ) , effectiveness ( parinama ) , inclination for sustenance ( jeevana ) difficulty ( klishta ) and strength ( shakti ) . these can be understood by inference or by reading treatises . one is memory based knowledge ( smriti ) and another is experience based knowledge ( anubhava ) . memory ( smriti ) is defined as recollection of seen , heard and experienced things and develops due to eight reasons : perception of cause ( nimitta grahanat ) , visual perception ( roopa grahanat ) , similarity ( sadrashya ) , contrast ( viparyaya ) , mind getting in contact with objects ( satvanubandha ) , practice ( abhyasa ) , constant thinking ( jnana yoga ) , repetitive listening ( punah shruta ) . experience based knowledge ( anubhava ) is of two types , true experience ( yathartha ) and false experience ( ayathartha ) . true experience ( yatartha anubhava ) is again of four types - as told by reliable / authoritative people ( aptopadesha ) , one 's own sensory perception ( pratyaksha ) , inference ( anumana ) , and comparison ( upamana ) . false experience ( ayatartha anubhava ) is of three types namely , doubting or suspicion ( samshaya ) , illusion ( viparyaya ) and hypothetical argument ( tarka ) . hallucination is an illusional ( viparyaya ) type of false experience ( ayatartha anubhava ) , i.e. experience based knowledge ( jnana ) according to ayurvedic explanations . charaka observed that rajas and tamas are two factors which affect the mind and they produce symptoms of mental disorder . while describing pathogenesis of a mental disorder ( comparable to obsessive compulsive disorder ) , charaka says the mind ( manas ) and intellect ( buddhi ) get clouded with increase of rajas and tamas . hridaya ( seat of manas ) is disturbed by an imbalance of doshas ( tridosha ) . the man who has become stupid and weak in intelligence tends to formulate incorrect judgments concerning true and false , good and harmful . charaka describes eight essential psychological factors that are negatively affected in various ways in all psychiatric disorders . they are mind ( manas ) , cognition ( buddhi ) , orientation and responsiveness ( samjna jnana ) , devotion ( bhakti ) , habits ( sheela ) , psychomotor activity ( chesta ) and conduct ( achara ) . over activation of rajas ( action ) or tamas ( inertia ) causes the mind to lose balance which in turn affects tridosha ( vata , pitta , kapha ) . when this balance is totally lost , the result is a mental disorder , or if it tilts in any one direction conditions such as anger , anxiety , and nervousness occur , which are normal emotions but exhibited in an intensive manner . the doshas as they accumulate as toxins have negative emotional components like vata expressed as fear , pitta as anger and kapha as attachment . vata dosha in particular has strong psychological implications because the mind is part of the field of vata . unmada covers a wide range of symptoms and is synonymous with insanity , mania , mental derangement , mental disorders etc . , charaka defined unmada or mental disorder as instability of mind ( manas ) , intellect ( buddhi ) , deranged perception of all sensory stimulus or loss of orientation of time , situation and place ( sanjna jnana ) , memory ( smriti ) , inclination ( bhakti ) , mannerism ( sheela ) , activities ( chesta ) and conduct ( achara ) . all these terms were applied to that disordered state of mind in which an individual lost his power of regulating his actions and conduct according to the rules of the society . as per modern classifications , the term covers all the psychotic disorders . while explaining the prodromal symptoms of unmada , svanah karnayoh is one symptom . indriyasthana ( section on signs of life and death ) , chapter i varna swareeyam indriyam , adhyaya deals with arishta lakshana . arishta lakshana are the entities , some related others unrelated to the patient , which forecast imminent death . these entities should to be examined by the physician to find out remaining span of life through direct perception ( pratyaksha ) , inference ( anumana ) , authoritative statements ( upadesha ) . these signs and many others not mentioned here - may be related to complexion , voice , smell , taste , touch , vision , auditory , olfactory , gustatory , tactile perceptions of the patient . these may be observed in patients suffering from any illness of tridosha origin , somatic or psychiatric . in charaka samhita , indriyasthana chapter iv , indriyaneekam indriyam adhyaya , charaka states that as these sensory organs transcend all the senses they should be examined directly by the treating physician . the perception pertaining to the sense organs which is abnormal and arisen without any apparent cause is indicative of death : the patient who sees the sky as solidified like earth or sees the earth like the sky , or both in contradiction attains death ( shloka/7)one who sees air moving in the sky but does not visualize the burning fire should be taken for lost ( shloka/8)if one sees a net in clear flowing or stagnant water where there is no net , his life span is ended ( shloka/9)while awake , if one visualizes ghosts and various demons or some other bizarre things , he is unable to live ( shloka/10)one who sees the normal colored fire as blue , lusterless , black or white dies on the seventh night ( shloka/11)if one sees rays of light without clouds , if one sees clouds in the sky though not there or if one sees lightning when there are no clouds , he attains death ( shloka/12)if one sees the sun or moon , like an earthen vessel covered in black cloth , he will not live long ( shloka/13)if a diseased person or healthy person visualizes solar eclipse or lunar eclipse though it is not there , it indicates death ( shloka/14)if one sees the sun during the night and moon during daytime , smoke rising without fire or lusterless fire in the night , he is going to die ( shloka/15)one who is about to die , sees bright objects as devoid of brilliance , or non - brilliant objects as brilliant and sees all objects devoid of their normal characteristics ( shloka/16)a person who is about to die , sees all objects as deformed , of different color , in different numbers without an apparent cause ( shloka/17).if a person sees nonvisible objects or does not see the visible ones , he dies soon ( shloka/18).one who hears sounds when there is none or does not perceive the real sound should be considered to be dead ( shloka/19)if one is not able to hear the fire burning sound when both ears are closed with fingers , then he should be considered to have very little life span left ( shloka/20)if one perceives good smell and bad smell in contrary , or does not perceive the smell entirely should be considered to have completed his life span ( shloka/21)if one does not perceive the taste even in the absence of any kind of inflammation in the mouth or perceives altered taste , he should be considered to die soon ( shloka/22)if one perceives touch sensations such as hot - cold , coarse - smooth , and soft - hard in an opposite way ( contrarily ) , he is about to die ( shloka/23)super sensory perceptions without any rigorous penance ( tapas ) or methodical yoga , indicate death ( shloka/24)if one perceives all senses correctly in spite of incapability of sense organs , it indicates death ( shloka/25)if a healthy person contrary to his intellect repeatedly perceives abnormal sensations though there is no object of stimulus , it indicates death ( shloka/26 ) . the patient who sees the sky as solidified like earth or sees the earth like the sky , or both in contradiction attains death ( shloka/7 ) one who sees air moving in the sky but does not visualize the burning fire should be taken for lost ( shloka/8 ) if one sees a net in clear flowing or stagnant water where there is no net , his life span is ended ( shloka/9 ) while awake , if one visualizes ghosts and various demons or some other bizarre things , he is unable to live ( shloka/10 ) one who sees the normal colored fire as blue , lusterless , black or white dies on the seventh night ( shloka/11 ) if one sees rays of light without clouds , if one sees clouds in the sky though not there or if one sees lightning when there are no clouds , he attains death ( shloka/12 ) if one sees the sun or moon , like an earthen vessel covered in black cloth , he will not live long ( shloka/13 ) if a diseased person or healthy person visualizes solar eclipse or lunar eclipse though it is not there , it indicates death ( shloka/14 ) if one sees the sun during the night and moon during daytime , smoke rising without fire or lusterless fire in the night , he is going to die ( shloka/15 ) one who is about to die , sees bright objects as devoid of brilliance , or non - brilliant objects as brilliant and sees all objects devoid of their normal characteristics ( shloka/16 ) a person who is about to die , sees all objects as deformed , of different color , in different numbers without an apparent cause ( shloka/17 ) . if a person sees nonvisible objects or does not see the visible ones , he dies soon ( shloka/18 ) . one who hears sounds when there is none or does not perceive the real sound should be considered to be dead ( shloka/19 ) if one is not able to hear the fire burning sound when both ears are closed with fingers , then he should be considered to have very little life span left ( shloka/20 ) if one perceives good smell and bad smell in contrary , or does not perceive the smell entirely should be considered to have completed his life span ( shloka/21 ) if one does not perceive the taste even in the absence of any kind of inflammation in the mouth or perceives altered taste , he should be considered to die soon ( shloka/22 ) if one perceives touch sensations such as hot - cold , coarse - smooth , and soft - hard in an opposite way ( contrarily ) , he is about to die ( shloka/23 ) super sensory perceptions without any rigorous penance ( tapas ) or methodical yoga , indicate death ( shloka/24 ) if one perceives all senses correctly in spite of incapability of sense organs , it indicates death ( shloka/25 ) if a healthy person contrary to his intellect repeatedly perceives abnormal sensations though there is no object of stimulus , it indicates death ( shloka/26 ) . chapter 28 entitled panchaendriyartha pratipattinam adhyaya of the sushruta samhita sutrasthana ( section of fundamentals ) deals with clinical aspects related to the five senses . a reference is made to abnormal sensations ( hallucinations ) , which when seen in persons indicate death : patient hears different noises which are actually not there , pertaining to divine worship , like verses of gods ( patha ) , singing ( geeta ) , noise of musical instruments ( vadya ) being played by celestial beings ( siddha , kinnara , gandharva , etc . ) . sometimes hears the noise of roaring of the sea , which is absent if hears the noise of forest while he is in the middle of the city or hears noises of the city while he is in a quiet place , if he starts loving noises which he hated earlier and gets angry with earlier loved noises ( shloka 4 - 6)perceives hot objects as cold ones and cold objects as hot ones . he feels his feet are covered with sand and mud even though they are perfectly clean . sometimes he feels that he is dirty and flies are attracted to him even though he has taken a bath and is clean ( shloka 7 - 10)if he perceives tastes opposite to what actually is eaten . sweet tastes like sour , etc . if he likes tastes which are not advisable to him or he can not perceive the taste at all ( shloka 11 - 12)if he perceives good smell as bad and dirty smell as good , if he can not perceive the smell of burning lamp or can not perceive any kind of smell at all even though he is not suffering from olfactory related diseases ( shloka 13 - 14)if the person sees stars sparkling in the sky during daytime , sees bright sun in the sky during the night and moon during daytime . even though , the sky is clear of clouds , if he sees lightning and rainbows he dies within 3 months ( shloka 15 - 16)if the person perceives heat / cold , time , season and direction as opposites to the real , if he sees everything covered in smoke without suffering from any eye ailmentsin the well - lit place if the person sees things as if submerged in water , if he sees the earth in eight lines , if the person is not able to see the stars namely the arundhati constellation and dhruv ( north star ) and different galaxies , one must understand his life span is overif a person is not able to see his own shadow , if he sees his normal limb as deformed or feels he has lost his limbs he will dieif the person sees the spirits of dogs , crows or eagles , if he sees celestial bodies ( yaksha , rakshasa , pishacha ) and horrifying things , he will definitely dieif a person sees the normal colored fire as peacock blue , lusterless , black or white , then death is certain . patient hears different noises which are actually not there , pertaining to divine worship , like verses of gods ( patha ) , singing ( geeta ) , noise of musical instruments ( vadya ) being played by celestial beings ( siddha , kinnara , gandharva , etc . ) . sometimes hears the noise of roaring of the sea , which is absent if hears the noise of forest while he is in the middle of the city or hears noises of the city while he is in a quiet place , if he starts loving noises which he hated earlier and gets angry with earlier loved noises ( shloka 4 - 6 ) perceives hot objects as cold ones and cold objects as hot ones . he feels his feet are covered with sand and mud even though they are perfectly clean . sometimes he feels that he is dirty and flies are attracted to him even though he has taken a bath and is clean ( shloka 7 - 10 ) if he perceives tastes opposite to what actually is eaten . sweet tastes like sour , etc . if he likes tastes which are not advisable to him or he can not perceive the taste at all ( shloka 11 - 12 ) if he perceives good smell as bad and dirty smell as good , if he can not perceive the smell of burning lamp or can not perceive any kind of smell at all even though he is not suffering from olfactory related diseases ( shloka 13 - 14 ) if the person sees stars sparkling in the sky during daytime , sees bright sun in the sky during the night and moon during daytime . even though , the sky is clear of clouds , if he sees lightning and rainbows he dies within 3 months ( shloka 15 - 16 ) if the person perceives heat / cold , time , season and direction as opposites to the real , if he sees everything covered in smoke without suffering from any eye ailments in the well - lit place if the person sees things as if submerged in water , if he sees the earth in eight lines , if the person is not able to see the stars namely the arundhati constellation and dhruv ( north star ) and different galaxies , one must understand his life span is over if a person is not able to see his own shadow , if he sees his normal limb as deformed or feels he has lost his limbs he will die if the person sees the spirits of dogs , crows or eagles , if he sees celestial bodies ( yaksha , rakshasa , pishacha ) and horrifying things , he will definitely die if a person sees the normal colored fire as peacock blue , lusterless , black or white , then death is certain . during ancient times , severe mental disorders could probably not be treated effectively hence death was thought to be inevitable . the above mentioned instances may not end in death today with effective diagnosis and management . what is important is that these false sensory perceptions were noted , studied and were given great importance as an indicator of prognosis of diseased condition in ayurveda .

the ancient indian system of medicine ayurveda is a compendium of various health related theories and practices and explained the abnormal state of mind , i.e. , psychopathology in various contexts . hallucinations were deemed abnormal . in ayurvedic classics , hallucinations were called false perceptions ( mithyajnana ) , illusions ( maya ) , infatuations ( moha ) , or confusion ( bhrama ) . hallucinations were not independent but a symptom of mental disorder ( manasa roga ) . hallucinations of different sensory organs were observed and explained . these symptoms could be observed in patients suffering from any illness of tridosha origin , organic disease or psychiatric disorder . false perceptions observed in patients were used as tools to understand the prognosis of the condition . this article may help provide preliminary insight and encourage interdisciplinary study toward understanding one of the main symptoms of schizophrenia .

cliched as this sounds , it is worth remembering this point , since systems models are typically developed for objectives quite different from those for standard pkpd models . prior knowledge the model is built on , understanding of the physiology , the biological scale of the problem , the source of the model parameters , etc . in the study of demin et al . , the main objectives of the effort were to understand the mechanism behind the two - phase bronchodilatory response of zileuton , a 5-lo inhibitor , and to compare the relative bronchodilatory efficacy of 5-lo inhibition and leukotriene receptor blockade . sufficient literature exists on the mechanism of action of 5-lo to formulate the system of equations ; however , accurate human in vivo parameter values are not known . therefore , the mathematical model structure , with parameters estimated from in vitro and ex vivo human and animal data , was considered sufficient for these objectives , in spite of their unknown uncertainty . thus , model calibration and evaluation of predictive capability were not performed . for a more quantitative objective ( e.g. , study design ) , more accurate parameter values would be required and consequently , a robust data set would be necessary . it is important that , along with stating the objective of the modeling exercise , the model evaluation procedure is also justified up - front . for many pkpd models , the structure is usually well accepted within the modeling community , and justification of the final model structure only requires support of the parsimony principle i.e . , that the model provides a sufficient goodness - of - fit of the data using the fewest number of parameters . selection of a systems model structure is usually based on different considerations on the basis of prior knowledge of the underlying mechanism only , available data and/or prior knowledge of the mechanism , or a desire to explore the ability of unknown mechanisms to describe data . prior knowledge of the underlying pharmacology is , therefore , critical to the justification of the model structure . simple representations of the model that capture the essence of the underlying pharmacology are critical to justify the model to reviewers , especially when the model is complex or when the model structure is being reported for the first time . another practice that greatly helps facilitate this interaction is if the full model code and all parameter values were made available to the reviewers and readers in a software - independent format ( e.g. , systems biology markup language ) while submitting the manuscript for review , as is indeed required by this journal . for cases where exploratory models are evaluated , the choice of alternative models should also be clearly justified , since the number of alternative models can be large . in many cases , the overall model is composed of individual subcomponents each describing different pharmacological interactions integrated either within the same scale or across multiple scales . the component submodels can be relatively simple ( e.g. , emax type models ) or more complex . standard model development criteria goodness - of - fit plots , visual predictive check , etc can be applied to the evaluation of simple models , as is done by demin et al . individual parameter sensitivity analyses can also be used to evaluate the impact of the uncertainty in the assumed parameter value on the emergent properties of the submodel . the final systems model is typically used in one or more of the following ways : ( i ) to explore experimentally untested input output response in a system ; ( ii ) to understand complex behavior of a system ; and ( iii ) to develop a quantitative understanding of all markers in a system . objectives ( i ) and ( ii ) are semi - quantitative , therefore , previously listed model evaluation steps are considered sufficient to qualify the model as fit for these purposes even if the predictions and observations do not entirely agree . in our publication , this was the case , the predicted maximum bronchodilation for zileuton was ~2025% , whereas the reported value is ~15% . no effort was made to further refine model parameters to ensure a more accurate prediction . if an accurate and unbiased description of the data is required , further refinement of model structure and parameter values may be required . if so , the choice of parameter to be reestimated and the modifications to the model structure should be justified . recently , hendriks initiated a discussion on some pitfalls in the evaluation of systems models by methods commonly applied to empirical pkpd models . the intent of this commentary is to contribute to this emerging debate by illustrating how a systems model of 5-lo inhibitor was evaluated , based on other recent systems pharmacology publications . empirical pkpd models are often confirmatory ; therefore , a precise unbiased description of data is necessary to achieve this . for example , to confirm dose adjustment in a subpopulation , an accurate and unbiased description of the variability in exposure is required . however , a systems model may be useful even while providing an inaccurate description of data because such an inaccurate description could provide insights into false assumptions regarding the underlying pharmacology . therefore , standard diagnostics ( parsimony , goodness - of - fit plots , objective function value , visual predictive check , etc ) should not be the sole criteria for evaluating systems models , even though correspondence between prediction and observation is necessary to engender confidence in the model . this dichotomy between subjective and objective criteria is at the root of the confusion in how to evaluate these models . in the author 's experience , a clear statement of the objectives of modeling exercise can go a long way in both identifying and helping to justify the evaluation procedure . if objective criteria such as the ones commonly used are not applicable , then what ? justification of a systems model on the basis of plausible structures and parameter values based on prior mechanistic knowledge and qualitative comparison of prediction vs. observations all constitute subjective criteria and should be acceptable for systems model justification . for example , in the study by demin et al . , the predicted bronchodilation for zileuton was 2025% vs. ~15% observed . however , the conclusions were still considered valid because of a weight - of - evidence approach : accurate prediction of biomarker and bronchodilation changes with montelukast , the known assumptions in the model , and the qualitative rather than quantitative objective of the effort . because of these limitations , it is prudent to often consider a systems model as a plausible and useful mathematical description of a system and not as the definitive description of the system . hopefully , further discussion of this topic will result in a more formal guidance for the modeling community . in the meanwhile , the set of points to consider in this commentary may help provide a starting point for obtaining convergence between authors and reviewers of systems models and help map out a way forward for authors and peer reviewers to reach a compromise in evaluating these models .

based on the author 's recent experience , there appears to be some confusion regarding the steps required to qualify a systems pharmacology model as adequate for the intended purpose . this manuscript outlines the model evaluation approach used in the author 's recent publication1 on the systems pharmacology of a 5-lipoxygenase inhibitor and is an attempt to generate discussion on this topic within the pharmacometrics and systems pharmacology community .

strokes and movement disorders are quite common diseases and come to the attention of all physicians , but most often neurologists , emergency physicians , and primary care providers . stroke - related ( vascular ) movement disorders , however , are relatively uncommon and represent a recognized complication of either ischemic or hemorrhagic strokes . although the prevalence of post - stroke movement disorders is unclear because of the lack of prospective studies , data from retrospective studies have estimated the overall prevalence to range from 1% to 4% of all strokes with both sexes affected equally.13 the spectrum of post - stroke movement disorders is broad and includes both hypo - and hyperkinetic syndromes . the latter often present as variable combinations of hyperkinetic movements ( dyskinesias ) and can be broadly classified into three main phenotypes : choreiform dyskinesias ( ballism , chorea , and athetosis ) , dystonia , and non - choreo - dystonic dyskinesias ( eg , tremor , asterixis , and myoclonus ) ( figure 1 ) . the prevalence of vascular dyskinesias remains uncertain . in the lausanne stroke registry from switzerland , 29 ( 1% ) of 2,500 patients admitted to the registry over 14 years developed hyperkinetic movement disorders with an estimated incidence of 0.08% per year however , the large loss to follow - up has likely underestimated the prevalence , particularly for delayed movement disorders.1 in another stroke registry from ecuador , 56 ( 3.7% ) of 1,500 patients admitted to the registry over 9 years developed post - stroke movement disorders within the first year after stroke : 50 patients ( 3.3% ) had hyperkinetic movements and 6 patients ( 0.4% ) parkinsonism.2 although data from these two studies suggest that vascular hyperkinetic movement disorders are likely to regress spontaneously , it remains unknown whether that decreases their prevalence over time as the incidence of delayed vascular dyskinesias is largely unidentified . this review summarizes the current knowledge about the complex pathophysiology , overlapping phenomenology , and treatment options for common post - stroke hyperkinetic movement disorders . we searched for related published literature via pubmed through june 2016 by using the search terms stroke , vascular , dyskinesia , movement disorders , basal ganglia circuit , cerebellar circuit , and motor control . stroke is broadly defined as an episode of neurological dysfunction caused either by a focal infarction of the central nervous system ( cns ) or by a nontraumatic intracerebral or subarachnoid hemorrhage.4 although acute focal neurological deficits ( eg , hemiparesis ) are the clinical hallmark of stroke presentation , nonfocal deficits ( eg , impaired consciousness ) or positive neurological phenomena ( eg , hemidyskinesia ) can occasionally be the initial manifestation of stroke resulting in diagnostic uncertainty and delayed treatment.46 movement disorders are a group of basal ganglia and/or cerebellar circuit disorders characterized by impaired ability to control or coordinate movements . they are usually classified first based on an accurate description of the abnormal movement ( phenomenology ) , and then subdivided according to the underlying cause ( etiology ) . the phenomenological classification divides movement disorders into the following types : 1 ) hypokinetic ( parkinsonian ) disorders , dominated by poverty ( hypokinesia ) or slowness ( bradykinesia ) of movement , 2 ) hyperkinetic ( dyskinetic ) disorders , characterized by excessive , abnormal involuntary movements , and 3 ) other movement disorder syndromes , can not easily be grouped under the previous two categories , such as ataxia and akathisia . focal if only one body region is involved , segmental if 2 adjacent body regions are affected , the etiological classification of movement disorders , on the other hand , subdivides them into the following types : 1 ) primary ( genetic or idiopathic ) disorders , without an identifiable secondary cause ; 2 ) secondary ( symptomatic ) disorders , due to a known acquired etiology such as vascular , toxic , or metabolic abnormalities ; and 3 ) psychogenic ( functional ) disorders , commonly due to conversion , somatic symptom , or factitious disorders.7 although dyskinesia is a broad term that can indicate any hyperkinesia , it is often employed in clinical practice to indicate mixed , unusual , or complex hyperkinetic movements , especially when these movements are challenging to describe ; nevertheless , a more specific phenomenological categorization should be used whenever possible . for example , variable mixtures of ballism , chorea , and athetosis are often seen simultaneously in the same patient and are commonly referred to as not infrequently , dystonia coexists with choreiform movements and the term choreo - dystonic dyskinesia is often used in this clinical setting . interestingly , the choreiform dyskinesias are considered variants within the same phenotypic spectrum , with ballism and athetosis representing its fastest and slowest motor phenotypes , respectively ( table 1).711 finally , the term dyskinesia is also used more specifically to indicate certain movement disorders such as levodopa - induced dyskinesia ( due to chronic exposure to levodopa in parkinson s disease patients ) and tardive dyskinesia ( due to prolonged exposure to dopamine - blocking agents).12,13 motor control is a complex process that is governed by a sophisticated motor circuitry involving both pyramidal ( cortical ) and extrapyramidal ( basal ganglionic and cerebellar ) circuits . motor commands are generated in the motor cortex , but basal ganglia and cerebellum closely refine these signals by acting as feedback loops to allow for smooth , accurate , coordinated movements . while the cortico - basal loop ( basal ganglia circuitry ) provides a tonic inhibitory output to the thalamus and thus the motor cortex , the output of the cortico - cerebellar loop ( cerebellar circuitry ) is tonically excitatory . glutamate is the major excitatory neurotransmitter and gamma - aminobutyric acid ( gaba ) is the major inhibitory neurotransmitter in this motor network ( figure 2).1418 despite the significant overlap in their functions , the basal ganglia are particularly important for gross motor skills , whereas the cerebellum is crucial for fine or complex motor skills ( eg , speech and writing ) in which accurate performance is necessary.1418 the basal ganglia are a large collection of subcortical nuclei , which play a central role in modulating motor cortical activity through the selection and execution of appropriate motor programs.16 almost all inputs to the basal ganglia circuitry arrive via the striatum ( caudate and putamen ) . the motor signals are then processed through the pallidum ( globus pallidus interna gpi and externa gpe ) , motor thalamus ( ventral anterior va and ventral lateral vl nuclei ) , and then back to the motor cortex . the normal function of this motor loop is influenced by two additional nuclei : substantia nigra and subthalamic nucleus , which regulate the activity of the striatum and pallidum , respectively.1620 while glutamate and gaba are the major excitatory and inhibitory neurotransmitters , respectively , dopamine and acetylcholine act as neuromodulators in this circuit that regulate striatal activity . dopamine is the transmitter of the nigrostriatal pathway , whereas acetylcholine is a major transmitter of striatal interneurons ( figure 3).21,22 the striatum is the largest component of the basal ganglia motor circuit , and the vast majority of its neurons are gabaergic medium - sized spiny neurons whose axons comprise the striatal output pathways . these neurons are divided into two subsets of approximately equal numbers and provide two projection systems that have opposite effects on movement , the direct and indirect pathways . the direct pathway ( go pathway ) originates from dopamine d1 receptor - expressing neurons and projects directly to the gpi . it is an excitatory pathway because its net effect is to disinhibit the thalamus and thus the motor cortex . the indirect pathway ( no - go pathway ) originates from d2 receptor - expressing neurons and projects to the gpe and subthalamic nucleus before terminating in the gpi . it is an inhibitory pathway because its net effect is to inhibit the thalamus and thus the motor cortex . dopamine has differential effects on these two pathways : it activates d1 receptors and facilitates the direct pathway , but it inhibits d2 receptors and suppresses the indirect pathway . therefore , the net effect of dopamine is to facilitate voluntary movements by promoting transient interruptions of the tonic inhibitory output of the basal ganglia via the simultaneous activation and suppression of the direct and indirect pathways , respectively.1620 the cerebellum consists of a cortex , white matter , and deep cerebellar nuclei . it acts as a sensorimotor information processor by receiving information from all parts of the nervous system and comparing the motor commands of the cortex with the proprioceptive information coming from joints and muscles . this enables the cerebellum to detect errors in muscle contractions during active movements and thus to contribute to motor accuracy and coordination.15 two cerebellar feedback loops are critical for this task , the dentato - rubro - olivary loop and the cortico - cerebellar loop . the dentato - rubro - olivary circuit ( guillain - mollaret triangle ) connects the dentate nucleus in the cerebellum with the contralateral red nucleus and inferior olivary nucleus in the brainstem via the superior cerebellar peduncle , the central tegmental tract , and the inferior cerebellar peduncle , respectively.23 this subcortical circuit is itself part of the larger cerebellar motor network , the cortico - cerebellar circuitry , in which the cerebral and cerebellar cortices are connected together indirectly . the motor cortex projects to the cerebellar cortex ( via pontine nuclei ) which , in turn , projects primarily to the vl nuclei of thalamus and then back to the cerebral cortex ( figure 4).14,15,23 as the inferior olivary nucleus receives collateral inputs from all afferent pathways projecting to the cerebellar cortex via mossy fibers , it compares intended with executed movements and conveys error signals to the cerebellar cortex via climbing fibers.14,15,24 the pathogenesis of stroke - related dyskinesias is still incompletely understood but suggested mechanisms include post - synaptic denervation hypersensitivity , trans - synaptic neuronal degeneration , as well as aberrant axonal and dendritic plasticity ( remodeling ) after the cerebrovascular injury.3 post - stroke dyskinesias are relatively rare even with marked lesions , but they can arise after any stroke subtype at any level within the motor circuitry and after any interval period . furthermore , no specific anatomical locations in the motor circuitry are reliably predictive of a particular dyskinesia , whereas the same dyskinesia can be caused by lesions in different locations in this circuitry . due to this overlap , stroke - related movement disorders can not be predicted from the location , size , or number of vascular insults.1,2,19 interestingly , though the pathogenesis of vascular and primary movement disorders is different , they share similar underlying pathophysiology . if symptomatic , vascular lesions involving the basal ganglia circuitry usually present with contralateral abnormal involuntary movements , but there have been rare case reports of ipsilateral dyskinesias.25 the most commonly involved areas in this regard are the striatum followed by the thalamus , but other reported locations include the fronto - parietal cortex , caudate , subthalamic nucleus , corona radiata , internal capsule , and pons.13,19 these vascular dyskinesias are believed to arise from underactivity of the indirect pathway ( and/or overactivity of the direct pathway ) leading to a decreased pallidal inhibitory output to the thalamus . the resultant thalamic disinhibition releases the motor cortex and allows movements that are normally suppressed.2,19,26,27 vascular parkinsonism , on the other hand , is usually caused by diffuse or multiple insults in the basal ganglia loops and presents with non - tremulous , lower - body parkinsonism ( ie , predominantly affecting the legs).2,19,28 it is believed to arise from overactivity of the indirect pathway ( and/or underactivity of the direct pathway ) leading to an increased pallidal inhibitory output to the thalamus . the resultant thalamic inhibition suppresses the motor cortex and leads to hypokinetic movements.3,23,29 the cortico - cerebellar and dentato - rubro - olivary circuits are critically involved in tremor genesis.30 insults to the various structures of this network can give rise to cerebellar tremor , palatal tremor ( previously known as palatal myoclonus ) , or holmes tremor ( also known as thalamic , rubral , midbrain , or cerebral outflow tremor ) . the latter occurs when the lesion involves both the nigrostriatal and cerebellar pathways resulting in resting and action tremor , respectively.3133 dystonic and parkinsonian tremors are special forms of tremor that occur in association with vascular dystonia and parkinsonism , respectively . acute focal , segmental , or hemibody dyskinesia should always raise a suspicion of structural lesions involving the motor circuitry.11,33 although stroke is the most common cause after the age of 50 years , other structural pathologies have also been reported , including tumors , arteriovenous malformation , cerebral abscess , encephalitis , and multiple sclerosis.11,3336 non - structural causes , especially hypoglycemia and nonketotic hyperglycemia , can occasionally present with dyskinesia.3741 interestingly , hemichorea - hemiballism is the most commonly reported dyskinesia arising secondary to either structural ( eg , vascular ) or non - structural ( eg , dysglycemic ) etiologies.34,36,38,41 a careful history ( including medical , drug , and family history ) and identifying the dominant phenomenology remain the first step in the clinical assessment , as that steers the differential diagnosis and the subsequent diagnostic trajectory . neuroimaging studies , such as computed tomography ( ct ) or magnetic resonance imaging ( mri ) , are important to confirm the diagnosis of stroke or to evaluate for structural stroke mimics . post - stroke dyskinesias often present as variable combinations of hyperkinetic movements and can sometimes be challenging to classify.1,19 nevertheless , identification of the dominant abnormal movement , whenever possible , allows for a more specific phenomenological categorization . furthermore , the relative predominance of one type of phenomenology might be state dependent ( eg , a resting athetosis might evolve into ballism with voluntary movements or when the patient is stimulated or excited ) . hemichorea - hemiballism is the most common vascular dyskinesia , but other reported mixed movements include chorea - ballism - dystonia , chorea - athetosis - dystonia , and dystonia - athetosis with or without action tremor or myoclonic jerks.1,11,33,4244 tremor , asterixis , and myoclonus are generally far less common than choreo - dystonic dyskinesias.1 post - stroke chorea usually presents as a hemichoreiform dyskinesia ( hemichorea , hemiballism , and/or hemiathetosis ) on the contralateral side of the vascular insult . the concomitant hemiparesis might initially prevent the expression of hemichorea , but improvement of the weakness unmasks this dyskinesia.1,2 hemichorea can occasionally be the presenting sign of acute stroke , though careful examination often reveals an underlying mild hemiparesis with or without other subtle deficits.45,46 post - stroke dystonia usually affects the contralateral limb , and it is often associated with hypertonia due to underlying spasticity ( spastic dystonia).2,3,33 interestingly , dystonia in combination with spasticity is usually intensified by muscle activation in the affected limb or elsewhere ( overflow phenomenon ) . cervical dystonia ( eg , torticollis ) and cranial dystonias ( eg , blepherospasm or oromandibular dystonia ) are rarely observed in association with cerebrovascular insults.4749 post - stroke tremor is a heterogeneous group of movement disorders characterized by various tremor phenomenologies , but isolated tremor unaccompanied by other abnormal movements is rare . action tremor is by far the most common form , but rest tremor can occasionally be seen ( eg , in vascular parkinsonism ) . cerebellar tremor , the prototype vascular tremor , is primarily a slow intention tremor ( < 5 hz ) with frequent postural component.50 holmes tremor is usually a low - frequency tremor ( < 5 hz ) with rest , postural , and intention components in the affected upper extremity.32 palatal tremor is a slow tremor of the soft palate ( < 5 hz ) in which patients may complain of a disturbing clicking sound generated by palatal muscle contractions causing opening and closing of the eustachian tube.31 dystonic tremor is usually a focal postural and/or kinetic tremor that occurs in association with dystonia , which might be subtle or overshadowed by the tremor . despite being arrhythmic with variable frequencies , dystonic oscillations are traditionally referred to as tremor.50 post - stroke myoclonus and asterixis are usually focal or segmental with corresponding lesions reported in numerous , mostly contralateral , brain regions . myoclonus is not an uncommon component of mixed vascular movement disorders , but isolated myoclonus is rare . dystonic myoclonus ( myoclonic dystonia ) has been described in patients with vascular dystonia secondary to thalamic infarcts.2,3,19 the natural history of post - stroke dyskinesias is variable . early / acute , occurring shortly after stroke , or delayed / chronic , emerging months to years later . on the other hand , their course can be transient , recurrent , persistent , or progressive.1,43,44 although the latency interval seems to depend partially on the dyskinesia type , it still varies widely within each vascular hyperkinetic movement disorder.2,3 while dystonia is frequently delayed in onset , hemichorea - hemiballism usually occurs shortly after stroke and occasionally represents the initial manifestation of acute cerebral ischemia or intracerebral hemorrhage.2,33,45,51 post - stroke dyskinesias are usually self - limited and resolve within 6 to 12 months of onset , but the overall long - term prognosis of the affected patients is similar to that of other stroke patients.1,2,46,52 although post - stroke dyskinesias tend to resolve spontaneously , a short - term treatment might sometimes be required for symptom control.1,2 as in all cases of secondary movement disorders , treatment of the underlying etiology is of paramount importance . the discussion of stroke prevention and management , however , is beyond the scope of this article . symptomatic pharmacotherapy might be necessary for severe dyskinesias , but periodic trials of therapeutic withdrawal ( ie , for patients with controlled symptoms ) are required due to the high likelihood of spontaneous regression . medications should be started at low doses and gradually titrated up until an effective and tolerable dosage is reached . although there are no established treatment guidelines , most of the treatment options are similar to those for primary movement disorders based on similar underlying pathophysiology . the symptomatic pharmacotherapy for post - stroke choreiform dyskinesias consists mainly of anti - dopaminergic therapy with typical or atypical antipsychotics ( neuroleptics ) . blockade of striatal dopamine d2 receptors is believed to be responsible for their anti - dyskinetic activity , as d2 receptor antagonists disinhibit the indirect pathway and , therefore , suppress abnormal involuntary movements . unfortunately , dopaminergic blockade carries the risk of acute dystonic reactions , tardive dyskinesia , and drug - induced parkinsonism . atypical antipsychotics ( eg , risperidone ) , however , are less likely to cause these side effects compared to typical antipsychotics ( eg , haloperidol ) and , therefore , are generally preferred for this use.53,54 tetrabenazine , a presynaptic dopamine depletor with weak postsynaptic d2 receptor blocking activity , is a reasonable alternative for patients who are intolerant or unresponsive to dopamine receptor antagonists.55 tetrabenazine acts primarily as a reversible inhibitor of the vesicular monoamine transporter 2 ( vmat2 ) in the presynaptic nerve terminals , thus exposing dopamine and other monoamines to monoamine oxidase and leading to their depletion.56 unlike dopamine receptor blockers , tetrabenazine rarely causes tardive dyskinesia because of its dominant presynaptic anti - dopaminergic properties.3,57 non - dopaminergic drugs have been tried in the management of vascular choreiform dyskinesias with varying success . case reports and small case series have suggested a potential beneficial effect of antiepileptic drugs including levetiracetam , topiramate , gabapentin , clonazepam , and valproate.5862 chemodenervation with botulinum toxin ( btx ) injections is the cornerstone of the symptomatic treatment of focal or segmental post - stroke dystonia.3 btx is a neurotoxic protein with several serotypes that cleaves the synaptic proteins ( snare ) in the presynaptic nerve terminals , thereby blocking the release of acetylcholine at the neuromuscular junction . snare proteins are required for the fusion of presynaptic storage vesicles containing acetylcholine with the presynaptic membrane.63 btx injections are given intramuscularly , often under electromyography ( emg ) guidance , and need to be repeated every 3 to 6 months.64 vascular dystonia usually has a poor response to oral pharmacotherapy , including dopamine blocking and depleting agents , anticholinergic drugs , baclofen , and benzodiazepines . oral medications , however , are widely used in generalized dystonia , dystonia mixed with other movement disorders , or as an adjuvant therapy in focal or segmental dystonia when there is an unsatisfactory response to btx.6467 post - stroke tremor is particularly refractory to pharmacotherapy . trials of medications with gaba - agonistic activity ( eg , clonazepam , valproate , topiramate , or primidone ) , alone or in combination , may be effective in individual cases . because of the dopaminergic ( nigrostriatal ) system involvement in holmes tremor , treatment with levodopa or dopamine agonists seems to be useful.31 propranolol , one of the first - line treatments in essential tremor , is usually of limited benefit in vascular tremor . dystonic and parkinsonian tremors are treated as vascular dystonia and parkinsonism , respectively.3,19 post - stroke myoclonus and asterixis usually improve spontaneously and do not require pharmacotherapy . when interfering with the patient s functional abilities , like eating or writing , myoclonus is most frequently treated with gabaergic medications ( eg , clonazepam and valproate ) , but levetiracetam or piracetam can be very useful . monotherapy should be attempted first , although eventually several drug combinations might be required.3,19,53 dystonic myoclonus is treated as vascular dystonia . finally , stereotactic functional neurosurgery , whether ablative or deep brain stimulation ( dbs ) , should be considered for patients with severe and persistent dyskinesias ( arbitrarily defined as duration longer than 1 year).3,19,53 while lesioning procedures ( eg , pallidotomy or thalamotomy ) are carried out using radiofrequency ablation or gamma knife radiosurgery , dbs ( eg , pallidal or thalamic dbs ) uses high - frequency electrical stimulation of the targeted nuclei . although the therapeutic mechanisms of action are not completely understood , ablative surgeries are believed to destroy abnormally hyperactive circuits in deep brain nuclei , while dbs suppresses abnormally excessive activity in the motor circuitry without significant destruction of the brain tissue.68,69 neurostimulation , therefore , has largely replaced neuroablation to treat refractory dyskinesias because it is less invasive , adjustable for maximal symptomatic benefit , and reversible in case of adverse effects . stereotactic functional neurosurgery depends on the accurate identification of anatomically and functionally distinct deep brain structures to maximize therapeutic benefits and to minimize adverse neurological events of any surgical intervention . the contralateral motor thalamus or internal pallidum are the usual neurosurgical targets , but it is yet to be determined which target is more effective for specific dyskinesias.53,70 the ventrolateral nuclei , including ventralis intermedius and ventralis oralis posterior , are the most common nuclei targeted within the motor thalamus.71 potential candidates with vascular dystonia should undergo surgery prior to the development of contractures and fixed deformities that may limit functional improvement as the dystonia improves.66 stroke is broadly defined as an episode of neurological dysfunction caused either by a focal infarction of the central nervous system ( cns ) or by a nontraumatic intracerebral or subarachnoid hemorrhage.4 although acute focal neurological deficits ( eg , hemiparesis ) are the clinical hallmark of stroke presentation , nonfocal deficits ( eg , impaired consciousness ) or positive neurological phenomena ( eg , hemidyskinesia ) can occasionally be the initial manifestation of stroke resulting in diagnostic uncertainty and delayed treatment.46 movement disorders are a group of basal ganglia and/or cerebellar circuit disorders characterized by impaired ability to control or coordinate movements . they are usually classified first based on an accurate description of the abnormal movement ( phenomenology ) , and then subdivided according to the underlying cause ( etiology ) . the phenomenological classification divides movement disorders into the following types : 1 ) hypokinetic ( parkinsonian ) disorders , dominated by poverty ( hypokinesia ) or slowness ( bradykinesia ) of movement , 2 ) hyperkinetic ( dyskinetic ) disorders , characterized by excessive , abnormal involuntary movements , and 3 ) other movement disorder syndromes , can not easily be grouped under the previous two categories , such as ataxia and akathisia . focal if only one body region is involved , segmental if 2 adjacent body regions are affected , the etiological classification of movement disorders , on the other hand , subdivides them into the following types : 1 ) primary ( genetic or idiopathic ) disorders , without an identifiable secondary cause ; 2 ) secondary ( symptomatic ) disorders , due to a known acquired etiology such as vascular , toxic , or metabolic abnormalities ; and 3 ) psychogenic ( functional ) disorders , commonly due to conversion , somatic symptom , or factitious disorders.7 although dyskinesia is a broad term that can indicate any hyperkinesia , it is often employed in clinical practice to indicate mixed , unusual , or complex hyperkinetic movements , especially when these movements are challenging to describe ; for example , variable mixtures of ballism , chorea , and athetosis are often seen simultaneously in the same patient and are commonly referred to as not infrequently , dystonia coexists with choreiform movements and the term choreo - dystonic dyskinesia is often used in this clinical setting . interestingly , the choreiform dyskinesias are considered variants within the same phenotypic spectrum , with ballism and athetosis representing its fastest and slowest motor phenotypes , respectively ( table 1).711 finally , the term dyskinesia is also used more specifically to indicate certain movement disorders such as levodopa - induced dyskinesia ( due to chronic exposure to levodopa in parkinson s disease patients ) and tardive dyskinesia ( due to prolonged exposure to dopamine - blocking agents).12,13 motor control is a complex process that is governed by a sophisticated motor circuitry involving both pyramidal ( cortical ) and extrapyramidal ( basal ganglionic and cerebellar ) circuits . motor commands are generated in the motor cortex , but basal ganglia and cerebellum closely refine these signals by acting as feedback loops to allow for smooth , accurate , coordinated movements . while the cortico - basal loop ( basal ganglia circuitry ) provides a tonic inhibitory output to the thalamus and thus the motor cortex , the output of the cortico - cerebellar loop ( cerebellar circuitry ) is tonically excitatory . glutamate is the major excitatory neurotransmitter and gamma - aminobutyric acid ( gaba ) is the major inhibitory neurotransmitter in this motor network ( figure 2).1418 despite the significant overlap in their functions , the basal ganglia are particularly important for gross motor skills , whereas the cerebellum is crucial for fine or complex motor skills ( eg , speech and writing ) in which accurate performance is necessary.1418 the basal ganglia are a large collection of subcortical nuclei , which play a central role in modulating motor cortical activity through the selection and execution of appropriate motor programs.16 almost all inputs to the basal ganglia circuitry arrive via the striatum ( caudate and putamen ) . the motor signals are then processed through the pallidum ( globus pallidus interna gpi and externa gpe ) , motor thalamus ( ventral anterior va and ventral lateral . the normal function of this motor loop is influenced by two additional nuclei : substantia nigra and subthalamic nucleus , which regulate the activity of the striatum and pallidum , respectively.1620 while glutamate and gaba are the major excitatory and inhibitory neurotransmitters , respectively , dopamine and acetylcholine act as neuromodulators in this circuit that regulate striatal activity . dopamine is the transmitter of the nigrostriatal pathway , whereas acetylcholine is a major transmitter of striatal interneurons ( figure 3).21,22 the striatum is the largest component of the basal ganglia motor circuit , and the vast majority of its neurons are gabaergic medium - sized spiny neurons whose axons comprise the striatal output pathways . these neurons are divided into two subsets of approximately equal numbers and provide two projection systems that have opposite effects on movement , the direct and indirect pathways . the direct pathway ( go pathway ) originates from dopamine d1 receptor - expressing neurons and projects directly to the gpi . it is an excitatory pathway because its net effect is to disinhibit the thalamus and thus the motor cortex . the indirect pathway ( no - go pathway ) originates from d2 receptor - expressing neurons and projects to the gpe and subthalamic nucleus before terminating in the gpi . it is an inhibitory pathway because its net effect is to inhibit the thalamus and thus the motor cortex . dopamine has differential effects on these two pathways : it activates d1 receptors and facilitates the direct pathway , but it inhibits d2 receptors and suppresses the indirect pathway . therefore , the net effect of dopamine is to facilitate voluntary movements by promoting transient interruptions of the tonic inhibitory output of the basal ganglia via the simultaneous activation and suppression of the direct and indirect pathways , respectively.1620 the cerebellum consists of a cortex , white matter , and deep cerebellar nuclei . it acts as a sensorimotor information processor by receiving information from all parts of the nervous system and comparing the motor commands of the cortex with the proprioceptive information coming from joints and muscles . this enables the cerebellum to detect errors in muscle contractions during active movements and thus to contribute to motor accuracy and coordination.15 two cerebellar feedback loops are critical for this task , the dentato - rubro - olivary loop and the cortico - cerebellar loop . the dentato - rubro - olivary circuit ( guillain - mollaret triangle ) connects the dentate nucleus in the cerebellum with the contralateral red nucleus and inferior olivary nucleus in the brainstem via the superior cerebellar peduncle , the central tegmental tract , and the inferior cerebellar peduncle , respectively.23 this subcortical circuit is itself part of the larger cerebellar motor network , the cortico - cerebellar circuitry , in which the cerebral and cerebellar cortices are connected together indirectly . the motor cortex projects to the cerebellar cortex ( via pontine nuclei ) which , in turn , projects primarily to the vl nuclei of thalamus and then back to the cerebral cortex ( figure 4).14,15,23 as the inferior olivary nucleus receives collateral inputs from all afferent pathways projecting to the cerebellar cortex via mossy fibers , it compares intended with executed movements and conveys error signals to the cerebellar cortex via climbing fibers.14,15,24 the pathogenesis of stroke - related dyskinesias is still incompletely understood but suggested mechanisms include post - synaptic denervation hypersensitivity , trans - synaptic neuronal degeneration , as well as aberrant axonal and dendritic plasticity ( remodeling ) after the cerebrovascular injury.3 post - stroke dyskinesias are relatively rare even with marked lesions , but they can arise after any stroke subtype at any level within the motor circuitry and after any interval period . furthermore , no specific anatomical locations in the motor circuitry are reliably predictive of a particular dyskinesia , whereas the same dyskinesia can be caused by lesions in different locations in this circuitry . due to this overlap , stroke - related movement disorders can not be predicted from the location , size , or number of vascular insults.1,2,19 interestingly , though the pathogenesis of vascular and primary movement disorders is different , they share similar underlying pathophysiology . if symptomatic , vascular lesions involving the basal ganglia circuitry usually present with contralateral abnormal involuntary movements , but there have been rare case reports of ipsilateral dyskinesias.25 the most commonly involved areas in this regard are the striatum followed by the thalamus , but other reported locations include the fronto - parietal cortex , caudate , subthalamic nucleus , corona radiata , internal capsule , and pons.13,19 these vascular dyskinesias are believed to arise from underactivity of the indirect pathway ( and/or overactivity of the direct pathway ) leading to a decreased pallidal inhibitory output to the thalamus . the resultant thalamic disinhibition releases the motor cortex and allows movements that are normally suppressed.2,19,26,27 vascular parkinsonism , on the other hand , is usually caused by diffuse or multiple insults in the basal ganglia loops and presents with non - tremulous , lower - body parkinsonism ( ie , predominantly affecting the legs).2,19,28 it is believed to arise from overactivity of the indirect pathway ( and/or underactivity of the direct pathway ) leading to an increased pallidal inhibitory output to the thalamus . the resultant thalamic inhibition suppresses the motor cortex and leads to hypokinetic movements.3,23,29 the cortico - cerebellar and dentato - rubro - olivary circuits are critically involved in tremor genesis.30 insults to the various structures of this network can give rise to cerebellar tremor , palatal tremor ( previously known as palatal myoclonus ) , or holmes tremor ( also known as thalamic , rubral , midbrain , or cerebral outflow tremor ) . the latter occurs when the lesion involves both the nigrostriatal and cerebellar pathways resulting in resting and action tremor , respectively.3133 dystonic and parkinsonian tremors are special forms of tremor that occur in association with vascular dystonia and parkinsonism , respectively . acute focal , segmental , or hemibody dyskinesia should always raise a suspicion of structural lesions involving the motor circuitry.11,33 although stroke is the most common cause after the age of 50 years , other structural pathologies have also been reported , including tumors , arteriovenous malformation , cerebral abscess , encephalitis , and multiple sclerosis.11,3336 non - structural causes , especially hypoglycemia and nonketotic hyperglycemia , can occasionally present with dyskinesia.3741 interestingly , hemichorea - hemiballism is the most commonly reported dyskinesia arising secondary to either structural ( eg , vascular ) or non - structural ( eg , dysglycemic ) etiologies.34,36,38,41 a careful history ( including medical , drug , and family history ) and identifying the dominant phenomenology remain the first step in the clinical assessment , as that steers the differential diagnosis and the subsequent diagnostic trajectory . neuroimaging studies , such as computed tomography ( ct ) or magnetic resonance imaging ( mri ) , are important to confirm the diagnosis of stroke or to evaluate for structural stroke mimics . post - stroke dyskinesias often present as variable combinations of hyperkinetic movements and can sometimes be challenging to classify.1,19 nevertheless , identification of the dominant abnormal movement , whenever possible , allows for a more specific phenomenological categorization . furthermore , the relative predominance of one type of phenomenology might be state dependent ( eg , a resting athetosis might evolve into ballism with voluntary movements or when the patient is stimulated or excited ) . hemichorea - hemiballism is the most common vascular dyskinesia , but other reported mixed movements include chorea - ballism - dystonia , chorea - athetosis - dystonia , and dystonia - athetosis with or without action tremor or myoclonic jerks.1,11,33,4244 tremor , asterixis , and myoclonus are generally far less common than choreo - dystonic dyskinesias.1 post - stroke chorea usually presents as a hemichoreiform dyskinesia ( hemichorea , hemiballism , and/or hemiathetosis ) on the contralateral side of the vascular insult . the concomitant hemiparesis might initially prevent the expression of hemichorea , but improvement of the weakness unmasks this dyskinesia.1,2 hemichorea can occasionally be the presenting sign of acute stroke , though careful examination often reveals an underlying mild hemiparesis with or without other subtle deficits.45,46 post - stroke dystonia usually affects the contralateral limb , and it is often associated with hypertonia due to underlying spasticity ( spastic dystonia).2,3,33 interestingly , dystonia in combination with spasticity is usually intensified by muscle activation in the affected limb or elsewhere ( overflow phenomenon ) . cervical dystonia ( eg , torticollis ) and cranial dystonias ( eg , blepherospasm or oromandibular dystonia ) are rarely observed in association with cerebrovascular insults.4749 post - stroke tremor is a heterogeneous group of movement disorders characterized by various tremor phenomenologies , but isolated tremor unaccompanied by other abnormal movements is rare . action tremor is by far the most common form , but rest tremor can occasionally be seen ( eg , in vascular parkinsonism ) . cerebellar tremor , the prototype vascular tremor , is primarily a slow intention tremor ( < 5 hz ) with frequent postural component.50 holmes tremor is usually a low - frequency tremor ( < 5 hz ) with rest , postural , and intention components in the affected upper extremity.32 palatal tremor is a slow tremor of the soft palate ( < 5 hz ) in which patients may complain of a disturbing clicking sound generated by palatal muscle contractions causing opening and closing of the eustachian tube.31 dystonic tremor is usually a focal postural and/or kinetic tremor that occurs in association with dystonia , which might be subtle or overshadowed by the tremor . despite being arrhythmic with variable frequencies , dystonic oscillations are traditionally referred to as tremor.50 post - stroke myoclonus and asterixis are usually focal or segmental with corresponding lesions reported in numerous , mostly contralateral , brain regions . myoclonus is not an uncommon component of mixed vascular movement disorders , but isolated myoclonus is rare . dystonic myoclonus ( myoclonic dystonia ) has been described in patients with vascular dystonia secondary to thalamic infarcts.2,3,19 early / acute , occurring shortly after stroke , or delayed / chronic , emerging months to years later . on the other hand , their course can be transient , recurrent , persistent , or progressive.1,43,44 although the latency interval seems to depend partially on the dyskinesia type , it still varies widely within each vascular hyperkinetic movement disorder.2,3 while dystonia is frequently delayed in onset , hemichorea - hemiballism usually occurs shortly after stroke and occasionally represents the initial manifestation of acute cerebral ischemia or intracerebral hemorrhage.2,33,45,51 post - stroke dyskinesias are usually self - limited and resolve within 6 to 12 months of onset , but the overall long - term prognosis of the affected patients is similar to that of other stroke patients.1,2,46,52 although post - stroke dyskinesias tend to resolve spontaneously , a short - term treatment might sometimes be required for symptom control.1,2 as in all cases of secondary movement disorders , treatment of the underlying etiology is of paramount importance . the discussion of stroke prevention and management , however , is beyond the scope of this article . symptomatic pharmacotherapy might be necessary for severe dyskinesias , but periodic trials of therapeutic withdrawal ( ie , for patients with controlled symptoms ) are required due to the high likelihood of spontaneous regression . medications should be started at low doses and gradually titrated up until an effective and tolerable dosage is reached . although there are no established treatment guidelines , most of the treatment options are similar to those for primary movement disorders based on similar underlying pathophysiology . the symptomatic pharmacotherapy for post - stroke choreiform dyskinesias consists mainly of anti - dopaminergic therapy with typical or atypical antipsychotics ( neuroleptics ) . blockade of striatal dopamine d2 receptors is believed to be responsible for their anti - dyskinetic activity , as d2 receptor antagonists disinhibit the indirect pathway and , therefore , suppress abnormal involuntary movements . unfortunately , dopaminergic blockade carries the risk of acute dystonic reactions , tardive dyskinesia , and drug - induced parkinsonism . atypical antipsychotics ( eg , risperidone ) , however , are less likely to cause these side effects compared to typical antipsychotics ( eg , haloperidol ) and , therefore , are generally preferred for this use.53,54 tetrabenazine , a presynaptic dopamine depletor with weak postsynaptic d2 receptor blocking activity , is a reasonable alternative for patients who are intolerant or unresponsive to dopamine receptor antagonists.55 tetrabenazine acts primarily as a reversible inhibitor of the vesicular monoamine transporter 2 ( vmat2 ) in the presynaptic nerve terminals , thus exposing dopamine and other monoamines to monoamine oxidase and leading to their depletion.56 unlike dopamine receptor blockers , tetrabenazine rarely causes tardive dyskinesia because of its dominant presynaptic anti - dopaminergic properties.3,57 non - dopaminergic drugs have been tried in the management of vascular choreiform dyskinesias with varying success . case reports and small case series have suggested a potential beneficial effect of antiepileptic drugs including levetiracetam , topiramate , gabapentin , clonazepam , and valproate.5862 chemodenervation with botulinum toxin ( btx ) injections is the cornerstone of the symptomatic treatment of focal or segmental post - stroke dystonia.3 btx is a neurotoxic protein with several serotypes that cleaves the synaptic proteins ( snare ) in the presynaptic nerve terminals , thereby blocking the release of acetylcholine at the neuromuscular junction . snare proteins are required for the fusion of presynaptic storage vesicles containing acetylcholine with the presynaptic membrane.63 btx injections are given intramuscularly , often under electromyography ( emg ) guidance , and need to be repeated every 3 to 6 months.64 vascular dystonia usually has a poor response to oral pharmacotherapy , including dopamine blocking and depleting agents , anticholinergic drugs , baclofen , and benzodiazepines . oral medications , however , are widely used in generalized dystonia , dystonia mixed with other movement disorders , or as an adjuvant therapy in focal or segmental dystonia when there is an unsatisfactory response to btx.6467 post - stroke tremor is particularly refractory to pharmacotherapy . trials of medications with gaba - agonistic activity ( eg , clonazepam , valproate , topiramate , or primidone ) , alone or in combination , may be effective in individual cases . because of the dopaminergic ( nigrostriatal ) system involvement in holmes tremor , treatment with levodopa or dopamine agonists seems to be useful.31 propranolol , one of the first - line treatments in essential tremor , is usually of limited benefit in vascular tremor . dystonic and parkinsonian tremors are treated as vascular dystonia and parkinsonism , respectively.3,19 post - stroke myoclonus and asterixis usually improve spontaneously and do not require pharmacotherapy . when interfering with the patient s functional abilities , like eating or writing , myoclonus is most frequently treated with gabaergic medications ( eg , clonazepam and valproate ) , but levetiracetam or piracetam can be very useful . monotherapy should be attempted first , although eventually several drug combinations might be required.3,19,53 dystonic myoclonus is treated as vascular dystonia . finally , stereotactic functional neurosurgery , whether ablative or deep brain stimulation ( dbs ) , should be considered for patients with severe and persistent dyskinesias ( arbitrarily defined as duration longer than 1 year).3,19,53 while lesioning procedures ( eg , pallidotomy or thalamotomy ) are carried out using radiofrequency ablation or gamma knife radiosurgery , dbs ( eg , pallidal or thalamic dbs ) uses high - frequency electrical stimulation of the targeted nuclei . although the therapeutic mechanisms of action are not completely understood , ablative surgeries are believed to destroy abnormally hyperactive circuits in deep brain nuclei , while dbs suppresses abnormally excessive activity in the motor circuitry without significant destruction of the brain tissue.68,69 neurostimulation , therefore , has largely replaced neuroablation to treat refractory dyskinesias because it is less invasive , adjustable for maximal symptomatic benefit , and reversible in case of adverse effects . stereotactic functional neurosurgery depends on the accurate identification of anatomically and functionally distinct deep brain structures to maximize therapeutic benefits and to minimize adverse neurological events of any surgical intervention . the contralateral motor thalamus or internal pallidum are the usual neurosurgical targets , but it is yet to be determined which target is more effective for specific dyskinesias.53,70 the ventrolateral nuclei , including ventralis intermedius and ventralis oralis posterior , are the most common nuclei targeted within the motor thalamus.71 potential candidates with vascular dystonia should undergo surgery prior to the development of contractures and fixed deformities that may limit functional improvement as the dystonia improves.66 stroke is the leading cause of focal or segmental limb dyskinesia as well as hemidyskinesia in elderly patients , but other structural and non - structural brain abnormalities should also be considered in the differential diagnosis . post - stroke dyskinesias , which can arise from either ischemic or hemorrhagic cerebrovascular insults , are often mixed and variable with several components of hyperkinetic movements . fortunately , post - stroke dyskinesias tend to resolve spontaneously within 6 to 12 months . a short - term symptomatic pharmacotherapy may be required in some patients , whereas surgical treatment is reserved for persistent , disabling , and medically intractable cases .

strokes , whether ischemic or hemorrhagic , are among the most common causes of secondary movement disorders in elderly patients . stroke - related ( vascular ) movement disorders , however , are uncommon complications of this relatively common disease . the spectrum of post - stroke movement disorders is broad and includes both hypo- and hyperkinetic syndromes . post - stroke dyskinesias are involuntary hyperkinetic movements arising from cerebrovascular insults and often present with mixed phenotypes of hyperkinesia which can sometimes be difficult to classify . nevertheless , identification of the most relevant motor phenotype , whenever possible , allows for a more specific phenomenological categorization of the dyskinesia and thus helps guide its treatment . fortunately , post - stroke dyskinesias are usually self - limiting and resolve within 6 to 12 months of onset , but a short - term pharmacotherapy might sometimes be required for symptom control . functional neurosurgical interventions targeting the motor thalamus or globus pallidus interna might be considered for patients with severe , disabling , and persistent dyskinesias ( arbitrarily defined as duration longer than 12 months ) .

contrast - induced nephropathy ( cin ) has been recognized as a serious complication of percutaneous coronary intervention ( pci ) , and associated with increased short - term and long - term morbidity or mortality.1 - 4 ) although many risk factors associated with the development of cin have been reported,5 - 7 ) decreased renal function and increased systemic exposure of contrast media ( cm ) are considered the most potent risk factors of cin.8)9 ) considering the importance of the prevention of cin , it is important to determine the safe cm volume . accordingly , previous investigators have suggested a safe cm dose regarding renal function.10 - 13 ) the key concept of those suggestions was reducing or adjusting cm dosage according to individual renal function . therefore , the accurate estimation of renal function may be necessary to identify high - risk patients and to suggest a reliable pharmacotoxic model . the most accurate method to estimate renal function is measured glomerular filtration rate ( gfr ) using radioisotope or radioiodine . however , the measure is both time- and cost - consuming and comes with potential side effects . therefore , serum creatinine ( scr ) itself or estimated gfr ( egfr ) based on scr are the most widely used methods in the clinical assessment of kidney function.14 - 16 ) despite its validity , egfr based on scr has attracted criticism due to various confounding factors and its level of imprecision . recently , cystatin c ( cyc ) has received a lot of attention and appears to be a promising alternative to scr for estimating gfr.17)18 ) contrary to scr , the cyc level depends almost entirely on the gfr and is less dependent on age , diet , nutritional status , and muscle mass.19 - 22 ) moreover , it might be more useful to detect early stage renal dysfunction than scr.23 ) however , the efficacy of egfr based on cyc ( egfrcyc ) for the prediction of cin was not fully evaluated and there was no previous study regarding safe cm volume estimation using egfrcyc in patients with elective pci . thus , we conducted this prospective study to assess the usefulness of the cm volume / egfrcyc in predicting the risks of developing cin and to determine the safe level of cm volume in patients undergoing pci . this study was conducted in a single - institution setting of a tertiary university hospital from september of 2009 to august of 2011 . the eligibility criteria of the present study were an age of 19 years or older and a referral for coronary angiography ( cag ) and pci . exclusion criteria were as follows : cardiogenic shock , pulmonary edema , emergent pci , end - stage renal disease requiring dialysis , and a previous administration of cm within 72 hours of pci . this study was approved by the ethics committee of our hospital , and all participating patients provided written informed consent . baseline egfr was calculated as creatinine clearance by a modification of diet in the renal disease ( mdrd ) study16 ) equation [ egfr=175{scr ( mg / dl)}{age ( years ) } ] . a correction factor of 0.85 was used for women and the cyc based equation { egfr=66.8(cyc ) } suggested by rule et al.21 ) respectively . a two dimensional echocardiography was performed before pci , and left ventricular ejection fraction ( lvef ) was calculated by the biplane modified simpson 's method . all the patients were given the same hydration regimen with intravenous isotonic saline at a rate of 1 ml / kg / hr for 12 hours prior to and after pci . additional prophylactic medications for cin ( e.g. , n - acetylcysteine ) were not permitted to be administered to the patients . in diabetic patients , metformin was discontinued on the cag day and withheld for the following 48 hours . after informed consent was obtained , all procedures were performed at the operator 's discretion . cag and pci were performed using the standard technique , via either a femoral or radial approach . they were also administered glycoprotein iib / iiia receptor blockers and heparin at the discretion of the operator . in the current study , we used either iso - osmolar , non - ionic cm , or iodixanol ( visipaque 320 mg i / ml ; ge healthcare korea , seoul , korea ) . the amounts of delivered cm were measured using an automated contrast delivery injection system ( acist ; acist medical systems , inc . , cin was defined as a 25% elevation or an absolute increase of > 0.5 mg / dl ( > 44 umol / l ) in the scr level compared to the baseline within 48 to 72 hours after pci , in accordance with the definition of the cin consensus working panel.2 ) all statistical analyses were performed with statistical package for the social sciences ( spss ) ( release 15.0 ; spss inc . , chicago , il , usa ) . to identify the correlations between cin and the clinical or laboratory parameters , a univariate analysis was performed using an unpaired t - test or mann - whitney u test for the continuous variables , and a chi - square test for the categorical variables , respectively . a multivariate logistic regression analysis was used to assess the correlation among the parameters , whose statistical significance was demonstrated on a univariate analysis at a level of p<0.05 and through previously well - known risk factors . models were developed with stepwise techniques , for which the results were expressed as odds ratios ( or ) with 95% confidence intervals ( ci ) . finally , the cm volume / egfrcyc was added to the model as a separate predictor to test for any interaction between cm - volume and egfr . an analysis of the receiver operating characteristic ( roc ) was conducted to determine the cut - off value and area under the curve ( auc ) of the cm volume / egfrcyc and cm volume / mdrd for predicting cin . this study was conducted in a single - institution setting of a tertiary university hospital from september of 2009 to august of 2011 . the eligibility criteria of the present study were an age of 19 years or older and a referral for coronary angiography ( cag ) and pci . exclusion criteria were as follows : cardiogenic shock , pulmonary edema , emergent pci , end - stage renal disease requiring dialysis , and a previous administration of cm within 72 hours of pci . this study was approved by the ethics committee of our hospital , and all participating patients provided written informed consent . baseline egfr was calculated as creatinine clearance by a modification of diet in the renal disease ( mdrd ) study16 ) equation [ egfr=175{scr ( mg / dl)}{age ( years ) } ] . a correction factor of 0.85 was used for women and the cyc based equation { egfr=66.8(cyc ) } suggested by rule et al.21 ) respectively . a two dimensional echocardiography was performed before pci , and left ventricular ejection fraction ( lvef ) was calculated by the biplane modified simpson 's method . all the patients were given the same hydration regimen with intravenous isotonic saline at a rate of 1 ml / kg / hr for 12 hours prior to and after pci . additional prophylactic medications for cin ( e.g. , n - acetylcysteine ) were not permitted to be administered to the patients . in diabetic patients , metformin was discontinued on the cag day and withheld for the following 48 hours . after informed consent was obtained , all procedures were performed at the operator 's discretion . cag and pci were performed using the standard technique , via either a femoral or radial approach . they were also administered glycoprotein iib / iiia receptor blockers and heparin at the discretion of the operator . in the current study , we used either iso - osmolar , non - ionic cm , or iodixanol ( visipaque 320 mg i / ml ; ge healthcare korea , seoul , korea ) . the amounts of delivered cm were measured using an automated contrast delivery injection system ( acist ; acist medical systems , inc . , cin was defined as a 25% elevation or an absolute increase of > 0.5 mg / dl ( > 44 umol / l ) in the scr level compared to the baseline within 48 to 72 hours after pci , in accordance with the definition of the cin consensus working panel.2 ) all statistical analyses were performed with statistical package for the social sciences ( spss ) ( release 15.0 ; spss inc . , chicago , il , usa ) . to identify the correlations between cin and the clinical or laboratory parameters , a univariate analysis was performed using an unpaired t - test or mann - whitney u test for the continuous variables , and a chi - square test for the categorical variables , respectively . a multivariate logistic regression analysis was used to assess the correlation among the parameters , whose statistical significance was demonstrated on a univariate analysis at a level of p<0.05 and through previously well - known risk factors . models were developed with stepwise techniques , for which the results were expressed as odds ratios ( or ) with 95% confidence intervals ( ci ) . finally , the cm volume / egfrcyc was added to the model as a separate predictor to test for any interaction between cm - volume and egfr . an analysis of the receiver operating characteristic ( roc ) was conducted to determine the cut - off value and area under the curve ( auc ) of the cm volume / egfrcyc and cm volume / mdrd for predicting cin . a total of 1640 patients were referred for cag and pci , between september 2009 and august 2011 . of these , the study group was composed of 450 men and 273 women , with a mean age of 63.710.4 years old . among all of the study patients , cin occurred in 29 patients ( 4.0% ) . patients were divided into two groups according the development of cin : the cin group ( n=29 ) and the non - cin group ( n=694 ) . compared to the non - cin group , clinical presentation as acute coronary syndromes was significantly more frequent in the cin group . the cin group showed significantly lower hemoglobin ( hb ) and egfr levels , and higher bun , scr , and cyc levels . they were also subjected to a higher cm volume , cm volume / egfrcyc , and cm volume / mdrd . as shown in table 2 , reduced lvef , hb level , and egfr using mdrd and cyc , increased cm volume , cm volume / mdrd and cm volume / egfrcyc were found to be significantly correlated with the development of cin in the univariate logistic regression analysis . clinical presentations as acute coronary syndrome , age , and diabetes were not within the range of statistical significance . in the multivariate analysis , decreased hb ( or 0.743 , 95% ci 0.566 - 0.975 , p=0.032 ) and increased cm volume / mdrd ( or 1.657 , 95% ci 1.156 - 2.374 , p=0.006 ) and cm volume / egfrcyc ( or 2.275 , 95% ci 1.496 - 3.461 , p<0.001 ) remained as significant independent risk factors for the development of cin . furthermore , the significantly highest incidence of cin was demonstrated in the 4th quartile of cm volume / egfrcyc compared with the 1st , 2nd , and 3rd quartiles ( p<0.001 ) ( fig . the roc curve was performed using cm volume / egfrcyc to assess its ability to predict the development of cin . it revealed a modest discrimination of cin at a cm volume / egfrcyc level of 4.493 ( auc=0.814 ; sensitivity=79.3% , specificity=80.0% ) ( fig . 2 ) . although there was no statistically significant difference between cm volume / egfrcyc and cm volume / mdrd , auc of the cm volume / mdrd ( 0.772 ) was lower than those of cm volume / egfrcyc . of those patients ( n=169 ) with cm volume / cyc egfr 4.493 , 13.6% ( 23/169 ) developed cin , while those of cm volume / cyc egfr < 4.493 evinced a significantly lower incidence of cin 1.1% ( 6/554 ; p<0.001 ) . a total of 1640 patients were referred for cag and pci , between september 2009 and august 2011 . of these , the study group was composed of 450 men and 273 women , with a mean age of 63.710.4 years old . among all of the study patients , cin occurred in 29 patients ( 4.0% ) . patients were divided into two groups according the development of cin : the cin group ( n=29 ) and the non - cin group ( n=694 ) . compared to the non - cin group , clinical presentation as acute coronary syndromes was significantly more frequent in the cin group . the cin group showed significantly lower hemoglobin ( hb ) and egfr levels , and higher bun , scr , and cyc levels . they were also subjected to a higher cm volume , cm volume / egfrcyc , and cm volume / mdrd . as shown in table 2 , reduced lvef , hb level , and egfr using mdrd and cyc , increased cm volume , cm volume / mdrd and cm volume / egfrcyc were found to be significantly correlated with the development of cin in the univariate logistic regression analysis . clinical presentations as acute coronary syndrome , age , and diabetes were not within the range of statistical significance . in the multivariate analysis , decreased hb ( or 0.743 , 95% ci 0.566 - 0.975 , p=0.032 ) and increased cm volume / mdrd ( or 1.657 , 95% ci 1.156 - 2.374 , p=0.006 ) and cm volume / egfrcyc ( or 2.275 , 95% ci 1.496 - 3.461 , p<0.001 ) remained as significant independent risk factors for the development of cin . furthermore , the significantly highest incidence of cin was demonstrated in the 4th quartile of cm volume / egfrcyc compared with the 1st , 2nd , and 3rd quartiles ( p<0.001 ) ( fig . 1 ) . the roc curve was performed using cm volume / egfrcyc to assess its ability to predict the development of cin . it revealed a modest discrimination of cin at a cm volume / egfrcyc level of 4.493 ( auc=0.814 ; sensitivity=79.3% , specificity=80.0% ) ( fig . although there was no statistically significant difference between cm volume / egfrcyc and cm volume / mdrd , auc of the cm volume / mdrd ( 0.772 ) was lower than those of cm volume / egfrcyc . of those patients ( n=169 ) with cm volume / cyc egfr 4.493 , 13.6% ( 23/169 ) developed cin , while those of cm volume / cyc egfr < 4.493 evinced a significantly lower incidence of cin 1.1% ( 6/554 ; p<0.001 ) . in this prospective and observational study , cm volume / egfrcyc was identified as a significantly independent predictor of cin compared with other previously well - known predictors . therefore , we were able to estimate the safe cm volume via measuring egfrcyc prior to cm administration . the cm volume / egfrcyc can be considered a simple and reliable indicator for determining the safe volume of cm based on the pre - procedural cyc level . although this simple model has a drawback in that many other variables are not included , this study also showed that other clinical and laboratory factors associated with cin and cm volume / egfrcyc remained independent predictors of cin after an adjustment for these confounding factors . many guidelines regarding the prevention of cin recommend the minimal use of cm.24 - 26 ) however , it is not easy to determine the acceptable cm amount . in the pci era , the use of cm is necessary to obtain satisfactory images and to perform pci . moreover , high - risk patients usually showed complex lesions and a proneness to the inevitable use of large amounts of cm . among a number of cin , cm volume can be changed and it can be potentially controlled at the discretion of the operator . therefore , we need to guide individual quantities of safe cm volume prior to pci . several studies have been trying to establish the risk of cin after exposure to cm via a similar concept using the relationship between the cm exposure and baseline renal function . in 1989 , cigarroa et al.10 ) proposed a simple formula called maximal acceptable cm volume ( cm volume / weight ) based on the scr level . in the retrospective study regarding high - risk patients ( scr > 2.0 mg / dl or cm volume > 300 ml ) , the cut off value of cm volume / egfr ( by cockcroft - gault equation ) was defines as 6.0.10 ) another study conducted in 3179 unselected patients undergoing pci reported that cm volume / egfr ( by mdrd equation ) of < 3.27 might be a useful indicator in determining the cm volume which might be considered safe from the cin.11 ) previously , we also reported safe cm volume using grams of iodine per egfr ( g - i / egfr ) at a level of 1.42.27 ) however , the abovementioned studies were based on scr or egfr using scr . although scr has been the most widely used biomarker of renal function , it has serious limitations in terms of imprecision in reflecting renal function . it is frequently affected by a lot of non - gfr factors , including muscle mass , dietary habit , and age.15)16)28 ) importantly , it has a limitation in detecting mild renal impairment.18 ) cystatin c is a non - glycosylated 13-kda basic protein of the cystatin super - family of cysteine proteinase inhibitors.17 ) cyc shows a consistent production rate and is eliminated by glomerular filtration . it is less dependent on age , sex , race , and muscle mass.19 ) therefore , cyc is considered to be a potential alternative to scr for estimating gfr , and cm volume / egfrcyc also suggested a better estimation of gfr that scr based egfr . the cm volume / cyc egfr represents both the amount of cm and individual renal function . it is obvious that an accurate measurement of renal function is required to make the model more reliable . from such a point of view , the method used in previous studies regarding safe cm volume estimation may have many limitations from the perspective of accuracy in gfr estimation . in this study , using cyc , we demonstrated that cm volume / egfrcyc could both represent individual renal function . in addition , both cm volume / mdrd and cm volume / egfrcyc could be identified as independent predictors of cin . apart from cm volume and renal function , another issue relating to cin is anemia . thus , anemia could be deemed a risk factor for mortality in cardiovascular disease.6 - 8)29 ) moreover , anemia is commonly combined with chronic kidney disease ( ckd ) and it can present as a complication of ckd . in this study , anemia was identified as an independent predictor of cin after an adjustment for other risk factors . yet , there remains limited data as to whether the correction of anemia can be helpful to prevent cin , whereas , periprocedural blood loss was considered as a potent risk factor of cin.30 ) therefore , further data on anemia and baseline renal function prior to pci is required to definitely determine their effects on the development of cin . therefore , a careful consideration of the safe cm volume would be mandatory for patients who are at an increased risk of developing cin based on a decreased egfr prior to the pci . moreover , pci treatment for complex lesions is often prone to large doses of cm administration . in these high - risk patients , we should consider the measurement of cyc prior to pci and pay attention to the safe cm volume , particularly based on the egfrcyc , to prevent cin when performing pci . our present study consisted of controlled patients who were scheduled for elective pci with appropriate hydration and had other nephrotoxic agents avoided . contrary to emergency situations , in scheduled pci , physicians can obtain sufficient information for a safe cm volume estimation prior to pci . therefore , results of the current study have more potent clinical implications than previous retrospective studies about unselected populations . although this study was a prospective controlled study , it can not be considered free from its limitation as a single institution study . furthermore , there remains debate regarding the differential safety of cm among various types of cm ( isoosmolar cm and low - osmolar cm , ionic cm and non - ionic cm ) . further studies with various types of cm are required to confirm the efficacy of this equation using egfrcyc . although the cyc based equation showed a better estimation of gfr compared with scr , there was little validated data about egfrcyc in asian populations . the delivered cm volume to egfr calculated from both scr and cyc , expressed as cm volume / mdrd and /egfrcyc , both showed a predictive value for the occurrence of cin . based on the current results , we could suggest a safe acceptable cm volume of 4.493 ml ( iodixanol 320 mg - i / ml ) per 1 u of egfr ( ml / min/1.73 m ) and a pre - interventional determination of acceptable cm volume based on egfrcyc , which might be helpful in avoiding cin . in comparison of the predictive value of the scr based and cyc based equation , many guidelines regarding the prevention of cin recommend the minimal use of cm.24 - 26 ) however , it is not easy to determine the acceptable cm amount . in the pci era , the use of cm is necessary to obtain satisfactory images and to perform pci . moreover , high - risk patients usually showed complex lesions and a proneness to the inevitable use of large amounts of cm . among a number of cin , cm volume can be changed and it can be potentially controlled at the discretion of the operator . therefore , we need to guide individual quantities of safe cm volume prior to pci . several studies have been trying to establish the risk of cin after exposure to cm via a similar concept using the relationship between the cm exposure and baseline renal function . in 1989 , cigarroa et al.10 ) proposed a simple formula called maximal acceptable cm volume ( cm volume / weight ) based on the scr level . in the retrospective study regarding high - risk patients ( scr > 2.0 mg / dl or cm volume > 300 ml ) , the cut off value of cm volume / egfr ( by cockcroft - gault equation ) was defines as 6.0.10 ) another study conducted in 3179 unselected patients undergoing pci reported that cm volume / egfr ( by mdrd equation ) of < 3.27 might be a useful indicator in determining the cm volume which might be considered safe from the cin.11 ) previously , we also reported safe cm volume using grams of iodine per egfr ( g - i / egfr ) at a level of 1.42.27 ) however , the abovementioned studies were based on scr or egfr using scr . although scr has been the most widely used biomarker of renal function , it has serious limitations in terms of imprecision in reflecting renal function . it is frequently affected by a lot of non - gfr factors , including muscle mass , dietary habit , and age.15)16)28 ) importantly , it has a limitation in detecting mild renal impairment.18 ) cystatin c is a non - glycosylated 13-kda basic protein of the cystatin super - family of cysteine proteinase inhibitors.17 ) cyc shows a consistent production rate and is eliminated by glomerular filtration . it is less dependent on age , sex , race , and muscle mass.19 ) therefore , cyc is considered to be a potential alternative to scr for estimating gfr , and cm volume / egfrcyc also suggested a better estimation of gfr that scr based egfr . the cm volume / cyc egfr represents both the amount of cm and individual renal function . it is obvious that an accurate measurement of renal function is required to make the model more reliable . from such a point of view , the method used in previous studies regarding safe cm volume estimation may have many limitations from the perspective of accuracy in gfr estimation . in this study , using cyc , we demonstrated that cm volume / egfrcyc could both represent individual renal function . in addition , both cm volume / mdrd and cm volume / egfrcyc could be identified as independent predictors of cin . apart from cm volume and renal function , another issue relating to cin is anemia . several studies also demonstrated a significant association of anemia and cin . thus , anemia could be deemed a risk factor for mortality in cardiovascular disease.6 - 8)29 ) moreover , anemia is commonly combined with chronic kidney disease ( ckd ) and it can present as a complication of ckd . in this study , anemia was identified as an independent predictor of cin after an adjustment for other risk factors . yet , there remains limited data as to whether the correction of anemia can be helpful to prevent cin , whereas , periprocedural blood loss was considered as a potent risk factor of cin.30 ) therefore , further data on anemia and baseline renal function prior to pci is required to definitely determine their effects on the development of cin . therefore , a careful consideration of the safe cm volume would be mandatory for patients who are at an increased risk of developing cin based on a decreased egfr prior to the pci . moreover , pci treatment for complex lesions is often prone to large doses of cm administration . in these high - risk patients , we should consider the measurement of cyc prior to pci and pay attention to the safe cm volume , particularly based on the egfrcyc , to prevent cin when performing pci . our present study consisted of controlled patients who were scheduled for elective pci with appropriate hydration and had other nephrotoxic agents avoided . contrary to emergency situations , in scheduled pci , physicians can obtain sufficient information for a safe cm volume estimation prior to pci . therefore , results of the current study have more potent clinical implications than previous retrospective studies about unselected populations . although this study was a prospective controlled study , it can not be considered free from its limitation as a single institution study . furthermore , there remains debate regarding the differential safety of cm among various types of cm ( isoosmolar cm and low - osmolar cm , ionic cm and non - ionic cm ) . further studies with various types of cm are required to confirm the efficacy of this equation using egfrcyc . although the cyc based equation showed a better estimation of gfr compared with scr , there was little validated data about egfrcyc in asian populations . the delivered cm volume to egfr calculated from both scr and cyc , expressed as cm volume / mdrd and /egfrcyc , both showed a predictive value for the occurrence of cin . based on the current results , we could suggest a safe acceptable cm volume of 4.493 ml ( iodixanol 320 mg - i / ml ) per 1 u of egfr ( ml / min/1.73 m ) and a pre - interventional determination of acceptable cm volume based on egfrcyc , which might be helpful in avoiding cin . in comparison of the predictive value of the scr based and cyc based equation ,

background and objectivesthe risk of contrast - induced nephropathy ( cin ) is significantly influenced by baseline renal function and the amount of contrast media ( cm ) . we evaluated the usefulness of the cystatin c ( cyc ) based estimated glomerular filtration rate ( egfrcyc ) in the prediction of cin and to determine the safe cm dosage.subjects and methodswe prospectively enrolled a total of 723 patients who received percutaneous coronary intervention ( pci ) and investigated the clinical factors associated with the development of cin . renal function was calculated as egfrcyc and a modified diet in the renal disease ( mdrd ) equation , respectively . systemic exposure of cm was calculated as cm volume to egfr ratio . we conducted a regression analysis to evaluate the predictive role of cm volume to egfrcyc for the risk of cin.resultsthe incidence of cin was 4.0% ( 29/723 ) . the patients with cin had a lower hemoglobin level , decreased renal function , and a higher cyc value , and had greater cm exposure . through multivariate regression analyses , hemoglobin { odds ratio ( or ) 0.743 , p=0.032 } , cm volume / egfrcyc ( or 1.697 , p=0.006 ) and cm volume / mdrd ( or 2.275 , p<0.001 ) were found to be independent predictors for cin . in the receiver operating characteristic curve analysis , fair discrimination for cin was found at a cm volume / egfrcyc level of 4.493 ( c - statics=0.814 ) , and at this value , the sensitivity and specificity were 79.3% and 80.0% , respectively.conclusionboth the cm volume / mdrd and cm volume / egfrcyc method would be simple , useful indicators for determining the safe cm - dose based on egfr value before pci . however , there was no significantly different predictive value between creatinine and cyc based gfr estimations .

we have recently described the l1tcrz , an hepatitis delta virus ( hdv)-like ribozyme that resides within the 5-end of the rna of both the l1tc and nartc retrotransposons of t. cruzi ( fig . the ribozyme catalyzes the self - cleavage of the rna sugar - phosphate backbone of its own molecule , leaving a 5-hydroxyl end on the downstream product , and it is expected to leave a 2,3-clyclic phosphate end on the upstream cleavage product , similar to the hdv and other known hdv - like ribozymes . the cleavage position of l1tcrz is located immediately upstream of the + 1 nucleotide of both l1tc and nartc . the 5-end is such that the ribozyme and the internal promoter sequence is present in the mature element rna insuring their transmission to the new retrotransposon copy during tprt process . the three helixes p1 , p2 and p4 and the two pseudoknots p1.1 and p3 are colored . the proposed switching of the folding toward a supposed - ires after elongation is shown in ( b ) . rnafold software proposes for the first 110 nt of the l1tc rna a structure involving three stem - loops called sl1 , sl2 and sl3 . p1 and p4 helixes are conserved after the folding switching but the two pseudoknots and the p2 helix are not maintained . a 5-hydroxyl end is expected for sl1 to be occluded by the gc pair - rich p1 stem . co - transcriptional cleavage assays were used to map the l1tcrz to the first 77nt ( + 1 to + 77 ) of l1tc / nartc elements . this 77 nt sequence is predicted to fold into an hdv - like ribozyme , and can be fitted to the consensus hdv secondary structure with minimal divergences . assays where l1tcrz sequence is preceded by various l1tc target derived sequences indicate that these sequences upstream of the l1tcrz have the potential to modulate the l1tcrz activity . it has been shown that the 49 nt downstream of l1tcrz can induce an rna structural change to a conformation not capable of l1tcrz activity ( fig . the sequence located downstream of l1tcrz in nartc does not produce a conformational switch and consequently the ribozyme activity is preserved in nartc rna . it is possible that the downregulation effect of l1tc downstream sequences may be an artificial consequence of the transcriptional activity of the t7 rna polymerase used in the in vitro assays . t7 rna polymerase may transcribe the downstream sequence so quickly , prior to ribozyme folding , allowing alternative folding pathways to occur . the inhibition of the ribozyme activity may not occur with an eukaryotic rna polymerase , whose polymerase activity is slower than the t7 phage polymerase . other non - ltr retrotransposon has an hdv - like ribozyme in its 5-end for the purpose of processing the retrotransposon rna . l1tc was the second reported case of a non - ltr retrotransposon to be associated to an hdv - like ribozyme , being the r2 the first one . r2 , unlike l1tc , is a site - specific element whose copies are always found at the same position in the redundant rdna genes . the 5-end of the r2 retrotransposon lacks a promoter relying on being co - transcribed with the rrna . the r2 encoded hdv - like ribozyme is used to process the co - transcript , thus releasing the r2 mrna . like l1tcrz , non - ltr retrotransposons that lack a promoter , like r2 , necessarily rely on host promoters and co - transcription to replicate . target site specificity would ensure that a promoter - less element like r2 would always insert downstream of a host promoter . an rna endonuclease system , like an element encoded ribozyme or a signal recognized by a host rna endonuclease , is required to process the element rna away from the co - transcript . conversely , non - ltr retrotransposons that contain internal promoter may become more permissive for their insertion site and may , by the virtue of the internal promoter , generate unit length element rna without the need for processing by a ribozyme . l1tc is the first described retrotransposon that carries both internal promoter function ( pr77 ) as well as a ribozyme ( l1tcrz ) function , combining features expected for non - specific and site - unspecific elements , respectively . the l1tc element is widely distributed throughout the t. cruzi genome but there is some degree of conservation of the sequence upstream the insertion sites that flanks the l1tc element copies . a similar consensus sequence has been detected flanking other retrotransposons belonging to the ingi / l1tc clade . since the conservation of these nucleotides is considered to be a trace of the insertion site selection , it may be possible to hypothesize which retrotransposition machinery of an autonomous element is used by each non - autonomous element . however , this hypothesis is only supported by bioinformatic data as the function of the upstream sequence motifs remains unknown . the implications of the pr77/l1tcrz dual function in trypanosomes are explored in the next section . the coexistence of both , ribozyme and internal promoter systems within l1tc and nartc elements may be related to the genetic regulation of the host . trypanosomatid genomes are organized in large directional polycistronic clusters that are transcribed by rna polymerase ii and separated by the so - called strand switch regions ( ssrs ) . monocistronic mrnas are produced through trans - splicing of capped spliced leader ( sl ) sequence onto the 5-end of the individual coding sequences . most likely , the l1tcrz ensures the proper release of any l1tc ( or nartc ) elements that were co - transcribed as part of a polycistronic rna ( fig . functional roles of the ingi / l1tc clade 7779 bp signature : promoter - ribozyme duality . ( a ) transposable element mrna is released from policystronic transcripts by l1tcrz cleavage when the copy is located in sense orientation within a cluster . ( d ) the bi - directional launching of the rna polymerase ii ( pol ii ) for transcription of the large polycistronic clusters may be mediated by the pr77 promoter from the mobile elements accumulated within the ssrs . ( e ) pr77- mediated transcription of sense - inserted copies located within a cluster could prevent the transcriptional decay level of genes located far from the cluster transcription start site . ( f ) mobile element copies inserted in sense at the 3-utrs of somatic genes could be downregulating the mrna level reducing the rna stability after l1tcrz cleavage ( as the sider - dependent downregulation described in leishmania spp ) . code : te , transposable element ; same color boxes ( red or green ) , somatic genes within the same cluster ; blue boxes , copies of transposable elements ; black arrows , transcription start site and orientation ; black box , sider copy . code for the diagram of transcriptional reinforcement ( e ) : red , transcriptional level of the pol ii launched from the ssr ; blue , transcriptional level of the pol ii launched from dispersed pr77 ; dotted - line , global pol ii transcriptional level . the l1tc pr77 internal promoter may guarantee the transcription of the l1tc copies present in the antisense orientation with respect to the polycistronic clusters ( fig . 2b ) as well as those that lie outside them ( fig . 2c ) . the existence of many sider antisense - inserted copies in the intergenic regions within the clusters of leishmania genome supports this idea . in addition , only two rna polymerase ii - dependent promoters have been described in trypanosomatids : the sl rna external promoter and the l1tc pr77 internal one . it has been suggested that , due to the high density of l1tc and nartc copies found within ssrs , the initiation of the polycistronic transcription may be a result of the firing from the pr77 promoters ( fig . finally , the dispersed copies in sense orientation could also be preventing a decay of the transcription level of distal regions , ensuring the correct expression of the last genes of each cluster ( fig . the origin of the pr77-dependent transcription has been determined by primer extension using extracted rna from epimastigote cells and found to be located at the + 1 nucleotide . since the l1tcrz cleaves at the 5-side of the mentioned + 1 nucleotide of the element , the result of the primer extension previously referred above could be a consequence of the l1tc hdv - like ribozyme activity . in contrast to the hammerhead or hairpin ribozymes that cleave within their catalytic core , the particular cleavage characteristic of the hdv - like ribozymes allows to keep intact the catalytic core at the mrna 3-product , ensuring its complete preservation within the de novo synthesized copies given the single reverse transcription step of the tprt . moreover , the existence of l1tcrz enables the generation of l1tc mrnas from polymerase ii polycistronic transcripts . the l1tcrz would preserve the individuality of each l1tc / nartc copy , guaranteeing the accurate definition of the 5-end of the element and preventing the mobilization of host sequences located upstream of the parental copy . taking into account all these data , we propose that the l1tc mrnas lack in vivo sl sequences , since their 5-end start at the nucleotide + 1 of the element . many other cytoplasmic rnas are naturally uncapped , like trnas , 5s rrna or viral rnas of pestivirus and narnavirus , among others . for example , trnas are transcribed as longer precursors , and their 5- and 3-ends are maturated by rnase p and rnase z , respectively . the 5-end and the 3-end of trnas and 5s rrna are usually constrained in a gc pair rich helix like the 5-end of narnavirus rna . actually , the disruption of the gc pairs of the 5-stem loop of narnavirus rna makes it to be susceptible to exonuclease ski1/xrn1 degradation , thus reducing its half - life . the in silico analysis of the folding of the 5-utr of the l1tc and ingi rnas resulting from the l1tcrz cleavage predicts that it adopts a 5-stem loop rich in gc pairs ( sl1 of fig . thus , the l1tcrz could be responsible of some kind of 5-end rna maturation to ensure the stability of the uncapped l1tc mrna in vivo . as previously shown , the pr77-derived transcripts lack sl and are abundant although poorly translatable . the confirmation of an in vivo 5-hydroxyl end in the mature l1tc mrnas would point to a cap - independent translation in which other sequences , apart from pr77 , must be involved . the best known cap - independent translation system is the ires ( internal ribosome entry site ) . an ires consist of a complex rna structure able to mediate a cap - independent ribosome recruitment . however , in some cases , this recruitment may require additional cellular factors , associated or not , to cap - dependent translation . we have found an attenuator sequence downstream of the l1tcrz in l1tc ( fig . 1b ) , but not in nartc , revealing that the downstream sequences of l1tc and nartc induce different refoldings . the l1tc 5-utr rna , but not the nartc rna , adopts a trna - like structure susceptible of being cleaved by rnase p. the rnase p is a natural ribozyme responsible for the maturation of the 5-end of trnas by the catalysis of an endonucleolytic cleavage that removes the 5-leader sequence of pre - trnas . the variety of trnas strongly indicates that the recognition of the substrates is mediated by three - dimensional trna folding . interestingly , rnas of some ires are also recognized by rnase p , revealing the existence of a so - called trna - like structure within them . although other trna - like structures unrelated to ires have been described , the identification of one of them within the 5-utr rna of the coding l1tc element , and not in the 5-rna region of the non - coding nartc element , is consistent with the hypothesis of the existence of an ires in l1tc . thus , we propose a model in which after l1tcrz cleavage , the l1tc mrna elongation leads to a rna refolding toward an uncapped hidden 5-end and an ires structure ( fig . the pr77/l1tcrz sequence of 77 nt in length is also found in other trypanosomatid s non - ltr retrotransposons like ingi , rime , dires and siders with a high level of identity . consequently , it has been called the 7779 bp signature . however , there are no data reporting that the catalytic rna and the internal promoter functions of the 7779 bp signature are maintained across various members of the ingi / l1tc clade of retrotransposons . ingi is a supposedly active line element that resides within the t. brucei ( tbingi ) and t. vivax ( tvingi ) genomes like the l1tc element does in both the t. cruzi and t. congolense ( l1tco ) . rime is a truncated version of ingi ( like nartc of l1tc ) and also resides within t. brucei ( tbrime ) and t. vivax ( tvrime ) genomes . to date , there is not data reporting the existence of nartc homologous elements in the t. congolense genome . the presence of abundant stop codons and misreading mutations indicate that dires have not coding capacity . siders ( short interspersed degenerated retroposons ) are short non - coding degenerated elements of approximately 600 bp in length that reside , together with dires , in the trypanosomatid s genomes . dires and siders are thought to be mobilized in trans by the ingi / l1tc machinery . however , ingi / l1tc homologs in the genomes of various leishmania spp , where siders are especially abundant , have not been described . extensive phylogenetic analyses of the ingi / l1tc - related elements lineage of trypanosomatids have been preformed . the transposition mechanism of the non - ltr elements involves two single and asymmetric cleavages at the target insertion site that lead to the generation of a direct repetition ( of 1112 nt in l1tc ) flanking each element , called target site duplication ( tsd ) ( fig . the conservation degree of the tsds provides a hint of the elapsed time from the transposition , since ancient copies would have accumulated mutations at their tsds , while recent ones would not . based on that , it was established that tvsider1c , tcosider1 , tbsider1 , lmsider and lmdire have not been recently mobilized , suggesting they are not active elements . since the hdv - like ribozyme activity has low sequence requirements but needs a consistent folding , we have analyzed the predicted folding of the 77 bp signature of the ingi / l1tc clade members as an indicator of the element activity ( fig . the results indicate that the tvsider1c consensus sequence has severe deficiencies for a proper hdv - like ribozyme folding as it has two mispairings in the p1 helix and a complete extinction of the pseudoknot p1.1 ( fig . 3 ) . however , the tvsider1a and tvsider1b consensus sequence has an hdv - like ribozyme folding , although the latter bears a mispairing in the p3 pseudoknot ( fig . interestingly , all the foldings compatible with a functional hdv - like ribozyme correlate with the deduced active elements by the analysis of the tsds . folding predicted for the pr77-transcribed rnas corresponding to the consensus sequence of different families of the trypanosoma vivax sider1(a - c ) described by bringaud , f. et al . , 2011 . the tvsider1c consensus presents severe misfolding mutations like two mismatches at p1 helix , one mismatch at p2 and complete disappearance of the p1.1 pseudoknot . by contrast , the existence of a conserved ribozyme activity in a degenerated retrotransposon might mean that both the insertion and the catalytic activity would have been positively selected along the evolution as they provide some kind of favorable genetic regulation for the host . this may well be the case of sider copies inserted within the 3-utr of many genes of leishmania major and l. infantum . in fact , it has been shown that sider2 copies within the 3-utrs of two genes in l. major ( sider3810 and sider1270 ) and l. infantum ( sider4000 and sider1222 ) cause a downregulation of the mrnas via an endonucleolytic cleavage of such rnas without prior deadenylation . in the l. major sider2 copies two in vivo endonucleolytic cleavages were detected within the 77 bp signature ( so called signature ii in lmsider2 ) . the existence of an active ribozyme within this signature could explain the generation of these endonucleolytic cleavages that lead to the rna degradation ( fig .

the sequence corresponding to the first 77 nucleotides of the l1tc and nartc non - ltr retrotransposons from trypanosoma cruzi is an internal promoter ( pr77 ) that generates abundant , although poorly translatable , un - spliced transcripts . it has been recently described that l1tcrz , an hdv - like ribozyme , resides within the 5-end of the rna from the l1tc and nartc retrotransposons . remarkably , the same first 77 nucleotides of l1tc / nartc elements comprise both the pr77 internal promoter and the hdv - like l1tcrz . the l1tcrz cleaves on the 5-side of the + 1 nucleotide of the l1tc element insuring that the promoter and the ribozyme functions travel with the transposon during retrotransposition . the ribozyme activity would prevent the mobilization of upstream sequences and insure the individuality of the l1tc / nartc copies transcribed from associated tandems . the pr77/l1tcrz sequence is also found in other trypanosomatid s non - ltr retrotransposons and degenerated retroposons . the possible conservation of the ribozyme activity in a widely degenerated retrotransposon , as the leishmania siders , could indicate that the presence of this element and the catalytic activity could play some favorable genetic regulation . the functional implications of the pr77/l1tcrz dual system in the regulation of the l1tc / nartc retrotransposons and in the gene expression of trypanosomatids are also discussed in this paper .

obesity is increasingly prevalent among reproductive age women [ 13 ] and is associated with pregnancy complications such as gestational diabetes and hypertensive disorders of pregnancy [ 2 , 46 ] . obesity disproportionately impacts women of racial and ethnic minorities , with highest rates in black women , followed by hispanic and white women , and potentially contributes to racial and ethnic disparities in other chronic diseases such as diabetes , hypertension , and cardiovascular disease [ 79 ] . like preconception obesity , gaining excessive gestational weight disproportionately affects racial and ethnic minorities and is associated with pregnancy complications and future risk of long term overweight and obesity [ 10 , 11 ] . pregnancy provides an opportunity to identify unhealthy behaviors and promote healthy eating habits , which can be sustained beyond pregnancy . however , evidence is not yet clear as to whether the observed racial and ethnic differences in preconception obesity and gestational weight gain are associated with differences in obesity - related dietary behaviors [ 1216 ] . further , we found no studies specifically evaluating fast food and sugar - sweetened beverage intake in this population , two potentially modifiable dietary behaviors which are associated with obesity [ 1719 ] . previous studies used health behavior surveys to evaluate differences in dietary habits between black and hispanic women , but results did not show consistent differences between the racial and ethnic groups [ 1214 , 16 ] . the largest of these four studies excluded women with chronic medical comorbidities such as hypertension or diabetes or high risk pregnancies another large , prospective cohort of 2394 women used food frequency questionnaires to evaluate dietary differences among racial groups , but the study populations were primarily middle class . our primary objective was to address an important evidence gap : the association between race and ethnicity and differences in women 's obesity - related dietary behaviors during pregnancy and after delivery , among a socioeconomically diverse group of women . based on evidence from previous studies [ 13 , 12 , 14 ] , we hypothesized that black women would have higher odds of both fast food and sugar - sweetened beverage intake . we conducted a cross - sectional analysis using data collected in a convenience sample , using a one - time , self - administered questionnaire describing health behaviors among a sample of pregnant and postpartum women . this study was approved by the johns hopkins university school of medicine institutional review board . a total of 247 english or spanish speaking women , 18 years old , pregnant or within 1 year postpartum , who reported the ability to read the survey in english or spanish , completed the survey . women were recruited from 1 of 4 outpatient clinics ( including high risk obstetrics and pediatrics practices ) in 2 academic hospitals in baltimore , maryland , between january and april 2013 . participants completed a one - time , self - administered questionnaire at the time of their or their children 's appointments . of the 247 women , the 212 women who were identified as black , hispanic , or white were included in this secondary analysis . the 35 participants in the other racial categories were as follows : 4% were asian , 0.4% were hawaiian or pacific islander , 1.6% were american indian or native alaskan , and 3.3% described themselves as multiethnic . the diversity of the other racial / ethnic group limited our ability to make comparative inferences about their dietary habits , and these women were thus excluded from this analysis . we did not calculate the percentage of patients approached who agreed to participate in our study or evaluate the ways in which the participating women may differ from those women that chose not to participate . the survey , which included questions about sociodemographics and dietary behaviors , was adapted from validated national survey instruments [ 2022 ] . items on fast food were adapted from the coronary artery risk development in adults study : how many times in the past week did you eat out in a fast food restaurant such as mcdonald 's , burger king , wendy 's , arby 's , pizza hut , or kentucky fried chicken ? ( 1 ) never or less than once weekly , ( 2 ) 1 - 2 times per week , ( 3 ) more than 3 times per week but less than daily , or ( 4 ) at least daily . items related to sugar - sweetened beverages were adapted from the behavioral risk factor surveillance system : in the past 7 days , how often did you drink soda ( not diet ) or other sugar - sweetened beverages , like hawaiian punch , lemonade , or kool - aid ? ( 1 ) never or less than 1 can per week , ( 2 ) 1 - 2 cans per week , ( 3 ) more than 3 cans per week but less than daily , ( 4 ) about 1 can per day , or ( 5 ) 2 or more cans per day . a pilot study was performed to ensure that the questionnaire met criteria for a 5th - grade literacy level , as well as culture appropriateness , ease of understanding , and quick time to completion . participants were asked the following questions : are you hispanic or latino ? which of the following best describes your race ? check all that apply : asian , african american or black , caucasian / white / european american , native hawaiian or other pacific islander , american indian / alaska native , or multiethnic or mixed . we then categorized the racial and ethnic groups into african american / black , hispanic , caucasian , or other races / ethnicities . as above , women reporting other racial or ethnic categories were not included in the analysis . preconception body mass index ( bmi ) was calculated based on self - reported height and preconception weight for pregnant women and current weight for postpartum women and categorized into obese ( bmi 30 ) and nonobese ( bmi < 30 ) [ 23 , 24 ] . the primary outcomes were fast food frequency and sugar - sweetened beverage intake , both defined as less than once weekly versus once or more weekly . the rationale for these cut - points was based on the median intake in our sample . prior studies used similar cut - points and showed that the consumption of fast food two or more times per week was associated with weight gain and insulin resistance over 15 years , when compared to those who eat fast food less than twice weekly . notably , existing literature on sugar - sweetened beverage intake demonstrated greatest risk of weight gain and coronary heart disease [ 26 , 27 ] , with at least daily consumption of sugar - sweetened beverages . a frequency cut - point of once or greater per week was also deemed simple to assess clinically and to be potentially actionable . sociodemographic variables included age , language proficiency , marital status , education , employment , and income . english language proficiency was categorized as adequate if the respondent reported very good english language proficiency and limited for other responses , based on response categorization in the us census . a binary variable for marital status was created to assess differences between those who were married or living with a partner and those who were not . education level was divided into three variables including those with less than a high school education , those graduating high school or obtaining a ged , and those with one or more years of college . employment was assessed and categorized into employed ( full or part time ) , unemployed , maternity leave , home maker , disability , or student . we also assessed income and categorized the data into broad categories , as noted in table 1 . financial strain was a separate income variable , based on participant response to the survey question , in the past 12 months , was there ever a time when you did not have enough money to meet the daily needs of you and your family ? pregnancy status and medical comorbidities were binary variables based on responses to the question , have you been told that you have had any of these health problems ? check all that apply : overweight or obese , type 2 diabetes , gestational diabetes ( diabetes in pregnancy ) , high blood pressure , preeclampsia or toxemia , and none of the above . a binary variable was created for smoking , in which a yes response represents any smoking . descriptive analyses were used to explore the data by race and obesity categories and to describe the proportion who endorsed barriers to healthy behaviors . age , marital status , english language proficiency , presence of a child under age 5 at home , and education level were included in the model based on our review of the literature . financial strain was included as the financial variable , rather than income , due to concerns about differential bias as a result of the large percentages of black ( 23% ) and hispanic ( 37% ) participants who declined to answer the question on income . to evaluate the role of obesity in the relationship between race / ethnicity and dietary behaviors , we assessed effect modification using stratified samples by bmi 30 and < 30 . the rationale was that obesity may be the result of poor dietary behaviors but obese pregnant women may be more likely to receive behavioral counseling and thus make lifestyle changes . while we do not know of any specific data examining the role of race in these behaviors , there is data demonstrating racial and cultural differences in body image , which could potentially lead to modification of racial differences in fast food and sugar - sweetened beverage intake , based on obesity . first , we limited the sample to include only the pregnant women ( n = 179 ) as pregnant and postpartum women may report different behaviors . the percentage of postpartum women was so small ( 16% ) that we were unable to compare these two groups . we compared the results in just pregnant women to the results in the entire model to assess for differences . second , we changed the cut - point of sugar - sweetened beverage intake to assess daily not weekly intake , 1 versus < 1 sugar - sweetened beverage daily , and reevaluated our model . this sensitivity analysis was designed to address the difference between the cut - point we utilized in our study ( intake of 1 or more sugar - sweetened beverages weekly ) and that used in the literature pertaining to sugar - sweetened beverages ( intake of 1 or more sugar - sweetened beverages daily ) [ 2527 ] . table 1 shows the characteristics of the 212 women in our sample by race / ethnicity . figure 1 shows the preadjustment frequency of fast food ( figure 1(a ) ) and sugar - sweetened beverage ( figure 1(b ) ) intake by race / ethnicity . overall , 52% of women reported fast food intake less than once weekly or never , 39% reported intake 1 - 2 times weekly , and 9% reported intake 3 times weekly . in terms of sugar - sweetened beverage intake , the plurality ( 40.6% ) reported less than 1 serving weekly , while 29.3% reported 1 - 2 servings and 14.6% reported 36 servings weekly . table 2 shows the results of our adjusted logistic regression models . with respect to fast food intake , black women had 2.38 times higher odds of consumption once or more weekly , when compared to white women ( ci = 1.08 and 5.23 ) . we did not detect differences in fast food frequency between hispanic and white women ( ci = 0.45 and 2.70 ) . women aged 3034 had 2.6 times higher odds when compared to women 1824 years old ( ci = 1.02 and 6.62 ) . women reporting financial strain had 1.4 times greater odds of fast food intake than those who did not report financial strain ( ci = 1.01 and 1.93 ) . women who were married or lived with a partner had 0.4 reduced odds of consuming fast food ( ci = 0.21 and 0.85 ) , when compared to those without a spouse or live - in partner . in adjusted analyses , we did not detect racial / ethnic differences in sugar - sweetened beverage intake . compared to those without young children at home , women with a child under age 5 at home were 3.0 times more likely to drink sugar - sweetened beverages once or more weekly ( ci = 1.54 and 6.00 ) . married women and those living with a partner had reduced odds of drinking sugar - sweetened beverages ( or = 0.30 and ci = 0.13 , 0.68 ) compared with unmarried and single women . lastly , women aged 35 years or older had lower odds of sugar - sweetened beverage intake when compared to women 1824 ( ci = 0.15 and 0.99 ) . in stratified analyses , we did not detect racial / ethnic differences in fast food intake among obese women . however , nonobese black women had 4.66-fold greater odds of fast food intake once or more weekly , when compared to nonobese white women ( ci = 1.49 and 14.5 ) . there were no significant racial / ethnic differences in sugar - sweetened beverage intake in the stratified obese or nonobese subgroups . results were otherwise very similar to those seen in the analysis of the entire cohort . the first sensitivity analysis , in which we excluded postpartum women and examined the adjusted odds of fast food and sugar - sweetened beverage intake , showed that black women had 2.6 times higher odds of fast food intake once or more weekly when compared with white women ( ci = 1.10 and 6.06 ) , confirming our findings from the entire sample . in the second sensitivity analysis , we assessed a daily cut - point for sugar - sweetened beverage intake , comparing the 18% of our sample that reported daily versus nondaily sugar - sweetened beverage intake . even with this different cut - point , we confirmed a null association between race and ethnicity and the odds of drinking one or more sugar - sweetened beverages daily . in a cross - sectional analysis of 212 pregnant and postpartum women , 47.7% of women reported eating fast food one or more times weekly , but only 1.9% consumed it one or more times daily . in contrast , 59% reported drinking one or more sugar - sweetened beverages per week , with 15.6% drinking at least one can daily . we found significant racial and ethnic differences in fast food , but not sugar - sweetened beverage , intake . black women had 2-fold greater odds of fast food intake once or more weekly when compared with white women . the increased strength of the association among nonobese black women was interesting in light of previous studies demonstrating that normal and overweight women are at greater risk of excess gestational weight gain than obese women [ 2 , 16 ] . this emphasizes the need for inclusion of nonobese women in discussions around dietary habits , healthy gestational weight gain , and postpartum weight loss . our data provides new information about racial differences in dietary behaviors and highlights the need for interventions to target obesogenic dietary behaviors in pregnancy and postpartum , as failure to lose gestational weight during the first year postpartum is associated with worsened cardiovascular risk markers and overweight at 15 years postpartum [ 32 , 33 ] . marriage or living with a partner was associated with reduced odds of both fast food and sugar - sweetened beverage intake [ 3436 ] . our finding may represent increased financial means , improved social support , or factors not measured in our study . this data adds new information to existing literature on marriage and pregnancy related health behaviors . prior data has shown decreased use of tobacco and drugs during pregnancy , increased prenatal care , and improved pregnancy outcomes among women who have a good relationship with the father of their child , when compared to those without such a relationship . financial strain was associated with increased odds of both fast food and sugar - sweetened beverage intake when compared with women who did not report financial strain . this finding was expected given that healthier foods are often more expensive and less available in lower income neighborhoods lacking a grocery store with healthy food options . fast food and sugar - sweetened beverages may represent less expensive alternatives to grocery purchased food for low - income families . the finding that women having children under age 5 in the home were more likely to drink sugar - sweetened beverages was counter - intuitive . one possible explanation might be parental fatigue , leading to higher intake of caffeinated beverages that contain sugar . another possible explanation might be having the sugary drinks on hand for children , leading to increased intake on the part of the parent . the findings of increased fast food intake among women 3034 and decreased sugar - sweetened beverage intake per month among women 35 and over were unexpected . while the majority of data on postpartum weight loss interventions have focused on middle class white women [ 3740 ] , studies in the general population have shown that culturally tailoring interventions can result in significant weight loss in low - income and racial and ethnic minority groups [ 4144 ] . concern exists , however , that black women lose less weight than their white counterparts and drop - out rates for all participants remain high [ 40 , 46 ] . several qualitative studies have examined barriers to , and facilitators of , healthy lifestyles in pregnant and postpartum populations , and these results should be considered when designing dietary interventions [ 4749 ] . our results highlight the need to address sugar - sweetened beverage intake in all pregnant and postpartum women , while specifically targeting reduction in fast food intake among black women . a successful approach to changing high risk dietary behaviors among black women could involve recruitment from trusted community sources , such as churches and community centers , which have been shown to be helpful in weight loss . further study would be needed to see if this improves recruitment as well as retention in interventions . educational interventions , such as nutritional seminars or guided grocery shopping , could be practical methods to address educational barriers and enhance awareness of what constitutes a healthy diet and healthy body weight and healthy available alternatives to fast food [ 12 , 51 , 52 ] . finally , cultural adaptations of dietary recommendations have resulted in weight loss among african americans in nonpregnant populations [ 53 , 54 ] . further study is needed to determine what specific cultural adaptations would be most helpful in changing dietary habits among pregnant and postpartum women . in addition to interventions specifically for black women , we would also recommend broad interventions that would improve nutrition among all pregnant and postpartum women , regardless of race or ethnicity . these would include interventions to address time constraints and lack of social support [ 41 , 55 , 56 ] , as well as family preferences . this format could reinforce person educational activities and serve as a forum for recipe sharing and meal planning and provide ongoing motivation and peer support . concurrent policy initiatives should be employed to complement the clinical interventions , ensuring access to healthier , more affordable foods [ 5760 ] and decreasing access to unhealthy foods such as sugar - sweetened beverages and fast food through taxation as well as insurance reimbursement for successful weight loss programs [ 6265 ] . the major strengths of our study were in the diverse sample of black , hispanic , and white participants to evaluate racial and ethnic differences , while controlling for key socioeconomic variables and preconception bmi . some groups may have been under- or overrepresented as a result of not using probability samples . we are unable to report how many women declined to participate or how those who chose to participate differ from those who did not . this can introduce bias ; however , this also allowed us to evaluate real - world clinical populations . higher percentages of hispanic ( 41% ) and black ( 6.5% ) women did not answer the self - report question pertaining to preconception weight and height , as compared to 1.9% of white women . hispanic women were also less likely to be insured or have a pcp , which may lead to a lack of knowledge of weight and preconception medical diagnoses . these results suggest that the preconception rates of overweight and obesity may be higher in both groups than reported . to address this limitation , we compared our results to national survey data on obesity for women aged 2039 years and found similar obesity rates for black and white women but underestimated rates for hispanic women , likely as a result of missing data . likewise , incomplete responses to certain socioeconomic variables limited their use as covariates in our study . notably 36.5% of black women , 23.2% of hispanic women , and 11.1% of white women declined to answer the question about income . we found significantly increased odds of fast food intake among pregnant and postpartum black women when compared to white women , with an even stronger association among nonobese black women . these results suggest the need for nutritional counseling about fast food intake targeted at black women , including nonobese women , and about sugar - sweetened beverage intake in all women .

introduction . obesity is common among reproductive age women and disproportionately impacts racial / ethnic minorities . our objective was to assess racial / ethnic differences in obesity - related dietary behaviors among pregnant and postpartum women , to inform peripartum weight management interventions that target diverse populations . methods . we conducted a cross - sectional survey of 212 black ( 44% ) , hispanic ( 31% ) , and white ( 25% ) women , aged 18 , pregnant or within one year postpartum , in hospital - based clinics in baltimore , maryland , in 2013 . outcomes were fast food or sugar - sweetened beverage intake once or more weekly . we used logistic regression to evaluate the association between race / ethnicity and obesity - related dietary behaviors , adjusting for sociodemographic factors . results . in adjusted analyses , black women had 2.4 increased odds of fast food intake once or more weekly compared to white women ( ci = 1.08 , 5.23 ) . there were no racial / ethnic differences in the odds of sugar - sweetened beverage intake . discussion . compared with white or hispanic women , black women had 2-fold higher odds of fast food intake once or more weekly . black women might benefit from targeted counseling and intervention to reduce fast food intake during and after pregnancy .

there is convincing evidence that abdominal hysterectomy is associated with a less favourable outcome compared with a vaginal or laparoscopic approach . when an abdominal approach is chosen , the woman will experience reduced quality of life , a longer time before she returns to normal activity , and a longer hospital stay . the risk of complications such as wound infections is higher and the cosmetic result after abdominal hysterectomy is less favourable . despite this , abdominal hysterectomy is still very common . in a cross - section analysis of 518 828 hysterectomies in the united states from 2005 , 64% of the hysterectomies were performed abdominally whereas 14% were performed laparoscopically and 22% vaginally . in the national guidelines for norwegian gynecologists published by the norwegian gynecological association , no route or approach of hysterectomy is recommended . there are differences in how hysterectomies are managed in different hospitals throughout the country . in an audit evaluating the route of hysterectomy in all norwegian hospitals in women suffering from abnormal uterine bleeding and/or leiomyoma , significant variations in the percentage of abdominal hysterectomies between the hospitals were found . a reduction of the overall percentage of abdominal hysterectomies from 75% in 2003 to 62% in 2006 was found . in general , hospitals with low patient volumes tended to perform more abdominal hysterectomies compared to larger hospitals . in some small hospitals , the percentage of abdominal hysterectomy was 100% , whereas in the largest hospital , the percentage of abdominal hysterectomy performed by laparotomy was about 20% . this indicates that techniques for minimal invasive hysterectomies have not yet been adapted by most gynecologists in smaller departments , although the advantages and technique of such procedures have been frequently reported and described by colleagues from larger hospitals during the last decade . our hospital is a county hospital in norway , where approximately 120 hysterectomies are performed each year . our main route of hysterectomy has historically been abdominally . in order to perform less laparotomies during hysterectomy , laparoscopic supracervical hysterectomy was introduced in 2003 . despite this , the percentage of abdominal hysterectomy did not decrease . the percentage of abdominal hysterectomy remained above 50% until 2009 whereas a slight decrease in the percentage of vaginal hysterectomy from 30% to about 20% was seen . in order to reduce the number of abdominal hysterectomies , total laparoscopic hysterectomy was then introduced in april 2010 resulting in a shift towards a laparoscopic approach . the hospital policy was altered and laparoscopic hysterectomy , total or supracervical , was chosen to be the primary route of hysterectomy . in 2012 the percentage of hysterectomy performed laparoscopically was 69.8% whereas the percentage of vaginal and abdominal hysterectomy had declined to 12.8% and 17.4% , respectively . the aim of this study is to present how a shift towards minimal invasive hysterectomy was achieved in a relatively small gynecological department and present the results of the first 58 total laparoscopic hysterectomies . an experienced laparoscopic surgeon was invited to the hospital and he participated in the first procedures . the procedure was introduced to some consultants who performed all lsh procedures at our hospital for the first five years . prior to the first total laparoscopic hysterectomy ( tlh ) , excessive laparoscopic suture training was performed at a minimal access therapy training unit ( mattu ) where proper suture skills were learned from experienced laparoscopic surgeons . the two first authors performed further surgery , both with large experience in laparoscopy on an intermediate level including laparoscopic supracervical hysterectomy , salpingooophorectomy , cystectomy , and laparoscopic treatment of ectopic pregnancies and moderate endometriosis as well as single - port laparoscopy . total laparoscopic hysterectomy was performed in case of atypical endometrial hyperplasia and endometrial cancer , previous cervical intraepithelial neoplasia , and on patient request . only figo stage 1 endometrial cancer is treated in our unit , as more severe stages are referred to a university hospital for treatment according to national standards . lsh was introduced to the other consultants in 2012 making the department less vulnerable to doctors ' accessibility . if there was any preoperative uncertainty whether a planned hysterectomy could be performed by a laparoscopic approach , the case was discussed with the consultants with the largest laparoscopic experience . by placing a focus on minimal invasive hysterectomy , the threshold for performing an abdominal hysterectomy was increased . in case of a prolapsed uterus where a hysterectomy was indicated preoperatively , all women were given paracetamol 1.5 grams , diclofenac 100 mg , and oxycodone 10 mg as a single dose oral analgesics . under general anaesthesia and endotracheal intubation the patients were positioned in the dorsal lithotomy position with both legs supported in allen stirrups and their arms resting alongside the body . an orogastric tube was placed to decompress the stomach , a foley catheter was placed in the bladder , and 5 ml of 0.5% bupivacaine hydrochloride ( marcain ) was injected as local anaesthetic before an incision was made in the umbilicus . carbon dioxide was insufflated through this port with a pressure set at 12 mm hg . 5 ml of 0.5% bupivacaine hydrochloride ( marcain ) was injected at each of the places for three additional ports , versaport bladeless trocars ( covidien , norwalk connecticut , usa ) , placed in the lower right and left quadrant and in the midline , approximately 2 cm above the symphysis . prior to the total laparoscopic hysterectomies , a uterus manipulator ( colpo - probe vaginal fornix delineator ) was installed in the vagina . the bladder peritoneum was opened with a harmonic scalpel , and the bladder was pushed down over the uterus manipulator . the round ligament was divided with the harmonic scalpel and the peritoneum was opened downwards towards the bladder . the suspensory ligament of the ovaries was divided if the adnexa were removed ; otherwise the uteroovarian ligament and the proximal tube were divided . the uterus was removed through the vagina and the vaginal vault was sutured with three laparoscopic sutures polysorb 0 , extracorporeal suturing . for the laparoscopic supracervical hysterectomies , bipolar current and cold scissors were used to divide the uterus from its attachments including the uterine artery . morcellation was performed through the lower left quadrant using different reusable morcellators ( gynecare morcellex , ethicon and wisap , wisap medical technology gmbh ) and a single use morcellator ( lina xcise , lina medical ) . for both procedures the fascia at the accessory ports was sutured with polysorb 0 using the endoclose hook ( covidien , norwalk connecticut , usa ) if the incisions were larger than 10 mm . the fascia in the umbilicus was closed with the earlier placed suture and the skin was closed using abrupt intracutaneous caprosyn 3 - 0 sutures and/or steristrips . demographics and clinical data were collected from the patient 's medical record after the total laparoscopic hysterectomies and a retrospective analysis was performed . variables include bmi , parity , indication for hysterectomy , complications , and conversion to laparotomy . to evaluate possible risk factors for complications , a registration of previous abdominal surgery , intra - abdominal adhesions , and previous conisation due to cervical intraepithelial neoplasia ( cin ) were also performed . the data of complications was collected at least three months after surgery to ensure inclusion of possible late complications related to the performed surgery . the percentage of abdominal hysterectomy remained high ( 5070% ) until total laparoscopic hysterectomy was introduced in 2010 ( figure 1 ) . from 2010 , a significant decline in the percentage of abdominal hysterectomies occurred , resulting in only 17.4% abdominal hysterectomies in 2012 . the percentage of laparoscopic supracervical hysterectomy varied from 6.8% to 20.8% in the period from 2004 to 2009 . in 2010 the laparoscopic group included both lsh and tlh resulting in a remarkable increase in the total number of laparoscopic hysterectomies to 69.8% in 2012 . parallel with the increase in laparoscopic hysterectomies , a decrease in vaginal hysterectomies from 29.4% to 12.8% was observed . a total of 58 tlh procedures were performed from april 2010 to december 2012 . the majority of these procedures were performed on benign indications , where abnormal vaginal bleeding was the most common indication ( table 1 ) . the overall bmi was 27.0 ( sd 6.4 ) whereas a bmi of 31.6 ( sd 7.6 ) was found in women with endometrial cancer . no major complications such as major bleeding , bowel or urinary tract injury or vaginal cuff dehiscence occurred . the first patient was a 40-year - old woman who had a tlh performed due to uterine leiomyoma . during the operation , excision of endometriosis on the pelvic sidewall was performed . she was readmitted six weeks after the operation with abdominal pain that had occurred after sexual intercourse . a vaginal ultrasound was performed showing a 3 7 cm vaginal vault hematoma . the second case of a possible vaginal vault hematoma occurred in a 46-year - old woman who underwent tlh because of menorrhagia . the following day , a vaginal ultrasound performed by a consultant gynecologist showed no sign of a hematoma . one woman experienced a urinary tract infection together with a superficial wound infection at the suprapubic trocar incision site . we have described a successful change from abdominal to minimal invasive hysterectomy in a relatively small county hospital . a total change of department policy , dedicated gynecologists , and competence to perform both supracervical and total laparoscopic hysterectomies seemed necessary to achieve a shift from abdominal to laparoscopic hysterectomies . despite the recommendations in the literature , a large amount of the hysterectomies in many western countries is still performed abdominally . gynecologists are aware of the recommendations , but they still perform most hysterectomies through the not recommended abdominal route . senior gynecologists have performed hysterectomies through this route for years and they probably feel comfortable with this technique . secondly , lack of laparoscopic competence by senior gynecologists prevents trainees from learning minimal invasive procedures that over time may cause this unfavourable trend to be continued . thirdly , training courses in minimal invasive procedures are not that well established by the gynecological associations in many countries . it is consequently difficult and requires personal engagement as well as support from the hospitals and departments to achieve the needed competence to alter surgical praxis . since then , the laparoscopic approach for hysterectomy has been adopted in many university hospitals and minimal invasive units . however , the rate of abdominal hysterectomy remains high , indicating that the general adaption of laparoscopic hysterectomy among gynecologists is still disappointingly low . our study shows that change towards a laparoscopic approach even in smaller county hospitals is possible ; however it requires dedicated gynecologists who want to change the current practice . when laparoscopic supracervical hysterectomy was introduced in 2003 , the department possessed the knowledge to perform minimal invasive hysterectomies , but that is probably not sufficient if proper dedication is missing . generally , it could be assumed that it would be more difficult to introduce tlh rather than lsh , because tlh is a more advanced procedure that requires laparoscopic suture skills . but , as shown in our study , the complexity of the procedure was not the preventive issue counteracting further increase in the percentage of laparoscopic hysterectomies . it could be argued why not vaginal hysterectomy was established as the preferred route of hysterectomy in place of laparoscopic hysterectomy . as the cochrane review recommends , vaginal hysterectomy is the route of choice . on the other hand , if the complete field of gynecological surgery is evaluated , a large quantity of the operations could be performed laparoscopically . increasing laparoscopic skills by performing supracervical and total hysterectomies may contribute to further development of surgical competence . effort should be made to reduce the percentage of hysterectomies performed abdominally as the patients will benefit from a shift towards a less invasive procedure . it is possible for a county hospital to alter their praxis and perform mini invasive hysterectomies , but it requires dedicated gynecologists . this change to an advanced procedure like total laparoscopic hysterectomy could be achieved without patients suffering from major complications .

objective . to describe the route of hysterectomy in a county hospital and evaluate the shift towards a minimal invasive approach . design . retrospective cohort study . setting . a county hospital in norway . population . all women were scheduled for hysterectomy . methods . audit the route of hysterectomy in the period 20042012 . analyze the outcome of total laparoscopic hysterectomies . main outcome measures . complications after total laparoscopic hysterectomy . results . a shift towards a minimal invasive approach has been achieved during the study period . in 2012 only 17.4% of the hysterectomies were performed abdominally , compared to yearly percentages of above 50% in the period 20042009 . laparoscopic supracervical hysterectomy was introduced in 2003 , but the percentage of abdominal hysterectomy remained above 50% until total laparoscopic hysterectomy was introduced in 2010 . since the introduction of total laparoscopic hysterectomy in april 2010 , 58 procedures have been performed . there have been no major complications . two vaginal vault hematomas and one case of urinary tract infection were reported . conclusions . it is possible for a county hospital to alter their praxis and perform mini - invasive hysterectomies , but it requires dedicated gynecologists . this change to an advanced procedure like total laparoscopic hysterectomy could be achieved without patients suffering from major complications .

all m. smegmatis strains were isogenic to mc155 and were grown at 37 c in lb supplemented with 0.5% dextrose , 0.5% glycerol , and 0.05% tween 80 ( broth ) . for immunoprecipitation of card , rnap , and rnap , a card merodiploid strain was produced by integrating pmsg430smcard - ha ( constitutively expresses m. smegmatis c - terminal ha tagged card , kanamycin resistant ) into the attb site of m. smegmatis mc155 . allelic exchange experiments were performed with the card merodiploid strain using a dna donor sequence with homology to mc155 nucleotides 6141480 to 6142268 and 6140266 to 6141010 to delete all of the card gene except the nucleotides encoding the first 10 and last 3 amino acids from the endogenous locus , generating card attb::tetcard - ha . for immunoprecipitation of unfused ha peptide as a control , mc155 was transformed with pmsg431 , which integrates into the attb site of the genome and constitutively expresses ha peptide . cultures of m. smegmatis card attb::tetcard - ha and mc155 attb::pmsg431 strains were grown to late log phase ( od600 = ~ 1 ) before adding a final concentration of 2% formaldehyde and shaking at room temperature for 30 min to crosslink dna and proteins . the crosslinking was quenched by the addition of 0.25 ml of 2.5 m glycine per 5 ml of culture and incubated 5 min at 25 c with shaking . 5 ml ( ~ 2.5 10 mycobacterial cells ) of each culture was then collected by centrifugation . the cells were washed once with te and resuspended in 100 l of te supplemented with roche complete protease inhibitor cocktail . the cell suspension was lysed using a covaris focused - ultrasonicator so that the genomic dna was sheared into ~ 100 base pair ( bp ) fragments , as assessed by dna gel electrophoresis . the use of the covaris focused - ultrasonicator was critical for this step and other sonicator systems were unable to yield a comparable consistency and homogeneity of dna fragment distribution . the cell debris was spun down and the lysate was added to 400 l chip lysis buffer ( 50 mm hepes - koh [ ph 7.5 ] , 140 mm nacl , 1 mm edta , 1% triton x-100 ) plus roche complete protease inhibitor cocktail . protein nucleic acid complexes containing card - ha were immunoprecipitated from the m. smegmatis mc155 card attb::tetcard - ha strain cell lysate by adding 50 l of anti - ha agarose ( sigma ) . complexes containing unfused ha were immunoprecipitated from the mc155 attb::pmsg431 strain with the same anti - ha agarose . rnap and were immunoprecipitated from card attb::tetcard - ha with monoclonal antibodies specific for these subunits ( neoclone ; 8rb13 for , 2g10 for ) immobilized on gammabind g sepharose ( ge healthcare life sciences ) . each immunoprecipitation was performed in duplicate from two separate cultures , thus comprising two biological replicates . however , one of the rnap samples was lost during library preparation and , therefore , there is only data for one rnap replicate . the antibody matrix was washed 2 with chip lysis buffer , 2 with chip lysis buffer plus an additional 360 mm nacl , 2 with chip wash buffer ( 10 mm tris - hcl ph 8.0 , 250 mm licl , 0.5% np-40 , 0.5% sodium deoxycholate , 1 mm edta ) , and 2 with te , each time by rotating for 10 min at 4 c . complexes that co - precipitated with the respective antibody matrix were eluted twice by adding 100 l of chip elution buffer ( 50 mm tris - hcl ph 8.0 , 10 mm edta , 1% sds ) , incubating for 10 min at 65 c with agitation , spinning down the antibody matrix , and transferring the eluate to a new tube . wash and elution buffers were all supplemented with roche complete protease inhibitor cocktail . to reverse the crosslinks 15 l of each sample was removed for western blot analysis of proteins , while 100 g / ml of proteinase k was added to the rest of each sample and incubated at 37 c for 2 h before isolating nucleic acid by chloroform phenol extracting 2 times , ethanol precipitating and resuspending the dna pellet in 34 l of water . co - precipitated dna was sequenced using an ab solid 4 high - throughput genome sequencer ( life technologies ) and a 50 bp read length , which provided sufficient reads for over 100-fold coverage of the genome in each sample , wherein the m. smegmatis genome is 6,988,209 bp in length and the coverage of each sequencing reaction was over 800 mbp . table 1 shows the total number of reads and number of mapped reads for each sample . specifically , the normalized coverage ( or counts ) was determined by multiplying the raw ( sequenced ) coverage ( or counts ) in each sample by that sample 's size factor . the size factors are determined by taking the median of the ratios of observed counts . the normalized number of sequence reads per base pair was then expressed as a log2 value . if a read mapped with equal quality at multiple loci ( but not more than 3 ) , its contribution was distributed evenly among them . for example , the sequences of the 16s , 23s , and 5s ribosomal rna are identical in the m. smegmatis rrna and rrnb operons . therefore , the total number of reads for those sequences was split equally between the operons . if the number of mapping loci was higher than 3 , the read was discarded . the normalized number of reads for each base pair was saved as a wig file for each sample . we first determined how well replicate samples of the distribution of a given protein correlated to each other and how well the distribution of card correlated to the distributions of rnap and rnap ( table 2 , table 3 ) . the correlations were obtained by computing the pearson correlation of the genomic coverage profiles of each pair of samples . the coverage profiles were computed by summing the contributions of all mapped fragments , assuming they were 100 bp long , and then , in 20-bp steps along the entire genome , computing the average coverage of the surrounding 100-bp window . these data showed that individual replicates for a single immunoprecipitation condition correlated highly with one another ( bolded in table 2 ) and indicated that the distribution of card - ha or rnap was consistent between biological replicates . this consistency between replicates allowed us to average the pearson correlation values for each comparison to simplify the comparisons between immunoprecipitation conditions ( table 3 ) . the correlation between the distribution of card - ha and the distribution of rnap ( bolded in table 3 ) was almost as high as the correlation between the two card - ha replicates , indicating that the distribution of card - ha is very similar to that of rnap . to directly compare the genome distributions of card - ha , rnap , and rnap , the reads per base pair from the unfused ha peptide sample served as the background control and were subtracted from the other datasets . the rationale for this control was that as a non - dna binding protein , the ha peptide should be diffusely localized throughout the cell and serve as a readout for the background levels of nonspecific crosslinking to the dna . the normalized , background - corrected log2 reads per base pair were then smoothed over a 20-bp window and rnap and card - ha peaks were identified as described previously , . briefly , maxima and minima were assigned as inflection points where the values 10 bp were both lower or both higher , respectively . maxima within 20 bp were merged with the peak location assigned to the maximum with the highest absolute signal value . peaks were divided into 0.1 interval bins of peak heights with a lower cutoff of peak height of 0.4 log2 reads per base pair . starting with the lowest bin , we then calculated the distance of each peak to the nearest gene start and compared these distances to those computed using genome coordinates arbitrarily rotated 1 10 bp around the m. smegmatis genome . using the wilcoxon mann whitney ranksum test for nonsimilarity of distributions , , rnap peaks in the 1.11.2 peak - height bins and card - ha peaks in the 0.50.6 peak - height bins were statistically significant ( p values for similarity of the distributions < 0.0001 ) . in other words , for each peak - height bin , the two lists of peak - to - gene start distances ( actual and rotated by 1 10 bp ) were tested for whether they were from different populations using the wilcoxon mann peaks in the lowest peak - height bin for which peak - to - gene start distances differed from random with a p value of < 0.0001 plus all peaks with greater heights were then used as the statistically significant peaks . we then identified rnap peaks associated with each gene as the closest rnap peak upstream from the gene start and card - ha peaks associated with the rnap peaks as the closest card - ha peak to each selected rnap peak . to calculate average chip signals for the aggregate profiles ( fig . 1 ) , we selected a subset of 62 genes meeting the following criteria : ( i ) 300 bp in gene length , ( ii ) average rnap log2 chip signal 1.6/bp , ( iii ) associated with an rnap peak with log2 chip signal 3/bp , ( iv ) absence of other rnap peaks within 500 bp upstream or 1000 bp downstream of the associated rnap peak , ( v ) absence of an oppositely oriented gene with an average rnap log2 chip signal 1 upstream from the gene ( because an oppositely oriented gene could create a divergent promoter region with potential for overlapping rnap and card - ha chip signals ) , and ( vi ) absence of an upstream gene with average rnap log2 chip signal > 0 within 100 bp upstream from the gene ( because such an arrangement would indicate the gene is an internal member of an operon ) . the rnap , card - ha , and rnap signals from the 62 genes were then averaged using the distance from the center of the associated peaks to align the genes ( fig . 1 ) . for the gene alignments , the distance from the center of the associated peak served as a proxy for the transcriptional start site , since most transcriptional start sites are not mapped in m. smegmatis . this analysis showed that whereas rnap was found throughout transcribed regions of the genome , card - ha and rnap were primarily associated with promoter regions . these data matched the high correlation calculated for the distribution of card - ha and rnap ( table 2 , table 3 ) . levels of both card - ha and rnap dropped off immediately following the promoter sequences , suggesting that these proteins are lost from the rnap elongating complex after transcription initiation . the colocalization of rnap and card - ha led us to propose that in vivo , card associates with rnap initiation complexes at most promoters and is therefore a global regulator of transcription initiation . further analysis of the dataset also revealed that card was never present on the genome in the absence of rnap , suggesting that it may be targeted to the genome through its interaction with rnap . all m. smegmatis strains were isogenic to mc155 and were grown at 37 c in lb supplemented with 0.5% dextrose , 0.5% glycerol , and 0.05% tween 80 ( broth ) . for immunoprecipitation of card , rnap , and rnap , a card merodiploid strain was produced by integrating pmsg430smcard - ha ( constitutively expresses m. smegmatis c - terminal ha tagged card , kanamycin resistant ) into the attb site of m. smegmatis mc155 . allelic exchange experiments were performed with the card merodiploid strain using a dna donor sequence with homology to mc155 nucleotides 6141480 to 6142268 and 6140266 to 6141010 to delete all of the card gene except the nucleotides encoding the first 10 and last 3 amino acids from the endogenous locus , generating card attb::tetcard - ha . for immunoprecipitation of unfused ha peptide as a control , mc155 was transformed with pmsg431 , which integrates into the attb site of the genome and constitutively expresses ha peptide . cultures of m. smegmatis card attb::tetcard - ha and mc155 attb::pmsg431 strains were grown to late log phase ( od600 = ~ 1 ) before adding a final concentration of 2% formaldehyde and shaking at room temperature for 30 min to crosslink dna and proteins . the crosslinking was quenched by the addition of 0.25 ml of 2.5 m glycine per 5 ml of culture and incubated 5 min at 25 c with shaking . 5 ml ( ~ 2.5 10 mycobacterial cells ) of each culture was then collected by centrifugation . the cells were washed once with te and resuspended in 100 l of te supplemented with roche complete protease inhibitor cocktail . the cell suspension was lysed using a covaris focused - ultrasonicator so that the genomic dna was sheared into ~ 100 base pair ( bp ) fragments , as assessed by dna gel electrophoresis . the use of the covaris focused - ultrasonicator was critical for this step and other sonicator systems were unable to yield a comparable consistency and homogeneity of dna fragment distribution . the cell debris was spun down and the lysate was added to 400 l chip lysis buffer ( 50 mm hepes - koh [ ph 7.5 ] , 140 mm nacl , 1 mm edta , 1% triton x-100 ) plus roche complete protease inhibitor cocktail . protein nucleic acid complexes containing card - ha were immunoprecipitated from the m. smegmatis mc155 card attb::tetcard - ha strain cell lysate by adding 50 l of anti - ha agarose ( sigma ) . complexes containing unfused ha were immunoprecipitated from the mc155 attb::pmsg431 strain with the same anti - ha agarose . rnap and were immunoprecipitated from card attb::tetcard - ha with monoclonal antibodies specific for these subunits ( neoclone ; 8rb13 for , 2g10 for ) immobilized on gammabind g sepharose ( ge healthcare life sciences ) . each immunoprecipitation was performed in duplicate from two separate cultures , thus comprising two biological replicates . however , one of the rnap samples was lost during library preparation and , therefore , there is only data for one rnap replicate . the antibody matrix was washed 2 with chip lysis buffer , 2 with chip lysis buffer plus an additional 360 mm nacl , 2 with chip wash buffer ( 10 mm tris - hcl ph 8.0 , 250 mm licl , 0.5% np-40 , 0.5% sodium deoxycholate , 1 mm edta ) , and 2 with te , each time by rotating for 10 min at 4 c . complexes that co - precipitated with the respective antibody matrix were eluted twice by adding 100 l of chip elution buffer ( 50 mm tris - hcl ph 8.0 , 10 mm edta , 1% sds ) , incubating for 10 min at 65 c with agitation , spinning down the antibody matrix , and transferring the eluate to a new tube . wash and elution buffers were all supplemented with roche complete protease inhibitor cocktail . to reverse the crosslinks , 15 l of each sample was removed for western blot analysis of proteins , while 100 g / ml of proteinase k was added to the rest of each sample and incubated at 37 c for 2 h before isolating nucleic acid by chloroform phenol extracting 2 times , ethanol precipitating and resuspending the dna pellet in 34 l of water . co - precipitated dna was sequenced using an ab solid 4 high - throughput genome sequencer ( life technologies ) and a 50 bp read length , which provided sufficient reads for over 100-fold coverage of the genome in each sample , wherein the m. smegmatis genome is 6,988,209 bp in length and the coverage of each sequencing reaction was over 800 mbp . table 1 shows the total number of reads and number of mapped reads for each sample . specifically , the normalized coverage ( or counts ) was determined by multiplying the raw ( sequenced ) coverage ( or counts ) in each sample by that sample 's size factor . the size factors are determined by taking the median of the ratios of observed counts . the normalized number of sequence reads per base pair was then expressed as a log2 value . if a read mapped with equal quality at multiple loci ( but not more than 3 ) , its contribution was distributed evenly among them . for example , the sequences of the 16s , 23s , and 5s ribosomal rna are identical in the m. smegmatis rrna and rrnb operons . therefore , the total number of reads for those sequences was split equally between the operons . if the number of mapping loci was higher than 3 , the read was discarded . the normalized number of reads for each base pair was saved as a wig file for each sample . we first determined how well replicate samples of the distribution of a given protein correlated to each other and how well the distribution of card correlated to the distributions of rnap and rnap ( table 2 , table 3 ) . the correlations were obtained by computing the pearson correlation of the genomic coverage profiles of each pair of samples . the coverage profiles were computed by summing the contributions of all mapped fragments , assuming they were 100 bp long , and then , in 20-bp steps along the entire genome , computing the average coverage of the surrounding 100-bp window . these data showed that individual replicates for a single immunoprecipitation condition correlated highly with one another ( bolded in table 2 ) and indicated that the distribution of card - ha or rnap was consistent between biological replicates . this consistency between replicates allowed us to average the pearson correlation values for each comparison to simplify the comparisons between immunoprecipitation conditions ( table 3 ) . the correlation between the distribution of card - ha and the distribution of rnap ( bolded in table 3 ) was almost as high as the correlation between the two card - ha replicates , indicating that the distribution of card - ha is very similar to that of rnap . to directly compare the genome distributions of card - ha , rnap , and rnap , the reads per base pair from the unfused ha peptide sample served as the background control and were subtracted from the other datasets . the rationale for this control was that as a non - dna binding protein , the ha peptide should be diffusely localized throughout the cell and serve as a readout for the background levels of nonspecific crosslinking to the dna . the normalized , background - corrected log2 reads per base pair were then smoothed over a 20-bp window and rnap and card - ha peaks were identified as described previously , . briefly , maxima and minima were assigned as inflection points where the values 10 bp were both lower or both higher , respectively . maxima within 20 bp were merged with the peak location assigned to the maximum with the highest absolute signal value . peaks were divided into 0.1 interval bins of peak heights with a lower cutoff of peak height of 0.4 log2 reads per base pair . starting with the lowest bin , we then calculated the distance of each peak to the nearest gene start and compared these distances to those computed using genome coordinates arbitrarily rotated 1 10 bp around the m. smegmatis genome . using the wilcoxon mann whitney ranksum test for nonsimilarity of distributions , , rnap peaks in the 1.11.2 peak - height bins and card - ha peaks in the 0.50.6 peak - height bins were statistically significant ( p values for similarity of the distributions < 0.0001 ) . in other words , for each peak - height bin , the two lists of peak - to - gene start distances ( actual and rotated by 1 10 bp ) were tested for whether they were from different populations using the wilcoxon mann peaks in the lowest peak - height bin for which peak - to - gene start distances differed from random with a p value of < 0.0001 plus all peaks with greater heights were then used as the statistically significant peaks . we then identified rnap peaks associated with each gene as the closest rnap peak upstream from the gene start and card - ha peaks associated with the rnap peaks as the closest card - ha peak to each selected rnap peak . to calculate average chip signals for the aggregate profiles ( fig . 1 ) , we selected a subset of 62 genes meeting the following criteria : ( i ) 300 bp in gene length , ( ii ) average rnap log2 chip signal 1.6/bp , ( iii ) associated with an rnap peak with log2 chip signal 3/bp , ( iv ) absence of other rnap peaks within 500 bp upstream or 1000 bp downstream of the associated rnap peak , ( v ) absence of an oppositely oriented gene with an average rnap log2 chip signal 1 upstream from the gene ( because an oppositely oriented gene could create a divergent promoter region with potential for overlapping rnap and card - ha chip signals ) , and ( vi ) absence of an upstream gene with average rnap log2 chip signal > 0 within 100 bp upstream from the gene ( because such an arrangement would indicate the gene is an internal member of an operon ) . the rnap , card - ha , and rnap signals from the 62 genes were then averaged using the distance from the center of the associated peaks to align the genes ( fig the distance from the center of the associated peak served as a proxy for the transcriptional start site , since most transcriptional start sites are not mapped in m. smegmatis . this analysis showed that whereas rnap was found throughout transcribed regions of the genome , card - ha and rnap were primarily associated with promoter regions . these data matched the high correlation calculated for the distribution of card - ha and rnap ( table 2 , table 3 ) . levels of both card - ha and rnap dropped off immediately following the promoter sequences , suggesting that these proteins are lost from the rnap elongating complex after transcription initiation . the colocalization of rnap and card - ha led us to propose that in vivo , card associates with rnap initiation complexes at most promoters and is therefore a global regulator of transcription initiation . further analysis of the dataset also revealed that card was never present on the genome in the absence of rnap , suggesting that it may be targeted to the genome through its interaction with rnap . card modulates transcription through its direct interaction with rnap , . to determine at which stage of the transcription cycle ( initiation , elongation , or termination ) card acts , we used chip - seq to survey the distribution of card throughout the m. smegmatis chromosome . our data shows that card is localized to promoters throughout the m. smegmatis genome , indicating that card functions during transcription initiation . despite the previous finding that card has sequence non - specific dna binding activity , the chip - seq experiments also revealed that card was never present on the genome in the absence of rnap or rnap , suggesting that card is targeted to the genome through its interaction with rnap . the chip - seq data for the distribution of rnap also serves as a map of potential promoter elements throughout the m. smegmatis genome , which has never before been experimentally examined . compilation of the chip - seq data and previous microarray expression profiling analyses indicates that card is broadly distributed on promoters of most transcription units regardless of whether they were deregulated during card depletion . there is also the striking correlation between the distributions of card and rnap on the genome , despite the fact that no direct interaction between these proteins has been reported . the factors contributing to the enrichment of card at rnap containing holoenzymes as opposed to elongating rnap core complexes remain unknown and will be a topic of future study . all together , results from these experiments have provided invaluable information that will help direct the ongoing efforts in determining the mechanism of transcription regulation by card . in addition , this work serves as a framework for further investigations into rnap function in mycobacteria .

card is an essential mycobacterial protein that binds the rna polymerase ( rnap ) and affects the transcriptional profile of mycobacterium smegmatis and mycobacterium tuberculosis [ 6 ] . we predicted that card was directly regulating rnap function but our prior experiments had not determined at what stage of transcription card was functioning and at which genes card interacted with the rnap . to begin to address these open questions , we performed chromatin immunoprecipitation sequencing ( chip - seq ) to survey the distribution of card throughout the m. smegmatis chromosome . the distribution of rnap subunits and a were also profiled . we expected that rnap would be present throughout transcribed regions and rnap a would be predominantly enriched at promoters based on work in escherichia coli [ 3 ] , however this had yet to be determined in mycobacteria . the chip - seq analyses revealed that card was never present on the genome in the absence of rnap , was primarily associated with promoter regions , and was highly correlated with the distribution of rnap a . the colocalization of a and card led us to propose that in vivo , card associates with rnap initiation complexes at most promoters and is therefore a global regulator of transcription initiation . here we describe in detail the data from the chip - seq experiments associated with the study published by srivastava and colleagues in the proceedings of the national academy of science in 2013 [ 5 ] as well as discuss the findings from this dataset in relation to both card and mycobacterial transcription as a whole.the chip - seq data have been deposited in the gene expression omnibus ( geo ) database , www.ncbi.nlm.nih.gov/geo ( accession no . gse48164 ) .

a stem rot disease has been observed sporadically on asiatic dayflower at moist , shady forest edges and roadsides in geoje , korea in 2010 ( fig . , stems under the canopy are exposed to warm and highly humid conditions , which are favorable for the development of sclerotial stem rot . the disease started with small water - soaked lesions on the stem , which expanded and rotted . the heavily infected stems became rotted and blighted , and the plant eventually died ( fig . the freshly isolated pathogenic fungus grown on potato dextrose agar ( pda ) was examined microscopically . detailed microscopic examinations of a representative specimen were performed using a light microscope ( axioplan ; carl zeiss , jena , germany ) . small globoid sclerotia formed abundantly on the pda after 10 days of mycelial growth ( fig . the sclerotia , which were relatively uniform in size ( 1~3 mm ) , were white at first and became dark brown gradually . inoculums were prepared with mycelial mats and mixed with autoclaved soil for pathogenicity testing . then , healthy c. the first symptoms appeared at 7 days after inoculation , and typical symptoms developed into water - soaking lesions . then , white mycelia and sclerotia were observed on the rotted areas of stems ( fig . the causal pathogen was re - isolated from the lesions to confirm koch 's postulate . the complete its rdna region of the representative isolate was amplified and sequenced using the its1 ( 5'-tccgtaggtgaacctgcgg-3 ' ) and its4 ( 5'-tcctccgcttattgatatgc-3 ' ) primers as described by white et al . . a phylogenetic analysis was performed using mega4 software ( mega , tempe , az , usa ) , with the neighbor - joining method and the tajima - nei distance model . previously published its sequences of sclerotium rolfsii strains were included for reference . in the phylogenetic tree ( fig . 2 ) , the representative isolate was placed within a clade comprising reference isolates of s. rolfsii . based on symptoms , mycological characteristics , molecular data , and pathogenicity to the host plant , a representative culture of s. rolfsii has been deposited with the korean agricultural culture collection ( kacc 45483 ) , national academy of agricultural science , rural development administration , suwon , korea . to our knowledge , this is the first report of the presence of s. rolfsii on asiatic dayflower in korea . environmental conditions during the summer in korea are similar to subtropical conditions , which are favorable for development of this disease . the recent occurrence of this disease suggests the possibility that s. rolfsii is spreading widely .

stem rot was found for the first time on the asiatic dayflower plant ( commelina communis l. ) in korea . a detailed description of this korean specimen is given , along with its rdna internal transcribed spacer sequence . the fungus was identified as sclerotium rolfsii saccardo based on mycological characteristics and molecular data .

functional use of the upper extremity is fundamental for performing movements required in daily activities . upper extremity function is divided into two categories of motor skills : fine motor skills ( e.g. , feeding , dressing , and grooming ) and gross motor skills ( e.g. , crawling , walking , and postural control)1 . it usually involves a repetitious sequence of limb motions to simultaneously move a body segment and maintain its stability . in older adults , the gross motor skills ( e.g. , for reaching and grasping ) as well as fine motor skills ( e.g. , for manipulation ) are slower and less accurate than in normal young adults2 . therefore , whole - arm coordination , eye - head coordination , visual acuity , and muscle strength are important factors for well - coordinated upper extremity function3 . visual deficits and cognitive impairment are major problems resulting in changes in reach , grasp , and manipulation in community - dwelling older adults . upper extremity function , like other functional activities , is affected by age - related alteration in the visual system4 . many previous studies have indicated that decreased velocity in reaching movements or in repetitive tapping tasks are associated with age - related changes in visual processing4,5,6 . most of these studies used complex methods or equipment , such as kinematic measurement systems , to evaluate the relationship between upper extremity function and visual information4,5,6 . these measurement tools provide qualitative data on this relationship , but do not provide in - depth information on upper extremity function or the relationships among upper extremity function , visual deficits , and cognitive impairment . these tools are able to identify the scope of participants characteristics , and are appropriate for examination of the relationships among visual perceptual function , upper extremity function , and cognitive function . the purpose of this study was to investigate the relationships among visual perceptual function , upper extremity function , and cognitive function in community - dwelling older adults . fifty - eight community - dwelling older individuals , recruited from three senior complex community centers , participated in this study . this convenience sample was recruited using a leaflet that provided information regarding the purposes , procedures , rights of subjects , data usages , and other aspects of this study . the study was carried out in accordance with the international ethical guidelines and declaration of helsinki and was approved by the local institutional review board . the inclusion criteria were as follows : age > 60 years , absence of neurological deficits , absence of severe orthopedic diseases that could impact the procedures , and absence of significant cognitive impairments that would prevent understanding of verbal instructions . the exclusion criteria were as follows : serious visual impairment , dizziness or other vestibular impairment , and a history of severe alcohol or medication abuse . table 1table 1.clinical and demographic characteristics of the study participants ( n=58)variablesnumberpercentilegendermale2034.5female3865.5age ( yrs)616511.766701220.77175915.576801932.881851017.2 > 85712.1medical historyosteoarthritis5594.8hypertension4679.3diabetes4882.8 shows the clinical and demographic characteristics of the participants , including sex , age , and medical history . this was a cross - sectional study , in which the following four clinical tools were used : the mini - mental state examination ( mmse ) , 9 hole pegboard ( 9hp ) , box and block test ( bbt ) , and motor - free visual perception test , third edition ( mvpt-3 ) . two occupational therapists with clinical experience evaluated participants performance on all measurements and followed the standard method for administration of each clinical measure in a small tidy therapy room . the mmse is a valid , reliable , and extensive test of cognitive function ; it was developed as a to screening test for dementia and delirium by folstein and folstein in 1975 . the tool is a 30-point questionnaire , with scores ranging from 0 ( complete cognitive impairment ) to 30 ( no cognitive impairments ) and consists of 10 items grouped into five domains : orientation , registration , attention - calculation , recall , and language7 . the 9hp is a simple , reliable , and valid measurement of finger dexterity . the tool is administered by asking the individual to take pegs from a container , one by one , and place them in holes on a board as quickly as possible . participants must then remove the pegs from the holes , one by one , and replace them back into the container . the time ( seconds ) taken to complete the test is recorded as the score . a stopwatch , running from the moment the participant touches the first peg until the moment the last peg is placed in the container , is used to record the time8 . the participant is asked to grasp one block at a time , transport the block over a partition , and release it into the opposite compartment . the score is the number of blocks carried from one compartment to the other in one minute . the mvpt-3 , developed by chalfant and scheffelin is a representative measurement of visual perception and cognitive skill , the mvpt-3 includes five different areas : spatial relationships , visual discrimination , figure - ground , visual closure , and visual memory . individuals older than age 10 start with the example for item 14 . reliability of mvpt-3 scores was assessed with internal consistency and test - retest stability estimates . internal consistency estimates for the standardization sample ranged from 0.86 to 0.90 for ages 11 to > 85 years10 . the means and standard deviations of the dependent measures were analyzed using descriptive statistics . pearson correlations were used to determine the relationships between manual dexterity , static and dynamic balance , and visual perception . the collected data were analyzed using the pasw version 18.0 for windows ( spss inc . , chicago , il , usa ) , and the significance level was set at p < 0.05 . table 2table 2.mean and standard deviation of clinical outcome measures in this study ( n=58)variablesmeanstandard deviationmini - mental state examination ( scores)25.62.79 hole pegboard ( seconds)right 14.24.0left15.34.3box and block test ( scores)right63.110.6left59.311.8motor - free visual perception test - third edition ( scores)44.38.3 shows the means and standard deviations for the four clinical measures . the mmse correlated significantly and positively with the bbt on the dominant and non - dominant sides , with bbt on the non - dominant side , and with mvpt-3 . the mmse correlated significantly and negatively with 9hp on the dominant and non - dominant sides . the 9hp on the dominant side demonstrated a significantly positive correlation with 9hp on the non - dominant side as well as a significantly negative correlation with mmse and bbt on both the dominant and non - dominant sides . the 9hp on the non - dominant side had a significantly positive correlation with the 9hp on the dominant side and a significantly negative correlation with mmse and bbt on the dominant and non - dominant sides . the bbt on the dominant side had a significantly positive correlation with mmse , bbt on the non - dominant side , as well as with mvpt-3 , and had showed significantly negative correlation with 9hp . the bbt on the non - dominant side had a significantly positive correlation with mmse , with bbt on the dominant side , and with mvpt-3 , and a significantly negative correlation with 9hp , the mvpt-3 correlated significantly with mmse and bbt . however , the mvpt-3 did not correlate significantly with 9hp on either the dominant or non - dominant side ( table 3table 3.relationship among the clinical outcome measures in this study ( n=58)variablesmmse9hp - rt9hp - ltbbt - rtbbt - ltmvpt-3mmse0.4580.4190.4200.4230.7779hp - rt0.4580.9280.3570.4170.1719hp - lt0.4190.9280.3170.4320.148bbt - rt0.4200.3570.3170.9320.341bbt - lt0.4230.4170.4320.9320.322mvpt-30.7770.1710.1480.3410.322p<0.05 ; p<0.01 ; mmse : mini - mental state examination ; 9hp - rt : 9 hole pegboard - right ; 9hp - lt : 9 hole pegboard - left ; bbt - rt : box and block test - right ; bbt - lt : box and block test - left ; mvpt-3 : motor - free visual perception test - third edition ) . p<0.05 ; p<0.01 ; mmse : mini - mental state examination ; 9hp - rt : 9 hole pegboard - right ; 9hp - lt : 9 hole pegboard - left ; bbt - rt : box and block test - right ; bbt - lt : box and block test - left ; mvpt-3 : motor - free visual perception test - third edition this study examined the correlations among visual perception , manual dexterity , and cognitive impairment for community - dwelling older adults . the most important findings are as follows : ( 1 ) visual perceptual function had a negative correlation with the 9hp performance and a positive correlation with the bbt performance ; ( 2 ) visual perceptual function had a positive correlation with cognitive function had ; and ( 3 ) cognitive function showed a negative correlation with the 9hp score and a positive correlation with the bbt score . visual perception is necessary for basic and instrumental activities of daily living ( adls ) as well as for functional activities . previous studies have reported that age - related visual dysfunctions include generally decreased visual acuity and adaptability , reduction of depth perception , and reduction in peripheral vision11 , 12 . because of the close relationship between vision and perception , age - related visual changes influence perceptual abilities in older adults . older individuals demonstrate decreased visual discrimination , impaired figure - ground discrimination , decreased visual memory , decreased spatial relations , decreased pattern recognition , and poor attentional processing13 . visuo - perceptual dysfunction influences functional activities such as basic - adls , instrumental - adls , as well as work and recreational activities14 , 15 . brown et al . reported that the impairment of visual - motor integration in older adults leads to poor balance skills and to a higher incidence of falls and accidents16 . zheng et al . also reported that older adults with visual acuity impairment demonstrated instrumental - adls functional state decline and an increased risk of mortality17 . the results of this study suggest that improved visual perceptual function in older adults is associated with improved manual dexterity . this study also measured cognitive impairment in order to investigate the relationships among visual perception , cognitive function , and manual dexterity . although there is no universal effect of aging on learning , age - related changes in memory , processing ability , attention , and executive functions such as problem solving , mental flexibility , or abstraction are well documented18 . this study demonstrated that cognitive performance can affect manual dexterity in older adults . therefore , visual perceptual impairment and cognitive dysfunction can lead to decline in upper extremity function in older adults . age - related visual perceptual impairment and cognitive dysfunction should be considered in light of their effect on manual dexterity in older adults . the results of this study also suggest that there may be benefits to approaches that involve rehabilitation of upper extremity function , concurrent with visual perception and cognitive training for older adults . future studies are therefore needed to evaluate the therapeutic effects of visual perception and cognitive training programs on upper extremity function in older adults .

[ purpose ] the purpose of the present study was to examine the relationship between visual perceptual function and manual dexterity in community - dwelling older adults . [ subjects and methods ] fifty - eight participants were recruited by convenience sampling from local rehabilitation centers . this was a cross - sectional study that used the following four clinical tools : the mini - mental state examination , 9 hole pegboard , box and block test , and motor - free visual perception test , third edition . [ results ] the motor - free visual perception test , third edition and mini - mental state examination correlated significantly and positively with box and block test , and did correlate significantly and negatively with 9 hole pegboard . [ conclusion ] the results of this study suggest that visual perceptual impairment and cognitive dysfunction may influence manual dexterity in older adults , and rehabilitation of upper extremity function , along with visual perception and cognitive training , may be beneficial for this population .

adenomyosis is one of the most common disorders in women , and it is defined as the presence of endometrial and stromal glands in tissue , which can be accompanied by hyperplasia and hypertrophy of the myometrium ( 110 ) . while the etiology of adenomyosis is unknown , various factors have been found to influence it , such as previous caesarian section , dilation and curettage ( d&c ) , dilation and evacuation ( d&e ) , spontaneous abortion , endometrial hyperplasia , menarche age , parity , and gravidity ( 1 , 3 , 5 , 6 , 11 ) . adenomyosis can be accompanied by pelvic pain , menorrhagia , dysmenorrhea , dyspareunia , painful menstrual cycles , and infertility . however , painful menstrual cycles and infertility are less common in these individuals , and about 35% of them are asymptomatic . diagnosis of this disease is often done through pathology and histology after hysterectomy ( 1 , 2 , 46 , 8 , 10 ) . this incidence of this disease has been reported in in the range of 8.8 to 61.5% of women who had hysterectomies ( 2 ) . several studies have indicated that various factors influence adenomyosis , including previous caesarian section , dilation and curettage ( d&c ) , dilation and evacuation ( d&e ) , spontaneous abortion , endometrial hyperplasia , menarche age , and gravidity . however , the results obtained for the comparative effects of the various risk factors were different ( 14 , 6 ) . in a study on the incidence and investigation of risk factors of adenomyosis in denmark , researchers examined 549 patients and concluded that the incidence of adenomyosis varied from 10 to 18% according to different criteria and that there was no significant relationship between adenomyosis and uterine surgery , such as previous caesarean section , myomectomy , dilation and curettage ( d&c ) , dilation and evacuation ( d&e ) ( 2 ) . another study ( 2004 ) investigated pathology species of 873 patients , and the results indicated that 41.7% of patients had adenomyosis . in this study , no significant relationship was found between adenomyosis and experiences of uterine surgery , such as previous caesarean section , myomectomy , dilation and curettage ( d&c ) , and dilation and evacuation ( d&e ) ; however , experience of uterine surgery ( caesarian section , myomectomy , d&c , and d&e ) is one of the most common factors associated with the incidence of adenomyosis ( 2 ) . in most cases , adenomyosis is asymptomatic in patients and is only diagnosed after hysterectomy ( 12 ) . effective medical treatment has been not introduced for adenomyosis , and some treatments only partially reduce symptoms in patients ( 13 ) . the reported prevalence of adenomyosis has varied significantly among the various studies ( 12 , 1416 ) . also , there is little agreement among the studies concerning the relationship between previous surgery and the incidence of adenomyosis . some reports indicated that there was a strong relationship between previous cesarean section and the incidence of adenomyosis , but others reported no such relationship ( 1 , 2 , 11 , and 14 ) . in iran , where the prevalence of adenomyosis has been reported to be 31.3% , a study was conducted in which researchers examined 41 cases of adenomyosis , and they found that 97% of the patients in the study had undergone a previous cesarean section ( 17 ) . due to the different reports from many parts of the world and the controversy about the risk factors for adenomyosis , we focused our study on the potential relationship between prior uterine surgery and the subsequent incidence of adenomyosis . in this retrospective , cross - sectional study , the data that were necessary for the investigation and analyses were obtained from files of patients who had been hospitalized in the shariati hospital in bandar - abbas , iran , from 2001 through 2011 . in this cross - sectional study , we reviewed the medical records of all women who underwent hysterectomies for benign and non - emergency gynecological conditions this time period . all patients who had a hysterectomy for benign uterine reasons and had positive specimens for pathology were investigated . exclusion criteria of the study included women in whom endometriosis and uterine malignancies were reported in their pathology . the reason they were excluded was that the aim was to study adenomyosis ; therefore , other uterine malignancies were omitted from the study . in order to obtain information on the variables , such as age , gravidity , parity , spontaneous abortion , uterine size according to pelvic examination , experiences of prior hysterectomy , prior uterine surgery , caesarian section , myomectomy , and dilation and curettage ( d&c ) , we referred to the patients files at the shariati hospital in bandar - abbas . in addition , other data , including uterine weight , uterine leiomyoma , and pathologic reports also were obtained from the patients files . this study was approved by ethics committee of hormozgan university of medical sciences , and the confidentiality of the data in the patients files was protected at all times . the data were analyzed by using spss version 16 ( spss , inc . , chicago , il , united states ) , descriptive statistics , the student s t - test , and analysis of variance ( anova ) . in this study , we investigated 201 women who had hysterectomies at the shariati hospital in bandar - abbas from 2001 through 2011 . ten patients were excluded from the study because of incomplete files , so we evaluated 191 patients . the average age of the patients was 51.6612.46 ( ranging from 23 to 85 ) , and the average of the age of patients who had positive pathology was 50.29.6 , while it was 52.5213.8 for those with negative pathology . the student s t - test indicated that there was no statistically significant difference between the patients mean ages and adenomyosis ( p = 0.07 ) . also , the findings of the study indicated that there was not a significant relationship between menarche age and adenomyosis prevalence ( p = 0.75 ) . in addition , the relationship between gravidity and the prevalence of adenomyosis was not statistically significant ( p = 0.7 ) ( table 1 ) . also , no significant relationship was found between parity and adenomyosis prevalence ( p = 0.78 ) . furthermore , there was no significant relationship between uterine size and increased risk of adenomyosis ( p = 0.82 ) ( table 1 ) . in addition , the relationship between rate of abortion and adenomyosis was not statistically significant ( p = 0.74 ) ( table 1 ) . among the 191 patients that we studied , 117 cases ( 61.3% ) had not a history of previous uterine surgery . of the 74 patients ( 38.7% ) with a history of previous uterine surgery , 18 patients ( 9.2% ) had experienced caesarian section ( c / s ) , of which 5 patients ( 2.6% ) had experienced both dilation and curettage ( d&c ) and c / s . with regard to adenomyosis , 72 patients ( 37.7% ) were pathologically positive and 119 patients ( 62.3 percent ) were pathologically negative . seventy - nine cases ( 34.2% ) had hysterectomy due to abnormal uterine bleeding ( aub ) . aub was the only reason for hysterectomy in 74 of those who had hysterectomy due to aub ( table 2 ) . among the patients that we studied , 117 had not experienced surgery , of which 37 ( 31.62% ) were pathologically positive and 80 ( 62.3% ) were pathologically negative in terms of adenomyosis . eighteen cases ( 9.4% ) had a history of previous c / s , of which 12 cases ( 66.66% ) were pathologically positive and 6 cases ( 33.33% ) were pathologically negative in terms of adenomyosis . in addition , it was found that there was a significant relationship between a history of surgery and the positive results for adenomyosis , and cesarean surgery was the most prevalent type of surgery related to adenomyosis according to its higher statistics ( p = 0.01 ) ( table 3 ) . adenomyosis is one of the most common diseases in women , and it can be accompanied by various symptoms , such as dysmenorrhea , menometrorrhagia , dyspareunia , and chronic pelvic pain . since adenomyosis is prevalent , its complications can cause physical and mental problems in women , and it also can influence their fertility and childbearing ability . it seems that prevention is necessary , but the risk factors for adenomyosis must be identified in order to prevent it . it has been found in various studies that several factors can influence adenomyosis , including chronological age , menarche age , gravidity , parity , weight , uterine size , and previous uterine surgery ( 14 ) . the average age of the patients we studied was 50.25 9.66 , which was very close to that of other studies ( 16 , 11 ) . the prevalence of adenomyosis was found to be 37.7% in our study , which was similar to the results of several other studies ( 1 , 6 , and 7 ) . however , prevalence at this level was not found in other studies ( 2 , 4 ) . according to different studies , the prevalence of adenomyosis is variable and can result from differences in the populations that are studied , differences in the diagnostic criteria of adenomyosis , and incorrect diagnoses due to the lack of standard criteria for making such a diagnosis . in our study , there was not a significant relationship between menarche age and adenomyosis . our results were in agreement with those of vercellini in which no significant relationship between menarche age and adenomyosis was found ( 6 ) . the average number of parity in women who had adenomyosis was 6.62.64 , and it was 6.42.51 in women who did not have adenomyosis , thus , there was no a significant relationship between the number of parity and adenomyosis ( p = 0.712 ) . however , different from our results , other studies have demonstrated a significant relationship between parity and increased risk of adenomyosis ( 16 ) . the results of our study were in agreement with the results of bergholt s study , which showed that there was not a significant relationship between parity and adenomyosis ( 2 ) . in our study , there was not a significant relationship between adenomyosis and gravidity ( p = 0.712 ) , but some studies reported different results and concluded that women with adenomyosis had significantly higher gravidity than the group without adenomyosis ( 1 , 11 ) . we found a significant relationship between adenomyosis and prior uterine surgery ( p = 0.03 ) . panganamamul also reported a significant relationship between adenomyosis and prior uterine surgery ( 1 ) . our results were compatible with the results of these studies ; however , there was not a significant relationship between adenomyosis and prior uterine surgery in some studies ( 2 , 11 ) . our study did not show a significant relationship between uterine size and adenomyosis ( p = 0.8 ) , but , in some studies it was indicated that the sizes of the uterus were larger when adenomyosis was present than when it was not ( 3 , 4 , 7 ) ; our results were not in agreement with this . it seems that uterine sizes of cases with adenomyosis were smaller than those without adenomyosis . according to these studies , the lack of such a hypothetically significant relationship could be due to the fact that our study was a retrospective study , and the bimanual pelvic examinations were performed by different people . hence , prospective studies should be conducted with one person as the examiner with uterine weight included in the pathology result . in our study some studies reported that both spontaneous and induced abortions were risk factors for adenomyosis ( 1 , 2 , 6 , 11 ) . according to these studies , it could be concluded that the evidence favors the fact that , in general , abortion ( spontaneous and induced ) is a risk factor for adenomyosis . according to the results obtained in this study and their comparison with those of other studies in other parts of the world , it was found that adenomyosis is more prevalent in women of childbearing age , premenopausal women with high parity and gravidity , and women who have had prior surgeries or abortions . however , there were no results that implied that there was a relationship between adenomyosis and menarche age . the results of this study were different from those of related studies concerning the prevalence of adenomyosis . adenomyosis is a prevalent disease in women of childbearing age and can cause menometrorrhagia , iron deficiency anemia , dysmenorrhea , decrease in quality of life , and obstetrical complications , such as placenta acreta , placenta increta , atonia , and uterine rupture . therefore , it is important to be able to identify , treat , and prevent adenomyosis in order to decrease its possible complications . the data showed that adenomyosis could be reduced if unnecessary uterine surgeries were reduced . in order to decrease the number of unnecessary uterine surgeries , women should be trained to avoid unnecessary caesarian sections , and physicians should emphasize the importance of avoiding unnecessary caesarian sections . then advantages of vaginal delivery and the side effects of cesarean section should be explained to women . such training could be given through mass media , health centers , and health organizations . women should be reminded of the fact that abortion should not be induced when an unexpected pregnancy occurs ; also women should seek the advice and guidance of a physician when they intend to conceive so that cases of adenomyosis and the side effects of emergency cesarean sections can be reduced to the greatest extent possible . training should be provided for women of childbearing age to make them aware of the complications of multiple pregnancies .

background : adenomyosis is a common benign disorder in pre - menopausal women that presents with uterine enlargement , pelvic pain , heavy menstrual bleeding , and dysmenorrhea . however , its risk factors have not been clearly determined . the aim of this study was to determine whether prior uterine surgery is a risk factor for adenomyosis.methods:in this cross - sectional study , we reviewed the medical records of all women who had hysterectomies for benign and a non - emergency , gynecological condition during 20012011 at shariati hospital in bandar - abbas , iran . the variables in the study included age , gravidity , parity , abortion , prior uterine surgery , and the size of the uterus before hysterectomy . the data were analyzed by microsoft excel and spss version 16 , by conducting descriptive statistics , by the t - test , and by analysis of variance ( anova).results : of the 191 participants , 72 women ( 37.7% ) had pathologically - confirmed adenomyosis . women with adenomyosis reported a history of prior uterine surgery more frequently than women without adenomyosis ( p = 0.01 ) , but age , menarche , gravidity , parity , and history of abortion were similar between the two groups ( p > 0.05).conclusion : history of prior uterine surgery is a risk factor for adenomyosis .

within this report , we present a patient with difficulty of swallowing caused by an aberrant right subclavian artery . this is a congenital anomaly with the right subclavian artery originating from the dorsal part of the aortic arch and coursing through the mediastinum between the esophagus and the vertebral column . the diagnosis and treatment of this disorder is discussed based on the findings from the literature . usual causes of difficulty of swallowing or dysphagia in adults include malignancy , esophageal motility disorders , or esophageal strictures . the clinical syndrome of dysphagia in association with an aberrant right subclavian artery compressing the esophagus was first discovered by david bayford ( 17391790 ) and reported as dysphagia lusoria after lusus naturea ( freak of nature ) in 1787.1 an aberrant right subclavian artery is the most common embryologic abnormality of the aortic arch and occurs in 0.5% to 1.8% of the population , but it is usually asymptomatic.2,3 within this report , we present a patient with dysphagia lusoria and discuss the anatomical abnormality , diagnosis , and treatment of this rare entity . a 74-year - old woman was referred to our university hospital because of intermittent dysphagia with difficulty swallowing solid foods . her medical history included a breast carcinoma for which she underwent breast - conserving treatment 3 years earlier . she had been put on proton pump inhibitors since several years without relieving her symptoms . at physical examination , no abnormalities were found . by report from the referring hospital , an endoscopy revealed no abnormalities besides a hiatal hernia without the aspect of a barrett s esophagus or gastric inflammation . a barium contrast examination demonstrated a compression of the proximal esophagus at the level of the aortic arch suggesting of a mass compressing the esophagus ( fig . 1 ) . therefore , a computed tomography ( ct ) angiography scan was performed , which revealed an aberrant right subclavian artery arising from the aortic arch causing compression of the esophagus ( fig . 2 ) . because of the severity of symptoms and the inability to eat had caused weight loss , surgical treatment was indicated . figure 1a barium contrast examination revealing a filling defect at the level of the aortic arch . in the posterior anterior view , the parts of the esophagus proximal and distal to the defect are displaced to each other ( arrow , a ) . in the lateral projection , a wedge - shaped impression on the dorsal esophagus is seen ( arrow , b).figure 2a ct angiography which shows the aberrant origin of the right subclavian artery ( white arrow ) . ao aortic arch . a barium contrast examination revealing a filling defect at the level of the aortic arch . in the posterior anterior view , the parts of the esophagus proximal and distal to the defect are displaced to each other ( arrow , a ) . in the lateral projection , a wedge - shaped impression on the dorsal esophagus is seen ( arrow , b ) . a ct angiography which shows the aberrant origin of the right subclavian artery ( white arrow ) . she was placed in a half supine position and via a right supraclavicular approach , the right carotid and vertebral artery were identified . the aberrant right subclavian artery was dissected free from the esophagus and mobilized into the mediastinum . after administration of 5,000 iu of heparin intravenously , the proximal right subclavian artery was transected with an endostapler ( multifire endo ta 30 , covidien , ma , usa ) and cut . the distal artery was anastomosed end to side to the right aortic artery ( figs . the patient uneventfully recovered and was discharged 2 days after surgery . at 6 weeks of follow - up , figure 3an intraoperative view showing the aberrant right subclavian artery crossing dorsally to the common right carotid artery . scm sternocleidomastoid muscle.figure 4an intraoperative view showing the aberrant right subclavian artery ( asterisk ) crossing dorsally to the esophagus ( arrow).figure 5an intraoperative view showing the end - to - side anastomosis between the right subclavian artery ( a ) and the right common carotid artery ( c ) . b internal jugular vein.figure 6a 3d reconstruction of a postoperatively performed ct angiography showing the end - to - side anastomosis between the proximal right subclavian artery and the right common carotid artery . an intraoperative view showing the aberrant right subclavian artery crossing dorsally to the common right carotid artery . an intraoperative view showing the aberrant right subclavian artery ( asterisk ) crossing dorsally to the esophagus ( arrow ) . an intraoperative view showing the end - to - side anastomosis between the right subclavian artery ( a ) and the right common carotid artery ( c ) . b internal jugular vein . a 3d reconstruction of a postoperatively performed ct angiography showing the end - to - side anastomosis between the proximal right subclavian artery and the right common carotid artery . the right aortic arch disappears proximally to form the right subclavian and common carotid artery . these latter vessels fuse to form the brachiocephalic trunk ( or innominate artery ) which is usually the first branch of the aortic arch . abnormal involution of the right aortic arch with a persisting ( seventh ) intersegmental artery results in the evolution of an aberrant right subclavian artery.3 the aberrant right subclavian artery arises from the dorsal part of the aortic arch with a broad base formed by a remnant of the persisting primitive right aorta , the so - called kommerell s diverticulum.4 the aberrant subclavian or lusoria artery passes through the mediastinum between the esophagus and the vertebral column to reach the right axilla in the majority of cases.5,6 an aberrant right subclavian artery is in approximately one third of cases associated with carotid artery anomalies with a common origin of the left and right carotid artery ( bicarotid truncus).6 the majority of patients with an aberrant right subclavian artery will remain asymptomatic during lifetime.2,3,6 in symptomatic infants , it usually presents with respiratory signs . this is most likely due to the absence of tracheal rigidity , allowing its compression to lead to airway obstruction with recurrent pulmonary infection.57 it is not clear why dysphagia occurs in older patients . it could be attributed to increasing rigidity of the esophagus or arterial elongation and thickening due to atherosclerosis during lifetime.7 similarly , it is uncertain whether dysphagia occurs primarily due to esophageal impression or secondary to motility changes.7 others hypothesized that the coincidence of a common carotid origin and an aberrant right subclavian artery can give rise to compression of the esophagus between these vessels.6,7 in adults with dysphagia , an upper gastrointestinal endoscopy is usually performed . in case of an aberrant right subclavian artery , a pulsating impression can sometimes be seen , but it usually is normal.5,7 a barium contrast examination will show a filling defect at the level of the aortic arch , as illustrated in our patient ( fig . 1 ) . in the posterior anterior view , the parts of the esophagus proximal and distal from the defect appear displaced ( fig . 1a).6 in the lateral projection , a wedge - shaped impression on the dorsal esophagus is seen ( fig . 1b).6 ct or magnetic resonance ( mr ) angiography is considered as the gold standard for the diagnosis of an aberrant right subclavian artery.8 the management of these patients depends on the severity of their symptoms . janssen and colleagues reported three out of six patients with dysphagia lusoria who became free of symptoms after dietary changes or acid inhibition or promotility agents.7 in case of severe or persistent symptoms , surgical intervention is warranted to remove the aberrant vessel and reconstruct the vascular supply . the surgical approach depends on the vascular anatomy : an isolated supraclavicular approach can generally be used if the aortic arch is normal without aneurysm formation of the proximal aberrant right subclavian artery , as in the presented patient . in patients with associated lesions , a combined cervical and thoracic approach might be more appropriate.9 simple ligation and division of the aberrant subclavian artery is likely to cause ischemia of the upper limb or a subclavian steal syndrome ; therefore , subclavian carotid transposition is advocated ( reimplantation of the right subclavian artery to the right common carotid artery ) , as we used in our patient.10 the results of surgical treatment are excellent with relieve of symptoms in nearly all reported patients.7,9 in conclusion , dysphagia in an adult patient can be attributed to an anomaly of the right subclavian artery and this should be included in the differential diagnosis . a barium swallow examination can give a clue toward the diagnosis , but ct or mr imaging angiography is the golden standard for diagnosing this anomaly . in cases with severe symptoms resistant to medical therapy , surgical intervention should be considered . if no aneurysm or aortic disease is present , a supraclavicular approach with diversion of the proximal aberrant artery and an end - to - side anastomosis of the distal right subclavian artery to the right common carotid artery are recommended .

introductionwithin this report , we present a patient with difficulty of swallowing caused by an aberrant right subclavian artery.discussionthis is a congenital anomaly with the right subclavian artery originating from the dorsal part of the aortic arch and coursing through the mediastinum between the esophagus and the vertebral column . the diagnosis and treatment of this disorder is discussed based on the findings from the literature .

copd patients need for pulmonary rehabilitation , self - management support , and social support should be assessed and appropriate services offered throughout the disease trajectory . chronic obstructive pulmonary disease ( copd ) is the world s fourth leading cause of death and thus represents a substantial burden on society and those who suffer from it . persistent airflow limitation that is usually progressive and associated with an enhanced chronic inflammatory response in the airways and the lungs to noxious particles or gases . exacerbations and comorbidities contribute to the overall severity in individual patients.1 the typical symptoms are progressive and include chronic dyspnea , cough and sputum production , wheezing , and chest tightness . copd is a severe disease associated with poor health status , weight loss , fatigue , pain , and symptoms from comorbidities , including depression and anxiety.25 exacerbations are typical components of the disease trajectory , and as the disease progresses , acute exacerbations and hospitalization normally become more frequent , followed by a heavy patient burden and increased health care costs.6 unfortunately , there is no cure for the disease . however , appropriate patient - specific pharmacotherapy , smoking cessation , rehabilitation , patient education , and maintenance of physical activity can reduce the symptoms and exacerbations , improve quality of life , and prevent disease progression . as disease severity and lung function typically worsen over time , offering monitoring and follow - up services is essential . international and national guidelines for the management of copd have been developed with evidence - based recommendations for managing a stable copd condition ; in the guidelines , pulmonary rehabilitation and patient education are recommended , especially in the early stages of the disease . these interventions can improve health status and quality of life , increase maintenance of physical activity and exercise tolerance , and reduce hospital admissions and bed days.1,79 studies have demonstrated that the impact of copd on quality of life and self - management depends on the individual s condition ; however , dyspnea and low functional capacity are considered highly important.3,10 unpredictable breathlessness as well as gradual deterioration of functional capacity threatens bodily integrity , influences individuals stress levels and need for self - management support , and hinders activities of daily life.1114 in particular , through acute exacerbations and severe copd , breathlessness is associated with fear , anxiety , existential threats , and increased self - management demands.14,15 in everyday life , the disease affects social relationships , with experiences of guilt and shame and loss of social support,12,16 while social support seems important for optimizing health , quality of life , and self - management abilities.15,1719 those living with impaired health due to copd seem to expend a substantial amount of energy establishing strategies to avoid breathlessness and be in control while at the same time maintaining some spontaneity and independence in daily life , as well as in decision making concerning their treatment and health care.12,1921 through self - management education and strategies , persons living with copd can learn to cope functionally and be partners in the management of the disease . collaborative self - management as part of an integrated multidisciplinary care process is recommended.1,2224 self - management education programs of varying designs can be part of pulmonary rehabilitation or offered as independent services.1 the aim of self - management education is to increase the person s control over the disease through ownership , motivation , and involvement to manage the disease in collaboration with health care personnel . the key features of self - management are medical management , role management , and emotional management , and it includes skills regarding problem solving , decision making , symptom recognition , resource utilization and taking actions , as well as disease - specific self - health behaviors.21,25 self - management education improves quality of life , breathlessness , and patient activation and reduces hospital admissions.9,26 however , compared to regular health care services , comprehensive self - management programs alone have not shown long - term benefits in terms of quality of life.27,28 in norway , 250300,000 persons are estimated to be living with copd and are thus dependent on receiving diagnosis in order to be offered relevant treatment , monitoring , rehabilitation , patient education , and regular follow - up from health care services.24 studies have identified a lack of cohesive care pathways for persons living with copd in norway.24,2931 to improve these care services , norwegian national guidelines for the prevention , diagnosis , and treatment of copd were published in 2012.24 the guidelines emphasize the importance of early pulmonary rehabilitation and patient education and of regular follow - up services , and they outline the municipalities responsibilities in supporting persons living with copd . it is recommended for rehabilitation programs to include self - management education and smoking cessation support . until recently , pulmonary rehabilitation and self - management education programs in norway were offered by specialist health services . national white papers emphasize that the intention is to transfer these services to the municipalities.32,33 no publications have described norwegian copd patients health status , social provision , and self - management activation or general practitioner ( gp ) and specialist copd consultations or acute hospital admissions due to copd complications since the implementation of the 2012 guidelines for copd treatment . to map the health status , activation for self - management , and offers and use of particular copd health care services of persons living with copd in norway and to involve service users and health care personnel in the present work , a participatory research project was designed with the following main research question : as reported by norwegians living with copd , what are the vulnerable areas and untapped resources in copd care compared to the cohesive health care services offered and used ? as reported by norwegians living with copd , what are the vulnerable areas and untapped resources in copd care compared to the cohesive health care services offered and used ? this paper reports the results of a survey from a cohort of norwegians living with copd . the aim of this study is to describe the health status , offers and use of particular copd health care services , social support , and self - management activation of persons living with copd . the following research questions are answered : what are the characteristics of the health status , offers and use of particular copd health care services , activation for self - management , involvement satisfaction , and social provision of male and female persons with copd?to what degree does the number of years with copd influence health status , smoking status , use of particular copd health care services , activation for self - management , involvement satisfaction , and social provision?to what degree does health status , defined as low and high copd assessment test ( cat ) scores , affect smoking status , use of particular copd health care services , activation for self - management , involvement satisfaction , or social provision ? what are the characteristics of the health status , offers and use of particular copd health care services , activation for self - management , involvement satisfaction , and social provision of male and female persons with copd ? to what degree does the number of years with copd influence health status , smoking status , use of particular copd health care services , activation for self - management , involvement satisfaction , and social provision ? to what degree does health status , defined as low and high copd assessment test ( cat ) scores , affect smoking status , use of particular copd health care services , activation for self - management , involvement satisfaction , or social provision ? although this study uses a cross - sectional design,34 it incorporates aspects of participatory research,35 which includes core - searchers living with copd in the research group in order to systematically grasp their perspectives , select relevant instruments , and ensure the results are more useful and easier to implement.36,37 in this study , the research group included four copd service users , two health care professionals , and three health researchers . the coresearchers completed a seminar on research designs and methods , and all participated equally throughout the research process . four hundred persons registered in a hospital trust in the western part of norway with a primary or secondary diagnosis of copd were invited to participate . the coresearchers participated in developing the design , including the selection of relevant questionnaires , formulation of information sheets , provision of additional information to the public , discussion of the results , and preparation of the article . the regional committees for medical and health research ethics west ( 2013/1301 ) and frde hospital trust ( 2013/5651 ) approved the study . a coresearcher employed at the hospital trust selected the participants from the hospital s electronic patient records . the participants were contacted by mail and sent an information sheet , a written consent form , and the questionnaires to be returned by mail . data were collected by using four questionnaires : the norwegian version of the cat , the norwegian version of the 13-item patient activation measure ( pam 13 ) , insignia health pam 13 , the social provision scale ( sps ) and its revised 16-item version , and one ad hoc questionnaire including sociodemographic and clinical variables . the cat ( glaxosmithkline 2009 , approved translation into norwegian ) was used to assess self - reported health status . the questionnaire has eight items ( cough , phlegm , chest tightness , mobility , activities , confidence , sleep , and energy ) , with scoring options on a likert scale ( 05 ) and a total possible sum of 040 ( where high scores indicate poor health status ) . this validated questionnaire is considered quick and easy - to - use and to have qualities equal to the well - known st georges respiratory questionnaire.1 by using this questionnaire , a simple and reliable measure of the self - reported impact of copd was provided on persons health status.3842 scores > 30 indicate very high impact , > 20 high impact , 1020 medium impact , and < 10 low impact of copd on health status.43the norwegian version of the pam 13 , insignia health44,45 was used to assess the respondents self - reported activation for self - management , meaning the level of knowledge , skills , and confidence needed to manage the disease . the questionnaire was validated , and the instrument reflects a person s behavior and process of developing self - management abilities.4547 the questionnaire has previously been used in surveys including persons with copd.9,18 the instrument contains 13 questions and is scored using a likert scale ( values : strongly disagree , disagree , agree , strongly agree , and not applicable ) . the scores are converted into an interval scale ( 0100 ) , with the sum score conceptualized in four activation levels associated with increasing self - management engagement ( level 1 : 47.0 points , level 2 : 47.155.1 points , level 3 : 55.272.4 points , and level 4 : 72.5 points).48interpretation of the levels : may not yet believe that the patient role is important;lacks confidence and knowledge to take action;beginning to take action;difficulty maintaining behaviors over time.48the sps and its revised 16-item version , which was developed from the original 24-item instrument because of the high correlation between subscores , were translated into norwegian and validated by bondevik.49 the questionnaire was used to obtain self - reports of the extent to which the statements described the patient s current social network . the instrument originates from weiss theory of loneliness as separated into emotional and social loneliness , with specific types of social support attachment , social integration , opportunity of nurturance , reassurance of worth , sense of reliable alliance , and obtaining of guidance.50 the sps measures specific types of social support on a likert scale , with responses ranging from 1 ( strongly disagree ) to 4 ( strongly agree).demographic and clinical variables were self - reported using an ad hoc questionnaire comprising 27 items that addressed activity , social participation , smoking status , particular health care services linked to the diagnosis of copd : gp copd consultations ; specialist copd consultations ( pulmonary physician specialists or pulmonary nurse specialists ) ; acute hospital admissions because of pneumonia or other complications due to copd in the previous year , as well as offers and use of pulmonary rehabilitation and self - management education , and satisfaction with service user involvement . questions about health care services were constructed in accordance with the norwegian guidelines for the management of stable copd.24 the items response values vary from dichotomous alternatives to grading scales and include open spaces for comments . the questionnaire was developed with strong coresearcher involvement and an emphasis on user friendliness and relevance . the cat ( glaxosmithkline 2009 , approved translation into norwegian ) was used to assess self - reported health status . the questionnaire has eight items ( cough , phlegm , chest tightness , mobility , activities , confidence , sleep , and energy ) , with scoring options on a likert scale ( 05 ) and a total possible sum of 040 ( where high scores indicate poor health status ) . this validated questionnaire is considered quick and easy - to - use and to have qualities equal to the well - known st georges respiratory questionnaire.1 by using this questionnaire , a simple and reliable measure of the self - reported impact of copd was provided on persons health status.3842 scores > 30 indicate very high impact , > 20 high impact , 1020 medium impact , and < 10 low impact of copd on health status.43 the norwegian version of the pam 13 , insignia health44,45 was used to assess the respondents self - reported activation for self - management , meaning the level of knowledge , skills , and confidence needed to manage the disease . the questionnaire was validated , and the instrument reflects a person s behavior and process of developing self - management abilities.4547 the questionnaire has previously been used in surveys including persons with copd.9,18 the instrument contains 13 questions and is scored using a likert scale ( values : strongly disagree , disagree , agree , strongly agree , and not applicable ) . the scores are converted into an interval scale ( 0100 ) , with the sum score conceptualized in four activation levels associated with increasing self - management engagement ( level 1 : 47.0 points , level 2 : 47.155.1 points , level 3 : 55.272.4 points , and level 4 : 72.5 points).48 interpretation of the levels : may not yet believe that the patient role is important;lacks confidence and knowledge to take action;beginning to take action;difficulty maintaining behaviors over time.48 may not yet believe that the patient role is important ; lacks confidence and knowledge to take action ; beginning to take action ; difficulty maintaining behaviors over time.48 the sps and its revised 16-item version , which was developed from the original 24-item instrument because of the high correlation between subscores , were translated into norwegian and validated by bondevik.49 the questionnaire was used to obtain self - reports of the extent to which the statements described the patient s current social network . the instrument originates from weiss theory of loneliness as separated into emotional and social loneliness , with specific types of social support attachment , social integration , opportunity of nurturance , reassurance of worth , sense of reliable alliance , and obtaining of guidance.50 the sps measures specific types of social support on a likert scale , with responses ranging from 1 ( strongly disagree ) to 4 ( strongly agree ) . demographic and clinical variables were self - reported using an ad hoc questionnaire comprising 27 items that addressed activity , social participation , smoking status , particular health care services linked to the diagnosis of copd : gp copd consultations ; specialist copd consultations ( pulmonary physician specialists or pulmonary nurse specialists ) ; acute hospital admissions because of pneumonia or other complications due to copd in the previous year , as well as offers and use of pulmonary rehabilitation and self - management education , and satisfaction with service user involvement . questions about health care services were constructed in accordance with the norwegian guidelines for the management of stable copd.24 the items response values vary from dichotomous alternatives to grading scales and include open spaces for comments . the questionnaire was developed with strong coresearcher involvement and an emphasis on user friendliness and relevance . data from the survey package were anonymously registered into a data file in statistical program for social sciences ( spss ; ibm corporation , armonk , ny , usa ) software , version 23.0 . when the scoring options were presented with an equal distance , as with a likert scale , the variable was treated as continuous and t - tests were used for comparisons between the mean values for the groups studied . for variables with categorical values , frequencies , and percentages , although this study uses a cross - sectional design,34 it incorporates aspects of participatory research,35 which includes core - searchers living with copd in the research group in order to systematically grasp their perspectives , select relevant instruments , and ensure the results are more useful and easier to implement.36,37 in this study , the research group included four copd service users , two health care professionals , and three health researchers . the coresearchers completed a seminar on research designs and methods , and all participated equally throughout the research process . four hundred persons registered in a hospital trust in the western part of norway with a primary or secondary diagnosis of copd were invited to participate . the coresearchers participated in developing the design , including the selection of relevant questionnaires , formulation of information sheets , provision of additional information to the public , discussion of the results , and preparation of the article . the regional committees for medical and health research ethics west ( 2013/1301 ) and frde hospital trust ( 2013/5651 ) approved the study . a coresearcher employed at the hospital trust selected the participants from the hospital s electronic patient records . the participants were contacted by mail and sent an information sheet , a written consent form , and the questionnaires to be returned by mail . data were collected by using four questionnaires : the norwegian version of the cat , the norwegian version of the 13-item patient activation measure ( pam 13 ) , insignia health pam 13 , the social provision scale ( sps ) and its revised 16-item version , and one ad hoc questionnaire including sociodemographic and clinical variables . the cat ( glaxosmithkline 2009 , approved translation into norwegian ) was used to assess self - reported health status . the questionnaire has eight items ( cough , phlegm , chest tightness , mobility , activities , confidence , sleep , and energy ) , with scoring options on a likert scale ( 05 ) and a total possible sum of 040 ( where high scores indicate poor health status ) . this validated questionnaire is considered quick and easy - to - use and to have qualities equal to the well - known st georges respiratory questionnaire.1 by using this questionnaire , a simple and reliable measure of the self - reported impact of copd was provided on persons health status.3842 scores > 30 indicate very high impact , > 20 high impact , 1020 medium impact , and < 10 low impact of copd on health status.43the norwegian version of the pam 13 , insignia health44,45 was used to assess the respondents self - reported activation for self - management , meaning the level of knowledge , skills , and confidence needed to manage the disease . the questionnaire was validated , and the instrument reflects a person s behavior and process of developing self - management abilities.4547 the questionnaire has previously been used in surveys including persons with copd.9,18 the instrument contains 13 questions and is scored using a likert scale ( values : strongly disagree , disagree , agree , strongly agree , and not applicable ) . the scores are converted into an interval scale ( 0100 ) , with the sum score conceptualized in four activation levels associated with increasing self - management engagement ( level 1 : 47.0 points , level 2 : 47.155.1 points , level 3 : 55.272.4 points , and level 4 : 72.5 points).48interpretation of the levels : may not yet believe that the patient role is important;lacks confidence and knowledge to take action;beginning to take action;difficulty maintaining behaviors over time.48the sps and its revised 16-item version , which was developed from the original 24-item instrument because of the high correlation between subscores , were translated into norwegian and validated by bondevik.49 the questionnaire was used to obtain self - reports of the extent to which the statements described the patient s current social network . the instrument originates from weiss theory of loneliness as separated into emotional and social loneliness , with specific types of social support attachment , social integration , opportunity of nurturance , reassurance of worth , sense of reliable alliance , and obtaining of guidance.50 the sps measures specific types of social support on a likert scale , with responses ranging from 1 ( strongly disagree ) to 4 ( strongly agree).demographic and clinical variables were self - reported using an ad hoc questionnaire comprising 27 items that addressed activity , social participation , smoking status , particular health care services linked to the diagnosis of copd : gp copd consultations ; specialist copd consultations ( pulmonary physician specialists or pulmonary nurse specialists ) ; acute hospital admissions because of pneumonia or other complications due to copd in the previous year , as well as offers and use of pulmonary rehabilitation and self - management education , and satisfaction with service user involvement . questions about health care services were constructed in accordance with the norwegian guidelines for the management of stable copd.24 the items response values vary from dichotomous alternatives to grading scales and include open spaces for comments . the questionnaire was developed with strong coresearcher involvement and an emphasis on user friendliness and relevance . the cat ( glaxosmithkline 2009 , approved translation into norwegian ) was used to assess self - reported health status . the questionnaire has eight items ( cough , phlegm , chest tightness , mobility , activities , confidence , sleep , and energy ) , with scoring options on a likert scale ( 05 ) and a total possible sum of 040 ( where high scores indicate poor health status ) . this validated questionnaire is considered quick and easy - to - use and to have qualities equal to the well - known st georges respiratory questionnaire.1 by using this questionnaire , a simple and reliable measure of the self - reported impact of copd was provided on persons health status.3842 scores > 30 indicate very high impact , > 20 high impact , 1020 medium impact , and < 10 low impact of copd on health status.43 the norwegian version of the pam 13 , insignia health44,45 was used to assess the respondents self - reported activation for self - management , meaning the level of knowledge , skills , and confidence needed to manage the disease . the questionnaire was validated , and the instrument reflects a person s behavior and process of developing self - management abilities.4547 the questionnaire has previously been used in surveys including persons with copd.9,18 the instrument contains 13 questions and is scored using a likert scale ( values : strongly disagree , disagree , agree , strongly agree , and not applicable ) . the scores are converted into an interval scale ( 0100 ) , with the sum score conceptualized in four activation levels associated with increasing self - management engagement ( level 1 : 47.0 points , level 2 : 47.155.1 points , level 3 : 55.272.4 points , and level 4 : 72.5 points).48 interpretation of the levels : may not yet believe that the patient role is important;lacks confidence and knowledge to take action;beginning to take action;difficulty maintaining behaviors over time.48 may not yet believe that the patient role is important ; lacks confidence and knowledge to take action ; beginning to take action ; difficulty maintaining behaviors over time.48 the sps and its revised 16-item version , which was developed from the original 24-item instrument because of the high correlation between subscores , were translated into norwegian and validated by bondevik.49 the questionnaire was used to obtain self - reports of the extent to which the statements described the patient s current social network . the instrument originates from weiss theory of loneliness as separated into emotional and social loneliness , with specific types of social support attachment , social integration , opportunity of nurturance , reassurance of worth , sense of reliable alliance , and obtaining of guidance.50 the sps measures specific types of social support on a likert scale , with responses ranging from 1 ( strongly disagree ) to 4 ( strongly agree ) . demographic and clinical variables were self - reported using an ad hoc questionnaire comprising 27 items that addressed activity , social participation , smoking status , particular health care services linked to the diagnosis of copd : gp copd consultations ; specialist copd consultations ( pulmonary physician specialists or pulmonary nurse specialists ) ; acute hospital admissions because of pneumonia or other complications due to copd in the previous year , as well as offers and use of pulmonary rehabilitation and self - management education , and satisfaction with service user involvement . questions about health care services were constructed in accordance with the norwegian guidelines for the management of stable copd.24 the items response values vary from dichotomous alternatives to grading scales and include open spaces for comments . the questionnaire was developed with strong coresearcher involvement and an emphasis on user friendliness and relevance . data from the survey package were anonymously registered into a data file in statistical program for social sciences ( spss ; ibm corporation , armonk , ny , usa ) software , version 23.0 . when the scoring options were presented with an equal distance , as with a likert scale , the variable was treated as continuous and t - tests were used for comparisons between the mean values for the groups studied . for variables with categorical values , frequencies , and percentages , tests were reported . a total of 116 persons ( 29% of those invited ) , including 77 males and 36 females ( three did not report their gender ) , returned the questionnaires and a written consent . the mean age was 69.3 ( standard deviation [ sd ] 7.5 ) years , with a mean number of years with copd of 8.7 years ( sd 7.4 ) , and the mean cat score was 20.3 ( sd 7.7 ) ( representing a high impact of copd on health ) . of the copd participants in this study , 75 ( 65.2% ) were married or cohabitants ( table 1 ) . fifty ( 43.9% ) participants had been to gp consultations because of copd the previous year ; however , a total of 63.9% had acute hospital admissions due to copd complications during the same period . only 21 respondents ( 18.8% ) had received offers for pulmonary rehabilitation services , with 18 ( 94.7% ) of these respondents attending these services . copd self - management education was offered to 44 respondents ( 38.9% ) , with 38 ( 86.4% ) attending the course . sixty - six persons ( 75.9% ) reported partly or completely agreeing with the statement , i participate sufficiently in discussions about the content of my treatment and follow - up of health care services the mean pam 13 score for the sample was 58.1 ( sd 23.4 ) , corresponding to level 3 , and 65 participants ( 59.6% ) scores corresponded to levels 34 , which indicates that they were beginning to take action but were still having difficulty maintaining behavior over time ( table 1 ) . since copd is a progressive disease , this study explored how health status , smoking status , gp copd consultations , specialist copd consultations , acute hospital admissions due to copd complications , self - management activation , social support , and involvement satisfaction developed over time . by dichotomizing the number of years the respondents had copd using the median value as the cutoff , two groups emerged , representing patients with a short and long history of copd , that is , < 7.5 years and 7.5 years , respectively . the sps subscore for reassurance of worth showed a significant difference between the groups with a short and long history of copd ( p=0.046 ) . health status , self - reported offers and use of copd health care services , activation , and involvement satisfaction showed no difference with number of copd years ( table 2 ) . in this sample , 97 respondents ( 85.8% ) reported cat scores < 30 , and 16 respondents ( 14.2% ) assessed their health status with cat scores 30 , the last group indicating a very high impact of copd on health status . respondents with cat scores < 30 were compared with those with cat scores 30 regarding smoking status , gp copd consultations , specialist copd consultations , acute hospital admissions due to copd complications , involvement satisfaction , activation , and social support . significant differences ( p<0.001 ) were found between the groups for acute hospital admissions ( lowest number of admissions for respondents with cat scores 30 ) and a lower activation score was observed for those with scores 30 ( p=0.022 ) . additionally , the group with the highest disease impact scored significantly lower on the three sps subscores ( table 3 ) . this study aimed to identify the vulnerable areas and untapped resources of copd patient care compared to the cohesive health care services offered and used , as reported by a cohort of norwegians living with copd . according to the research question regarding health status , self - report of particular copd health care services , involvement satisfaction , activation for self - management , and social provision of male and female persons with copd , no variables were found with significant differences in the gender analysis . health status was characterized by a mean cat score that indicated a high impact of copd on health , as these persons were assumed to face impaired quality of life , comorbidities , and hindrances for participation in everyday life.2,3,10,11,13 thus , these participants may live with considerable self - management demands.12,14,20,21 it was interesting to note that 44 persons ( 40.4% ) had a pam 13 score in accordance with activation levels 12 , corresponding to having an unclear role in taking responsibility and a lack of confidence and knowledge to take action.48 this finding suggests the need for self - management support . self - management education is recognized as an essential form of support and a recommended part of pulmonary rehabilitation and cohesive chronic care , as these types of interventions can improve quality of life and reduce hospital admissions.9,21,26 however , in this cohort , only a limited number of copd patients were offered pulmonary rehabilitation or copd self - management education . this finding represents a contrast from the recommendations in the norwegian and international guidelines.1,24 furthermore , more than half of the participants had not visited their gp for copd consultations during the previous year , another discrepancy from the norwegian guidelines,24 which recommend gp copd consultations for persons diagnosed with copd once or twice a year . the municipalities have the actual responsibility for offering pulmonary rehabilitation and copd self - management education for persons living with copd , including new offers of pulmonary rehabilitation and health checkups after hospitalizations because of copd exacerbations . consequently , in this cohort , the self - reported data indicated that the national guidelines for the management of copd24 were not sufficiently implemented , as offers of copd self - management education , pulmonary rehabilitation , and gp copd consultations for the participants occurred less than recommended during the study period . this finding of inadequate offers of pulmonary rehabilitation , copd self - management education , and follow - up services is in accordance with and corresponds with studies published prior to the most recent national guidelines.30,31,51 furthermore , 63.9% of the respondents had acute hospital admissions because of copd complications in the previous year . rehabilitation and self - management education can reduce hospital admissions and number of bed days.1,8 the reported low frequency of offers of pulmonary rehabilitation , copd self - management education , and gp copd consultations in this cohort indicates that offers of these health care services are untapped resources for this group , despite being services with the potential to reduce hospital admissions1,8 and improve self - management activation and quality of life for persons living with copd.9,26 however , a positive result was the high compliance ( 94.7%86.4% ) with the offers of pulmonary rehabilitation and copd self - management education . these results conflict the reports of low adherence to pulmonary rehabilitation among copd patients.52 interestingly , most of the respondents also reported high satisfaction with service user involvement , an important aspect , since collaborative self - management as part of integrated care53 highly depends on service user involvement and empowerment . unfortunately , the impression from later qualitative interviews with a sub - sample of the respondents confirms that several respondents seemed unaware of the recommendations in the guidelines and the health care services available to them . as the number of years of experience with copd was expected to influence self - reported health status , use of particular health care services , activation for self - management , social provision , or involvement satisfaction , the variable years with copd was divided ( table 2 ) . after dichotomizing the respondents into groups of patients with < 7.5 years of experience with copd and those with 7.5 , the present study found no significant difference between the groups , with the exception of sps scores . a positive result was that the cat score , copd health consultations , or acute hospital admissions because of copd complications did not increase over time ; that is , in this cohort , these results indicated that health status did not necessarily decrease over time , despite the progressive nature of this disease . however , the sps subscore of reassurance of worth showed a significant difference between groups , indicating fewer opportunities for social support over years of having copd . since social support is important for both self - management abilities and health,17,19 this result represents a possible vulnerable area for persons living with copd . to assess the self - reported impact of copd on the participants health status , the cat variable was divided into low and high cat scores , particularly to identify its influence on gp copd consultations , specialist copd consultations , and acute hospital admissions due to copd complications , involvement satisfaction , smoking status , activation , and social provision ( table 3 ) . persons reporting a high impact of copd on their health reported poorer activation for self - management and poorer scores on the sps , which indicates higher vulnerability . these results correspond to those of a study by bos - touwen et al , which identified physical health status and social support as associated with activation for self - management in a population living with chronic diseases ( copd , type 2 diabetes mellitus , chronic heart failure , and chronic renal disease).18 however , in a later study of determinants of self - management in copd patients only , illness perception and gold stage , but not social support , were identified as explanatory determinants.54 this difference in results illustrates the complexity of self - management of copd . an integrative review of factors influencing copd self - management emphasized that a range of physical , social , cultural , psychological , and existential factors influences these persons self - management abilities and that the content of copd self - management might not yet be adequately characterized.19 another interesting result is that those reporting cat scores 30 also reported a lower incidence of copd - related acute hospital admissions in the previous year and no change in copd health consultations . a possible explanation of these findings could be that these respondents received more follow - up services such as home care and/or support from next - of - kin , thus receiving a higher level of self - management support in terms of other factors , for example , early detection and treatment of exacerbations at home . through the participatory research design , the coresearchers contributed valuably in the development of design and the ad hoc questionnaire , recruitment , in discussion of the study s results , and preparation of the article . copd sufferers , limiting the use of patient , and increasing the use of person and participants . the members of the collaborative team all approved of the final manuscript . this thorough collaborative approach is valued as a strength of this study . however , some limitations should be acknowledged . this cohort was small ( n=116 ) , the respondents represented a limited geographical area , and the response rate was low , at 29% , a challenge experienced also in other studies on persons living with copd.55 no data were available regarding noncompliance , and therefore why so few persons participated was not explainable . however , it may be speculated that this may be related to the high symptom load in this target group and the fact that completing a long questionnaire with many pages may be too great a challenge for some persons living with copd . furthermore , not much was known of the respondents home care services , information that might have been helpful to explain the results . due to the low number of respondents , only descriptive statistics were studied . a cohort of norwegians living with copd reported inadequate offers of health care services in comparison to the recommendations in the national guidelines for managing stable copd . furthermore , social provision decreased with increasing years of copd and poorer health status , and those living with a very high impact of copd on their health reported significantly lower activation . thus , social provision and self - management activation appear to be possible areas for improvement , indicating that , within this cohort , offers of follow - up services in accordance with the copd guidelines are untapped resources for improving health status and self - management and reducing hospital admissions . although copd is a progressive disease , health status , self - management activation , use of gp and specialist copd consultations , or acute hospital admissions due to copd complications did not vary with the number of years of having copd . the need for pulmonary rehabilitation , copd self - management support , and social support should be assessed among persons living with copd , and the services offered to these persons and their families throughout the entire course of the disease . more research is needed to explore the complexity of the implementation of guidelines as well as the self - management of copd . participatory research designs and the inclusion of persons living with copd as coresearchers have the potential to add quality to research designs and results .

purposeto describe a cohort of chronic obstructive pulmonary disease ( copd ) patients and perform a within - group comparison regarding self - management activation , social provision , and health status.patients and methodsa cross - sectional survey including 116 persons.resultsthe sample comprised 65 men and 38 women , mean age 69 years . fourteen percent reported very high impact of copd on their health ; 19% had received pulmonary rehabilitation offers , 39% had been offered self - management education , and 64% had acute hospital admissions due to copd complications in the past year . persons with copd assessment test ( cat ) scores 30 reported significantly poorer self - management activation and significantly lower social provision than those reporting cat scores < 30 . number of copd years had no significant influence on copd health care consultations or self - management activation.conclusionpersons with copd reported decreasing social provision with increasing copd years and poorer health status . although copd is a progressive disease , health status and self - management activation did not vary with number of copd years . those living with a very high copd impact on health reported significantly lower self - management activation but fewer acute hospital admissions.practice implicationscopd patients need for pulmonary rehabilitation , self - management support , and social support should be assessed and appropriate services offered throughout the disease trajectory .

congenital anomalies of the coronary arteries , though uncommon , are an important cause of chest pain and in some cases can produce hemodynamically significant abnormalities , and even sudden cardiac death . malignant anomalous right coronary , with an incidence of 0.03 - 0.17% of patients undergoing angiography , is a rare form of such congenital anomalies . the development of electrocardiography ( ecg ) gated multidetector row computed tomography ( mdct ) allows accurate and noninvasive detection of coronary artery anomalies . knowledge of normal ct appearances of various anomalies and an understanding of their clinical significance is essential for correct diagnosis . a 38 year old male presented with complaints of chest pain , aggravated by physical stress , of more than four months duration . ct coronary angiography was performed using a 64 slice ct scanner ( brilliance-64 , philips , the netherland ) . the following parameters were used : 120 kvp , 800 mas , rotation time : 0.4 s , pitch : 0.2 mm and collimation : 64 0.625 mm . using a dual head pressure injector ( mallinckrodt ) , 80 ml of nonionic iodinated contrast ( iopamiro 370 , bayer , germany ) was administered at the rate of 5 ml / sec , followed by 20 ml of saline . the total scan time was 8.6 seconds . ct coronary angiography showed the right coronary artery originating from the left coronary sinus , coursing between the aortic root and pulmonary artery with a narrowed proximal segment . the rest of the coronary arteries , including the left main lca , the left anterior descending and the left circumflex were normal in course . maximum intensity projection of top of heart showing both right coronary artey ( rca ) and left coronary artey ( lca ) originating from left coronary sinus . rca has a slit - like ostium and courses between pulmonary artery ( pa ) and aorta ( a ) volume rendered image of same showing anomalous , interarterial course of right coronary artey ( rca ) , between pulmonary artery ( pa ) and aorta ( a ) anomalies of coronary arteries may be found incidentally in 0.3 - 1% of healthy individuals . the coronary artery anomalies can be classified into anomalies of origin , course or termination or as hemodynamically significant or insignificant . hemodynamically significant anomalies are characterized by abnormalities of myocardial perfusion , leading to increased risk of myocardial ischemia or sudden death . these include an anomalous origin of either the lca or rca from the pulmonary artery , an anomalous course between the pulmonary artery and aorta ( inter - arterial ) of either the rca arising from the left sinus of valsalva or the lca arising from the right sinus of valsalva , and , occasionally myocardial bridging or congenital coronary artery fistula . the rca arising from the left sinus of valsalva as a separate vessel or as a branch of a single coronary artery has an incidence of 0.03 - 0.17% of patients undergoing angiography . the anomalous origin may have inter - arterial , retro - aortic , prepulmonic or septal ( subpulmonic ) course , the most common being inter - arterial . the incidence of sudden death with this anomaly is estimated at 25 - 40% and is associated with exercise in half of the reported cases . various theories have been proposed for this association , including slit like ostium , acute angulation at the origin and compression of the vessel between the aorta and pulmonary artery . the choice of treatment for this congenital anomaly is still controversial ; however , because of significant hemodynamic consequences and propensity to cause sudden death , most of the literature advocates definite surgical revascularization in all cases , and the different options available include coronary artery bypass grafting , reimplantation of the coronary ostia and unroofing of the coronary artery , the last being considered the best solution if anatomically feasible . in japan , the treatment for this condition is conservative with the patient being treated medically with beta blockers . ct coronary angiography was made possible with the introduction of mdct and development of ecg gated scanning . for several decades , the imaging of coronary arteries was done with conventional angiography . however , it has been reported that anomalies of coronary arteries detected on conventional angiography were only 35% of those detected on 16 slice mdct . although coronary artery anomalies are far less common than acquired coronary artery disease , their propensity to cause premature cardiac morbidity and mortality especially in young adults make them significant . in a study by eikart et al . , cardiac abnormality was responsible for 51% of sudden deaths in young adults , with coronary artery abnormality being the most common cardiac abnormality ( 61% ) . in conclusion , we describe a 38 year old man with an anomalous right coronary artery , arising from the left sinus of valsalva and coursing between the aortic root and pulmonary artery . this case report emphasizes the role of non - invasive ecg gated mdct coronary angiography in accurate detection of coronary artery anomalies .

we report a case of an anomalous origin of a right coronary artery from the left coronary sinus with an inter - arterial course , between the aorta and the main pulmonary artery . this variant has been called malignant because of its association with sudden death , especially in young asymptomatic athletes . although these variants are rare , knowledge of cross sectional anatomy of the coronary arteries and their variants is critical , especially because some variants are associated with sudden death .

the commonest reasons for high spread of regional blocks are large volumes of local anesthetics and/or high placement of epidural catheters . monoparesis following lumbar epidural block is a rare occurrence , with few cases reported in literature . a healthy , 63 kg , 30-year - old primigravida requested for labor analgesia . as per the institutional protocol , a combined spinal epidural technique ( csea ) was administered . lumbar puncture was done in l3l4 interspace and 25 mcg of fentanyl was injected intrathecally . the epidural space was identified with loss of resistance to saline , at a depth of 5 cm to the skin , and a catheter was fixed with the 10-cm mark at the entry point . the patient had satisfactory pain relief and received two epidural top - up doses of 6 ml 0.125% bupivacaine each , at 60 min intervals . about 45 min after the administration of the last dose , it was decided that a cesarean section be performed as the labor had not progressed . in the operating room , the block was extended with a mixture of 15 ml 2% lignocaine with 50 mcg fentanyl titrated over 10 min to achieve a t6 level block . neurological examination showed right upper limb power of 2/5 ( biceps flexion 2/5 , shoulder abduction 2/5 ) , full power of 5/5 of the left upper limb , and bilateral lower limb power of 4/5 . the sensory examination revealed loss of pin prick sensation at dermatomes c6 , c7 , c8 and t1 of the right arm . there was no involvement of cranial nerves . the epidural catheter was removed and the patient was kept under observation in the recovery room . the incidence of neurological complications in obstetric epidural anesthesia ranges from 0.3 to 2 in 10,000 blocks . commonly described complications of cnb include technical failure , nausea , pruritis , urinary retention , postdural puncture headache , hypotension , bradycardia , and cardiac arrest . jenkins , in a study comprising 145,550 epidurals , found the incidence of high or total spinal block to be 1 in 16,200 epidurals . scott and hibbard observed that neuropathy was the most common complication following epidural anesthesia in the obstetric population . scott and tunstall reported 8 reversible neuropathies in 14,856 obstetric spinal blocks and 38 reversible neuropathies in 108,133 obstetric epidural blocks . the cause of the above was postulated as either trauma due to needle / catheter or maternal obstetric palsies . the rate of permanent neurological injury after spinal and epidural anesthesia ranges from 0 to 4.2 per 10,000 and from 0 to 7.6 per 10,000 , respectively . other reported complications of cnb are cranial nerve palsies with or without horner 's syndrome and monoparesis . the incidence of cranial nerve palsy ranges from 1 to 3.7 in 100,000 obstetric regional anesthesia . involvement of optic nerve , trigeminal nerve , abducent nerve , facial nerve and vestibulocochlear nerve too has been reported . all these complications have been attributed to loss of cerebrospinal fluid ( csf ) or high spread of block after epidural anesthetic secondary to excessive cephalad spread , with full recovery within 45 h. horner 's syndrome following cnb is seen in obstetric practice and is not an uncommon complication , and its occurrence may be attributed to low anatomical origin of the ocular sympathetic supply , with fibers arising from as low as t4 . the block to this level is not uncommon in obstetrics practice , and it can lead to cranial nerve involvement . our patient had weakness in the right upper limb , involving both sensory and motor components at the end of surgery . though the volume of local anesthetic used in epidural was 15 ml , the patient complained of weakness of the right upper limb . the exact mechanism for the high spread to local anesthetic to cause monoparesis is difficult to define . a case of upper limb monoparesis following epidural anesthesia has been reported . according to the authors , the length of the epidural catheter of more than 6.5 cm in epidural space probably resulted in unilateral migration into a higher intervertebral foramen , leading to a high unilateral block . unilateral upper limb involvement may occur due to the presence of septa in the epidural space . epidural catheters placed 68 cm within the epidural space are less likely to get dislodged and require replacement than the catheters placed 24 cm when prolonged labor is anticipated . the volume of local anesthetic injected into the epidural space alone is not the reason for the high spread of local anesthetic . using epidurograms , showed that volumes greater than 40 ml injected in the lumbar or caudal region reach the thoracic and lower cervical segments . conversely , cranial nerve block after test dose through the epidural catheter has been reported . the present case shows that delayed extension of epidural block , though rare , can occur and the attending anesthesiologist must be aware of such a complication so that it can be managed effectively . the management includes reassuring the patient , and taking adequate care of the airway , breathing and circulation .

monoparesis following lumbar epidural block is a rare occurrence , with few cases reported in the literature . we report development of transient brachial monoparesis following epidural anesthesia in a parturient for cesarean section . the patient received a mixture of 15 ml of 2% lignocaine with 50 mcg fentanyl epidurally to achieve a blockade up to t6 level . she remained hemodynamically stable throughout the procedure , with no respiratory distress or desaturation . however , near the end of surgery , she developed weakness in the right upper limb . the weakness lasted for 90 min , followed by complete neurological recovery . subsequent hospital stay was uneventful .

evaluation of students competencies is one of the essential tools for assessing success of education programs in accomplishment of its objectives . on the other hand , evaluation is a major determinant of students learning patterns . in other words some believe that ethical evaluation of medical students should emphasize on ethical knowledge and moral reasoning while some others think that in addition to these objectives , compassion , respect and altruism are essential moral competences which need to be evaluated ( 3 ) . it means that although moral reasoning is an important goal , this competency does not necessarily end to ethical and professional practice of students . the other challenge in moral evaluation is that how it is possible to evaluate students ` attitude and values and ethical practice in a valid and reliable way ( 4 ) . in published literatures there are so many papers introducing and discussing different tools for evaluation of ethical attitude , reasoning and behavior ( 2 , 57 ) . defining issue test ( dit ) , sociomoral reflection measure ( srm ) , and moral judgment interview ( mji ) have been developed for evaluation of moral reasoning , not being enough specific they are used generally for research purpose though ( 8) . in practice , for evaluation of students medical ethics competency , methods such as multiple choice questions , short essay , portfolio , objective structured clinical examination ( osce ) , faculty evaluation and 360 degree evaluation are being used ( 2 , 7 ) . although there is no consensus among ethicists on the best evaluation method , they all agree that different methods should be used ( 7 ) . this method not only evaluates trainee s ethics knowledge but also assesses their moral reasoning and ethical behavior . nowadays increasing number of medical ethics osce stations are including in comprehensive exams in different educational phases in medical schools around the world ( 2 , 911 ) . in iran , there is a consensus about the necessity of evaluation of graduate s ethical competency among medical education leaders and they have mandated adding some medical ethics multiple choice questions ( mcq ) to pre - internship comprehensive exam , residency entrance exam , annual residency and board exam . since multiple choice question method alone is not enough for moral competency evaluation , we decided to assess practicality of evaluation of ethical competency by designing and conducting an osce station for medical ethics . success of this method could convince educational leaders to use other methods for evaluation of graduate s ethical competency . in this paper we present our experience of including one medical ethics station in seven endocrinology osce stations of endocrinology board exam . we used bioethics osce experience of toronto center for bioethics to design one station on truth telling . the scenario is about an inoperable pancreatic cancer patient whose wife asks physician not to tell the diagnosis to the patient . a standardized patient played the role of the patent s wife in this station and talked to examine physicians . we also used two global rating questions using likert scale to score their ethical decision making and communication skill . this station was pilot tested in a medical ethics education workshop in may 2009 which the standardized patient was trained for . in pilot conduct of this station one of workshop attendees participated as the examinee and other six participants rated his performance . all scores were very close to each other , indeed 5 out of six participants gave the same mark to the examinee and the reliability of station was 0.83 . participants also evaluated the face and content validity of the checklist . based on their suggestion the finalized station included in the osce part of the endocrinology board exam in september 2009 . time for each physician s interview with simulated patient in ethics station was 5 minutes . one rater ( first author ) was present in the station to fill checklists and rate examinees . most participants had good to excellent communication skill and only one participant did not communicate with the standardized patient and easily accepted her request ( table 1 ) . in their ethical decision making , % 26 ( 5 ) of participants had poor performance and only 3 of them had good or acceptable ethical encounter . most participants asked for the patient s wife reason for her request ( 15 people ) and almost all of them ( 18 people ) mentioned that patient had right to know about his condition . the most prevalent mentioned justification for patient s right was the need to plan for the rest of life ( 12 people ) . two people told that patient might ask about his condition and one participant gave several different reasons for refusing her request such as she is not telling lie to patient because it is unprofessional , finally patient would find out they were hiding something form him , also if he heards about his condition he would lose his trust on physicians and would be affected by a worse emotional stress . nine participants refused to withhold the truth from patient , however no one acknowledged the patient s right not to know . all of them believed that they had to give information to the patient in any case . there was a significant correlation between participant s score of communication skill and score of their ethical decision making ( r=0.48 , p=0.03 ) . although the mean score of board osce for those with good ethical performance was higher , this correlation was not significant ( table 2 ) . a positive correlation has been observed between participants osce score and their communication skill score but it was not significant . mean ethical performance score of female participants ( 2 , 17 ) was significantly higher than male ones ( 1.43 out of 5 ) ( p=0.01 ) , however there was no significant difference between their communication skill . small though , this experiment showed practicability of conducting and including an osce ethics exam in evaluation of trainees . low score of participant s ethical performance was due to lack of training in their educational course and we could not expect them to show good ethical competency just by reading ethics text books . osce is a good method for integrated evaluation of trainees knowledge and skills in facing with ethical dilemmas . if we just use multiple choice questions for evaluation of ethics competency , students learning will be limited to knowledge while using methods that could evaluate their moral judgment and behavior will encourage them to learn practical ethics competencies . moreover standardized patient could be used as a teaching method . in teaching medical ethics besides reflection and discussion on ethical dilemmas , students need to practice their ethical skills and receive feedbacks on their practice ( 13 ) . we should notice that osce method alone is not enough for precise evaluation of ethical practice . since the pattern of all ethics station could be recognized by students and they could get ready for showing good competency in those stations , being able to have ethical performance is not equal to having ethical practice ( 5 ) . in other words , thus other evaluation methods such as 360 degree evaluation are needed to assess this dimension of educational objectives in medical ethics ( 2 ) . furthermore osce method is very costly and having enough number of ethics station will pose logistical difficulties . in a study conducted by singer et al , 46 ethics station had internal consistency reliability ( cronbach s ) of 0.28 to 0.46 . they estimated that at least 41 ethical stations are needed in an osce test for achieving acceptable internal consistency of 0.8 ( 12 ) . although inter - rater reliability of our station in its pilot phase was good , because of small sample size in the pilot study , we should have evaluated its reliability in the main study . unfortunately due to logistical problems we were not able to have two raters , so we did not evaluate inter - rater reliability of the station . the other important pitfall of our study was that one station has not content validity for assessing ethical performance . in other words , we could not be sure of ethical competency of physicians in encountering with different types of ethical problems in their professional life through watching their conduct in facing with just one ethical problem . this study showed that it is practical to develop an ethics station in comprehensive medical exams in different phase of medical education course and this method could motivate medical students to learn ethical practice . despite all straight points of this evaluation method , it should be noticed that conducting one or two osce station in graduate comprehensive exam is not enough for evaluation of their ethical practice and other complementary evaluation methods such as continuous peer and faculty evaluation of students during their educational course are necessary .

in this study we discuss our experience of including an ethics objective structured clinical examination ( osce ) station in endocrinology board exam.one osce station on truth telling was developed and a standardized patient was trained for role playing in this station . based on a pilot study , the evaluation checklist got modified . then the finalized station added into the osce phase of endocrinology board exam.based on this experience , adding ethics station in board exams is practical and reasonable . since osce method could evaluate students ethical decision making and communication skill it could be used in combination with other kinds of evaluation in assessing ethics competency of graduates . using this method could push the ethics learning approach toward more practical and skill based ones .

dye sensitized solar cells ( dsscs ) are photoelectrochemical , alternative energy source devices that convert light energy into electricity , based on the photosensitization of wide - bandgap metal oxide semiconductors such as tio2 . photosensitization is a process whereby energy of absorbed light is transferred from a photosensitizer ( light - absorbing molecules such as dyes ) to an acceptor ( such as semiconductors ) [ 1 , 2 ] . successfully demonstrated by o'regan and grtzel in 1991 , the dssc devices have attracted a lot of interest towards development and improvement of new families of dyes and metal complexes . dsscs sensitized with dyes having heavy transition - metal complexes such as ruthenium based complex are the most efficient and have been recorded to operate with power conversions efficiency to 11 - 12% using nanoporous tio2 electrodes [ 46 ] . however , the high cost of ruthenium complexes and the long - term unavailability of these noble metals [ 48 ] switch the need to search for alternative photosensitizers to be used in tio2-based photovoltaic devices . accordingly , many studies have shown the possibilities of using natural dyes as sensitizers in dsscs [ 3 , 7 ] . it is the complexity associated with handling of environmental concerns of using synthetic and metal - based dyes that have encouraged researchers to explore alternative green sources of dyes from various natural resources . the study of natural photosynthesis process has highlighted the functionality of natural pigments in harnessing solar energy . this knowledge has further improved understandings on properties of dye pigments and has shown the pathways to the use of natural dyes or pigments for capturing solar radiation , converting it to more manageable forms of energy . these natural - derived sensitizers , which are commonly involved as light harvesting pigments , offer many advantages over the usage of rare transition metal complexes and other synthetic dyes . in the nature , these natural pigments serve as antenna complexes that collect light and transfer the energy to reaction centre complexes where the chemical oxidation and reduction conversion reactions to long term energy storage take place . chlorophyll is the well - known and dominant natural pigment in terms of absorbing specific wavelengths of the visible light when harnessing energy from the sun , converting sunlight to chemical energy and transferring of electrons . others such as carotenoids and anthocyanins are known as accessory pigments that absorb light at different wavelengths than that absorbed by chlorophyll . besides that they are known to have additional role as protective mechanisms against excess light . it is the energy collectively absorbed by these pigments that drives the light reactions in the process of photosynthesis in higher plants and other photosynthetic organisms . such natural pigments can easily be extracted from fruits , vegetables , plants , and flowers with minimal chemical procedures and hence attract a great interest in producing a low cost and yet easy to fabricate dssc photovoltaic device . their availability in large quantities in nature , convenient extraction with cheaper organic solvents , ability of application without fine purification , being environmentally friendly , and low production cost of the devices are some of the other competitive advantages of utilizing natural dyes [ 11 , 12 ] . this paper examines the synergistic performance of chlorophyll and xanthophyll pigments extracted from one type of filamentous freshwater green algae , cladophora sp . algae are used in the biofuel production and this research would increase its value addition where they could be cultured for their photosensitive pigments . green algae include unicellular and multicellular colonial flagellates and macroscopic seaweeds that are able to manufacture their own food material through photosynthesis . factors such as water , light , carbon dioxide , temperature , and minerals are necessary for algal growth [ 13 , 14 ] . algae could cope with a highly variable environment such as rapid attenuation of light with depth . besides , this absorbed light energy could also be reemitted as fluorescence or dissipated as heat rather than being utilized in the photosynthesis [ 9 , 15 ] . these algal derived photosynthetic pigments are known to work mutually in nature for the photosynthesis process by broadening the light absorption capacity as well as providing photoprotection through xanthophyll cycle which dissipate excess light energy [ 9 , 15 ] . furthermore , the photosynthesis process is linked through z - scheme via electron transport coperformed by photosystem i ( psi ) and photosystem ii ( psii ) . in order to increase light absorbing capacity of dsscs , however , mixed dye system would account for many possible types of interactions between dyes with various constituent presents . we hypothesized that , since the pigments were derived from a single species , desirable interactions between dyes could be achieved through synergistic effect in improving electron injection , light harvesting , and limitation of electron recombination . therefore , this paper discusses our experiment on cosensitization of dyes from a single natural source in evaluating the performance of dssc using mix chlorophyll and xanthophyll dyes . stability of the investigated natural pigments under the natural temperature and light intensity variations as well as electron kinetics that exist in the ambient of dssc was measured . all extraction procedures were carried out under dim light and glassware containing dyes were covered with aluminium foil to minimize photooxidation . , 20 g , were cleaned and rinsed using distilled water prior to extraction . the dye pigment was extracted by grinding the algae ( 20 g ) with absolute ethanol ( 100 ml ) and left overnight ( refrigerated at about 3c ) . the residual solids were filtered off and the filtrate was then centrifuged to separate any remaining solid content and extract containing both chlorophyll and xanthophyll was recovered . hot saponification method was adopted to isolate chlorophyll and xanthophyll pigments , by mixing 20% methanolic potassium hydroxide ( 5 ml ) into 50 ml of the recovered extract and kept overnight at 56c . petroleum ether ( 70 ml ) was then added and the mixture was shaken and left to stand until two layers of xanthophyll pigment ( yellow ) and chlorophyll pigment ( green ) were separated . the yellow coloured layer containing xanthophyll pigment was then washed with acetone in 1 : 1 v / v ratio . the presence of chlorophyll and xanthophyll was confirmed by using the uv - visible absorption spectroscopic techniques ( model : shimadzu uv-1800 ) . absolute ethanol ( 4.75 ml ) was added to each of the dye pigment ( 0.25 ml ) making a total volume of 5 ml . the effect of temperature and light were examined at 27c ( laboratory temperature ) and 31c ( average temperature under direct sunlight ) , with light source equivalent to 100 mw / m illumination . chlorophyll and xanthophyll concentrations were estimated and converted to percentage retention for accurate comparison among the treatments . tio2 paste solaronix ( nanoxide - t , colloidal anatase particles size : ~13 nm , ~120 m g ( bet ) , switzerland ) was used for photoelectrodes fabrication . the tio2 paste was coated using doctor blade method on precleaned fluorine - doped conducting tin oxide ( fto ) glasses ( ~7 sq ) . electrodes were then preheated at ~50c using a hair - drier and sintered at 450c for 30 minutes . the thickness of the tio2 electrodes used for this investigation is ~9 m ( scanning electron microscope ( sem ) ) [ 21 , 22 ] . the tio2 electrodes were subsequently dipped in the extracted chlorophyll , xanthophyll , and mixture of chlorophyll and carotenoid dyes ( 1 : 1 v / v ) for overnight . dsscs were assembled by introducing the redox electrolyte containing tetrabutylammonium iodide ( tbai ; 0.5 m)/i2 ( 0.05 m ) , in a mixture of acetonitrile and ethylene carbonate ( 6 : 4 , v / v ) between the dyed the tio2 electrode and platinum counter electrode . those dsscs were light soaked by placing the cells under irradiation of 100 mw / cm for about 4 hours after applying the electrolyte to obtain the best reading . the cells were then put under solar simulator ( model : dyesol lp-156b ) for current - voltage measurement . the power conversion efficiency ( ) was calculated using the following described relation : ( 1)=ffiscvocp , where isc is the short - circuit photocurrent density ( a cm ) , voc is the open circuit voltage ( v ) , p is the intensity of the incident light ( w cm ) , and ff is the fill factor defined as ff = imvm / iscvoc , in which i m and vm are the optimum photocurrent and voltage that can be extracted from the maximum power calculated from the i - v data [ 23 , 24 ] . electrochemical impedance spectroscopic measurement was carried out using computer controlled electrochemical interface ( si 1287 , solatron ) , and impedance / gain - phase analyzer ( si 1260 , solatron ) . the frequency range and the amplitude of alternative voltage were from 0.01 hz to 10 hz and 10 mv , respectively . the impedance measurements were performed at open circuit condition by applying the bias voltage conditions . impedance parameters and equivalent circuits were then obtained by fitting the spectra with zview software ( v3.3 , scribner associate inc . ) . absorption characteristics of the extracted dyes from filamentous freshwater green algae were recorded using a uv - vis spectrophotometer . chlorophyll is an important biomolecule for photosynthesis that absorbs light in the blue and red regions of the visible spectrum . figure 2 compares the absorption spectra of chlorophyll , xanthophyll , and dye cocktail ( mixture of chlorophyll and xanthophyll , 1 : 1 v / v ratio ) , where chlorophyll has two absorption peaks at ~420 nm and ~645 nm and xanthophyll is with absorption maxima of ~449 nm . the broader peak at the blue region ( ~400500 nm ) represents the mixture of chlorophyll and xanthophyll pigments . this broadening can be attributed to the ability of pigment cocktail harvesting a broader spectrum of solar energy that can in turn be used to produce a higher photocurrent . both pigments are known to be mutually responsible for increasing light harvesting efficiency in the filamentous freshwater green algae . the biological function of xanthophyll is generally accepted as to serve as essential accessory light - harvesting pigments by absorbing photons and transfer them to chlorophyll molecules . schematic diagram of light harvesting by xanthophyll and chlorophyll is depicted as in figure 3 . the absorption spectra of visible light pertaining to chlorophyll , xanthophyll , and mixed dye pigments ( cocktail ) on tio2 electrodes are shown in figure 4 . importantly , no obvious absorption peak was observed when these pigments were tested on tio2 electrodes . the absorption of mixed dye was stronger than that of those obtained for chlorophyll and xanthophyll pigments individually . this could be attributed to the interactions between tio2 and the dye molecules on the mesoporous tio2 electrode . loading of dyes onto tio2 electrodes was investigated by determining the difference in dye concentration before and after dipping the tio2 anode into dye solutions . concentration of the adsorbed xanthophyll pigments was found to be 2.5 10 g / ml , much lesser than that of chlorophyll pigments ( 2.2 g / ml ) . furthermore , it was found that more chlorophyll pigments from the mixed dye system ( 2.4 g / ml ) could adsorb onto tio2 film than the chlorophyll in the individual dye system . therefore , a better dssc performance could be expected from dssc sensitized using chlorophyll dyes and mixed dyes . since more chlorophyll from the mixed dye system was adsorbed onto the tio2 electrode , this might indicate that xanthophyll could act as coadsorbent and such synergistic behavior could lead to the enhancement of overall dssc performance by preventing recombination losses , thus promoting efficient electron injection to take place . stability of chlorophyll dye exposed to visible light under two different temperatures ( 27c and 31c ) expressed in terms of percentage retention are depicted in figure 5 . chlorophyll dye ( with no exposure to light ) was found to be more stable at ambient temperature of 27c . the percentage retention of chlorophyll was reduced from 91.7% to 40.3% when storage temperatures were changed from 27c to 31c . the rate of degradation of chlorophyll dye was significantly faster ( 15% retention at 27c and zero retention at 31c over 24 hours period ) when exposed to light ( 100 mw / cm ) , indicating the higher degree of photosensitivity of this biomolecule . combination of both high temperature and exposure of light increased the rate of degradation process of chlorophyll suggesting that it is highly susceptible to photodegradation . as shown in figure 6 , xanthophyll dye was found to be more stable ( shows low degradation over 24-hour period ) than chlorophyll under light . however , exposure to concurrent high temperature and continuous light made the xanthophyll more prone to photodegradation . the rate of photodegradation of xanthophyll was relatively slower ( higher percentage retention over time ) than those of chlorophyll ( lower percentage retention over time ) . this confirms the nature of xanthophyll in resisting degradation , thus showing its capability to enhance the light capturing processes in algae and plants . this can be explained by the unique ability of xanthophyll dye that undergoes conformational changes . in nature , xanthophyll pigments exert their photoprotective action by rapidly quenching the excited state of chlorophyll pigments . xanthophyll has the ability to dissipate excess energy through reversible conformational changes known as the xanthophyll cycle that leads quenching and heat dissipation [ 9 , 29 ] to protect cell system against photodamage . in the cycle , the conversion of violaxanthin to antheraxanthin and then to zeaxanthin happens under high light conditions . it is known that such conversion is beneficial as zeaxanthin is the most effective of the three xanthophyll derivatives in heat dissipation , where antheraxanthin is only half as effective as zeaxanthin and antheraxanthin make up to 60% of the total xanthophyll cycle pool in photosynthesizing organs that grow under full sunlight conditions , to absorb excess light energy and dissipate as heat thus preventing damage to the photosynthetic activity of chloroplast , which is commonly known as photoinhibition . therefore , the concurrent effect of high temperature and exposure to light leads to a higher rate of photodegradation of these light - harvesting active biomolecules which could be seen as limiting factors in the usage of natural pigments without structural modification in dssc . dsscs fabricated using these extracted dyes were studied to preliminarily determine their synergistic performances as sensitizers of this low cost technology from a green source . the current density to voltage ( i - v ) characteristics of the dsscs sensitized with the extracted pigments ( figure 7 ) is recorded as shown in table 1 . the performance of the dsscs sensitized with the chlorophyll dye showed conversion efficiency ( ) of 0.055% , with open circuit voltage ( voc ) of 0.585 v and short circuit current density ( isc ) of 0.145 ma cm , and with fill factor ( ff ) of 0.59 . xanthophyll dye displayed active photochemical activities on the mesoporous tio2 electrode with conversion efficiency ( ) of 0.038% , open circuit voltage ( voc ) of 0.61 v and short circuit current density ( isc ) of 0.104 ma cm , and fill factor ( ff ) of 0.54 . in nature , xanthophyll acts as accessory pigments to broaden light harvesting capability . however , the results of this work suggest that xanthophyll too has the ability to absorb light energy and transfers the excited electrons to the semiconductor tio2 . the mixture of chlorophyll and xanthophyll dyes ( mix dye ) was expected to perform better than individual dyes due to the broadening of the uv - vis spectrum of mix dye in the blue region ( see figures 2 and 4 ) . the results of power conversion efficiency indicated that mixed dye system performed ~1.5 times and ~2 times higher compared to individual pigment of chlorophyll and xanthophyll , respectively . similarly , an increase in both short circuit current and open circuit voltage in the mix dye system were observed . this indicates that mixed cosensitization with these two pigments could effectively transfer energy synergistically to the tio2 semiconductor . in furthering our understanding of the kinetic process of electrochemical and photoelectrochemical processes happening in the dssc system , electrochemical impedance spectroscopy ( eis ) experiment electron recombination resistant and effective electron lifetime were determined by fitting eis spectrum using suitable equivalent circuit that mimics the physical process in the dssc . figure 8(a ) shows nyquist plots of dsscs sensitized with individual chlorophyll , xanthophyll , and mix dyes , under bias voltage conditions . the first arc ( a ) corresponds to the impedance at pt electrode / electrolyte and the second arc ( b ) corresponds to the impedance at tio2/dye / electrolyte interfaces . these impedances were determined by fitting the eis data in terms of an appropriate equivalent circuit as shown as an inset in figure 8(a ) . the magnitude of the second arc of the nyquist plot can be used to deduce the charge transfer resistances related to the recombination of electrons ( rk ) in tio2/dye / electrolyte interface . the rk values were 205 , 97 , and 222 for dsscs sensitized with chlorophyll , xanthophyll , and mixed dye , respectively . low recombination resistance ( rk ) in tio2/dye / electrolyte interface directly affects decay of voc in dsscs due to high recombination process . energy transfer between chlorophyll and xanthophyll molecules via quenching effect could reduce the recombination of dark current . figure 8(b ) illustrates the bode plot of chlorophyll , xanthophyll , and mixed dye sensitized dsscs . peaks at the low frequency ( 1010 hz ) and the high frequency ( 1010 hz ) were associated with effective life - times of electrons on the interface of tio2/dye / electrolyte ( eff ) and on the platinum electrode / electrolyte interface , respectively . these peak frequencies were inversely proportional to the respective effective life - times of electrons . eff was calculated for chlorophyll , xanthophyll , and mixed dye sensitized dssc as 0.313 , 0.123 , and 0.250 seconds , respectively ( see table 2 ) . these results indicate a higher effective life - time of electrons ( eff ) when dssc was sensitized with chlorophyll and mixed dye as compared to that of xanthophyll . mixed dye with the highest electron recombination resistance ( rk ) in tio2/dye / electrolyte interface performed best among the tested dsscs . in order to obtain an efficient dssc , a reduction in charge transport resistance ( rw ) , while increase in the combined factors such as effective life - time of electrons ( eff ) , electron recombination resistance ( rk ) , electron density ( s ) and diffusion factor ( deff ) , in tio2/dye / electrolyte interface is required . therefore , the efficiency of dssc sensitized with mixed dye was in the agreement with the electrochemical impedance data . the results revealed that the synergistic effect of chlorophyll and xanthophyll dominantly increased the electron recombination resistance and therefore enhanced the photoconversion efficiency of dssc . dyes derived from a single natural source , cladophora algae , give positive interaction when employed , as a mix , in dssc as the sensitizer . the extracted chlorophyll and xanthophyll were used as mixed dye sensitizer to obtain an enhanced overall cell performance whereby the was increased up to 1.5 and 2 times as compared to the respective individual dyes . both temperature and light were found to be critical factors in the degradation of both chlorophyll and xanthophyll dyes . photodegradation was best indicated by individual chlorophyll dye , where total degradation was observed in less than 24 hours at higher temperature and the presence of light . however , quenching effect performed by xanthophyll through conformational changes ( xanthophyll cycle ) could contribute to the overall durability of the dssc . the synergistic performance of the mixed dye in the dssc photovoltaic system resulted in a reduction in electron recombination ( high electron recombination resistant ) process that contributed to increase in the overall cell performance .

chlorophyll and xanthophyll dyes extracted from a single source of filamentous freshwater green algae ( cladophora sp . ) were used to sensitize dye sensitized solar cells and their performances were investigated . a more positive interaction is expected as the derived dyes come from a single natural source because they work mutually in nature . cell sensitized with mixed chlorophyll and xanthophyll showed synergistic activity with improved cell performance of 1.5- to 2-fold higher than that sensitized with any individual dye . the effect of temperature and the stability of these dyes were also investigated . xanthophyll dye was found to be more stable compared to chlorophyll that is attributed in the ability of xanthophyll to dissipate extra energy via reversible structural changes . mixing the dyes resulted to an increase in effective electron life time and reduced the process of electron recombination during solar cell operation , hence exhibiting a synergistic effect .