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eclampsia is an acute neurological complication of preeclampsia characterized by seizures and/or consciousness disorders which can not be related to another neurological disease . while in developed countries , eclampsia worsens in 12% cases of preeclampsia ; however , it is a real public health problem in developing countries where eclampsia is an obstetric emergency and common intensive care . hence , its management requires a multidisciplinary approach , combining , according to the case , obstetricians , pediatricians , anesthetists , and intensive care and emergency agents . in sub - saharan africa , the statistics are alarming . it has been recorded about 20% of maternal mortality in abidjan and 35% in senegal . among the morbidity factors , this neurological disease was first described in 1881 by the discovery of cerebral hemorrhage in eclampsia . recent studies using computed tomography ( ct ) and magnetic resonance imaging ( mri ) helped to better understand brain lesions that may occur during an eclampsia . the aim of this study was to identify the types of neurological lesions observed on ct in eclampsia to determine their impact on the prognosis of the patients in terms of survival or death . we report 39 cases of brain lesions in patients admitted to the intensive care unit ( icu ) of the university hospital of cocody . this is a descriptive , prospective study carried out in collaboration with the medical imaging service . it was conducted from january 1 , 2012 , to june 30 , 2013 , that is 18 months . the brain ct was performed after parental consent of the patients in whom the diagnosis of eclampsia was confirmed . for each patient , we collected on a standardized form epidemiological data ( maternal age , maternity , parity , gestational age , and origin ) , clinical data ( state of consciousness , blood pressure , deficient signs , and infectious syndrome ) , biological data ( blood count and platelets , transaminases , prothrombin rate , thick smear , bilirubin , blood urea nitrogen , creatinine , proteinuria , and blood gases ) , interpretation of the brain ct ( type , location of lesions ) , therapeutic data ( previous treatment before admission in icu and that received in icu : diazepam , phenol - barbital , antihypertensive , tracheal intubation , mechanical ventilation , sedation , etc . , ) , and finally fate of patients in terms of length of hospitalization , survival , or success . the ct interpretation was made by the same physician in the imaging unit of the university hospital of cocody . we considered the obstetrical treatment time lapse between the occurrence of the first seizure and expulsion of the fetus and the time of admission of the patient in intensive care . the time of completion of the ct was comprised between the occurrence of the first seizure and the time of completion of the ct scan . the capture and analysis of the results were performed with epi info 2002r2 windows 9x , nt 4.0 , 2000 , xp of cdc atlanta . the quantitative variables were expressed as means matching their dispersion index . in 18 months , among the 54 patients admitted to the icu for eclampsia , 39 had brain lesions ( 72% ) . they had a mean gestational age of 34 2.55 weeks of amenorrhea ( range , 2739 wa ) . the admission time was 9.88 10.20 h ( range , 0248 h ) . among them , 24 patients underwent a caesarean section with general anesthesia . the time of care spent in the icu was 07.13 4.18 h ( range , 0320 h ) . they were dominated by a disturbance of consciousness with a glasgow score of < 10 ( 61.5% ) , high blood pressure > 160/100 mmhg ( 61.5% ) , subintrant seizures ( 51.3% ) , hellp syndrome ( 33.3% ) , pyramidal syndrome ( 2.5% ) . they received an antihypertensive treatment with nicardipine through infusion dose of 14 mg / h and diazepam through intravenous infusion at a dose of 0.10.3 mg / kg / h . the timeline for the completion of the ct scan was 42 30 h ( range , 672 h ) . these were ischemia : 10 cases ( 25.6% ) [ figures 1 and 2 ] , bruising intraparenchymal : 3 cases ( 7.7% ) [ figure 3 ] , and subarachnoid hemorrhage : 1 case ( 2.5% ) , cerebral edema : 9 cases ( 23% ) [ figure 4 ] of which 5 were isolated and 4 associated with ischemia . the locations were variously associated ( parietal , parietal - frontal , parieto - fronto - occipital , occipital , and middle cerebral artery . well systematized in the left occipital with deletion of grooves opposite ( red arrow ) corresponding to ischemic lesion in the area of the posterior cerebral in an eclamptic patient of 27 years cortico - subcortical hypodensity in internal parietal associated with hemorrhagic reshuffle ( red arrow ) with deletion of cortical grooves opposite , in the framework of an affection of the posterior branch of the anterior cerebral in an eclamptic patient of 27 years old hyperdense oval spontaneously homogeneous left - hemispheric centro ( red arrow ) associated with subarachnoid component ( yellow arrow ) accompanied by a peripheral hypodense halo under acute cerebral subarachnoid hemorrhage in an eclamptic patient of 25 years old diffuse deletion of cortical sulci and basal cisterns accompanied by a collapse of the ventricular system with gray - white substance dedifferentiation substance diffuse cerebral edema in an eclamptic patient of 17 years old the ct came back normal in 20 patients ( 51.3% ) . in icu , antihypertensive treatment with nicardipine ( 24 mg / h ) was pursued by a syringe pump . the treatment was based on anticonvulsant phenobarbital ( 200400 mg intramuscularly ) parenteral route or sodium valproate ( 1.5 g/24 h ) by oral route . a tracheal intubation was performed in 30 patients ( 76.9% ) associated with mechanical ventilation in 18 cases . among them , 4 received neurosedation ( midazolam + fentanyl ) and the other 14 with mild sedation ( midazolam + tramadol ) . the hospitalization timeline varied from 1 to 14 days with an average of 6.16 3.26 days [ table 1 ] . caractristiques cliniques et volutifs des patients selon le type de lsions neurologiques other complications of preeclampsia were hellp syndrome ( 13 cases ) , acute pulmonary edema ( 2 cases ) , and renal malfunction ( 7 cases ) of which 2 required dialysis sessions . we recorded four deaths ( 10.2% ) and they all had hellp syndrome . among these deaths , 3 were characterized by a hemorrhagic stroke and 1 by an ischemic stroke [ table 1 ] . our study suffers some limitations related to the low rate of performance of brain ct ( 72% ) and completion timelines that differ significantly ( 672 h ) . however , it has the advantage of a prospective study as it included a larger number of patients ( 39 cases ) than those reported by other authors ( 1934 cases ) . this observation was found in a series in canada , which observed 21 cases of cerebral ischemia in 34 patients . indeed , in france , it has been observed a prevalence of edematous lesions whereas the taiwanese series , found a prevalence of hemorrhagic stroke . the occurrence of ischemic lesions in eclampsia may have its explanation in the pathophysiology of preeclampsia . indeed , complex phenomena in the placenta resulting in placental ischemia may be the cause of ischemic disorders in several organs . however , the most frequently cited cause is vasospasm which is the cause of hypoperfusion causing ischemia and/or cerebral edema . the absence of lesions in ct of 20 comatose patients does not exclude the possibility of brain lesions that could have been diagnosed by mri . in fact , in the french study , the mri exhibited some edematous brain lesions in eclamptic patients who had a normal ct scan . in the unit , the antihypertensive treatment with nicardipine . the fight against seizures was based on phenobarbital or sodium valproate because we do not have magnesium sulfate which has a higher efficiency . tracheal intubation was performed in thirty patients associated with mechanical ventilation in 18 cases due to severe vital distress . among them , 4 received neurosedation and the other 14 received a mild sedation ( midazolam + tramadol ) . in the course of administration of this treatment , 4 patients ( 10.2% ) died . the literature reported that hemorrhagic strokes during eclampsia have a bad prognosis compared to ischemic stroke . all patients who died in our series also had hellp syndrome , a severe complication of preeclampsia . hellp syndrome associated with stroke is a factor of bad prognosis in patients with eclampsia . coagulation disorders encountered in this condition associated with severe hypertension could explain the occurrence of the stroke h. further , this mortality rate can be explained by the clinical features . patients with hemorrhagic stroke admitted to the icu exhibited a deeper consciousness disorder ( glasgow score < 9 ) , and the timeline for completion of the ct was relatively long ( 72 h ) which was a delay in the adequate care . eclamptic patients admitted to the icu often suffer from various intracerebral lesions . performing a ct scan brain scans have an undeniable advantage in the management of eclamptic patients provided that it is performed early .

the aim of this study was to identify the encephalic lesions in the eclampsia occurrences . within a period of 18 months , computed tomography ( ct ) of the brain was performed in all patients admitted in intensive care for eclampsia . these cts were analyzed and intracerebral lesions were identified . thirty - nine patients were included . we noted 10 cases of ischemic stroke , 9 cases of cerebral edema , and 3 cases of hemorrhagic stroke and subarachnoid hemorrhage . the ct scan came back to normal in 20 eclamptic patients . overall , delays in obstetric and intensive care and time of completion of the ct were long . ct has allowed highlighting in patients with eclampsia varied intracerebral lesions . the early performance of the ct is therefore essential for a better support of patients .

nowadays , an increasing amount of gene expression data sets is available through public repositories ( e.g. , ncbi geo , arrayexpress ) , which might contain the necessary clues for the discovery of new findings , leading to the development of new treatments or therapies . it is one of the most recent challenges to unlock the hidden potential of these data , by using it in large - scale analysis pipe - lines . integrating this vast amount of data originating from different but independent studies could be beneficial for the discovery of new biological insights by increasing the statistical power of gene expression analysis [ 3 , 4 ] . with integrative analysis we mean combining the information of multiple and independent studies , designed to study the same biological problem , in order to extract more general and more reliable conclusions . to this purpose , two approaches exist : meta - analysis and analysis by data merging . in the meta - analysis approach the results of individual studies ( e.g. , p values , ranks , classification accuracies , etc . ) in contrast , the merging approach integrates microarray data at the expression value level after transforming the expression values to numerically comparable measures . the first step , selecting and retrieving all appropriate data sets , is the same for both scenarios . the main difference between meta - analysis and the integrative analysis via data merging is visible in the next two steps : according to the meta - analysis approach ( figure 1(a ) ) each data set is analyzed individually and the final results are combined , while according to the merging approach ( figure 1(b ) ) the data sets are first combined in a unique , much larger data set and then analyzed . combining information from multiple existing studies can increase the reliability and generalizability of results . through the integrative analysis of microarray data the sample size increases and with it the statistical power to obtain a more precise estimate of gene expression results . this immediately overcomes the problem of low sample sizes , which is the main limitation for individual microarray studies . at the same time the heterogeneity of the overall estimate is assessed , making the results more generalizable . this way we avoid the danger of study - specific findings or artifacts . integrative analysis is also a relatively easy and inexpensive way of gaining new biological insights since it makes comprehensive use of already available data accumulated through the years by various groups all over the world . in [ 5 , 6 ] several issues for integrative analysis are reported , and while being specific for meta - analysis , many of them are also relevant for merging . most of these issues are however related to the retrieval and preprocessing of the data and can be solved by using an appropriate data acquisition approach , as we show later in this work ( see section 2 ) . for the merging approach an additional issue has to be mentioned . before combining the expression values of different studies since the use of different experimentation plans , platforms , and methodologies by different research groups introduces undesired batch effects in the gene expression values [ 7 , 8 ] an additional transformation of the data to remove those effects is needed . to the best of our knowledge no real comparison of both approaches can be found in the literature . in a merging approach is preferred to find a robust prognostic marker for breast cancer using multiple microarray data sets , based on the hypothesis that [ ] deriving separate statistics and then averaging is often less powerful than directly computing statistics from aggregated data . there is however no empirical evidence to validate this statement yet . in this work we investigate both approaches in the context of the identification of lung cancer - related differentially expressed genes ( degs ) or biomarkers , that is , genes that have a discriminating profile in normal tissues versus lung cancer tissues . those genes can provide new insights into the biological mechanisms of lung cancer and of cancer in general and can lead to promising biomarkers and new directions for drug development or treatments . we select a number of already published and publicly available data sets containing both normal and cancer tissues and identify robust and stable degs using both approaches for integrative analysis . to retrieve relevant publicly available data sets we used the insilico db as a starting point . insilico db is a web - based central warehouse containing ready - to - use , consistently preprocessed , and expert - curated genome - wide data sets ( https://insilicodb.org/app/ ) . a list of potential data sets was programmatically retrieved from the insilico db using the getdatasetlist function from the r / bioconductor insilicodb package . this list was further restricted by defining the following constraints.only frozen rma ( frma , ) processed studies were considered , that is , studies for which the original cel files were available and which were consistently preprocessed by the internal insilico genomic pipeline.each study should contain at least 30 samples in order to be statistically relevant.each study should contain both samples from normal tissue and from lung cancer tissue , more or less equally distributed . in order to achieve this we looked at the disease keyword which is available in most curations and filtered on lung cancer | adenocarcinoma values and control values for lung cancer and normal samples , respectively.only studies assayed on affymetrix human genome u133a ( gpl96 ) and affymetrix human genome u133 plus 2.0 ( gpl570 ) were taken into consideration . this search resulted in a list of six studies , summarized in table 1 . for each data set a new curation these curations contain the disease keyword with control and lung cancer as keywords and are used as such through the rest of this paper . only frozen rma ( frma , ) processed studies were considered , that is , studies for which the original cel files were available and which were consistently preprocessed by the internal insilico genomic pipeline . each study should contain both samples from normal tissue and from lung cancer tissue , more or less equally distributed . in order to achieve this we looked at the disease values for lung cancer and normal samples , respectively . only studies assayed on affymetrix human genome u133a ( gpl96 ) and affymetrix human genome u133 plus 2.0 ( gpl570 ) a very important application of microarray studies is the identification of genes that are consistently and significantly differentially expressed in one group of samples compared to another , according to a target biological variable of interest . these genes are called informative genes , biomarkers , or differentially expressed genes ( degs ) . many methods and approaches to find degs exist and here we opted for the r / bioconductor limma package . recent and detailed overviews of possible alternative methods can be found in [ 19 , 20 ] . after applying limma we call every gene significantly differentially expressed ifit has an adjusted p value lower than 0.05;it has a log fold change higher than 2 . in order to ensure the robustness of the found deg lists and to encounter false - positive discoveries we implemented an extra resampling step on top of the limma method . in each iteration , we arbitrarily keep 90% of the samples and apply limma to obtain a deg list fulfilling the two above - mentioned criteria . after n resampling iterations we obtained n different deg lists and our final deg list will be the intersection of those lists . taking the intersection might seem rather strict but we empirically confirmed a convergence of the number of degs after around 50 resampling iterations , depending on the quality of the study . it has an adjusted p value lower than 0.05 ; it has a log fold change higher than 2 . the general workflow for both meta - analysis and merging approaches was already visualized in figure 1 . for the meta - analysis approach ( see figure 1(a ) ) , we first obtain a robust deg list as described above for each of the six studies individually and then combine the results by taking the intersection of those deg lists . this final list of degs will contain all genes that were found to be informative across all studies consistently . for the merging approach ( see figure 1(b ) ) , we first merge all six studies into one global data set using the following batch effect removal methods : none ( no batch effect removal ) , bmc ( batch - mean centering , ) , combat ( empirical bayes , ) , dwd ( distance - weighted discrimination , ) , and xpn ( cross - platform normalization , ) . then we applied to each merged data set the same procedure to find robust degs . we first look at the results of the meta - analysis approach by looking at the number of degs obtained from the individual data sets as listed in table 2 . in the second column the number of degs without using resampling is shown , followed by the number of degs after applying resampling as explained in the previous section . we notice that using this resampling strategy leads to a decrease in the number of degs for all data sets ( ratios between 60 and 80% depending on the specific data set ) . the rationale behind this extra validation step for the biomarkers discovery is the fact that a stable biomarker should be identified even by making small perturbations in the data set , caused by removing systematically random samples . another observation that can be made is the higher number of degs for the last three data sets . this difference is probably due to the difference in platform : gpl96 for the first three studies and gpl570 for the last three studies ; see table 1 . since the latter platform has more than 7000 genes more than the former platform , a higher chance of finding degs is obvious . also note from table 1 that the average sample size for platform gpl96 is around 74 , while for platform gpl570 it is around 122 ; this also can have a minor effect on the robustness of degs . the final list of degs in the meta - analysis approach can be obtained by taking the intersection of all single - study deg lists . this list of 25 genes consists of genes that are consistently differentially expressed in all six studies and can be considered as the most promising list of biomarkers for lung cancer , based on our input data . this list can be found in supplementary information ( s1 ) available online at http://dx.doi.org/10.1155/2014/345106 . in the merging approach , all six data sets are first merged into one global data set and thus only one deg list is finally retrieved . within this study we applied six different batch effect removal methods resulting in six different lists of degs . the results are presented in table 3 by listing the number of degs found for every batch effect removal method . we still notice a need for resampling as it clearly helps to remove false positives , although the difference in number of degs with and without resampling is less prominent than in the meta - analysis approach ( ratios between 85 and 89% for the different batch effect removal methods ) . as a first remark from table 3 we can observe a relatively low impact of using batch effect removal for this particular study . with the exception of the xpn method , the methods of bmc , combat , and dwd are not able to find more degs than when no batch effect removal at all is performed . however , the six lists have 102 genes in common , which is quite a significant result . the final list of degs in the merging approach is obtained by taking the intersection of all batch effect removal methods . similar results of none and the other batch effect removal methods represent a surprising result that we investigated more in detail since there are clearly batch effects present , as is demonstrated in a multidimensional scaling plot of the merged data set with no batch effect removal ( none ) in figure 2 . from this mds plot we can see that the biggest source of variation is rather technical than biological since all samples are clustered in two groups corresponding to the two different platforms they were assayed on . based on our results this is however not the case and the explanation lies in the fact that mds plots provide a global view on the data , while the identification of degs is more based on local effects , that is , the specific expression of one gene in certain conditions . the explanation for this apparently paradoxical result is in the fact that not all genes are affected by batch effect removal in the same way . genes which are differentially expressed in individual studies could still remain differentially expressed after data merging with no batch effect correction , if their expression is not much affected by batch effects . moreover , even if a gene is affected by batch effects we observed that the difference between the two modes or conditions of the gene ( in our case control versus lung cancer ) is almost always preserved over all samples of the merged data set . to illustrate the local effect of batch effect removal methods we will inspect two genes in detail . first , gene adrb1 is one of the genes that was only identified as a deg if no batch effect removal was applied , raising the question if batch effect removal method is distorting the biological signal of this gene . we can look at the boxplots of this specific gene in figure 3 . on the top left plot we can notice that this gene is only differentially expressed in three studies and those three studies are from the same gpl570 platform . for the other studies from the gpl96 platform , the situation is completely different with an almost stable expression of the adrb1 gene . the difference in expression in the three studies is however big enough to bias the global expression as being differentially expressed ( fc > 2 ) , as can be seen in the bottom left plot . if we apply batch effect removal , all samples from both platforms are brought closer together , thereby decreasing the influence of the differential expression of platform gpl570 . this results in a global expression that is not differentially expressed anymore ( fc < 2 ) see bottom right plot . from one point of view combat ( arbitrarily chosen , other batch removal methods are similar ) indeed removes a biological relevant signal that is present in the data , or at least part of the data , but one can argue that this signal is not consistent across all individual studies and can be due to a technical , platform - dependent artifact . we also investigate the gene lrrn3 which , in contrast , was only identified as deg if batch effect removal was applied . if we compare the top left and top right plots from figure 4 we can see that combat ( again arbitrarily chosen , other batch removal methods are similar ) nicely removes the batch effect between the different studies for this gene and creates a clear and consistent differential expression profile across all samples . this leads to a situation in which this gene is labeled as differentially expressed by the combat method , but not by the none method since it , just slightly , fails in the log fold change requirement . in this case , instead of a technical artifact , it is actually the batch effect that distorts the global expression profile of the lrrn3 gene . both meta - analysis and merging approaches are able to find differentially expressed genes ( degs ) consistently expressed in all individual data sets . in both approaches a resampling or bootstrapping framework is needed to avoid false positives and to ensure robust gene lists . although batch effects were clearly present when merging the different data sets this surprising finding is however not generalizable to the clustering or classification of tasks since it is very depending on the specific application . if we compare the final degs for the meta - analysis approach with the list obtained in the merging approach we can conclude that significantly more degs are identified through merging . moreover , all 25 identified degs through meta - analysis are also identified in the merging approach . most genes in both lists were scanned in the literature and showed to play a role or at least be involved in the development of lung cancer and can be further validated and used in clinical applications .

an increasing amount of microarray gene expression data sets is available through public repositories . their huge potential in making new findings is yet to be unlocked by making them available for large - scale analysis . in order to do so it is essential that independent studies designed for similar biological problems can be integrated , so that new insights can be obtained . these insights would remain undiscovered when analyzing the individual data sets because it is well known that the small number of biological samples used per experiment is a bottleneck in genomic analysis . by increasing the number of samples the statistical power is increased and more general and reliable conclusions can be drawn . in this work , two different approaches for conducting large - scale analysis of microarray gene expression data meta - analysis and data merging are compared in the context of the identification of cancer - related biomarkers , by analyzing six independent lung cancer studies . within this study , we investigate the hypothesis that analyzing large cohorts of samples resulting in merging independent data sets designed to study the same biological problem results in lower false discovery rates than analyzing the same data sets within a more conservative meta - analysis approach .

black lipid membranes ( blms ) are widely used for recording the activity of incorporated ion channel proteins . however , blms are inherently unstable structures that typically rupture within a few hours after formation . here , stabilized blms were formed using the polymerizable lipid bis - dienoyl phosphatidylcholine ( bis - denpc ) on glass pipettes of 10 m ( i.d . ) . after polymerization , these blms maintained steady conductance values for several weeks , as compared to a few hours for unpolymerized membranes . the activity of an ion channel , -hemolysin , incorporated into bis - denpc blms prior to polymerization , was maintained for 1 week after blm formation and polymerization . these lifetimes are a substantial improvement over those achievable with conventional blm technologies . polymerized blms containing functional ion channels may represent an enabling technology for development of robust biosensors and drug screening devices .

prostate carcinoma is one of the most common malignant diseases in men . in croatia , introduction of prostate - specific antigen ( psa ) testing and , particularly , transrectal ultrasound ( trus)-guided prostate biopsy have greatly improved the detection of prostate cancer . biopsy can be performed in different patient positions , i.e. , the lithotomy position , the lateral decubitus position , or the knee - chest position , and typically , 10 , 12 , or even more core samples are taken . sampling of a greater number of biopsy cores causes some degree of discomfort or pain in most patients . one randomized trial indicated that intrarectal application of 2% lidocaine gel prior to biopsy was a simple , safe , and effective analgesic method for this purpose . however , a recent meta - analysis of 5 other randomized trials comparing intrarectal lidocaine gel with a placebo gel or no treatment and two subsequent trials comparing lidocaine gel with no treatment failed to detect a pain - relieving effect of this intervention . also , a recent randomized trial suggested that pain perception in patients undergoing prostate biopsy might be less profound in the left lateral decubitus ( lld ) position than in the lithotomy position , whereas another one did not . our standard procedure has been systematic 12-core prostate biopsy in the lithotomy position with the intrarectal placement of 10 ml of 2% lidocaine gel 5 minutes prior to the procedure . considering the conflicting reports on pain perception in the lateral decubitus vs. lithotomy position and the analgesic effects of intrarectal anesthetic gel [ 7 - 10 ] , the primary objective of the present study was to evaluate pain perception in patients undergoing prostate biopsy in the lld position compared with the lithotomy position , and the secondary objective was to evaluate the analgesic effect of intrarectal 2% lidocaine gel . this prospective , randomized , single - center , open - label trial ( with blinded data analysis ) was performed between february 2008 and july 2010 in general hospital varazdin , croatia . consecutive eligible patients were randomly assigned to one of three groups : group 1 , biopsy performed in the lld position without topical lidocaine ( " test " treatment ) ; group 2 , biopsy performed in the lithotomy position with intrarectal application of 10 ml of 2% lidocaine gel 5 minutes before the procedure ( our standard procedure , " positive control " treatment ) ; and group 3 , biopsy performed in the lithotomy position without topical lidocaine ( " negative control " treatment ) . in the five trials embraced by the recent meta - analysis and two subsequent trials , pain scores on a 10-point visual analogue scale ( vas ) in patients undergoing prostate biopsy with or without intrarectal anesthetic gel averaged between 3 and 5.6 with standard deviations ( sds ) between 1.5 and 2.7 . in an analysis of variance with 3 treatment groups , assuming a mean vas score of 5 under the null hypothesis and an sd of 2.5 , a total of 126 patients ( 42 per group ) would provide 81% power at the overall 0.05 alpha level to detect a mean difference of 1.5 vas score points between any two treatments . the targeted detectable difference ( 1.5 points ) was set arbitrarily but accounted for the fact that the effect of the periprostatic nerve block vs. placebo has been estimated at around 2.1 score points and at around 1.5 score points as compared with intrarectal lidocaine . considering a possible imprecision of the variability estimate , we decided to enroll a total of 150 patients ( 50 per group ) to ascertain at least 80% power for detection of the targeted difference . the randomization list was generated by using the permutated block method ( block size 6 ) and was concealed from the patient - enrolling investigators ( confined with a registered nurse assisting in the procedure but not participating in the study ) . when a patient met the inclusion and exclusion criteria , the assigned treatment was disclosed . inclusion criteria were as follows : 1 ) indication for a prostate biopsy ( elevated psa or psa velocity 0.75 ng / ml ) or an abnormal digital rectal finding , 2 ) no previous prostate biopsy , and 3 ) informed consent . exclusion criteria were : 1 ) concomitant anorectal disease , 2 ) current treatment with analgesics , 3 ) impaired intellectual ability ( inability to understand the informed consent ) , and 4 ) contraindications for the lithotomy position ( e.g. , hip joint disease or a contracture ) . all biopsies were performed by the same investigator ( a qualified urologist ) on a siemens sonoline sl-1 ultrasound device with siemens endo - p sonde biplane , us biopsy 18 g biopsyneedle , and promag 2.2 automatic biopsy system and were systematic 12-core biopsies ( all cores were obtained from the lateral areas of the prostate , 2 from the base , 2 from the mid - lobe , and 2 from the apex on each side ) . antibiotic prophylaxis ( 2500 mg oral ciprofloxacin / day ) was started the night before the procedure and was extended for 4 days . immediately after the procedure , patients ' pain perception was assessed by using a 10-point vas ( 0 , no pain ; 10 , excruciating pain ) . all other visits or procedures were performed on an " as - needed " basis . blinded data analysis was performed by an investigator not included in the clinical part of the trial . considering the trial objectives , the comparisons of interest were between group 1 ( lld position , " test " ) and groups 2 and 3 ( lithotomy position with or without lidocaine gel , i.e. , " positive control " and " negative control " control , respectively ) and between group 2 and group 3 . univariate comparisons were performed by using kruskal - wallis nonparametric analysis of variance with adjustment for three pair - wise comparisons ( dunn 's test ) . because in a multivariate analysis ( adjustment for age , psa value , prostate volume , pathohistological diagnosis , and digital rectal finding ) , residuals of the vas scores showed a normal distribution , a general linear model was fitted to the data to obtain adjusted ( least - square , ls ) means and ls - mean differences between the groups with tukey - kramer adjustment for multiple comparisons . because the ls - means for group 2 and group 3 were practically identical , an additional analysis was done in which group 1 ( lld position ) was compared with groups 2 and 3 cumulatively ( lithotomy position ) . we used stata ver . 9.0 ( statacorp lp , college station , tx , usa ) software . this prospective , randomized , single - center , open - label trial ( with blinded data analysis ) was performed between february 2008 and july 2010 in general hospital varazdin , croatia . consecutive eligible patients were randomly assigned to one of three groups : group 1 , biopsy performed in the lld position without topical lidocaine ( " test " treatment ) ; group 2 , biopsy performed in the lithotomy position with intrarectal application of 10 ml of 2% lidocaine gel 5 minutes before the procedure ( our standard procedure , " positive control " treatment ) ; and group 3 , biopsy performed in the lithotomy position without topical lidocaine ( " negative control " treatment ) . in the five trials embraced by the recent meta - analysis and two subsequent trials , pain scores on a 10-point visual analogue scale ( vas ) in patients undergoing prostate biopsy with or without intrarectal anesthetic gel averaged between 3 and 5.6 with standard deviations ( sds ) between 1.5 and 2.7 . in an analysis of variance with 3 treatment groups , assuming a mean vas score of 5 under the null hypothesis and an sd of 2.5 , a total of 126 patients ( 42 per group ) would provide 81% power at the overall 0.05 alpha level to detect a mean difference of 1.5 vas score points between any two treatments . the targeted detectable difference ( 1.5 points ) was set arbitrarily but accounted for the fact that the effect of the periprostatic nerve block vs. placebo has been estimated at around 2.1 score points and at around 1.5 score points as compared with intrarectal lidocaine . considering a possible imprecision of the variability estimate , we decided to enroll a total of 150 patients ( 50 per group ) to ascertain at least 80% power for detection of the targeted difference . the randomization list was generated by using the permutated block method ( block size 6 ) and was concealed from the patient - enrolling investigators ( confined with a registered nurse assisting in the procedure but not participating in the study ) . when a patient met the inclusion and exclusion criteria , the assigned treatment was disclosed . inclusion criteria were as follows : 1 ) indication for a prostate biopsy ( elevated psa or psa velocity 0.75 ng / ml ) or an abnormal digital rectal finding , 2 ) no previous prostate biopsy , and 3 ) informed consent . exclusion criteria were : 1 ) concomitant anorectal disease , 2 ) current treatment with analgesics , 3 ) impaired intellectual ability ( inability to understand the informed consent ) , and 4 ) contraindications for the lithotomy position ( e.g. , hip joint disease or a contracture ) . all biopsies were performed by the same investigator ( a qualified urologist ) on a siemens sonoline sl-1 ultrasound device with siemens endo - p sonde biplane , us biopsy 18 g biopsyneedle , and promag 2.2 automatic biopsy system and were systematic 12-core biopsies ( all cores were obtained from the lateral areas of the prostate , 2 from the base , 2 from the mid - lobe , and 2 from the apex on each side ) . antibiotic prophylaxis ( 2500 mg oral ciprofloxacin / day ) was started the night before the procedure and was extended for 4 days . immediately after the procedure , patients ' pain perception was assessed by using a 10-point vas ( 0 , no pain ; 10 , excruciating pain ) . all other visits or procedures were performed on an " as - needed " basis . blinded data analysis was performed by an investigator not included in the clinical part of the trial . considering the trial objectives , the comparisons of interest were between group 1 ( lld position , " test " ) and groups 2 and 3 ( lithotomy position with or without lidocaine gel , i.e. , " positive control " and " negative control " control , respectively ) and between group 2 and group 3 . univariate comparisons were performed by using kruskal - wallis nonparametric analysis of variance with adjustment for three pair - wise comparisons ( dunn 's test ) . because in a multivariate analysis ( adjustment for age , psa value , prostate volume , pathohistological diagnosis , and digital rectal finding ) , residuals of the vas scores showed a normal distribution , a general linear model was fitted to the data to obtain adjusted ( least - square , ls ) means and ls - mean differences between the groups with tukey - kramer adjustment for multiple comparisons . because the ls - means for group 2 and group 3 were practically identical , an additional analysis was done in which group 1 ( lld position ) we used stata ver . 9.0 ( statacorp lp , college station , tx , usa ) software . two patients randomly assigned to group 3 ( lithotomy position , no lidocaine ) decided not to participate in the trial ; hence , a total of 148 patients were included . patients in the three groups were generally comparable regarding age , psa levels , prostate volume , digital rectal findings , and pathohistological diagnosis ( table 1 ) . in the univariate analysis , vas pain scores in group 1 ( lld position , no lidocaine ; median , 2.95 ) were significantly lower than in either group 2 ( lithotomy position+lidocaine ; median , 4.95 ) or group 3 ( lithotomy , no lidocaine ; median , 4.60 ) , whereas the difference between groups 2 and 3 was insignificant ( table 2 ) . after adjustment for the factors depicted in table 1 , vas pain scores in group 1 remained significantly lower than in group 2 or group 3 , whereas there was practically no difference between group 2 and group 3 ( table 3 ) . two patients in group 3 experienced minor rectal bleeding during the post - biopsy observational period . one patient in group 1 and one in group 3 developed a febrile uroinfection , which responded well to antibiotics and subsided within 2 weeks after the procedure . hematuria was observed in 17/50 patients ( 34% ) in group 1 , 20/50 patients ( 40% ) in group 2 , and 15/48 patients ( 31% ) in group 3 , all resolving within 2 weeks after biopsy . trus - guided prostate biopsy has greatly improved the diagnosis of prostate cancer because it is a highly sensitive and specific method that is generally ( regardless of the performance modalities ) safe and well tolerated . the major disadvantages of the procedure are anal discomfort , largely due to the insertion of the ultrasound probe ; pain , predominantly induced by the needle penetration through the prostate capsule ; and a certain level of psychological stress , largely related to the nature of the procedure ( e.g. , patient 's position , anal insertion of the probe , the possibility of a malignant disease ) . regarding pain management , the guidelines of the european association of urology suggest periprostatic block as a state of the art method . periprostatic block is more effective and apparently at least equally as safe and well tolerated as application of topical anesthetic gel or no local analgesic treatment . nevertheless , it may have complications , such as repeated injections during the biopsy , systemic lidocaine toxicity , urinary incontinence , and , occasionally , degradation of the image resolution due to anesthetic injection . therefore , other methods may still be considered legitimate , particularly because the level of pain induced by the biopsy is , at worst , moderate in most patients . topical intrarectal application of anesthetic gels ( e.g. , 2% lidocaine ) has been traditionally considered to relieve anal discomfort , but a recent meta - analysis of randomized trials and two subsequent trials indicated a lack of an analgesic effect . the evaluation of lidocaine gel in the present trial would have attained a higher level of evidence had we used a placebo gel and a full 22 factorial design ( i.e. , combination of patient positions and gel ) , but this was not the primary objective of this study . from the methodological standpoint ( randomization , accounting for potential confounders ) , the present observations should be viewed as a fair contribution to the body of evidence [ 8 - 10 ] indicating a lack of efficacy of topical lidocaine in this setting . the main finding of the present trial is a lower intensity of pain in patients undergoing prostate biopsy in the lld position than in the lithotomy position ( with or without intrarectal lidocaine gel ) as assessed on a 10-point vas immediately after the procedure . by size , the observed adjusted differences ( around 1.4 score points vs. the lithotomy position with 2% lidocaine gel , around 1.2 score points vs. the lithotomy position without lidocaine , and around 1.3 score points vs. cumulative lithotomy position ) are similar to those between periprostatic nerve block and intrarectal anesthetic gel ( around 1.5 score points ) and should therefore be considered practically relevant . similar observations were reported from a recent trial in which 340 patients were randomly assigned to undergo prostate biopsy either in the lld or lithotomy position without further topical analgesic treatment , but another smaller trial ( total n=70 ) reported less pain in the lithotomy position than in the lld position . there appears to be no plausible explanation for the discrepancy between the current and some of the published vs. other published data , except for the fact that all three trials were single - center trials and that under such conditions , observations in smaller trials could be by chance ( due to sampling variability ) . it should be noted , however , that in the present trial the " effect of patient position " was consistently significant in statistical terms and practically relevant by size in both univariate comparisons and after adjustment for potentially relevant covariates . taken together , the present and the published data strongly suggest that patients ' pain perception during trus - guided prostate biopsy in the lld position might be relevantly lower than in the lithotomy position , and this topic thus deserves further well - designed multicentric evaluations . the present data do not allow for a straightforward explanation of the beneficial " effect " of the lld position . perception of pain is a highly subjective psychological phenomenon and the methods commonly used in this setting ( e.g. , vas scoring systems ) evaluate exactly that - the subjective interpretation of painful stimuli that may be affected by a number of other psychological phenomena , particularly anxiety and depression , which are highly prevalent among patients undergoing prostate biopsy . the lithotomy position could be more unpleasant or embarrassing than the lld position , and therefore , the pain perception could be intensified ; it is considered " gynecological " and " exposing " as compared to the lld position , which is a more relaxed , physiological position typically used for sleep and rest . therefore , it could be viewed as a stress - relieving position that could be beneficial during this relatively unpleasant procedure . unfortunately , neither the present nor the published studies evaluated the levels of depression or anxiety concomitantly with the pain evaluation . we suggest that such evaluations in future studies could help us to understand the underlying mechanisms of the observed phenomena . in conclusion , the present results support the view that the subjective perception of pain during prostate biopsy is less profound in patients in the lld position than in the lithotomy position and that intrarectal application of 2% lidocaine gel has no analgesic effect in this setting .

purposethe primary objective was to assess whether transrectal ultrasound ( trus)-guided prostate biopsy in the left lateral decubitus ( lld ) position differed from the procedure in the lithotomy position regarding patients ' pain perception . the secondary objective was to assess the analgesic effect of intrarectal 2% lidocaine gel in this setting.materials and methodsthis single - center , open - label trial enrolled 148 men undergoing prostate biopsy . then men were randomly assigned to group 1 ( lld position , no lidocaine , n=50 , " test " ) , group 2 ( lithotomy position+lidocaine , n=50 , " positive control " ) , and group 3 ( lithotomy position , no lidocaine , n=48 , " negative control " ) . twelve - core samples were taken in each biopsy set . pain was assessed by using a 10-point visual analogue scale ( vas).resultsacross the groups , patients were comparable regarding age , prostate - specific antigen levels , prostate volume , digital rectal examination findings , and pathohistological diagnosis . vas scores were lower in group 1 ( median , 2.95 ) than in group 2 ( median , 4.95 ; p<0.001 ) or group 3 ( median , 4.60 ; p<0.001 ) . the difference between group 2 and group 3 was insignificant ( p=0.268 ) . the adjusted mean differences ( with adjustment for the above covariates ) were as follows : group 1 vs. group 2 , -1.43 ( 95% confidence interval [ ci ] : -2.25 to -0.60 ; p<0.001 ) ; group 1 vs. group 3 , -1.22 ( 95% ci : -2.04 to -0.41 ; p=0.001 ) ; group 2 vs. group 3 , 0.20 ( 95% ci , -0.63 to 1.04 ; p=0.836 ) ; and group 1 vs. groups 2 and 3 , -1.33 ( 95% ci , -1.92 to -0.73 ; p<0.001 ) . the procedure was comparably well tolerated across the groups.conclusionspain perception during prostate biopsy was lower in the lld position than in the lithotomy position . intrarectal 2% lidocaine gel does not seem to affect pain perception .

some autoimmune diseases can be considered a result of incomplete thymic negative selection or peripheral tolerance , resulting in the activation of t cells specific for autologous proteins . in evaluating these issues , we have been examining the chemistry of peptides selected by class ii mhc molecules during antigen processing ( summarized in reference 1 ) . we identified two sets of t cells that differed in their recognition of the same peptide segment 4861 of hen - egg white lysozyme ( hel ) 2345 . a type a t cell is the conventional t cell that recognizes the mhc - bound peptide resulting from the processing of the protein in the processing vesicles . it also recognizes the peptide when offered to apcs as an exogenous peptide , i.e. , a form that does not require intracellular processing . t cell recognizes only the exogenous peptide but not that derived from the processed protein . using hel as a protein antigen , we have shown that type b t cells escaped thymic negative selection in hel transgenic mice , thus raising the issue that these t cells could be involved in autoimmunity if an autologous protein is partially proteolyzed in tissues 4 . one explanation for the features of type b t cells is that they are recognizing changes in the exogenous peptide not found in the naturally processed peptide . these changes could occur during the handling of the peptide by the apc or from its synthesis or postsynthetic modifications . indeed , peptide modifications are important in the context of t cell recognition ( for examples , see references 6 and 7 ) , and one such change involves the deamidation of asparagines in proteins or peptides to yield aspartic acid and isoaspartic acid . reported the important finding that autologous peptides containing an isoasp modification were immunogenic : immunization of mice with murine cytochrome c peptide , residues 90104 , did not trigger t cell proliferation , but immunization with the isoasp containing peptide was effective 8 . furthermore , immunization with a peptide derived from a nuclear ribonucleoprotein having the isoasp also led to antibody production that cross - reacted to the unmodified protein . these findings led mamula et al . to postulate the possible involvement of such modifications in the autoimmunity of the lupus type . a recent report claims that the type b t cells described in our previous papers may react with a modification of the exogenous peptide brought about by the spontaneous conversion of asn to asp or isoasp 9 . asn in the peptide segment 5260 of hel is an important tcr contact amino acid . deamidation of asn in proteins and peptides yields isoasp and asp derivatives in the ratios of 4:1 10111213 . we demonstrate that an isoasp residue can be specifically recognized by t cells , confirming the mamula et al . , as the evaluation of a number of t cell clones indicates that the recognition of asp or isoasp is not the basis for the difference in the type a and type b reactivity . hel 4861 , ( dgstdygilqinsr ) , referred to as 4861 n59 , the segment in bold binds to i - a 14 , was synthesized using f - moc chemistry on a synergy 430a peptide synthesizer ( applied biosystems ) . hel 4861 with isoasp or asp at 59 referred to as 4861 d59 and hel 4861 d59 , respectively , were provided by dr . the binding of each peptide to purified i - a molecules was done as reported 15 using baculovirus produced molecules in a competitive binding assay . the following hybridomas were generated as described previously : 3a9 16 , alv 11 , alv48 , and dav21 3 . the cp series of hybridomas was generated by immunization of b10.br mice with 10 nmoles of hel or 4861 in cfa . hybridomas mla11.2 and mlc10.22 were generated by immunization of ml-5 transgenic mice 45 , expressing a secreted form of hel with 10 nmoles of the tryptic fragment of hel , 4661 in cfa . hybridoma b10a6.5 was generated by immunization of b10.br mice with 10 nmoles of the same tryptic fragment . the iso series of hybridomas were generated by immunization of b10.br mice with 10 nmoles of hel4861d59 . we have used both male and female b10.br mice which were raised in the small animal facility of washington university in st . m12.c3.f6 , a b lymphoma expressing i - a molecules ( 5 10 ) was incubated with decreasing doses of peptide in the presence of t cell hybridomas ( 5 10 ) in a total volume of 200 l in mem with 10% fetal calf serum . incubation was for 18 h , followed by transfer of supernatants to wells containing ctll-2 , an il-2dependent cell line . ctll-2 were cultured for 40 h , with [ h]thymidine added for the last 24 h. [ h]thymidine incorporation was determined using a wallac counter . most of the t cell hybridomas were tested against 4861 peptide ( batches 1255 , 1387 , or abbt , with comparable results ) . the isoaspartate content of peptides was determined by a procedure based on the incorporation of radioactive methyl groups into isoaspartate residues , using the enzyme protein isoaspartyl methyltransferase and s - adenosyl l-(methyl)-h methionine ( obtained from amersham pharmacia biotech ) . we used and followed exactly the instructions in the isoquant kit from promega ( lot number 116482 , catalog number ma1010 ) . we tested several batches of peptide that were used for several years and kept stored in water at ph 3 . hel 4861 , ( dgstdygilqinsr ) , referred to as 4861 n59 , the segment in bold binds to i - a 14 , was synthesized using f - moc chemistry on a synergy 430a peptide synthesizer ( applied biosystems ) . hel 4861 with isoasp or asp at 59 referred to as 4861 d59 and hel 4861 d59 , respectively , were provided by dr . the binding of each peptide to purified i - a molecules was done as reported 15 using baculovirus produced molecules in a competitive binding assay . the following hybridomas were generated as described previously : 3a9 16 , alv 11 , alv48 , and dav21 3 . the cp series of hybridomas was generated by immunization of b10.br mice with 10 nmoles of hel or 4861 in cfa . hybridomas mla11.2 and mlc10.22 were generated by immunization of ml-5 transgenic mice 45 , expressing a secreted form of hel with 10 nmoles of the tryptic fragment of hel , 4661 in cfa . hybridoma b10a6.5 was generated by immunization of b10.br mice with 10 nmoles of the same tryptic fragment . the iso series of hybridomas were generated by immunization of b10.br mice with 10 nmoles of hel4861d59 . we have used both male and female b10.br mice which were raised in the small animal facility of washington university in st . m12.c3.f6 , a b lymphoma expressing i - a molecules ( 5 10 ) was incubated with decreasing doses of peptide in the presence of t cell hybridomas ( 5 10 ) in a total volume of 200 l in mem with 10% fetal calf serum . incubation was for 18 h , followed by transfer of supernatants to wells containing ctll-2 , an il-2dependent cell line . ctll-2 were cultured for 40 h , with [ h]thymidine added for the last 24 h. [ h]thymidine incorporation was determined using a wallac counter . most of the t cell hybridomas were tested against 4861 peptide ( batches 1255 , 1387 , or abbt , with comparable results ) . the isoaspartate content of peptides was determined by a procedure based on the incorporation of radioactive methyl groups into isoaspartate residues , using the enzyme protein isoaspartyl methyltransferase and s - adenosyl l-(methyl)-h methionine ( obtained from amersham pharmacia biotech ) . we used and followed exactly the instructions in the isoquant kit from promega ( lot number 116482 , catalog number ma1010 ) . we tested several batches of peptide that were used for several years and kept stored in water at ph 3 . previous structural and immunological studies indicated that in the core segment of the 5260 peptide of hel , asn 59 is a solvent exposed residue that contacts the tcr 141718 . table indicates that immunization with 4861 d59 generated t cells specific for 4861 d59 peptide that did not recognize the wild - type 4861 n59 peptide . these t cells reacted with as little as 0.1 to 0.3 m of peptide , and yet failed to be triggered by as much as 30 m of 4861 n59 ( see iso.187 of fig . we also isolated t cells that recognized specifically the 4861 d59 and the 4861 d59 peptides ( table ) . a type a t cell is defined as reacting with both hel as well as to 4861 34 . a type b t cell will preferentially recognize the peptide and will react poorly , if at all , to hel . a small response can be obtained at the high levels of hel in culture , of 30 to 100 m . in table , all type a t cells were triggered by 0.1 m hel , while none of the type b were triggered up to 10 m . when a number of type a and type b t cells were examined for their response to the three peptides , we found two patterns of reactivity : the first set responded to the three peptides with slight differences in the sensitivities for each ( cp4.1 and cp3.43 ; fig . the second set only recognized the 4861 n59 peptide , but not the isoasp or asp derivatives ( 3a9 , b61.6 , and b10.a6.5 ; fig . two of four type a t cells only recognized the 4861 n59 , while the other two did not discriminate among the asn , asp , and isoasp 59 residues . likewise , 5 of 16 type b t cells were completely specific for the n59 tcr contact residue , while the remaining 11 had variable degrees of responses among the three peptides . studies are now being done to examine the use of the various tcr contact residues among these different t cells . the finding that the wild - type peptide , 4861 n59 , was recognized uniquely by either a and b types while , conversely , the 4861 d59 specific t cells failed to recognize it , argues strongly that spontaneous conversion of the asn 59 tcr contact residue in the 4861 n59 peptide is not the explanation for the type b specificity . we also tested for the degree of isoasp conversion in each of the peptides used in the past 6 yr . these peptides were repeatedly used and kept in the laboratory under various temperature conditions . using the incorporation of radioactive methyl groups , we failed to find significant levels of isoaspartyl conversion in 6/7 preparations that were tested ( table ) . whether the asn in these peptides is converted during the assay becomes irrelevant in light of the results with t cells . indeed , based on the sensitivities of the t cells indicated above , the amount of spontaneous isoasp conversion of the 4861 n59 peptide must be limited to < 1% in the 18 h of the assay . finally , we found only slight differences in binding of the three peptides to purified i - a molecules , with ic 50% of 0.12 , 0.24 , and 0.82 m for 4861 n59,4861 d59 , and 4861 d59 , respectively . in summary , our results extend the report by mamula et al . , calling attention to the fact , using cloned cells , that t cells can make an important discrimination between asn , asp , and isoasp residues in a peptide . this discrimination is important in the context of changes that may take place in autologous proteins in tissues , as they correctly speculated . it is also important in the context of peptides made in the laboratory 8 : as noted , one of our peptides ( 1317 in table ) had 17% of conversion and could have induced specific t cells . in our case , the importance of this issue is highlighted because asn 59 has been documented as a solvent exposed residue available for t cell recognition of the 5260 core segment of the hel peptide . from a thorough analysis of a large panel of t cell hybridomas , it is obvious that the recognition of asp or isoasp at the tcr contact residue 59 , which is p8 in the peptide , is independent of the type a and type b states and is not the feature that distinguishes them . paper we contend that conclusions are difficult to make on one or two t cells , particularly when the issue of sensitivity in the various manipulations of the assays are not taken into consideration . also , to note that other hel peptides in which we found the type a and type b t cells did not have the sequence of asn followed by the ser or gly , the destabilizing residues that favor the deamidation . we made the distinction between type a and b t cells based on our identification of the natural processed peptides of hel , from which we identified and quantitated their amounts displayed after processing . type b which was not displayed by the endogenous processing of hel . as the linear sequence was identical ( and not explained by postsynthetic modifications ) , the best explanation for the type b state of a peptide - mhc complex is that it results from a unique conformation 34 formed when exogenous peptides bind to class ii molecules in early endosomal compartments or on plasma membrane , as described in reference 19 . this conformation is distinct from the type a that results when peptides bind from a processed protein to a nascent class ii molecule in the processing compartments . as mentioned , the importance of type b t cells is that many escape thymic negative selection and peripheralize , representing a set of t cells that can potentially react with peptides formed at sites of inflammation 45 .

we have analyzed a panel of t cell hybridomas specific for the chemically dominant epitope of hen egg - white lysozyme 4861 which has asparagine 59 as an important t cell receptor contact residue . a number of t cells recognize 4861 with asparagine at position 59 , but not the aspartic acid or isoaspartic acid derivatives . conversely , we find t cells that specifically recognize 4861 bearing an isoaspartic acid at residue 59 , but not asparagine . for other t cells , asparagine , aspartic acid , or isoaspartic acid at residue 59 is irrelevant . we present evidence that our previous distinction between type a and type b t cells is not explained by asparagine deamidation at residue 59 .

reagents were of reagent grade and solvents were of hplc grade . tpc was purchased from porphyrin systems ( lbeck , germany ) and 10% palladium on charcoal was from aldrich ( st . tpp was synthesized by standard procedures from pyrrole and benzaldehyde and was metallated by refluxing it ( 2 mg in 15 ml of dry dimethylformamide ( dmf ) ) with 15 mg of anhydrous palladium(ii ) chloride for 30 min . the catalyst was filtered off by chromafil rc-20/25 disks ( machery - nagel , dren , germany ) . absorbances were measured on a uv vis spectrophotometer ( varian cary 50 bio ) and reaction mixture analysis was carried on an hplc ( agilent 1100 ) with rp-18 lichrospher column ( 2504 mm , 5 m ) and diode array uv vis detector and isocratic acetonitrile / thf ( 9:1 , v / v ) as eluent . conversions were obtained from the peak areas at 419 nm in the hplc chromatograms , after calibration using authentic samples of tpp and tpc . retention times ( tr ) were the following : chlorin 6.7 min , porphyrin 7.2 min , and pd(ii)tpp 9.4 min . ten milliliters of 2.40 m solution of tpp in thf was stirred at 25c for 1 h , with the amounts of catalyst ranging from 27 to 750 mg . the same procedure was repeated using toluene . as an example , 10 ml of 0.1 mm solution of tpp in toluene was stirred with 1 ml of 0.26 m toluene solution of formic acid , 0.4 ml of triethylamine and 50 mg of catalyst at 50c . at intervals , samples were withdrawn , filtered and injected on the hplc . as an example , 10 ml of 0.1 mm solution of tpp in toluene and 5 ml of aqueous 5 mm solution of sodium phosphinate ( 25 eq ) were refluxed in the presence of 50 mg of catalyst . reagents were of reagent grade and solvents were of hplc grade . tpc was purchased from porphyrin systems ( lbeck , germany ) and 10% palladium on charcoal was from aldrich ( st . tpp was synthesized by standard procedures from pyrrole and benzaldehyde and was metallated by refluxing it ( 2 mg in 15 ml of dry dimethylformamide ( dmf ) ) with 15 mg of anhydrous palladium(ii ) chloride for 30 min . the catalyst was filtered off by chromafil rc-20/25 disks ( machery - nagel , dren , germany ) . absorbances were measured on a uv vis spectrophotometer ( varian cary 50 bio ) and reaction mixture analysis was carried on an hplc ( agilent 1100 ) with rp-18 lichrospher column ( 2504 mm , 5 m ) and diode array uv vis detector and isocratic acetonitrile / thf ( 9:1 , v / v ) as eluent . conversions were obtained from the peak areas at 419 nm in the hplc chromatograms , after calibration using authentic samples of tpp and tpc . retention times ( tr ) were the following : chlorin 6.7 min , porphyrin 7.2 min , and pd(ii)tpp 9.4 min . ten milliliters of 2.40 m solution of tpp in thf was stirred at 25c for 1 h , with the amounts of catalyst ranging from 27 to 750 mg . as an example , 10 ml of 0.1 mm solution of tpp in toluene was stirred with 1 ml of 0.26 m toluene solution of formic acid , 0.4 ml of triethylamine and 50 mg of catalyst at 50c . at intervals as an example , 10 ml of 0.1 mm solution of tpp in toluene and 5 ml of aqueous 5 mm solution of sodium phosphinate ( 25 eq ) were refluxed in the presence of 50 mg of catalyst . although ctr presents practical advantages , it requires very fine tuning of several experimental parameters solvents , donors , catalysts in order to render the process efficient . key features in our approach are ( i ) the establishment of conditions to quantitatively analyze reaction progress with identification of side products , ( ii ) the study of adsorption isotherms , which are crucial in any heterogeneously catalyzed reaction , in order to predict which conditions are more likely to produce the best results , and ( iii ) screening and adjustment of hydrogen donors and other conditions to optimize yields . uv / vis is broadly used to monitor the reaction progress in porphyrin chemistry , but mainly due to spectral similarities of the involved species , hplc with diode array detection proved to be a more reliable quantitative methodology , with many reports on its application to the above compounds existing in the literature.[2023 ] therefore , some efforts were devoted to the establishment of elution conditions . in the measurement of the adsorption of porphyrins onto the catalyst , various solutions of known concentration of porphyrin were stirred with different amounts of the catalyst , under controlled conditions , and the decrease in porphyrin concentration was monitored by uv / vis [ figure 1 ] . lin ear plots of the langmuir adsorption isotherms for thf ( o ) and toluene ( ) tpp solutions on 10% palladium over charcoal initial attempts to reduce tpp were made using formic acid as hydrogen donor and thf or toluene as solvents . conversions to the desired product tpc were below 40% [ figure 2 ] along with the formation of metalloporphyrin pd(ii)tpp ( not quantified ) and trace amounts of bacteriochlorin and isobacteriochlorin . the metalloporphyrin is formed by complexation of pdions present on the catalyst , in spite of the reducing conditions , and was identified by comparison with an authentic sample . the latter compounds are due to reduction of the formed chlorin 2 , and were identified simply from their visible spectra characteristic bands . although the preparation of an authentic sample of pd(ii)tpc was attempted , the product of reaction between tpc and pdcl2 exhibited the same tr and visible spectrum as pd(ii)tpp , probably due to oxidation during the reflux and , therefore , the possibility of its formation can not be discarded . percent conversion ( reduction ) of tpp with time , in toluene , with formic acid ( o ) and with aqueous solution of sodium phosphinate ( ) in the second approach , a biphasic system consisting of a toluene solution of tpp and an aqueous solution of sodium phosphinate was used . product distribution was found to be dependent on donor concentration : when 25 equivalents of the latter were used , the desired product tpc was obtained in nearly 40% yield ; for other substrate : donor ratios ( 1:5 , 1:15 , 1:35 , 1:50 and 1:100 ) , an unidentified product was formed ( tr = 4.9 min ) . in all cases , the metalloporphyrin was observed , but none of the other side products were obtained . a plausible explanation for the stagnation of hydrogenation below 50% conversion is catalyst poisoning by the product , as although a double bond was reduced , most of the system responsible for the adsorption remained untouched , preventing product desorption . further work will be required to tackle this problem , but so far , it has been demonstrated that tpp could be reduced to tpc in a single step , although in moderate yields , using either ( i ) formic acid as hydrogen donor in a thf or toluene solution or ( ii ) a biphasic system of aqueous sodium phosphinate and toluene as solvent for the substrate , and 10% palladium on charcoal as catalyst .

a number of new porphyrin - based photosensitizers have been developed for photodynamic therapy ( pdt ) in recent years . chlorins , which are a reduced form of porphyrins , show better potential of application since they have a stronger absorption band on the red region of the visible spectrum and , hence , a deeper penetration into tissues . we found that by using heterogeneous catalytic transfer reduction ( ctr ) , meso - tetraphenylporphyrin ( tpp ) could be hydrogenated , although in modest yields , to meso - tetraphenylchlorin ( tpc ) in a single reaction step . best reaction conditions were attained using formic acid or sodium phosphinate / water as hydrogen donors , tetrahydrofuran ( thf ) or toluene as solvent and 10% palladium on charcoal as catalyst .

recently , the health care community has recognised the importance of using qol measurement as an essential component of a treatment modality 's efficacy . at every stage of disease , treatment choices may involve modalities that differ in side effects and impact upon qol . quality - of - life assessment can be helpful in weighting the risks and benefits of treatment options , particularly when differences in survival among the options are small or non - existent . quality of life is subjective in nature , therefore there is a wide agreement that health - related quality of life should be conceptualised as a multidimensional construct . physical functioning , disease - and treatment - related symptoms , psychological / emotional well being , and social interactions are critical domains that are included in most efforts to measure overall quality of life . when considering quality of life in advanced cancer patients one of the treatment choices is a palliative one , therefore we consider of great importance the inclusion of a new dimension when measuring quality of life in such a population , that of " choice of treatment " . recent studies and new articles clearly indicate that physicians must be educated to routinely ask patients about their wishes for medical care and to recognise that they are legally and morally bound to honour those requests . such communication is especially pressing in the context of advanced illness , when the achievement of a peaceful death assumes priority over inappropriate prolongation of dying . many valid assessment instruments have been developed that measure qol such as eortc , the functional assessment of cancer treatment ( fact ) . in 1986 , the european organisation for research and treatment initiated a research program to develop an integrated , modular approach for evaluating the quality of life of patients participating in international clinical trials . eortc with its clinical focus and its multicultural orientation provides a rather unique context for developing and testing quality of life questionnaires . the aim of our study was to assess the psychometric properties of a new quality of life instrument on a hellenic sample of terminally ill cancer patients receiving only palliative treatment , which is called pqli ( palliative care quality of life instrument ) . it was found to be concise , quantitative and easily used ; it has been designed primarily for use by the patients themselves ; it was based on the existing literature [ 7,9 - 11 ] and the items that the patients consider as most important to what they perceive as " quality of life " , the latter was elicited by means of qualitative research . it became evident from the qualitative assessment on the patients ' description on their qol that there is a need to participate in the treatment process ; this would give them a sense of control over their fatal disease . patients want a voice in their end - of - life care , and participation in treatment choices encompasses the psychosocial outcomes that these may have in their lives . when considering quality of life in advanced cancer patients the treatment choice is a palliative one , therefore we consider of importance the inclusion of a new dimension when measuring quality of life in such a population , that of " choice of treatment " . the lack of a questionnaire that examines the quality of life specifically in a palliative care setting , the individuals that form their support system , as well as the unique needs that these patients have , was the driving force for a measurement like this to be developed . patients with symptomatic incurable cancer disease were selected for study by means of palliative treatment modality . no restrictions were placed with regard to histologic type of cancer , age , or performance status . all patients attended the " pain relief and palliative care unit " of areteion hospital where the study took place , between january 2002 and october 2002 . criteria for inclusion were : age > 18 years , no cerebral metastases , no known psychiatric disorder , to be cognitively capable of filling in the questionnaire , fluent in the hellenic language , and off anticancer treatment for 3 months . from 630 cancer and non - cancer patients that were treated in the unit that period , 144 advanced cancer patients were drawn using stratified random sampling , according to the performance status , and were judged eligible to enter the study . from them , 24 patients ( 16.7% ) were excluded due to refusal to participate in the study . the final sample was consecutive and consisted of 120 responding patients from whom written informed consent was obtained . demographic and clinical data of sample the pqli is a 28-item questionnaire , composed of six multi - item scales ( 2 functional scales , 1 symptom scale , 1 choice of treatment scale , 1 psychological scale ) and a single item scale ( overall quality of life ) . each scale was accompanied by a title . the questionnaire development involved the following phases : first , literature search that identified the relevant qol issues , and the existing questionnaires , second , a provisional list of items was presented to 3 experts for feedback on appropriateness of content and breadth of coverage , third , the list was administered to patients from the target group to determine the extent to which they have experienced these problems , while they were asked to choose a number of issues that troubled them the most ( figure 1 ) . next , the resulting list of items was reviewed for clarity and overlap by other experts , and finally , the questionnaire was pretested by administering it to some patients ( n = 20 ) from the target population , and through structured interviews with each patient individually after the completion of the questionnaire . in these interviews , the patients rated the questionnaire scales within a range of " 1 " ( i.e. first choice ) to " 7 " ( i.e. seventh choice ) . the researchers , then , evaluated whether scores from the resulting pqli corresponded well with those independently obtained ratings . variables , which -according to patients -affect their quality of life . in the final questionnaire format six of the scales were presented into three optional statements to be scored 1 , 2 and 3 respectively . in " choice of treatment " scale the patients were asked to choose the item that is " most " important in the choice of treatment and rate it 1 ; then choose the item that is " next " important and rate it 2 ; and so on , the last item which is the " least " important , the patients were asked to rate it as 5 . the single item scale " overall quality of life " has the form of a bi - polar numerical scale . this rather short interval was chosen because of the imminent risk of sudden changes in their health status . the instrument was designed primarily to be a self - assessment but where the patient 's condition would not permit it the researcher assisted him / her . { in the appendix - see additional file 1 ] is presented the questionnaire in the english language . two independent translators translated the pqli in english and then another two independent translators translated it back to greek . the same translation method has already been used in the validity and reliability of the eortc qlq c-30 ( v.3 ) in greek . a range of analyses was carried out to establish scale reliability , and to evaluate empirically the validity of the questionnaire scales . higher mean scores from 0 to 100 represent a better level of functioning and qol on the scales of " activity " , " self - care " , " support " , " communication " , " psychological affect " , and " overall quality of life " , and higher mean values on the health status scale , represent more symptomatology and worse quality of life . the current procedures for scoring the pqli reflect the multidimensionality of the quality of life domain . internal consistency of the questionnaire before and during palliative treatment was assessed by cronbach 's alpha and was considered acceptable for group comparisons if the coefficient exceeded 0.70 , as recommended by nunnally . cronbach 's alpha tests whether the items in a questionnaire have a homogeneous content with respect to the construct of interest . test - retest reliability of patients ' ( n = 120 ) responses was evaluated by comparing the scores recorded on two occasions , an average of seven days apart ( spearman - rho test ) . the patient 's clinical stage did not change between the first and the second completion , and the status of the patients was stable between test and retest . due to its hierarchical nature , the intertest reliability of the ranking statements ( choice of treatment ) was established by using the " kendall's - w " test . five indirect methods to evaluate validity were adopted : first , by comparing the scale scores with patients with poorer and better eastern cooperative oncology group ( ecog - the clinical variable assessed ) performance status using the mann - whitney u non - parametric test . second , by assessing the statistical difference of the questionnaire - scales before and during treatment in terms of wilcoxon rank test between scales for related subjects . third , by exploratory factor analysis , using principal components with non - orthogonal ( direct oblimin ) rotation , was used to assess the validity of the pqli . as a fourth process , correlations were calculated between pqli items and those of two others instruments , the assessment of quality of life in palliative care ( aqel ) , and the european organisation for research and treatment of cancer ( eortc ) core quality of life questionnaire ( qlq - c30 , version 3.0 ) . the aqel is focusing in patients undergoing palliative treatment , includes 22 items in seven scales , from physical , psychological , social and existential domains , while it has proven to be reliable and valid . in addition , the eortc qlq - c30 is a widely used second - generation questionnaire designed to measure cancer patients ' physical , psychological and social functions . it is a psychometrically established 30-item questionnaire , incorporated in nine multi - item , and several single - item scales . concurrent related validity was performed with correlations among the scales of pqli ( inter - scale correlations ) . accordingly , the seven factors obtained from the interview from the 120 patients were rated and coded from 1 to 7 according to the patients ' choice : 1 , 2 , 3 , 4to 7choice . the closeness of the hypothetical model of pqli to the empirical data of interviewing rating scores is evaluated statistically through gamma test . to evaluate whether scores from the pqli instrument corresponded well with those independently obtained ratings , we first performed a factor analysis in which the seven latent variables from the pqli form were intercorrelated with the seven forms from the interview . we then tested a predictive model to observe whether constructs from the pqli instrument could predict analogous measured constructs from the interview measurements . initially , all possible predictive paths were included simultaneously and non - significant paths were dropped gradually . this procedure was a test of both the convergent and discriminative validity of the pqli instrument ( i.e. , variables on the pqli should be related to corresponding variables on the interview and not to non - corresponding variables ) and the criterion - related validity of the pqli form ( i.e. , the ability of the pqli to predict an independent criterion variable ) . the whole statistical analysis was conducted using the spss version 8.0 statistical package ( spss inc , chicago , il ) . the pqli is a 28-item questionnaire , composed of six multi - item scales ( 2 functional scales , 1 symptom scale , 1 choice of treatment scale , 1 psychological scale ) and a single item scale ( overall quality of life ) . the questionnaire development involved the following phases : first , literature search that identified the relevant qol issues , and the existing questionnaires , second , a provisional list of items was presented to 3 experts for feedback on appropriateness of content and breadth of coverage , third , the list was administered to patients from the target group to determine the extent to which they have experienced these problems , while they were asked to choose a number of issues that troubled them the most ( figure 1 ) . next , the resulting list of items was reviewed for clarity and overlap by other experts , and finally , the questionnaire was pretested by administering it to some patients ( n = 20 ) from the target population , and through structured interviews with each patient individually after the completion of the questionnaire . in these interviews , the patients rated the questionnaire scales within a range of " 1 " ( i.e. first choice ) to " 7 " ( i.e. seventh choice ) . the researchers , then , evaluated whether scores from the resulting pqli corresponded well with those independently obtained ratings . variables , which -according to patients -affect their quality of life . in the final questionnaire format six of the scales were presented into three optional statements to be scored 1 , 2 and 3 respectively . in " choice of treatment " scale the patients were asked to choose the item that is " most " important in the choice of treatment and rate it 1 ; then choose the item that is " next " important and rate it 2 ; and so on , the last item which is the " least " important , the patients were asked to rate it as 5 . the single item scale " overall quality of life " has the form of a bi - polar numerical scale . this rather short interval was chosen because of the imminent risk of sudden changes in their health status . the instrument was designed primarily to be a self - assessment but where the patient 's condition would not permit it the researcher assisted him / her . { in the appendix - see additional file 1 ] is presented the questionnaire in the english language . two independent translators translated the pqli in english and then another two independent translators translated it back to greek . the same translation method has already been used in the validity and reliability of the eortc qlq c-30 ( v.3 ) in greek . a range of analyses was carried out to establish scale reliability , and to evaluate empirically the validity of the questionnaire scales . higher mean scores from 0 to 100 represent a better level of functioning and qol on the scales of " activity " , " self - care " , " support " , " communication " , " psychological affect " , and " overall quality of life " , and higher mean values on the health status scale , represent more symptomatology and worse quality of life . the current procedures for scoring the pqli reflect the multidimensionality of the quality of life domain . internal consistency of the questionnaire before and during palliative treatment was assessed by cronbach 's alpha and was considered acceptable for group comparisons if the coefficient exceeded 0.70 , as recommended by nunnally . cronbach 's alpha tests whether the items in a questionnaire have a homogeneous content with respect to the construct of interest . test - retest reliability of patients ' ( n = 120 ) responses was evaluated by comparing the scores recorded on two occasions , an average of seven days apart ( spearman - rho test ) . the patient 's clinical stage did not change between the first and the second completion , and the status of the patients was stable between test and retest . due to its hierarchical nature , the intertest reliability of the ranking statements ( choice of treatment ) was established by using the " kendall's - w " test . five indirect methods to evaluate validity were adopted : first , by comparing the scale scores with patients with poorer and better eastern cooperative oncology group ( ecog - the clinical variable assessed ) performance status using the mann - whitney u non - parametric test . second , by assessing the statistical difference of the questionnaire - scales before and during treatment in terms of wilcoxon rank test between scales for related subjects . third , by exploratory factor analysis , using principal components with non - orthogonal ( direct oblimin ) rotation , was used to assess the validity of the pqli . as a fourth process , correlations were calculated between pqli items and those of two others instruments , the assessment of quality of life in palliative care ( aqel ) , and the european organisation for research and treatment of cancer ( eortc ) core quality of life questionnaire ( qlq - c30 , version 3.0 ) . the aqel is focusing in patients undergoing palliative treatment , includes 22 items in seven scales , from physical , psychological , social and existential domains , while it has proven to be reliable and valid . in addition , the eortc qlq - c30 is a widely used second - generation questionnaire designed to measure cancer patients ' physical , psychological and social functions . it is a psychometrically established 30-item questionnaire , incorporated in nine multi - item , and several single - item scales . concurrent related validity was performed with correlations among the scales of pqli ( inter - scale correlations ) . accordingly , the seven factors obtained from the interview from the 120 patients were rated and coded from 1 to 7 according to the patients ' choice : 1 , 2 , 3 , 4to 7choice . the closeness of the hypothetical model of pqli to the empirical data of interviewing rating scores is evaluated statistically through gamma test . to evaluate whether scores from the pqli instrument corresponded well with those independently obtained ratings , we first performed a factor analysis in which the seven latent variables from the pqli form were intercorrelated with the seven forms from the interview . we then tested a predictive model to observe whether constructs from the pqli instrument could predict analogous measured constructs from the interview measurements . initially , all possible predictive paths were included simultaneously and non - significant paths were dropped gradually . this procedure was a test of both the convergent and discriminative validity of the pqli instrument ( i.e. , variables on the pqli should be related to corresponding variables on the interview and not to non - corresponding variables ) and the criterion - related validity of the pqli form ( i.e. , the ability of the pqli to predict an independent criterion variable ) . the whole statistical analysis was conducted using the spss version 8.0 statistical package ( spss inc , chicago , il ) . internal consistency of the questionnaire before and during palliative treatment was assessed by cronbach 's alpha and was considered acceptable for group comparisons if the coefficient exceeded 0.70 , as recommended by nunnally . cronbach 's alpha tests whether the items in a questionnaire have a homogeneous content with respect to the construct of interest . test - retest reliability of patients ' ( n = 120 ) responses was evaluated by comparing the scores recorded on two occasions , an average of seven days apart ( spearman - rho test ) . the patient 's clinical stage did not change between the first and the second completion , and the status of the patients was stable between test and retest . due to its hierarchical nature , the intertest reliability of the ranking statements ( choice of treatment ) was established by using the " kendall's - w " test . five indirect methods to evaluate validity were adopted : first , by comparing the scale scores with patients with poorer and better eastern cooperative oncology group ( ecog - the clinical variable assessed ) performance status using the mann - whitney u non - parametric test . second , by assessing the statistical difference of the questionnaire - scales before and during treatment in terms of wilcoxon rank test between scales for related subjects . third , by exploratory factor analysis , using principal components with non - orthogonal ( direct oblimin ) rotation , was used to assess the validity of the pqli . as a fourth process , correlations were calculated between pqli items and those of two others instruments , the assessment of quality of life in palliative care ( aqel ) , and the european organisation for research and treatment of cancer ( eortc ) core quality of life questionnaire ( qlq - c30 , version 3.0 ) . the aqel is focusing in patients undergoing palliative treatment , includes 22 items in seven scales , from physical , psychological , social and existential domains , while it has proven to be reliable and valid . in addition , the eortc qlq - c30 is a widely used second - generation questionnaire designed to measure cancer patients ' physical , psychological and social functions . it is a psychometrically established 30-item questionnaire , incorporated in nine multi - item , and several single - item scales . concurrent related validity was performed with correlations among the scales of pqli ( inter - scale correlations ) . accordingly , the seven factors obtained from the interview from the 120 patients were rated and coded from 1 to 7 according to the patients ' choice : 1 , 2 , 3 , 4to 7choice . the closeness of the hypothetical model of pqli to the empirical data of interviewing rating scores is evaluated statistically through gamma test . to evaluate whether scores from the pqli instrument corresponded well with those independently obtained ratings , we first performed a factor analysis in which the seven latent variables from the pqli form were intercorrelated with the seven forms from the interview . we then tested a predictive model to observe whether constructs from the pqli instrument could predict analogous measured constructs from the interview measurements . initially , all possible predictive paths were included simultaneously and non - significant paths were dropped gradually . this procedure was a test of both the convergent and discriminative validity of the pqli instrument ( i.e. , variables on the pqli should be related to corresponding variables on the interview and not to non - corresponding variables ) and the criterion - related validity of the pqli form ( i.e. , the ability of the pqli to predict an independent criterion variable ) . the whole statistical analysis was conducted using the spss version 8.0 statistical package ( spss inc , chicago , il ) . the 28 items were all acceptable to the participants . they encompassed physical , social , health , and psychological aspects of life . the 61.7% of the participants regarded as most important the variable of long - term quality of life , while the 76.7% of the respondents regarded as least important the variable " effects on sexual life " . the distributions of the respondents in each category , for example , were 51.7% of the respondents are not working , a 40% can fully care for themselves , a 66.7% reported pain , 66.7% of the patients reported support from their friends and relatives , the 64.2% stated that they do not discuss their family problems with their doctor , and a 49.2% answered that they do not feel fear of death . from this figure the clinical profile of patients can be seen . although restricted to a limited cultural setting , this data was considered quite interesting for clinicians . table 2 shows the means and standard deviations for the multi - item measures , before and during treatment . from the descriptive statistics matrix , the cronbach 's alpha for each scale was found to be greater than the critical value of 0.70 , while the overall cronbach 's alpha was 0.787 . the test - retest reliability ( table 3 ) of scales and items as well showed that all the coefficients of agreement were greater than 0.82 ( p < 0.001 in all cases ) . due to the nature of the " choice of treatment " scale the reliability was calculated by performing the kendall 's coefficient of concordance and was found 0.353 with a p - value < 0.0001 . the correlation matrix of the scales within the pqli - pre - and - on - treatment is displayed in table 4 . the agreements are strong , consistent and statistically significant at the 0.005 or 0.001 levels . as expected , the strongest correlations were observed between the " activity " , " self - care " , " health status " , and " choice of treatment " . the " overall quality of life " ( oqol ) , correlated substantially with " activity " , " health status " , " self - care " , " choice of treatment " , " communication " , " support " , and " psychological affect " . correlations among the pqli scales . * before treatment under the diagonal ; during treatment above the diagonal . exploratory non - orthogonal factor analysis ( principal axis factoring extraction with direct oblimin rotation ) was carried out to further explore the validity of the pqli instrument . the bartlett test of sphericity was 3042.7 and it was significant ( p < 0.0001 ) . the kaiser - meyer - olkin measure of sampling adequacy was equal to 0.81 showing that the data is suitable for factor analysis . principal axis factoring extraction was used to analyse the underlying structure of the questionnaire , yielding seven independent factors accounting for 79.7 % of the variance . this seven - factor solution was deemed appropriate by examining the magnitude and rate of change in eigenvalues . based on the rule that meaningful factors should be associated with eigenvalues greater than 1.0 and a marginal change occur after seven factors ( scree test ) , the seven - factor solution is appropriate . for the interpretation of the factor solution direct oblimin rotation the variables constituting the seven factors are marked in bold fonts . by performing an orthogonal rotation using varimax , the same 7 factors were identified without any material difference confirming the results from the non - orthogonal rotation . factor 1 : activity ( keep working , house chores , enjoyment , hobbies ) , factor 2 : self - care ( driving or transportation , self - sufficient ) , factor 3 : health status ( pain , nausea / vomiting , lack of appetite , weak / tired , dyspnoea , stool disturbances , sleep disturbances ) , factor 4 : choice of treatment ( like to choose , able to choose ) , factor 5 : support ( relatives / friends , health care team , nursing stuff ) , factor 6 : communication ( social relationships , economic / professional problems , family problems ) , and factor 7 : psychological affect ( calm , optimistic , blue , control of the situation , fears of death ) . as shown in table 3 , the correlations under the diagonal among the scales of pqli were significantly associated , giving evidence of concurrent related validity . following , the pqli model was significantly associated with the empirical model deriving from the patients ' interview ( gamma = 0.78 , se = 0.11 , p < 0.001 ) . correlations between the ratings derived from the interview and the pqli factors are reported in table 6 . we allowed covariances ( correlations ) among the predictor variables and significant covariances among the error residuals of the outcome variables . the fit indices for this final path model reflected an excellent fit ( p < 0.001 , chitest ) . we found that the pqli constructs significantly predicted analogous interviewing scores by the patients . in most cases , there was considerable discriminative validity between similar observed and reported variables , except that pqli " health " also predicted " activity " from the interview rating . to refine these results , we needed to determine whether the path from the pqli " health " factor to the interviewing " health " rating was significantly larger than the path from pqli " health " to the " activity " interviewing rating variable . therefore , we ran a model that constrained these paths to equivalence and then examined the chi - difference test between these nested models . the difference test revealed that the paths were significantly different in magnitude ( p < 0.01 ) , thereby providing additional evidence of the discriminant validity of the pqli . correlations between pqli factors and interview scores for the 120 patients ( criterion related validity ) . significant regression paths among latent variables pqli model predicting interviewing ratings ( n = 120 ) . regression coefficients are standardized ( p < 0.001 , p < 0.01 , p # 60 ; 0.05 ) between the pqli and the assessment of quality of life in palliative care instrument ( aqel ) the correlations were , generally , strong in all the scales , ranging from 0.44 to 0.94 . the strongest correlations were found between the items of " insomnia " and " sleep disturbances " ( 0.94 ) and also in the items of " pain " ( 0.93 ) ( table 7 ) . moreover , there were significant correlations between the eortc qlq c30 and the relevant items of pqli , ranging from 0.79 to 0.97 , especially between the items of " pain " ( 0.97 ) , and " lack of appetite " ( 0.97 ) ( table 8) . there were significant correlations between the scales of pqli and the relevant scales of aqel and eortc qlq c30 , as shown in table 9 . the distinction between patients with low or high ecog performance status showed significant relationship between ecog scores and instrument scale scores . as we see in table 10 , patients with a better ecog performance status reported significantly higher scores in all the scales of the instrument . correlations between the european organization for research and treatment of cancer ( eortc ) core quality of life questionnaire ( qlq - c30 , version 3.0 ) and pqli items . correlations between relevant scales of the pqli and the scales of aqel as well as the european organization for research and treatment of cancer ( eortc ) core quality of life questionnaire ( qlq - c30 , version 3.0 ) . scores by ecog status , combined validity samples ( n = 120 ) . * mann - whitney u test table 2 shows the means and standard deviations for the multi - item measures , before and during treatment . from the descriptive statistics matrix , the cronbach 's alpha for each scale was found to be greater than the critical value of 0.70 , while the overall cronbach 's alpha was 0.787 . the test - retest reliability ( table 3 ) of scales and items as well showed that all the coefficients of agreement were greater than 0.82 ( p < 0.001 in all cases ) . due to the nature of the " choice of treatment " scale the reliability was calculated by performing the kendall 's coefficient of concordance and was found 0.353 with a p - value < 0.0001 . descriptive statistics and scale reliability before and during treatment . test retest correlations for all pqli scales and items . the correlation matrix of the scales within the pqli - pre - and - on - treatment is displayed in table 4 . the agreements are strong , consistent and statistically significant at the 0.005 or 0.001 levels . as expected , the strongest correlations were observed between the " activity " , " self - care " , " health status " , and " choice of treatment " . the " overall quality of life " ( oqol ) , correlated substantially with " activity " , " health status " , " self - care " , " choice of treatment " , " communication " , " support " , and " psychological affect " . correlations among the pqli scales . * before treatment under the diagonal ; during treatment above the diagonal . exploratory non - orthogonal factor analysis ( principal axis factoring extraction with direct oblimin rotation ) was carried out to further explore the validity of the pqli instrument . the bartlett test of sphericity was 3042.7 and it was significant ( p < 0.0001 ) . the kaiser - meyer - olkin measure of sampling adequacy was equal to 0.81 showing that the data is suitable for factor analysis . principal axis factoring extraction was used to analyse the underlying structure of the questionnaire , yielding seven independent factors accounting for 79.7 % of the variance . this seven - factor solution was deemed appropriate by examining the magnitude and rate of change in eigenvalues . based on the rule that meaningful factors should be associated with eigenvalues greater than 1.0 and a marginal change occur after seven factors ( scree test ) , the seven - factor solution is appropriate . for the interpretation of the factor solution direct oblimin rotation the variables constituting the seven factors are marked in bold fonts . by performing an orthogonal rotation using varimax , the same 7 factors were identified without any material difference confirming the results from the non - orthogonal rotation . factor 1 : activity ( keep working , house chores , enjoyment , hobbies ) , factor 2 : self - care ( driving or transportation , self - sufficient ) , factor 3 : health status ( pain , nausea / vomiting , lack of appetite , weak / tired , dyspnoea , stool disturbances , sleep disturbances ) , factor 4 : choice of treatment ( like to choose , able to choose ) , factor 5 : support ( relatives / friends , health care team , nursing stuff ) , factor 6 : communication ( social relationships , economic / professional problems , family problems ) , and factor 7 : psychological affect ( calm , optimistic , blue , control of the situation , fears of death ) . as shown in table 3 , the correlations under the diagonal among the scales of pqli were significantly associated , giving evidence of concurrent related validity . following , the pqli model was significantly associated with the empirical model deriving from the patients ' interview ( gamma = 0.78 , se = 0.11 , p < 0.001 ) . correlations between the ratings derived from the interview and the pqli factors are reported in table 6 . we allowed covariances ( correlations ) among the predictor variables and significant covariances among the error residuals of the outcome variables . the fit indices for this final path model reflected an excellent fit ( p < 0.001 , chitest ) . in most cases , there was considerable discriminative validity between similar observed and reported variables , except that pqli " health " also predicted " activity " from the interview rating . to refine these results , we needed to determine whether the path from the pqli " health " factor to the interviewing " health " rating was significantly larger than the path from pqli " health " to the " activity " interviewing rating variable . therefore , we ran a model that constrained these paths to equivalence and then examined the chi - difference test between these nested models . the difference test revealed that the paths were significantly different in magnitude ( p < 0.01 ) , thereby providing additional evidence of the discriminant validity of the pqli . correlations between pqli factors and interview scores for the 120 patients ( criterion related validity ) . significant regression paths among latent variables pqli model predicting interviewing ratings ( n = 120 ) . regression coefficients are standardized ( p < 0.001 , p < 0.01 , p # 60 ; 0.05 ) between the pqli and the assessment of quality of life in palliative care instrument ( aqel ) the correlations were , generally , strong in all the scales , ranging from 0.44 to 0.94 . the strongest correlations were found between the items of " insomnia " and " sleep disturbances " ( 0.94 ) and also in the items of " pain " ( 0.93 ) ( table 7 ) . moreover , there were significant correlations between the eortc qlq c30 and the relevant items of pqli , ranging from 0.79 to 0.97 , especially between the items of " pain " ( 0.97 ) , and " lack of appetite " ( 0.97 ) ( table 8) . there were significant correlations between the scales of pqli and the relevant scales of aqel and eortc qlq c30 , as shown in table 9 . the distinction between patients with low or high ecog performance status showed significant relationship between ecog scores and instrument scale scores . as we see in table 10 , patients with a better ecog performance status reported significantly higher scores in all the scales of the instrument . correlations between the european organization for research and treatment of cancer ( eortc ) core quality of life questionnaire ( qlq - c30 , version 3.0 ) and pqli items . correlations between relevant scales of the pqli and the scales of aqel as well as the european organization for research and treatment of cancer ( eortc ) core quality of life questionnaire ( qlq - c30 , version 3.0 ) . scores by ecog status , combined validity samples ( n = 120 ) . * mann - whitney u test the purpose of this study was to design and evaluate a method of collecting information about the quality of life of advanced ill cancer patients receiving palliative care treatment . although the primary intent of this project was to establish the basic reliability and validity of the pqli measurement system , we also hoped to demonstrate the sensitivity to change of this instrument by incorporating it into a clinical trial using repeated measures design . the questionnaire was simple to administer and score , and was well accepted by the responding patients . in average , it required 8 minutes to complete and , in most cases , could be filled with little or no assistance . this is a further proof that the proposed instrument is appropriate for this patient population , bearing in mind that one of the reasons for developing qol tools for palliative care is to meet the needs of patients who can not use traditional measures because they are frail and very ill . patients seemed to be genuinely pleased that an interest in their quality of life was a component of their overall care . two aspects of this instrument development are worth noting : the opportunity given to the patients to actively participate in this process , and the incorporation , for the first time , of the patients ' beliefs in their involvement in treatment decision and what this encompasses . the latter consists a major advantage of the pqli over the already existing ones in quality of life . the internal consistency coefficients for the pqli subscales are all beyond the acceptable level required for making group comparisons when evaluating changes in scores over time . the scales that showed too significant associations were those of " activity " , " self - care " , and " health status " . the careful construction of the test deriving from the patients ' interview as well as the setting of pass / fail scores through the exploratory factor analysis enhance the content and construct validity of the instrument . the correlation between the latent factors of pqli and the empirical factors of patients ' interview ( table 6 ) provide empirical evidence for concurrent criterion - related validity . additionally , the latter was strongly indicated by the significant regression coefficients as shown in figure 2 . although the pqli was related to both " health " and " activity " item of the interviewing rating score , this was not unexpected . indeed , the high intercorrelation between the " health " and " activity " items supports the clinical observation that a regressive health status often occurs in the context / of regressive activity . moreover , the significant correlation among the variables of pqli indicates that performance is related across test components as might be expected . furthermore , the factor analysis showed that the various components do assess unique and independent domains , confirming the discriminative validity of the measures . an important aspect that arises from this study concerns the doctor - patient communication and the relations of patients with their families and the important role of the latter . concealing a diagnosis of cancer from patients themselves , unfortunately , is still common practice in greece . in " psychological affect " , and specifically in the item concerning the " fear of death " , 56% of the patients said that they do n't feel fear of death , while reporting that many have been kept in the dark about the details of their illness diagnosis and prognosis . while most greek doctors nowadays favour disclosing a cancer diagnosis and prognosis to the patient directly , the relatives often veto this decision , since the role of the family is very powerful in the greek culture . nevertheless , they reported a high percentage ( 84.2% ) on the item of support . regarding the nature of their communication with their physicians on issues like social relationships , financial , and family relationships were very low ( 21.7% , 5.0% , and 12.5% respectively ) . quality of life research can provide the researcher and the clinician with a clearer view of the impact that a cancer treatment has on a patient 's life . this is clearly shown on this instrument from the scale that examines the attitude of patients ' toward treatment selection . the participant patients were receiving palliative treatment , which aims are psychological support and symptom relief both in short and long term , so the findings are consistent with the nature of the treatment they are receiving . moreover , another important aspect that arises from this study is the participation of the patients in the treatment choice . half of the responding population ( 50% ) reported that they want to choose the treatment they are going to receive , while a 47.5% said that they actually do choose their treatment . the latter is quite a large percentage considering they had no information on their diagnosis and prognosis . the psychometric testing of the pqli provided evidence that the elements of the measurement appropriately reflect quality of life in terminal cancer patients since the tests of validity and reliability yielded consistent results . indeed , research initiatives must rely on established quality of life instruments with proved records of statistical reliability and validity administered by objective parties . these results must be confirmed in larger multicenter trials to decrease any possible selection bias . moreover , when conducting a study in quality of life , since it is subjective in nature , we must remain cautious in our findings , since numbers may be inappropriate to adequately describe all aspects of patients ' lives . inclusion of health related quality of life measures in current and future prospective studies would be necessary to provide a database that is richer and more useful to patients and physicians . taken together , these results lend considerable support to the clinical validity of the pqli , and could be used as an audit in clinical studies involving patients with advanced stage cancer . in future studies we will continue to examine the validity of quality of life measurements obtained with the pqli using a variety of research strategies including corroboration of additional sources of independent evidence .

backgroundthis paper describes the development of a new quality of life instrument in advanced cancer patients receiving palliative care.methodsthe palliative care quality of life instrument incorporates six multi - item and one single - item scale . the questionnaire was completed at baseline and one - week after . the final sample consisted of 120 patients.resultsthe average time required to complete the questionnaire , in both time points , was approximately 8 minutes . all multi - item scales met the minimal standards for reliability ( cronbach 's alpha coefficient .70 ) either before or during palliative treatment . test - retest reliability in terms of spearman - rho coefficient was also satisfactory ( p < 0.05 ) . validity was demonstrated by inter - item correlations , comparisons with ecog performance status , factor analysis , criterion - related validation , and correlations with the assessment of quality of life in palliative care instrument ( aqel ) , and the european organisation for research and treatment of cancer ( eortc ) core quality of life questionnaire ( qlq - c30 , version 3.0).conclusionthe pqli is a reliable and valid measure for the assessment of quality of life in patients with advanced stage cancer .

subjects were recruited by advertising in local newspapers in the area of erlangen - nrnberg , germany , and eligible subjects were enrolled consecutively in one of two subpopulations , according to their clinical characteristics . written informed consent was obtained prior to study inclusion . study protocols were approved by the local ethics committee ( university of erlangen - nrnberg ) , and the study was conducted in accordance with the declaration of helsinki and the principles of good clinical practice guidelines . the main inclusion criteria of study population a were having type 2 diabetes and arterial hypertension defined as mean seated systolic blood pressure ( sbp ) 130179 mmhg and/or diastolic blood pressure ( dbp ) 80109 mmhg or being on antihypertensive medications . other inclusion criteria were normoalbuminuria or microalbuminuria and glomerular filtration rate ( gfr ) > 1.34 ml / s . the main exclusion criteria were having poor diabetes control ( a1c > 9% ) ; receiving thiazolidinediones and/or the initiation of statins for at least 4 weeks prior to the commencement of the study ; having hypertension - related end - organ damage , such as proliferative retinopathy and symptomatic cardiovascular disease ; and being a smoker . study population b was composed of patients with hypercholesterolemia defined as having a fasting ldl cholesterol level of > 4.14 mmol / l and < 6.58 mmol / l , while not receiving statins for at least 4 weeks before study inclusion . the main exclusion criteria included having diabetes , micro- or macroalbuminuria , arterial hypertension , homozygous familial hypercholesterolemia , and hyperlipoproteinamia type iii ; showing evidence of having cardiovascular or renal disease ; and being a smoker . all had an estimated gfr of > 1.34 ml / s and were free of any cardiovascular medications . all subjects were studied in the supine position at the same time in the morning in a quiet and temperature - controlled room . this urine was discarded . a constant infusion ( 250 ml / h ) of saline was administered throughout the protocol . after 115120 min of rest , patients were asked to urinate and basal urine was obtained . thereafter , the nos inhibitor l - nmma ( clinalfa , lufelingen , switzerland ) was administered intravenously as a bolus infusion ( 3 mg / kg body wt ) over 5 min , followed by constant infusion over 25 min with a rate of 0.05 mg / kg / min . therefore , the total dose was 4.25 mg / kg body wt . at the end of the l - nmma phase , urine was collected again ( l - nmma sample ) . for safety reasons , an infusion of 100 mg / kg body wt of l - arginine ( 6% l - arginine hydrochloride ; university hospital pharmacy , erlangen , germany ) was infused to counteract l - nmma induced vasoconstriction . in parallel , renal hemodynamic parameters were determined by the constant - infusion input - clearence technique with inulin ( inutest ; fresenius , linz , austria ) and sodium p - aminohippurate ( clinalpha , basel , switzerland ) for gfr and renal plasma flow ( rpf ) , respectively , as previously outlined in detail ( 24 ) . in brief , after a bolus infusion of inulin and p - aminohippurate over a 15-min period and a subsequent constant infusion over 105 min , a steady state between the input and renal excretion of the tracer substances was reached , and , in addition , the administration of the above - mentioned experimental substance ( l - nmma ) was started . renal vascular resistance was calculated as the product of mean arterial pressure ( map ) and 1-hematocrit divided by rpf . throughout the infusion protocol , blood pressure ( sbp , dbp , and map ) and heart rate were monitored at fixed intervals ( every 5 min ) by means of an oscillometric device ( dinamap ; criticon , norderstedt , germany ) . all samples were centrally measured by the laboratory of the university of erlangen - nrnberg , according to established methods . in brief , the urine albumin - to - creatinine ratio ( uacr ) was calculated by dividing urinary albumin by urinary creatinine . normal distribution of data was confirmed by a kolmogorov - smirnow test before further analysis . normally distributed data were compared by paired student t tests and are expressed as means sd . data of uacr were not normally distributed ; therefore , medians and interquartile ranges are reported . uacr values were log transformed before statistical analysis . where indicated , a multiple stepwise regression analysis with significance levels of 0.05 for entry and 0.10 for removal of a variable at each forward step was conducted . all analyses were performed using spss version 15.0 ( spss , chicago , il ) . the main inclusion criteria of study population a were having type 2 diabetes and arterial hypertension defined as mean seated systolic blood pressure ( sbp ) 130179 mmhg and/or diastolic blood pressure ( dbp ) 80109 mmhg or being on antihypertensive medications . other inclusion criteria were normoalbuminuria or microalbuminuria and glomerular filtration rate ( gfr ) > 1.34 ml / s . the main exclusion criteria were having poor diabetes control ( a1c > 9% ) ; receiving thiazolidinediones and/or the initiation of statins for at least 4 weeks prior to the commencement of the study ; having hypertension - related end - organ damage , such as proliferative retinopathy and symptomatic cardiovascular disease ; and being a smoker . study population b was composed of patients with hypercholesterolemia defined as having a fasting ldl cholesterol level of > 4.14 mmol / l and < 6.58 mmol / l and fasting triglycerides < 4.0 mmol / l , while not receiving statins for at least 4 weeks before study inclusion . the main exclusion criteria included having diabetes , micro- or macroalbuminuria , arterial hypertension , homozygous familial hypercholesterolemia , and hyperlipoproteinamia type iii ; showing evidence of having cardiovascular or renal disease ; and being a smoker . all had an estimated gfr of > 1.34 ml / s and were free of any cardiovascular medications . all subjects were studied in the supine position at the same time in the morning in a quiet and temperature - controlled room . before the experimental protocol was started , patients were advised to empty their bladder . this urine was discarded . a constant infusion ( 250 ml / h ) of saline was administered throughout the protocol . after 115120 min of rest , patients were asked to urinate and basal urine was obtained . thereafter , the nos inhibitor l - nmma ( clinalfa , lufelingen , switzerland ) was administered intravenously as a bolus infusion ( 3 mg / kg body wt ) over 5 min , followed by constant infusion over 25 min with a rate of 0.05 mg / kg / min . therefore , the total dose was 4.25 mg / kg body wt . at the end of the l - nmma phase , urine was collected again ( l - nmma sample ) . for safety reasons , an infusion of 100 mg / kg body wt of l - arginine ( 6% l - arginine hydrochloride ; university hospital pharmacy , erlangen , germany ) renal hemodynamic parameters were determined by the constant - infusion input - clearence technique with inulin ( inutest ; fresenius , linz , austria ) and sodium p - aminohippurate ( clinalpha , basel , switzerland ) for gfr and renal plasma flow ( rpf ) , respectively , as previously outlined in detail ( 24 ) . in brief , after a bolus infusion of inulin and p - aminohippurate over a 15-min period and a subsequent constant infusion over 105 min , a steady state between the input and renal excretion of the tracer substances was reached , and , in addition , the administration of the above - mentioned experimental substance ( l - nmma ) was started . renal vascular resistance was calculated as the product of mean arterial pressure ( map ) and 1-hematocrit divided by rpf . throughout the infusion protocol , blood pressure ( sbp , dbp , and map ) and heart rate were monitored at fixed intervals ( every 5 min ) by means of an oscillometric device ( dinamap ; criticon , norderstedt , germany ) . all samples were centrally measured by the laboratory of the university of erlangen - nrnberg , according to established methods . in brief , the urine albumin - to - creatinine ratio ( uacr ) was calculated by dividing urinary albumin by urinary creatinine . normal distribution of data was confirmed by a kolmogorov - smirnow test before further analysis . normally distributed data were compared by paired student t tests and are expressed as means sd . data of uacr were not normally distributed ; therefore , medians and interquartile ranges are reported . uacr values were log transformed before statistical analysis . where indicated , a multiple stepwise regression analysis with significance levels of 0.05 for entry and 0.10 for removal of a variable at each forward step was conducted . all analyses were performed using spss version 15.0 ( spss , chicago , il ) . the baseline clinical characteristics of the participants in population a ( n = 62 ) are given in table 1 . this population comprised middle - aged patients with diabetes , hypertension , and partly elevated cholesterol levels , but all had normal renal function . of 62 subjects , 25 had low - grade albuminuria ( male subjects : > 10 mg / g creatinine ; female subjects > 15 mg / g creatinine ) and 6 had microalbuminuria ( 30299 mg / g creatinine ) , but none had macroalbuminuria . baseline characteristics of the participants of study population a the baseline clinical characteristics of the subjects in study populations b ( n = 22 ) are given in table 2 . in this group , patients had elevated ldl cholesterol levels , whereas blood pressure , fasting glucose , and other baseline parameters were in the normal range . only three patients had low - grade albuminuria , but none had micro- or macroalbuminuria . baseline characteristics of the participants of study population b infusion of l - nmma led to an increase in sbp ( population a : from 142 14 to 155 17 mmhg ; population b : from 127 17 to 141 20 mmhg ; both p < 0.001 ) and dbp ( population a : from 78 10 to 85 11 mmhg ; population b : from 75 9 to 81 10 mmhg ; both p < 0.001 ) and to a decrease in heart rate ( population a : from 66 10 to 62 10 bpm ; population b : from 58 7 to 54 7 bpm ; both p < 0.001 ) . map , which is considered to be a parameter of renal perfusion pressure , increased in population a ( from 100 10 to 108 11 mmhg ; p < 0.001 ) and in population b ( from 94 10 to 103 13 mmhg ; p < 0.001 ) . there was a significant increase in the uacr in response to the blockade of enos with l - nmma in the hypertensive patients with type 2 diabetes ( baseline : 12.3 mg / g creatinine [ 6.419.1 ] vs. l - nmma : 16.9 mg / g creatinine [ 8.928.3 ] ; p = 0.001 ) ( fig . 1 ) and in patients with hypercholesterolemia ( baseline : 7.7 mg / g creatinine [ 4.08.9 ] vs. l - nmma : 7.9 mg / g creatinine [ 6.114.7 ] ; p = 0.044 ) ( fig . 2 ) . uacr before and after systemic infusion of the no inhibitor l - nmma in study population a on a log - scaled axis . uacr before and after systemic infusion of the no inhibitor l - nmma in study population b. because increased blood pressure attributed to l - nmma infusion also may led to an increased renal perfusion pressure and thereby to elevated albumin excretion , we performed additional analyses of our data . to assess the influence of map changes attributed to l - nmma infusion as a potential confounding factor as well as altered renal hemodynamics , map change in response to l - nmma infusion was not related to the increase in log - transformed uacr attributed to l - nmma infusion in both study populations ( population a : = 0.235 , p = 0.304 , and population b : = 0.024 , p = 0.949 ) . similarly , changes of sbp and dbp also were not related to changes of log - transformed uacr after l - nmma infusion ( p > 0.20 , data not shown ) . furthermore , in both populations there was no relation between the change in rpf ( population a : = 0.006 , p = 0.975 , and population b : = 0.278 , p = 0.522 ) , change in gfr ( population a : = 0.124 , p = 0.698 , and population b : = 0.122 , p = 0.606 ) , change in filtration fraction ( gfr / rpf ) ( population a : = 0.165 , p = 0.237 , and population b : = 0.054 , p = 0.832 ) , and change in renal vascular resistance ( population a : = 0.119 , p = 0.772 , and population b : = 0.182 , p = 0.363 ) and the increase in log - transformed uacr in response to l - nmma infusion . although not fully determined , metabolic factors such as hyperglycemia , a1c , and hyperlipidemia may influence endothelial permeability . however , neither fasting blood glucose and a1c , respectively , nor elevated ldl cholesterol were related with either baseline uacr or the change of log - transformed uacr in response to l - nmma ( p > 0.20 , data not shown ) . infusion of l - nmma led to an increase in sbp ( population a : from 142 14 to 155 17 mmhg ; population b : from 127 17 to 141 20 mmhg ; both p < 0.001 ) and dbp ( population a : from 78 10 to 85 11 mmhg ; population b : from 75 9 to 81 10 mmhg ; both p < 0.001 ) and to a decrease in heart rate ( population a : from 66 10 to 62 10 bpm ; population b : from 58 7 to 54 7 bpm ; both p < 0.001 ) . map , which is considered to be a parameter of renal perfusion pressure , increased in population a ( from 100 10 to 108 11 mmhg ; p < 0.001 ) and in population b ( from 94 10 to 103 13 mmhg ; p < 0.001 ) . there was a significant increase in the uacr in response to the blockade of enos with l - nmma in the hypertensive patients with type 2 diabetes ( baseline : 12.3 mg / g creatinine [ 6.419.1 ] vs. l - nmma : 16.9 mg / g creatinine [ 8.928.3 ] ; p = 0.001 ) ( fig . 1 ) and in patients with hypercholesterolemia ( baseline : 7.7 mg / g creatinine [ 4.08.9 ] vs. l - nmma : 7.9 mg / g creatinine [ 6.114.7 ] ; p = 0.044 ) ( fig . 2 ) . uacr before and after systemic infusion of the no inhibitor l - nmma in study population a on a log - scaled axis . uacr before and after systemic infusion of the no inhibitor l - nmma in study population b. because increased blood pressure attributed to l - nmma infusion also may led to an increased renal perfusion pressure and thereby to elevated albumin excretion , we performed additional analyses of our data . to assess the influence of map changes attributed to l - nmma infusion as a potential confounding factor as well as altered renal hemodynamics , multiple linear regression analyses were performed . map change in response to l - nmma infusion was not related to the increase in log - transformed uacr attributed to l - nmma infusion in both study populations ( population a : = 0.235 , p = 0.304 , and population b : = 0.024 , p = 0.949 ) . similarly , changes of sbp and dbp also were not related to changes of log - transformed uacr after l - nmma infusion ( p > 0.20 , data not shown ) . furthermore , in both populations there was no relation between the change in rpf ( population a : = 0.006 , p = 0.975 , and population b : = 0.278 , p = 0.522 ) , change in gfr ( population a : = 0.124 , p = 0.698 , and population b : = 0.122 , p = 0.606 ) , change in filtration fraction ( gfr / rpf ) ( population a : = 0.165 , p = 0.237 , and population b : = 0.054 , p = 0.832 ) , and change in renal vascular resistance ( population a : = 0.119 , p = 0.772 , and population b : = 0.182 , p = 0.363 ) and the increase in log - transformed uacr in response to l - nmma infusion . although not fully determined , metabolic factors such as hyperglycemia , a1c , and hyperlipidemia may influence endothelial permeability . however , neither fasting blood glucose and a1c , respectively , nor elevated ldl cholesterol were related with either baseline uacr or the change of log - transformed uacr in response to l - nmma ( p > 0.20 , data not shown ) . almost two decades ago , deckert et al . ( 25 ) proposed what usually has been cited as the steno hypothesis , which states that microalbuminuria reflects generalized vascular damage . this hypothesis links impaired vascular endothelial function to vascular leakage of albumin that , in terms of the kidney , easily can be detected by measuring urinary albumin excretion . indeed , albuminuria was associated with an increased systemic permeability of albumin in both type 1 diabetes ( 26 ) and type 2 diabetes in some ( 27 ) but not in all ( 28 ) studies as well as in clinically healthy subjects ( 29 ) . the importance of no for intact vascular function and endothelial integrity has been thoroughly documented . because decreased bioavailability of no is a major contributor to endothelial function , we have investigated whether an acute reduction of basal no activity provokes albuminuria . by measuring albuminuria both before and after systemic infusion of l - nmma , we could demonstrate that impaired renal endothelial function leads to a significant increase of albuminuria in response to nos inhibition with l - nmma . our direct methodological approach proves , for the first time in humans , the previously proposed concept that impaired basal no activity , and thus endothelial dysfunction , is a major pathogenic mechanism that increases the permeability of the vascular wall and hence the genesis of albuminuria . using a before - and - after design without a control group may limit the assessment of the magnitude of the observed effect . nevertheless , our data suggest that the change of uacr in response to inhibition of no , resulting in a reduction of basal no activity , may serve as a marker of endothelial function in the kidney , which can be easily measured . it is noteworthy that these findings were evident in both hypertensive patients with type 2 diabetes and patients with hypercholesterolemia but without hypertension and type 2 diabetes . the change of map according to l - nmma was not related to the change of uacr according to l - nmma , even after adjustment for filtration fraction ( gfr / rpf ) , ruling out an impact of changes of blood pressure and renal perfusion pressure on increased albumin excretion . we are aware that multiple regression analysis is an indirect statistical approach to assess potential influence of systemic and renal hemodynamic changes on the change of log - transformed uacr attributed to l - nmma infusion , which , however , is one of the most accepted methods to account for potential confounding factors in vivo in humans . our findings are in line with animal models , genetic studies , and large studies with prospective character . it has been shown in a diabetic mouse model ( lepr ) that genetic eradication of enos expression ( enos ) resulted in an increase of albuminuria compared with control , lepr , or enos mice ( 21 ) . moreover , induction of diabetes in an inbred enos knockout animal model on the cf57/bl6 background , which is known to be resistant to nephropathy , has caused glomerular endothelial injury and resulted in overt albuminuria ( 30 ) . sharma et al . ( 31 ) have shown , using an in vitro approach and thereby ruling out the effects of altered hemodynamics or circulating cells of factors , that inhibition of nos activity resulted in an increased glomerular capillary permeability to albumin . it has been suggested that inhibition of nos could enhance o2-mediated oxidative injury , which can be attenuated by exogenous no and/or by o2 scavengers . these findings were supported by a similar approach , and it has been concluded that nos - catalyzed no production is an important mechanism in regulating glomerular permeability to albumin , which likely involves control of the bioavailability of superoxide ( 32 ) . in accordance , giner et al . ( 33 ) have reported elevated levels of markers of oxidative stress in hypertensive patients with microalbuminuria compared with those without microalbuminuria , and similar results were reported from type 2 diabetic patients ( 34 ) . genetic studies in humans have shown that enos polymorphisms that potentially impair enos gene transcription and activity are associated with an increased risk of diabetic nephropathy ( 22,23 ) . in a small japanese study ( 35 ) , ~200 diabetic patients with normo- , micro- , and macroalbuminuria were investigated . percentage change of flow - mediated dilation of the brachial artery , to be in part related to endothelial function and endogenous no production , was significantly decreased in micro- and macroalbuminuric compared with normoalbuminuric patients . furthermore , flow - mediated vasodilation was significantly correlated with the degree of albuminuria . in the hoorn study ( 36 ) , even after adjustment for age , sex , baseline arterial diameter , and other potential confounding factors , flow - mediated vasodilation was lower in the presence of microalbuminuria . there are many large , prospective trials evaluating the impact of therapeutic approaches to reduce microalbuminuria in diverse populations . these trials are focused on cardiovascular events or on the progression of renal disease and not on an improvement of endothelial function . interestingly , it was shown that the therapeutic approaches associated with lowering albuminuria ( e.g. , rev . in 37 ) also are known to improve endothelial function . in the two study populations , we found that nos inhibition , as assessed by measuring uacr both before and after the blockade of nos with systemic infusion of l - nmma provokes albuminuria that is unrelated to changes in the systemic circulation . interestingly , this finding was evident in patient groups prone to endothelial dysfunction and albuminuria , and , therefore , findings can not be extrapolated to the general population . thus , additional studies are necessary to evaluate whether acute deterioration of endothelial function by reducing no activity causes an increase of albuminuria also in the general population .

objectivethe barrier function of the glomerular filter has been studied for decades . albuminuria reflects a malfunction of this barrier , and in animals dysfunctional endothelial nitric - oxide ( no ) synthase results in albuminuria . we aimed to analyze the importance of no for the glomerular barrier function in humans.research design and methodsto assess the effect of endothelial dysfunction on albuminuria , we measured the urine albumin - to - creatinine ratio ( uacr ) both before and after the blockade of no synthases ( noss ) with systemic infusion of ng - monomethyl - l - arginine ( l - nmma ) in two distinct study populations . in population a , 62 hypertensive patients with type 2 diabetes and , in population b , 22 patients with hypercholesterolemia but without hypertension or type 2 diabetes were examined . all subjects had normal renal function.resultsthere was a significant increase in the uacr in response to nos inhibition with l - nmma in hypertensive patients with type 2 diabetes ( study population a ) and in patients with hypercholesterolemia ( study population b ) . linear regression analyses revealed that the change in mean arterial presssure in response to l - nmma was not related to the increase in the uacr in response to l - nmma in either population , even after adjusting for filtration fraction.conclusionsnos inhibition provokes albuminuria that is unrelated to changes in blood pressure . it is noteworthy that this finding was evident in patient groups prone to endothelial dysfunction and albuminuria . thus , acute deterioration of endothelial function by reducing no activity causes an increase in albuminuria .

it is estimated that 80 million unexpected pregnancies yearly occur in the world of which 45 million end in abortion . in this direction , appropriate application of family planning is very helpful through reduction of mortality as a result of abortion , achievement to success in the promotion of maternal health , enhancement of women 's social position and overall social - economic development . the major causes of unexpected pregnancies are the failure in contraception methods and lack of selecting an efficient contraception method . on the other hand , appropriate and constants use of contraception methods are under the influence of having sufficient information about these methods and their side - effects . the best way to leave these problems out or to modify them is conducting counseling during the selection of a contraception method . appropriate counseling given by health services staffs can help to clients for select of the most efficient method and guarantee their achievement to their objectives in family planning and maternal health as well as the client 's satisfaction with the quality of these services through making a change in their awareness , skill and attitude toward pregnancy and contraception methods . therefore , it can be concluded that one of the basic principles to succeed in contraception methods is an appropriate family planning . previous qualitative research in family planning has introduced numerous obstacles , such as organizational and structural obstacles . mugisha and reynolds ( 2008 ) reported that organizational and structural factors such as availability of contraception methods , the related equipments and work overload are as effective elements on quality of family planning services . baraitser et al . ( 2003 ) in their study pointed out to lack of privacy , tiresome environment and unsuitable clients waiting room as the obstacles of clients participation in receiving family planning services . ( 2009 ) in their investigation on the obstacles in family planning services quality , reported that mismanagement and limitation of time act as the influencing factors . although qualitative research has detected these obstacles , these obstacles have not been studied with a proper sample size in a quantitative research in iran . detection of the most important and efficient obstacles , can speed up the initial steps to delete or modify these obstacles more efficiently and enhance the quality of family planning counseling . on the other hand , comparison of viewpoints in three groups of managers , staffs and clients the dissimilarity of these obstacles in viewpoints of the various individuals may lead to conflicts in their function either to delete or control these obstacles and eventually , result in failure of family planning counseling . therefore , in order to improve the quality of present counseling and to access the family planning goals and with regard to the importance of concurrent measurement of these viewpoints , the present study aimed to compare managers , staffs and clients viewpoints about organizational and structural obstacles in family planning counseling in health care centers in isfahan in 2012 . the present study is comparative descriptive cross - sectional one - step three - group study on 295 subjects including 59 managers , 110 staffs and 126 clients . a total of 20 health - care centers were selected through a lottery to recruit their staffs and clients . sampling was a census for staffs and convenient random sampling for a group of clients . inclusion criteria were married women aged 15 - 49 years who were using one of the contraception methods . the managers were selected through census sampling from 44 health - care centers as well as managers and family planning experts in provincial health centers and health centers number 1 and 2 in isfahan . the first section contained questions on managers , staffs and clients personal and fertility variables and the second section included a viewpoints survey questionnaire of organizational and structural obstacles in family planning . the viewpoints about organizational and structural obstacles in family planning were investigated by mean obtained score of answering 21 five - point likert scale questions in the questionnaire of viewpoint survey . the answers were scored as absolutely disagree ( grade 0 ) , disagree ( grade 1 ) , no idea ( grade 2 ) , agree ( grade 3 ) and absolutely agree ( grade 4 ) . organizational and structural obstacles were the obstacles such as lack of privacy , inadequate educational equipments and inadequate various supplies of contraceptives in health - care centers [ table 1 ] . content validity was used to confirm the validity of the questionnaire , prepared by referring to textbooks and valid articles . reliability of the questionnaire was confirmed through cronbach 's alpha test ( = 0.91 ) . data were collected through a self - report questionnaire in two groups of managers and staffs and self - report or questioning in the group of clients . data were analyzed by descriptive and inferential statistical methods one - way anova through spss . frequency distributions ( % ) of the responses to viewpoint surrey in managers , staffs and clients based on organizational and structural obstacles with regard to personal and fertility characteristics of the managers , there were 59 subjects ( 33 female and 26 male ) with a mean age of 43 5.5 years , with managerial experience of an official post for 10 6 years , of whom 79.7% were general physicians and 54.2% had two children . about 49.2% of the managers were relatively satisfied with continuing education programs concerning family planning counseling . with regard to personal and fertility characteristics in the group of staffs , there were 110 subjects with a mean age of 39.7 6.2 years with 13 6.7 years work experience in family planning counseling of whom 54.6% had bs of midwifery and 50.9% had two children . with regard to the staffs responses , mean daily number of clients referring to the center to receive counseling was calculated 10 6 and the staffs had spent 10 - 20 min for each counseling session . furthermore , 72.7% of the staffs were relatively satisfied with continuing education programs in relation with family planning counseling . with regard to personal and fertility characteristics of the clients , there were 126 subjects with a mean age of 29.7 5.8 years , 88.1% repetitive referrals , of whom 46.8% had education , lower than diploma , 92.1% were housewife and 56.1% had only child . about 41.3% of the clients reported the average time of waiting to receive family planning counseling as 10 - 20 min and 64.3% reported average length of time for each counseling session less than 10 min . on the other hand , 50.8% were relatively satisfied with the received family planning counseling in health - care centers . the obtained results with regard to subjects viewpoints reveal that the mean score of viewpoint about organizational and structural obstacles in family planning counseling in three groups of managers , staffs and clients were 49.60 16.40 , 49.50 17.15 and 48.30 13.15 respectively . one - way anova showed no significant difference in comparison of managers , staffs and clients viewpoints mean scores with regard to organizational and structural obstacles in family planning counseling ( p = 0.677 ) . as presented in table 2 , based on the obtained scores from organizational and structural obstacles questionnaire and their classification in five sub - groups , most of the managers ( 40.7% ) , staffs ( 48.2% ) and clients ( 56.3% ) reported the existence of organizational and structural obstacles in family planning counseling in moderate level . frequency distribution of viewpoint scores in three groups of managers , staffs and clients in relation with organizational and structural obstacles in family planning counseling table 1 shows that the frequency distribution of the responses to questions on organizational and structural obstacles are different in three groups of managers , staffs and clients . in the present study , most of the managers , staffs and clients reported that organizational and structural obstacles act as the obstacle in family planning process in a moderate level . in direction with the obtained results , akers et al . ( 2010 ) stated that there are numerous obstacles in family planning counseling from the side of health providers and health services system . in the present study , despite the agreed viewpoints of most of the managers and staffs with regard to staffs work overload and lack of adequate time for family planning counseling , most of the clients disagreed with this issue . research in quality of services and family planning centers reported that health providers are busy and this act as a factor for reduction of quality . this difference in viewpoints may be possibly due to the fact that clients did not observe so much services given by staffs to clients in their waiting time to assume they were working busily . staffs work overload from managers and staffs viewpoints can also be interpreted as spending a lot of time to record data in the files . therefore , the findings show the necessity of a clients appointment management system to relieve daily workload as well as running an electronic system to record the data to speed up provision of services . previous researches in relation with quality of family planning services and the quality of the centers providing these services have reported problems such as clients problems in receiving family planning services , staffs ignoring the clients and not spending adequate time on them despite having free time , staffs inappropriate behavior , long waiting time to receive family planning counseling , low interested and motivated staffs , lack of staffs commitment in the provision of family planning counseling properly , ignoring clients suggestions and desires during counseling and low experience of family planning counselors . in the present study , most of the managers , staffs and clients disagreed with the existence of these obstacles in family planning counseling . kols and sherman ( 1998 ) stated that clients may pretend being satisfied with the services as a result of pleasing the staffs , fear of missing services in future and cultural reasons . on the other hand , as the present study is quantitative while the studies from which the primary concepts were extracted were qualitative , it can be supposed that these problems also existed in the study population of the present study although most of the clients disagreed with the existence of such obstacles in family planning counseling process . , it may reveal the necessity of planning to reduce these problems , even in a low amount . in the other research on the quality of family planning services and the centers providing these services , reported factors such as inadequate various supplies of contraceptives in the health - care centers and limitations in the provision of some contraception methods despite being selected for the subjects in family planning counseling in all physical conditions for example absence of mense to insertion of intrauterine devices ( iud ) , lack of educational facilities and privacy as the factors to reduce the quality of services . these factors were the obstacles in conducting counseling from the viewpoint of most of the managers , staffs and clients in the present study . therefore , it is essential to modify the shortage of various supplies of contraceptives in health centers and educate health providers , prepare educational facilities and educate the staffs to use these facilities during family planning counseling as well as to provide a room for family planning counseling in health - care centers to respect clients privacy . in the present study , opposite to viewpoints of most of the staffs and managers , most of the clients preferred to refer to private health centers . it possibly shows that the clients accepted to tolerate higher costs to receive higher quality care in those centers . as mohammad - alizadeh et al . ( 2009 ) reported that clients prefer to refer to private centers due to low quality services of governmental centers . therefore , service providers should investigate the cause that makes the clients leave out of charge services in governmental centers and refer to other centers . with regard to viewpoints of most of managers concerning lack of sufficient information provision in the centers , most of the staffs and clients denied the existence of this problem as an efficient obstacle in family planning counseling . ( 2007 ) introduced this factor as a reducing element for quality of family planning services . it can be argued that this response from clients is as a result of their lack of awareness form the amount of word , the staffs may have denied this obstacle , due to supporting their position and their other colleagues and existence of their personal beliefs in the concept of adequacy of information provision . however , managers agreement with this issue can be due to factors such as lack of their trust in adequacy of information provided by the staffs , lack of their proper supervision on the manner of staffs services and the managers belief in staffs work overload and their disability to provide the information adequately . it seems that for detection of these obstacles more precisely , conducting more studies in relation with the evaluation of level and quality of transferred information to clients is essential . baraitser et al . ( 2003 ) argued that teenage clients were dissatisfied with the suitability of the waiting room in their family planning counseling . in addition , with regard to negative viewpoints of most of the managers and clients concerning unsuitability of the waiting room , most of the staffs accepted the existence of such obstacle in family planning counseling , which can be as a result of clients low expectation and preservation of managers prestige in position of the authorities to supervise the appearance and physical atmosphere of the centers . it is notable that the staffs as the persons attending the centers more than the managers and clients may detect problems in the centers , which need more attention . the other finding is agreement of the managers and the staffs on the fact that the staffs care making files for clients to respond to their problems and not provision of sexual counseling related to preconception methods , for which most of the clients disagreed . in qualitative study of mohammad - alizadeh ( 2008 ) , the clients referring to health centers complained of ignoring their sexual problems related to contraception methods and claimed that the staffs pay more attention to completing their medical file related to their problems . the clients dissatisfaction may be related to lack of clients expectations and respect to health providers , fear of responses to receive the services and their outcomes , not feeling comfortable to talk in relation with sexual subjects . on the other hand , it should be noted that acceptance of most of the managers , staffs and a number of clients concerning these obstacles revealed the existence of such problems in the health system and necessitates planning to solve these problems through making electronic clients files to provide them with the needed care and reduce waste of time and staffs concern for recording patients manually in order to have more time and peace to provide clients with services . in the present study , most of the staffs , opposite to other two groups , agreed that forbid to attendance of men in counseling sessions is an obstacle in family planning counseling process . ( 2008 ) reported the same limitation for men to attend the session during receiving family planning services . the reason for this difference is possibly in their viewpoint in not experiencing their spouses attendance due to working shift of the centers ( just mornings ) . therefore , in order to involve men in family planning counseling more , these problems should be solved through planning and educating the staffs to let women attend with their spouses in family planning counseling as the staffs accepted to have this obstacle in health - care centers . in general , the results of the study showed that the mean viewpoint score of the managers , employees , and clients in the field of organizational and structural obstacles have been different . the findings showed that organizational and structural factors are not considered so important by the clients as the health system planners believe . more investigation and administration of a need assessment are essential to detect the existing basic clients concerns through group discussion sessions with the presence of three major components in services provision receive system and all problems should not be related just to the health system .

background : organizational and structural obstacles are a group of major obstacles in achievement of appropriate family planning counseling . detection of these obstacles from the viewpoint of managers , staffs and clients who are key members in health services providing system is a major step toward appropriate planning to modify or delete this group of obstacles . the present study was conducted with the goal of comparing managers , staffs and clients viewpoints about organizational and structural obstacles in family planning counseling in health - care centers in isfahan in 2012.materials and methods : this is a cross - sectional one - step three - group comparative descriptive study conducted on 295 subjects including 59 managers , 110 staffs and 126 clients in medical health - care centers in isfahan in 2012 . managers and the staffs were selected by census sampling and the clients were recruited through convenient random sampling . the date collection tool was a researcher made questionnaire , which was designed in two sections of fertility and personal characteristics and viewpoint measurement . descriptive and inferential statistical test were used to analyze the data.results:the obtained results showed no significant difference between mean scores of viewpoints in three groups of managers , staffs and clients concerning organizational and structural obstacles in family planning counseling ( p = 0.677 ) . in addition , most of the managers , staffs and clients reported organizational and structural obstacles as the obstacles in the process of family planning in moderate level.conclusion:the results showed the necessity of health services managers planning to modify or delete organizational and structural obstacles especially the agreed obstacles from the viewpoint of managers , staffs and clients .

myosin binding protein c ( also known as c - protein : mybp - c ; cardiac isoform abbreviated : cmybp - c ) was discovered in 1973 ( offer et al . 1973 ) and is located on 79 stripes of 43 nm spacing in each half of the a - band ( cross bridge bearing zone , c - region ) of the sarcomere in skeletal and cardiac muscles ( fig . 1 ) . to date three different mybpc proteins are known , each encoded by different genes ( mybpc 13 ) , where slow ( mybpc1 , 1141 aa ) and fast ( mybpc2 1141 aa ) twitch muscle isoforms are restricted to skeletal muscle tissues and whereas mybpc3 ( 1273 aa ) is expressed exclusively in cardiac myocytes ( furst et al . 1992 ; gautel et al . 1995 ; weber et al . 1993 ) . all mybpc molecules share common architectural features : they are composed of seven immunoglobulin domains ( ig ) and three fibronectin type iii domains ( fniii ) called c110 where a 105 amino acid myosin binding protein motif is localized between c1 and c2 . in addition , a proline and alanine rich ( pa ) domain is localized near the aminoterminus ( fig . 2 ) . domains c9c10 interact with titin and c10 interacts with light meromyosin ( lmm ) ( freiburg and gautel 1996 ) . the actin and s2 ( head region ) myosin heavy chain binding regions are located at the aminoterminus ( fig . 2 ) . skeletal muscle mybpc also interacts with nebulin , but this interaction is not well studied ( jin and wang 1991 ) ( for a review ( kontrogianni - konstantopoulos et al . 2009 ) ) . another novel interaction between the cardiac specific c0 domain and the regulatory light chain ( rlc ) has been analyzed by ratti et al . 2depicts the structure of mybpc3 as well as highlights important structural domains and indicates mybpc3 interacting proteins . please note : the number of phosphorylation sites localized in the mybpc motif is species dependent . ig immunoglobulin , p / a proline / alanine , fn fibronectin ) electron micrograph of rat cardiac muscle labelled with a mybpc antibody . 2008 ) ( with dr pradeep luther s kind permission ) depicts the structure of mybpc3 as well as highlights important structural domains and indicates mybpc3 interacting proteins . please note : the number of phosphorylation sites localized in the mybpc motif is species dependent . ig immunoglobulin , p / a proline / alanine , fn fibronectin ) in addition , domains in the mid - region ( c5c8 ) have been hypothesized to interact with one another , forming a trimeric collar that constrains the thick filament ( moolman - smook et al . also , a slightly different model of mybpc organisation within the sarcomere has been proposed by squire et al . these authors due to structural considerations and because domains c7c10 are also reported to interact with titin , which is unlikely to run other than axially along the myosin filament , propose a model whereby the carboxyterminal end of mybpc is aligned axially , which contrasts the collar alignment structure ( fig . 3).fig . 3summarizes the major differences between the mybpc models proposed by ( moolman - smook et al . 2003 ) ( b ) . please note in a mybpc forms a trimeric collar that constrains the thick filament ( moolman - smook et al . top : schematic diagram showing a single mybpc protein interacting with myosin ( top view ) , bottom : side view . whereas in b the carboxyterminal end of mybpc is aligned axially ( squire et al . 2003 ) . top view : a thick filament is represented along with six surrounding actin filaments ( in striated muscle ) , the broken circle represents the c0 domain in the mybpc3 isoform . transparent ghosts to indicate their position ( grey circles : mybpc3 with green indicating the p / a rich domain ; yellow / brown : myosin , dark grey : actin ; figures are from : ( squire et al . 2003 ) with kind permission from the authors and the publisher ( journal of molecular biology ) ) summarizes the major differences between the mybpc models proposed by ( moolman - smook et al . please note in a mybpc forms a trimeric collar that constrains the thick filament ( moolman - smook et al . top : schematic diagram showing a single mybpc protein interacting with myosin ( top view ) , bottom : side view . whereas in b the carboxyterminal end of mybpc is aligned axially ( squire et al . 2003 ) . top view : a thick filament is represented along with six surrounding actin filaments ( in striated muscle ) , the broken circle represents the c0 domain in the mybpc3 isoform . ghosts to indicate their position ( grey circles : mybpc3 with green indicating the p / a rich domain ; yellow / brown : myosin , dark grey : actin ; figures are from : ( squire et al . 2003 ) with kind permission from the authors and the publisher ( journal of molecular biology ) ) however three major differences exist between the cardiac and skeletal muscle isoforms : mybpc3 consists of an additional immunoglobulin domain ( c0 ) at its amino - terminus , the domain c5 contains a proline rich 25 residue insertion and several phosphorylation sites are species dependent localized in the myosin binding motif ( gautel et al . mybpc3 mutations were first reported in 1995 , which was an important discovery ( bonne et al . 2006 ) and depending on the population analyzed , mybpc3 mutations are found in up to 4050% of the genotyped hcm patients ( richard et al . to date about 200 different mybpc3 mutations have been reported ( marston 2011 ; schlossarek et al . mybpc3 mutations are associated with a slightly lower penetrance , later onset of disease and with milder forms of disease progression in comparison to other hcm causing mutations located for example in the beta myosin heavy chain ( myh7 ) gene ( niimura et al . however this general statement may not necessarily be true for all mybpc3 mutations and indeed some mybpc3 mutations are associated with a poor prognosis . for example a recently discovered deletion in intron 32 in mybpc3 ( nt 21348 - 21372 , accession no . u91629 ) , leading to a pre - terminal stop codon , has been found at a frequency of about 4% in india and south east asia , with significant impact on human heart failure ( dhandapany et al . 2009 ; waldmuller et al . 2003 ) . while this mutation has been initially described in hcm patients , it is important to note that this mutation is also associated with other types of cardiomyopathy such as dcm . other mybpc3 mutations primarily found in dcm patients have also been reported , for example the asn948thr missense mutation ( daehmlow et al . 2002 ) . in addition the mybpc3 arg502trp mutation with a frequency of about 2.4% is the most common hcm - causing mutation among individuals of european descent in the usa ( saltzman et al . however other mutations may be prevalent in different european populations , such as in the netherlands , where three different founder mutations are present : ( i ) the c.2373_2374insg mybpc3 which is present in the great majority of hcm patients ( up to 25% ) and where ( ii ) the c.2864_2865delct and ( iii ) the c.2827c > 2003 ; christiaans et al . 2010 ) . beside the important mutations in mybpc3 , mybpc1mutations are a cause of distal arthrogryposis type 1 ( da1 ) , a disease characterized by congenital contractures of the hands and feet ( gurnett et al . although by now mybpc3 mutations are well known causes of hcm and dcm including associated heart failure , the underlying molecular mechanisms are not well defined , some of which will be discussed in the next chapters . to gain more insight into the underlying molecular mechanisms , mybpc3 has been deleted in genetically altered mouse models by two independent groups . loss of this protein is not associated with any embryonic lethality and mybpc3 also is not essential for sarcomere formation but its absence results in profound eccentric hypertrophy in the homozygous animals ( carrier et al . hemodynamic analysis in the mice generated by the carrier group , where exons 1 and 2 have been deleted , revealed the presence of normal contractility but severe diastolic defects . in addition heterozyogous animals develop septal hypertrophy , a hallmark of hcm ( carrier et al . 2004 ) . however these animals were engineered so that they harbour a complete ablation of the gene and therefore are useful to identify basic mechanisms , but the overwhelming majority of human mutation carriers express mutant mrnas and probably proteins , which makes it difficult to relate these data directly to the situation in patients . therefore , in addition to the pure knockout models , wildtype mybpc3 and different mybpc3 mutants have been overexpressed in various models such as an amino - terminal truncated mybpc3 , which mimics a certain type of human mutations which lead to the loss of the carboxyterminal domains including the titin and myosin binding sites . overexpression of the mutant , but not the wildtype protein , caused major features of hcm including hypertrophy and an increase in calcium sensitivity ( yang et al . whereas above mentioned transgenes were well expressed at the mrna and protein levels , overexpression of a mybpc3 mutant lacking only the myosin binding domain resulted in the expression of only very modest levels of mutant protein ( i.e. about 5% ) which led to a mild hypertrophy and heart failure phenotype ( yang et al . two additional knock in mouse models have been engineered to carry mutations found in patients and which affect titin and myosin binding . interestingly animals homozygous for these mutations develop a dcm like phenotype with depressed contractility and hypertrophy ( mcconnell et al . another knock in mouse model was generated such that the mutant protein did not contain the aminoterminal myosin binding domain . the mutant protein was readily integrated into the sarcomeres of heterozygous and homozygous animals , was pka phosphorylatable and major structural defects could not be detected . however this mutation was associated with a significant increase in calcium sensitivity ( witt et al . an additional mouse knock in model , based on a g > a transition located on the last nucleotide of exon 6 and which was found in a patient with hcm , has been generated by lucie carrier s group . interestingly this mutation gives rise to three different mrnas : ( i ) missense mutation ( ii ) nonsense due to exon skipping , frameshift and premature stop codon and ( iii ) deletion / insertion as nonsense but with additional partial retention of a downstream intron which restores the reading frame and which leads to an almost full length protein . homozygous animals develop hypertrophy , interstitial fibrosis and decreased myocardial function whereas heterozygous animals do not have any obvious phenotype ( vignier et al . additional genetically altered mouse models have been generated to study mybpc3 phosphorylation , which will be discussed in the next chapter . human mybpc3 contains at least four phosphorylation sites which are localized inside the myosin binding motif ( serines 275 , 284 , 304 , with an additional phosphorylation site not unequivocally identified ) ( copeland et al . 2010 ) ) , the mouse myosin binding motif contains three to four sites ( serines 273 , 282 , 302 , ( 305 ) ) and five sites have been identified in the canine genome as well as three sites in the rat genome ( jia et al . these phosphorylation sites are targets of protein kinase a ( pka ; serines 273 , 282 , 302 ) ( gautel et al . 1995 ) , protein kinase c ( pkc ; serines 273 , 302 ) ( mohamed et al . 1998 ) , calmodulin dependent kinase ii ( camkii ; serine 302 ) ( gautel et al . 1995 ) , protein kinase d ( pkd ; serine 302 ) ( bardswell et al . 2010 ) and ribosomal s6 kinase ( rsk ; serine 282 ) ( cuello et al . 2011 ) . mybpc3 phosphorylation also has been studied in various transgenic mouse models , for example lines have been established where the phosphorylation sites ( ser273 , ser282 , and ser302 ) , along with two adjacent sites that could be potentially phosphorylated ( thr272 , thr281 ) , were converted to alanines . while overexpression of wildtype mybpc3 was able to rescue the mybpc3 null phenotype , the non - phosphorylatable mybpc3 was not ( sadayappan et al . 2005 ) . also a similar approach has been used to analyze mybpc3 phosphorylation by generating animals whereby only the three known phosphorylation sites were converted into alanines ( t3sa ) . again these animals showed , when crossed into the mybpc3 null background , in addition to hypertrophy systolic and diastolic defects , further supporting the notion that pka phosphorylation of mybpc3 is important for myocardial function ( tong et al . 2008 ) . these animals showed an increase in contractility and relaxation of about 22 and 25% , respectively . an increase in the phosphorylation of the remaining mybpc3 and other proteins such as troponin i and phospholamban was also observed ( yang et al . 2001 ) . the reverse experiment , i.e. overexpression of a phosphomimetic mybpc3 in a mybpc3 null background , has also been performed , which resulted in subtle changes in sarcomeric ultrastructure characterized by increased distances between the thick filaments which may indicate that phosphomimetic mybpc3 affects thick thin filament relationship . the phosphomimetic mybpc3 also prevented interaction with myosin heavy chain in vitro as analyzed by yeast two hybrid and pull down assays . this effect is difficult to explain , but it may well be that phosphomimetic mybpc3 prevents an increase of thick filament packing density which is associated with reduced calcium activated force generation . an alternative explanation could be that phosphorylated mybpc3 is protected from calpain ( see also later ) mediated proteolysis ( sadayappan et al . it is probably important to mention , that loss of mybpc3 phosphorylation also has been observed in failing human hearts and strategies to increase its phosphorylation may have cardio - protective effects ( copeland et al . 2006 ) . above mentioned studies have been intensified by generating mouse models overexpressing various single or double phosphomimetic or non phosphorylatable serines in the mybpc3 knockout background ( i.e. the cmybp - c , cmybp - c , cmybp - c ; on position s-273 , s-282 and s-302 , respectively ) . one major conclusion of these studies is that s-282 has a unique regulatory role in that its phosphorylation is critical for the subsequent phosphorylation of s-302 , but that all residues play a role in regulating the contractile response to -agonist stimulation ( sadayappan et al . 2011 ) . although there is now compelling evidence supporting the notion that mybpc3 phosphorylation modulates contractility by controlling the proximity of the myosin heads to actin , however the precise molecular mechanism remains unclear ( reviewed in ( barefield and sadayappan 2010 ; schlossarek et al . 2011 ) ) . in particular when using in vitro systems it was shown that mybpc3 phosphorylation can abolish the ability of mybpc3 to interact with the s2 region of the myosin heavy chain but may enhance mybpc3 interactions with the thin filament . vice versa dephosphorylation results in strong binding of mybpc3 to the myosin head , probably preventing its force generating strong interaction with actin ( kulikovskaya et al . however recent data also indicate that mybpc3 may act synergistically with the myosin rlc to enhance cross - bridge formation by altering the interaction of the myosin head with actin . it may well be that this interaction depends on phosphorylation of either mybpc3 or rlc and may well be able to provide another mechanism how phosphorylation of sarcomeric proteins may affect protein / protein interactions and kinetics of force development ( colson et al . although it is well known that the distance between thick and thin filaments is a major determinant of calcium sensitivity and that loss of mybpc3 is associated with increased calcium sensitivity , the precise molecular mechanism of how mybpc3 phosphorylation affects this system is unknown . part of this problem is that pka phosphorylation decreases calcium sensitivity via troponin i phosphorylation which leads to a decrease in tropinin c calcium sensitivity . however mybpc3 phosphorylation , which decreases calcium sensitivity , has no effect on interfilament spacing ( colson et al . future studies , based on various transgenic animals , will certainly help to answer this question . in general two different molecular mechanisms may account for the observed pathologic effects of any mutation : ( i ) poison peptide ( functional integration of a mutant protein ) ( ii ) haploinsufficiency ( loss of one allele ) . however , any mutation may also cause disturbances in the degradation of such a product , including its mrna . for example , the mrna might be degraded via nonsense mediated mrna decay or the protein may be degraded via the ubiquitin proteasome system ( ups ) with implications for protein degradation in general , including autophagy . most of the more than 600 known hcm causing mutations located in other sarcomeric protein genes are point mutations or single amino acid deletions ( van driest et al . 2004 ) , hence the poison peptide mechanism seems to be the likely mechanism for these mutations . in contrast , the most frequent type of mybpc3 mutation affects splice acceptor and donor sites or are deletions or insertions predicted to result in truncated mybpc3 proteins ( van driest et al . 2004 ) . however , multiple studies were unable to detect or document the presence of the predicted truncated mybpc3 molecules . for example van dijk and colleagues analyzed the c.2373dupg and the c.2864_2865delct mutations and found a significant decrease in mutant mybpc3 mrna . also they detected a significant loss of mybpc3 protein but were unable to detect the truncated proteins ( van dijk et al . marston and colleagues studied mybpc3 protein expression in two patients carrying the glu258lys and the arg502trp missense mutations in addition to seven other mutations which are predicted to lead to premature terminations and truncated proteins . however no truncated protein was detectable and in all samples studied significant lower amounts of mybpc3 protein was detected ( marston et al . another complex mutation where a t to a transition occurred on position 2604 together with a deletion of c at 2605 was predicted to lead to a truncated protein ( 1 - 868 + 13 nonsense amino acids with a premature stop codon in c7 ) was also studied in great detail , but no truncated mybpc3 protein could be detected ( jacques et al . 2008 ) . in summary , several different studies failed to detect truncated mybpc3 proteins in mybpc3 mutation carriers , which makes the poison peptide mechanism unlikely to occur in the studied mutations / individuals . also these studies documented loss of mybpc3 protein in mutations carriers , with only 6775% of the normal protein present , which indicates haploinsufficiency may contribute to the phenotype . myosin interaction and by binding to both proteins it may directly affect calcium sensitivity . by employing skinned myocytes from patients with hcm mutations , a variety of studies showed that mybpc3 mutations are associated with higher calcium sensitivity . however , these data may also result from low levels of troponin i ( tni ) phosphorylation present in the myectomy tissue rather than the mutation itself ( hoskins et al . nevertheless an increase in calcium sensitivity in myocardial samples obtained from patients affected by mybpc3 mutations is likely because removal of mybpc3 from myocardial samples under experimental conditions is associated with an increase in calcium sensitivity ( hofmann et al . 2003b ) and an increase in calcium sensitivity has also been shown in mybpc3 deficient myocardium ( rybakova et al . 2011 ) . calcium sensitivity depends on the rate of calcium binding to or calcium release from troponin c and kinetic studies suggest that for hcm causing mutations the rate of calcium binding to tnc is increased , but it remains to be elucidated whether this is a general phenomenon or whether other determinants of calcium sensitivity such as the tnc calcium dissociation rate may also be affected and whether this holds true for mybpc3 mutations ( dong et al . therefore , mybpc3 mutations may primarily increase calcium sensitivity ( gain of function ) which may cause faster cross bridge cycling ( hypercontractility ) , but which may also cause incomplete relaxation , a cause of diastolic dysfunction frequently observed in hcm . this concept also is supported by hemodynamic data obtained from mybpc3 deficient animals , where no systolic but severe diastolic dysfunction has been observed ( carrier et al . it might well be that mybpc3 mutations are primarily linked to an increase in calcium sensitivity , but it is difficult to link this event directly to the secondary effects such as myocardial hypertrophy including myofibrillar disarray , fatal arrhythmias and sudden cardiac death ( fig . 4 ) . one hypothesis proposes energy deficiency due to reduced thermal efficiency in hcm or another hypothesis states that troponin c functions as an intracellular calcium buffer or calcium store ( bers 2008 ) . an increase in calcium sensitivity could affect calcium transients , which indeed have been found to be increased in myh6 arg403gln knock in animals ( gao et al . an increase in calcium transients may also have effects on calcium dependent enzymes such as calcineurin , calmodulin dependent kinase , and protein kinase c all of which have been shown to be important for the initiation of myocardial hypertrophy ( kubis et al . 2003).fig . 4summary of possible pathologic events caused by a primary increase in calcium sensitivity , which has been shown to be present in various mybpc3 transgenic animal models as well as in patients affected by mutations in the mybpc3 gene . camkii calmodulin dependent kinase ii , pkc protein kinase c summary of possible pathologic events caused by a primary increase in calcium sensitivity , which has been shown to be present in various mybpc3 transgenic animal models as well as in patients affected by mutations in the mybpc3 gene . camkii calmodulin dependent kinase ii , pkc protein kinase c the hypothesis that sarcomeric hcm mutations lead to inefficient atp utilization was proposed by the watkins group and is an attractive model , because it is able to explain many different features found in hcm ( ashrafian et al . according to this theory a lower atp / adp + p ratio may contribute to energy depletion in critical compartments , which may affect calcium re - uptake and lead to an increase in calcium concentrations and thus activation of calcium dependent enzymes such as calcineurin . another consequence is the activation of adenosine monophosphate kinase ( ampk ) , which is also able to induce hypertrophy and yet another consequence is the increase in mitochondrial demands which may lead to an increase in reactive oxygen species , mitochondrial abnormalities and apoptosis all of which have been observed in various patients affected by hcm ( ishikawa et al . as pointed out above most mybpc3 mutations lead to haploinsufficiency but some missense mutations might escape targeted removal and might well be expressed in the myocardium . this might particularly be true for the reported aminoterminal mutations g5r , r35w and t59a in the human genome ( sarcomere protein gene mutation data base ) as well as for the a31p mutation observed in maine coon cats ( meurs et al . 2005 ) , all of which are linked to hcm . ratti and co - workers ( ratti et al . 2011 ) studied above mentioned missense mutations . while g5r had only mild effects , a31p could not be expressed as a soluble protein and t59a was not chosen as in many other species a threonine residue is substituted by an alanine and no extreme effects were expected , the r35w seemed to be important for binding to rlc . mybpc3 mutations by modulating calcium sensitivity may also have effects , either directly or indirectly , on mechanosensation and mechanotransduction . for example , changes in calcium transients caused either via an increase in calcium sensitivity or via inefficient atp usage , may have effects on calcium dependent enzymes which may in turn affect mechanosensory effects . for example , calcineurin mediated nfat and gata4 activation , protein kinase c mediated effects on mybpc3 itself or calmodulin kinase ii effects on phospholamban and or ryanodine receptor phosphorylation may contribute to changes in gene expression and myocardial remodelling . myocardial hypertrophy and remodelling itself may lead to changes in bio - mechanical properties of the heart , which feeds back to changes in intracellular signalling ( knll et al . the sarcomeric z - disc is a central nodal point for signalling ( knll et al . 2011a ) and changes in z - disc structure or composition may well affect its function . in this context , the e3 ubiquitin ligase atrogin 1 has been found to colocalize at the sarcomeric z - disc with a truncated mybpc3 which may have consequences for z - disc mediated signalling ( mearini et al . however it remains unclear whether above mentioned changes in intracellular signalling , if not alone able to cause effects , will sensitize the myocardium at predisposed locations to maladaptation . it also remains to be seen whether z - disc mediated apoptotic events play a role in mybpc3 related hcm ( knll et al . it remains also unclear , how mybpc3 mutations are linked to fibrosis as well to intracellular and cellular myofibrillar disarray . it might well be that fibrosis , which is probably an early event in hcm , causes myofibrillar disarray by displacing existing cardiac myocytes , but it remains unclear how intracellular disarray develops . in this context , myofibrillar disarray contributes certainly to inefficient energy use ( i.e. cardiac myocytes are not aligned properly ) . a recent study on hcm myocardial biopsies , including those from patients affected by mybpc3 mutations , supports the view that fibrosis is an early event and is present even before the onset of hypertrophy ( ho et al . inhibition of fibrosis , either via anti tgf antibodies or the angiotensin ii type 1 receptor antagonist losartan prevented onset of hypertrophy , non myocyte proliferation and fibrosis in genotype positive but hypertrophy negative ( pre - hypertrophic ) animals ( teekakirikul et al . 2010 ) . these data point as well to the importance of other cell types than cardiac myocytes which are inherent in the myocardium and which contribute significantly to the phenotype ( knll et al . 2011b ) . a novel link between an increase in myofibrillar calcium sensitivity and arrhythmias has been uncovered recently . an increase in calcium sensitivity led to a change in the shape of the ventricular action potentials in mice which results in shorter effective refractory periods , greater beat to beat variability of action potential durations and increased dispersion of ventricular conduction velocities at higher heart rates all of which may predispose to the occurrance of arrhythmias . these effects were greatest in hcm causing troponin t mutant animals and most importantly , these effects were reproduced when the calcium sensitizer emd57033 was used and they were reversible when blebbistatin , a calcium de - sensitizer , was employed . another important finding of this study is that the degree of myocardial hypertrophy does not correlate with the arrhythmia risk ( baudenbacher et al . other mechanisms may involve above mentioned energy depletion with consecutive effects on energy dependent ion exchanges ( ashrafian et al . 2003 ) , loss of cardiac myocytes via apoptosis , necrosis or autophagy which may lead to electric isolation of single cardiac myocytes and fibrosis ( teekakirikul et al . another important approach has been used by lucie carrier s group , when they analyzed the degradation process of mybpc3 . this group found that the e3 ubiquitin ligase atrogin 1 interacts with both , mutant ( a 32 kda mybpc3 truncated protein , based on a human mutation and which comprises only of domains c0 and parts of c1 termed m7t - cmybp - c ) and wildtype mybpc3 , but only the mutant mybpc3 was targeted for degradation . this makes atrogin 1 also a novel mybpc3 interacting protein , but whether the interaction of atrogin 1 and full length wildtype mybpc3 is physiologically relevant remains to be seen . interestingly atrogin 1 and m7t - cmybp - c co - localize at the sarcomeric z - disc , which points to the importance of this structure in protein degradation processes ( mearini et al . their data also indicated that muscle ring finger protein 1 ( murf1 ) regulates mybpc3 mrna and protein via a posttranscriptional mechanism ( mearini et al . also , calpain 1 , a ubiquitously expressed calcium dependent protease and which localizes to the sarcomeric z - disc and i - band via interactions with titin , interacts with mybpc3 as well and might contribute to mybpc3 turnover ( jiang et al . 2002 ; kontrogianni - konstantopoulos et al . 2009 ) . in addition , the lysosomal inhibitor bafilomycin a1 caused an increase in truncated mybpc3 proteins , which points to the possible involvement of autophagy in mybpc3 related degradation processes . as briefly mentioned above , domains c5c8 have been hypothesized to interact with one another , forming a trimeric collar that constrains the thick filament . interestingly , mutations r654h and n755k , both are hcm causing and located in c5 , decreased the affinity to c8 about 2-fold and by at least 10-fold , respectively ( moolman - smook et al . disturbances of intra- mybpc3 interactions by mutations are a completely novel approach to explain the complex phenotype observed in genetically altered animal models as well as in human patients . mybpc3 mutations like the majority of all other cardiomyopathy causing mutations are associated with incomplete , age and gender dependent penetrance . powerful epigenetic and environmental factors are thought to have a major impact on the phenotype , which also implies the possibility of effective therapeutical interventions . if increased calcium sensitivity is the primary event which leads to hcm in mybpc3 mutation carriers then calcium desensitizers might be able to influence positively disease progression , which has been shown in animal models ( baudenbacher et al . however dcm is associated with decreased calcium sensitivity and precise calibration of calcium sensitivity is certainly important and needs to be monitored properly , if applied in therapy . another although still very experimental approach is to use short oligonucleotides to intervene in mrna processing such that exon skipping events can be used therapeutically to prevent the synthesis of pathologic mrnas or to use sirnas / mirnas to suppress pathologic mrnas calcium antagonists such as diltiazem have been successfully used to suppress hcm in the r403q alpha mhc transgenic mouse ( semsarian et al . 2002 ) as well as in a mouse model carrying the tnt - i79 n mutation ( westermann et al . . however it will be important to find out , whether mybpc3 mutation positive patients will respond appropriately to this type of treatment . 2010 ) as well as parvalbumin overexpression in a glu180gln tropomyosin animal model rescued the phenotype in both of these lines , which points to the possibility of manipulating the calcium metabolism in order to treat the disease ( coutu et al . however while phospholamban ablation in muscle lim protein deficient ( minamisawa et al . 1999 ) and in glu180gly tropomysin transgenic animals ( alves et al . 2011 ) is favourable , ablation of the very same gene in mybpc3 transgenic animals is not ( song et al . it remains to be determined whether these differences are due to the well known differences between human and mouse physiology or whether these differences reflect differences in the underlying molecular mechanisms . another possibility might be to intervene at the level of energy metabolism via perhexiline which shifts substrate utilization away from free fatty acids to carbohydrates by inhibiting the enzyme carnitine palmitoyl transferase . perhexiline improved cardiac energetics , normalized exercise diastolic dysfunction and increased exercise capacity in patients ( abozguia et al . it remains also to be seen whether moderate physical activity in mybpc3 mutation carriers is favourable , as suggested by animal experimentation ( konhilas et al . indeed anti - apoptotic gene expression has been observed when animals were subjected to limited physical exercise ( konhilas et al . also calcineurin inhibition may have favourable effects as suggested by animal experimentation ( sussman et al . 1998 ) , but application of available calcineurin inhibitors such as cyclosporine a and tacrolimus , at doses needed to affect calcineurin activity in the heart , is associated with significant side effects such as hypertension , nephro - toxicity and diabetes , which precludes their application in human individuals . another way to inhibit hypertrophy is to use statins ( 3-hydroxy-3-methyglutarylcoenzyme a reductase inhibitors ) , which act via reducing membrane bound ras and thus reducing perk44/42 and erk1/2 activation which reduced hcm related symptoms in a r403q beta mhc transgenic rabbit model ( patel et al . however human pilot studies based on these animal experiments remained disappointing ( bauersachs et al . therefore it remains to be determined whether this approach is not applicable in general or whether a genotype phenotype specific analysis will identify patient subgroups more susceptible to this type of treatment . although mybpc3 has been the subject of intense research for almost over four decades , our understanding of its function in physiology and patho - physiology is still limited . however , mybpc3 mutations are a major cause of human cardiomyopathy and associated heart failure , with some disease causing mutations having frequencies of up to 4% in various populations . this may explain major features observed in patients such as the hypercontractile phenotype , changes in calcium transients , myocardial hypertrophy , the defect in relaxation and effects on remodelling including the development of myofibrillar disarray as well as the link to live threatening arrhythmias ( fig . future research will focus on the identification of pathways leading from a single mybpc3 mutation to the complex disease phenotype in patients . although challenging and certainly difficult to achieve , this approach will undoubtedly result in the development and design of patient specific , novel therapies . inducible pluripotent stem cells ( ips ) , gene therapy approaches , whole genome sequencing and determination of the explicit genotypes , as well as medicinal chemistry will have to be combined to increase our knowledge in this field .

myosin binding protein c ( mybpc ) is a crucial component of the sarcomere and an important regulator of muscle function . while mutations in different myosin binding protein c ( mybpc ) genes are well known causes of various human diseases , such as hypertrophic ( hcm ) and dilated ( dcm ) forms of cardiomyopathy as well as skeletal muscular disorders , the underlying molecular mechanisms remain not well understood . a variety of mybpc3 ( cardiac isoform ) mutations have been studied in great detail and several corresponding genetically altered mouse models have been generated . most mybpc3 mutations may cause haploinsufficiency and with it they may cause a primary increase in calcium sensitivity which is potentially able to explain major features observed in hcm patients such as the hypercontractile phenotype and the well known secondary effects such as myofibrillar disarray , fibrosis , myocardial hypertrophy and remodelling including arrhythmogenesis . however the presence of poison peptides in some cases can not be fully excluded and most probably other mechanisms are also at play . here we shall discuss mybpc interacting proteins and possible pathways linked to cardiomyopathy and heart failure .

current guidelines recommend that pregnant women be treated with highly active antiretroviral therapy ( haart ) regimens for prevention of perinatal transmission of hiv , even if therapy is not yet recommended for treatment of maternal hiv infection . these therapies are generally welltolerated during pregnancy and have resulted in rates of maternal - to - child transmission ( mtct ) of under 2% among nonbreastfeeding populations . antiretroviral therapy is often discontinued after delivery if prepregnancy therapy was not indicated for maternal health . studies of scheduled treatment interruption showing poorer outcomes among subjects randomized to treatment interruption rather than continuous therapy have raised the question of whether discontinuing therapy after delivery among women receiving haart for prevention of mtct may be harmful to the mother . several small studies using various treatment schedules have not suggested harm from scheduled treatment interruptions , although all have shown lower cd4 + lymphocyte counts at the end of the study in treatment interruption groups [ 46 ] . recently however , results from other studies have shown higher rates of morbidity and mortality among subjects assigned to a treatment interruption arm compared to those provided with continuous therapy . the cd4-guided therapy arm of the trivacan trial in africa was stopped early because of a significantly increased rate of serious morbidity in the interruption arm ( 15.2/100 person - years ) compared to the continuous therapy arm ( 6.7/100 person - years , rr 2.27 , 95% ci 1.154.76 ) . at enrollment , all subjects had cd4 + cell counts above 350 cells / ul and hiv rna below 300 copies / ml . the largest trial reported to date , the smart study , used similar inclusion and therapy interruption / reinstitution guidelines and included 5472 subjects . in smart , the rate of opportunistic disease or death was 3.3/100 person - years in the therapy interruption group and 1.3/100 person - years in the continuous therapy group ( hr 2.6 , 95% ci 1.93.7 for interruption compared to continuous group ) . unexpectedly , the hazard ratio for major cardiovascular , renal , and hepatic disease was 1.7 ( 95% ci 1.12.5 ) for the interruption compared to the continuous group , despite less overall antiretroviral drug exposure in the interruption group . while the results of trivacan and smart raise concerns regarding the potential risk of stopping therapy after use in pregnancy for prevention of perinatal transmission , both of these trials used lower cd4 + lymphocyte count thresholds for resumption of therapy than would be used based on clinical treatment guidelines . data on outcomes after antiretroviral therapy ( art ) limited to pregnancy compared to similar women not receiving therapy or continuing therapy are limited and primarily reflect use of zidovudine ( zdv ) monotherapy rather than haart [ 1015 ] . given the paucity of data on the long - term maternal effects of short - term haart regimens for prevention of perinatal transmission of hiv and the concerns regarding therapy interruption raised by the smart and trivacan studies , we compared clinical and laboratory outcomes after delivery among women enrolled to the women and infants transmission study ( wits ) , a longitudinal cohort study , who either stopped or continued antiretroviral therapy at delivery that had been started for prevention of mtct of hiv . laboratory markers of immune activation and potential cardiovascular and metabolic risk were included . wits is a multicenter prospective observational study of pregnant women and their infants . beginning in december ( 1989 ) , pregnant hiv-1-infected women were recruited at centers in chicago , massachusetts ( boston and worcester ) , new york city , and san juan . the study was approved by each site 's institutional review board , and all women provided informed consent for enrollment of themselves and their infants . women were enrolled during pregnancy or within 7 days after delivery , and infants were enrolled within 7 days of birth . for women monitored during pregnancy , study visits occurred at or before 20 weeks ' gestation , at 25 2 weeks , at 32 2 weeks , and at delivery . women were followed for one year after delivery with visits at two , six , and twelve months . at each visit , a detailed medical and behavioral questionnaire was administered , and a physical examination and phlebotomy were performed . antiretroviral treatment was not prescribed as part of the study but detailed data on its use was collected . therapy during pregnancy was categorized as zdv monotherapy or combination therapy , consisting of at least two nucleoside reverse transcriptase inhibitors with or without nonnucleoside reverse transcriptase inhibitors or protease inhibitors . clinical events were categorized using the 1993 cdc revised surveillance case definition . to be eligible for inclusion in this analysis , women had to be enrolled after 6/1/1994 when use of zdv for prevention of hiv transmission became standard therapy for pregnant women and before 6/30/2006 to allow a year of followup postpartum . in addition , they had to be enrolled by 32 weeks of gestation to allow time for antiretroviral therapy to be administrated and be art nave at enrollment . they had to have a cd4 + lymphocyte count above 350 cells / ul at enrollment so that art was being prescribed primarily for prevention of mtct and not for maternal health indications . of the 3297 women enrolled to wits by 6/30/2006 , 2777 had a confirmed delivery , 1165 were art nave at enrollment , and 1091 of these were enrolled before the delivery visit . of these , 512 received art during pregnancy , 261 of whom had a cd4 + lymphocyte count above 350 cells / ul at enrollment during pregnancy , and 206 were enrolled after 6/1/1994 . the 206 women included did not differ significantly from the 3091 women not included in age , race / ethnicity , history of cdc class b or c condition , or hiv rna levels . based on inclusion criteria , included women had higher cd4 + lymphocyte counts ( mean 603 cells / ul versus 467 cells / ul , p < .001 ) and lower gestational age at enrollment ( 16.1 weeks versus 21.6 weeks , p < .001 ) than the excluded women . to assess hiv disease progression , we used knotted splines and mixed effect models to compare the post delivery slope of the cd4 + lymphocyte counts , cd4 + lymphocyte percents , and hiv rna levels between women continuing or stopping art at delivery using [ 18 , 19 ] . rates of development of new cdc class b or c events were compared using cox proportional hazard models . the marginal mean values for laboratory testing postpartum were compared between the above listed groups using a general estimating equation . when indicated , some data were log - transformed to produce a normal distribution . cd4 + lymphocyte counts were determined by flow cytometry at daids immunology quality assurance program certified laboratories . lymphocyte phenotyping was done using a facscan ( becton dickinson , san jose , ca ) equipped with lysis ii software as previously described . plasma hiv-1 rna was measured in stored specimens using the roche amplicor hiv-1 monitor test ( roche diagnostic systems , branchburg , nj ) , as described in . specimens were assayed for routine metabolic analytes ( total cholesterol , high - density lipoprotein ( hdl ) cholesterol , triglycerides , high sensitivity c - reactive protein ( hscrp ) ) at quest diagnostics , baltimore , md and for specialty analytes ( leptin , lipoprotein - associated phospholipase a2 ( lp - pla2 ) , interleukin 6 ( il-6 ) ) at quest diagnostics nichols institute , san juan capistrano , ca . aliquots of frozen serum were shipped to quest diagnostics on dry ice for batch testing of each analyte . low - density lipoprotein cholesterol ( ldl ) was calculated using the friedewald equation for those samples exhibiting triglyceride values below 400 mg / dl . the laboratory utilized the fda - cleared cholesterol esterase / oxidase method of olympus ( olympus america , inc . , melville , ny ) on an automated testing platform for total cholesterol quantitation . for hdl cholesterol , the laboratory utilized the fda - cleared , hdl - cholesterol plus 2nd generation assay from roche diagnostics ( indianapolis , in ) on an automated testing platform . for triglyceride quantitation , the laboratory utilized the fda - cleared triglyceride reagents from olympus on an automated testing platform . serum high sensitivity crp levels were measured using the fda - cleared , automated bn ii in vitro diagnostic system ( dade behring , inc . , this method uses a particle enhanced immunonephelometric assay with a sensitivity of 0.2 mg / l . leptin was measured using the research use only human leptin ria kit from linco research , inc . this method is a competitive radioimmunoassay that utilizes a fixed concentration of iodine-125-labeled human leptin tracer antigen with a constant dilution of human leptin antiserum . the concentration of unknown human leptin in the study subject 's samples is determined against a calibration curve that is set up with each assay run that includes increasing concentrations of standard unlabeled human leptin antigen . human il-6 was measured with the research use only quantikine hs human il-6 immunoassay kit from r&d systems , inc . this assay utilizes a quantitative sandwich enzyme immunoassay technique to estimate the concentration of il-6 in serum specimens . lp - pla2 was measured with the fda - approved plac test kit from diadexus ( south san francisco , ca ) . of the 206 women eligible for inclusion , 147 continued therapy after delivery and 59 discontinued . women stopping therapy at delivery were slightly younger and had higher cd4 + lymphocyte counts than women continuing therapy , but the groups were similar in race / ethnicity , gestational age at enrollment , history of class b or c illness , and hiv rna levels . zdv monotherapy was used by 103 women during pregnancy , 41 of whom stopped therapy at delivery , and 62 of whom continued . three additional women received monotherapy with other drugs , one with nevirapine and two with didanosine . one hundred women took two or more antiretroviral drugs during pregnancy , 18 of whom stopped therapy at delivery , and 82 of whom continued . of the women on combination therapy , 27 took two nrti 's , two took triple nrti regimens including abacavir , 11 took two nrti 's plus an nnrti , 55 took two nrti 's with one or more pi 's , four took triple class regimens , and one took a regimen of one nrti with an nnrti . we compared the risk of hiv disease progression between women who stopped and women who continued therapy after delivery in several ways . the slopes of cd4 + lymphocyte counts and percentages and of hiv rna levels were compared between two and six months and six and twelve months postpartum to assess for rebound changes after stopping therapy . as shown in figure 1 and table 2 , the rate of change in cd4 + cell measures and hiv rna was not significantly different between the two groups . to evaluate further by type of therapy during pregnancy , the changes in cd4 + lymphocyte count and percentage and hiv rna level were compared among those stopping or continuing zdv monotherapy and those stopping or continuing combination regimens . table 2 shows the difference in slope for each parameter between all women who stopped or continued therapy , women who stopped or continued zdv monotherapy , and those who continued or stopped combination therapy . for example , over two to six months after delivery , women in the entire group who stopped therapy had a 12.2 cells / month loss in cd4 + lymphocyte count compared to women continuing therapy , but the difference was not significant . no significant differences between slopes were found for any of the time periods or any of the therapy groups . clinical progression was also assessed . none of the women developed a new class c condition during the first year postpartum . among the entire group , the hazard ratio ( hr ) for development of a new class b condition was 2.09 ( 95% ci 0.795.58 , p = .14 ) for women who stopped therapy at delivery compared to those who continued . for the zdv monotherapy group , the hr was 1.24 ( 95% ci 0.314.95 , p = .76 ) and for the combination therapy group the hr was 2.93 ( 95% ci 0.6413.36 , p = .16 ) . class b events among women stopping therapy included cervical dysplasia / carcinoma in situ ( cis ) in four , recurrent pneumonia in one , and vaginal yeast infection for over 28 days in one . class b events among women continuing therapy included cervical dysplasia / cis in three , oropharyngeal candidiasis in two , other infections in two , and other gastrointestinal problem , vaginal yeast infection for over 28 days , and pelvic inflammatory disease in one each . since treatment interruption studies have suggested an increased risk of morbidity not specifically related to hiv disease progression , we evaluated several laboratory markers of immune activation and of potentially increased risk of cardiovascular disease ( table 3 ) . cd4 + and cd8 + counts and percentages are shown for all women in the analysis and each subgroup . lymphocyte activation markers were available for a subset of women . of note , the cd8+cd38 + t lymphocyte and cd8+dr+ t lymphocyte percentages were higher in women stopping therapy than in those continuing in the overall group and the zdv group . results were available in too few women in the combination therapy group to allow meaningful analyses . hs - crp , lipid levels , leptin , and interleukin-6 levels were not different between the groups among all women and those receiving zdv . lp - pla2 levels were significantly different in the total group and the combination therapy group , but levels were higher in the women continuing therapy compared to those stopping . overall , the data on changes in cd4 + lymphocyte count , hiv rna levels , and clinical progression are reassuring over the first year postpartum among women with cd4 + lymphocyte counts above 350 cells / ul who choose to stop antiretroviral therapy after delivery compared to those who continue . one recent retrospective review found an increased risk of death or opportunistic infection among women stopping therapy after delivery , but the group was more heterogeneous than in the current study with 46% having previous arv exposure and 36% having a pre - arv cd4 + cell count below 350 cells / ul . the number of class b events in our study was low for both the continuation and noncontinuation groups , making it difficult to further define the risk of developing these events according to treatment continuation . the relative risk estimate for combination therapy is consistent with other studies and is consistent with changes in activated cd8 + counts and percents that was seen in this study . however , when taken in the context of the overall low numbers of events , and insignificant changes in cd4 + count and cd8 + count slopes there is little evidence that stopping therapy increases a woman 's risk of progression . among those enrolled to the smart study , divergence of the curves in the risk of opportunistic infection or death were seen by 12 months after enrollment between the drug conservation and viral suppression groups , even among those who were therapy nave or not on antiretroviral therapy at delivery [ 8 , 23 ] . however , women included in the current study were all therapy nave with nadir cd4 + lymphocyte counts above 350 cells / ul and baseline cd4 + cell counts of 603 cells / ul , compared to the smart study in which the median nadir cd4 + lymphocyte count was 250 cells / ul and participants had initiated antiretroviral therapy a median of six years before enrollment . among subjects in smart who were antiretroviral nave , the nadir cd4 + lymphocyte count was 376 cells / ul and the baseline was 437 cells / ul , also lower than in the current study . thus , differences in disease progression in those stopping therapy compared to those continuing may take longer to manifest among women with higher baseline cd4 + cell counts and no prior antiretroviral therapy . a concerning laboratory finding between the groups was higher levels of cd8 + cell activation among women stopping therapy at delivery compared to those continuing . pregnancy has been shown to lead to increased levels of cd8+dr+cd38 + lymphocytes in hiv - uninfected women compared to nonpregnant women , but levels return to baseline after delivery [ 21 , 24 ] , while levels are much higher during pregnancy in hiv - infected women and remain elevated postpartum in the absence of antiretroviral therapy . the current data suggest that continuing antiretroviral therapy after pregnancy may reduce levels of cd8 + cell activation among hiv - infected women overall and among women on zdv compared to women who stop therapy , but lymphocyte activation results were available for only two women stopping combination regimens , precluding a comparison in this group . increased levels of cd8+dr+cd38 + cells have been associated with both an increased risk of disease progression and reduced cd4 + cell gains on haart . thus persistently high levels postpartum may predict more rapid disease progression over time in those stopping therapy , but longer followup is required to assess this hypothesis . in addition , lymphocyte activation should be compared between women stopping and continuing haart regimens after delivery in larger numbers of women . elevated hscrp has recently been shown to predict hiv disease progression among untreated hiv - infected women postpartum . we did not observe significant differences in crp levels between women stopping versus continuing therapy postpartum , suggesting against differential risk in disease progression . one of the areas of increased morbidity and mortality noted in subjects in the drug conservation group of the smart trial was cardiovascular outcomes including myocardial infarction , coronary artery disease requiring surgery , and stroke . since the median age in the smart trial was 43 years compared to 27 years in the current study , we evaluated surrogate markers of cardiovascular risk since cardiovascular events were not expected . median values of laboratory markers that have been associated with an increased risk of cardiovascular outcomes , including elevated cholesterol , low density lipoproteins , triglycerides , and high sensitivity c - reactive protein , and decreased high density lipoproteins were not different between groups continuing versus stopping therapy . these results do not suggest an increased risk of cardiovascular morbidity among women stopping therapy but the predictive value of these markers in hiv - infected populations has primarily been assessed in populations of older men , and gender differences have been noted in some analytes in the general population . the only analyte found to differ between the groups was lp - pla2 , a macrophage - derived enzyme that has been an independent predictor of cardiovascular events in healthy populations [ 30 , 31 ] . lp - pla2 is not an acute phase reactant and is only minimally correlated with systemic inflammatory and hemostatic markers , suggesting that it may be useful for prediction of cardiovascular risk in hiv - infected groups , although further study is required . of note , lp - pla2 levels were higher among women continuing therapy , possibly indicating higher risk associated with continued art exposure or possibly related to the older age . the median values in both groups were lower than the proposed cutoff of below 235 ng / ml , so the clinical implications of the differences require further study . the overall results however do not suggest a major difference in cardiovascular risk between the groups , and especially not increased risk among women stopping therapy . while the smart study clearly showed benefits to continuing art once initiated , not all sti studies have shown differences in outcome . negative studies had smaller sample sizes but also used higher thresholds for restarting art . studies using a threshold of 350 cells / ul for reinitiating art tended to show fewer differences in outcomes between continuous versus interrupted therapy [ 46 ] . these studies are more consistent with the current standard of care in developing countries , with initiation of therapy recommended at a cd4 + cell count of 350 cells / ul , which would apply to pregnant women stopping art used for prevention of pmtct . in developing countries , therapy is usually initiated at a lower cd4 + cell count of 200250 cells / ul , more similar to the situation in the smart study . in addition , if studies currently in progress confirm reduced transmission with maternal haart during breastfeeding , women in developing countries may have longer periods of haart for prevention of pmtct before discontinuation , potentially increasing the risk of adverse outcomes with discontinuation . assessment of the effects of discontinuing art used for pmtct must be included in studies in resource limited settings . the current results are generally reassuring that stopping art used solely for pmtct does not increase the short - term risk of hiv disease progression or of abnormalities in common cardiovascular risk markers . however , followup was only for one year postpartum , and longer followup may be needed to detect differences in this relatively healthy population . women were not randomized , but chose whether or not to continue therapy in consultation with their health care provider . thus , there may be unmeasured confounders that affected the outcomes and mitigated differences . finally , the numbers included in this study were relatively low , especially in the group stopping combination therapy , thus limiting power . overall , the data do not indicate major differences in short - term outcome between women with cd4 + cell counts above 350 cells / ul who choose to stop or continue art that was initiated for pmtct . however , declines in lymphocyte activation markers among women continuing art are consistent with large treatment interruption studies demonstrating improved outcomes with continuous rather than intermittent art . as use of combined art regimens for prevention of pmtct expands , especially in developing countries where thresholds for reinitiation of therapy are lower and duration of preventive therapy may be longer if used during breastfeeding , further evaluation , in randomized trials , of the risks and benefits of continuing therapy , is required .

objective . to assess clinical progression and inflammatory markers among women stopping or continuing antiretroviral therapy ( art ) after pregnancy . methods . art - nave women with cd4 + lymphocyte counts > 350 cells / ul initiating art during pregnancy had clinical events and laboratory markers compared over one year postpartum between those stopping ( n = 59 ) or continuing ( n = 147 ) art . results . slopes in cd4 count and hiv rna did not differ between groups overall and in subsets of zdv or combination therapy . the hazard ratio ( hr ) of a new class b event was 2.09 ( 95% ci 0.795.58 ) among women stopping art , 1.24 ( 0.314.95 ) in those stopping zdv , and 2.93 ( 0.6413.36 ) among those stopping combination therapy . women stopping art had increased immune activation . no significant differences were seen in c - reactive protein , lipids , leptin , or interleukin-6 . conclusions . while changes in cd4 and hiv rna levels over one year were similar between women stopping or continuing art postpartum , higher immune activation among women stopping therapy requires further study .

diabetic macular edema ( dme ) is one of the manifestations of diabetic retinopathy , which has an impact on central visual acuity . it is considered the main cause of visual loss and impairment in eyes with diabetic retinopathy.1,2 dme is characterized by the presence of exudation and accumulation of extracellular fluid in the retinal layers , which are caused by increase in the permeability of retinal blood vessels . vascular endothelial growth factor ( vegf ) is associated with the breakdown of the blood retinal barrier , causing increased vascular permeability that results in the pathogenesis and development of macular edema.3,4 vegf is a protein that promotes the angiogenesis and increases the vascular permeability . vegf is a main cause of vascular growth and edema , which are present in many vascular retinal disorders such as diabetic retinopathy and dme.5,6 many studies showed that the inhibition of vegf with intravitreal injection of anti - vegf agents is helpful in the treatment of dme.7 there are three commonly used intravitreal anti - vegf agents : aflibercept , bevacizumab , and ranibizumab . they are beneficial and safe for the treatment of dme . only ranibizumab and aflibercept are approved by the food and drug administration ( fda ) for this indication.810 ranibizumab is a recombinant humanized monoclonal antibody fragment that binds to all the isoforms of human vegf - a . intravitreal ranibizumab reduces dme with subsequent visual improvement , thus it replaced laser photocoagulation in the treatment of dme.11,12 aflibercept is a recombinant fusion protein that binds all isoforms of vegf - a , vegf - b , and placental growth factor . the drug is currently approved for the treatment of neovascular age - related macular degeneration , macular edema associated with vein occlusion , and dme.13,14 it was shown to be more effective than ranibizumab and bevacizumab in regaining lost visual acuity in a large randomized controlled trial.15 the aim of this study is to compare intravitreal aflibercept and ranibizumab for the treatment of dme in eyes with moderate visual loss . a total of 70 eyes of 42 diabetic patients were included in this prospective randomized study . the study was approved by the ethical committee of the mitghamr lasik and eye center ( approval number 212015 ) , and all patients provided written informed consent to participate according to the tenets of the declaration of helsinki . the patients were randomly divided into two groups ; patients in group i ( 35 eyes ) were treated with intravitreal aflibercept injection and patients in group ii ( 35 eyes ) were treated with intravitreal ranibizumab injection . patients with type i or ii diabetes , dme in eyes as diagnosed clinically and with oct , patients with best corrected visual acuity ( bcva ) ranged from 0.1 to 0.25 ( moderate visual loss ) and with edema affecting the central 1 mm of the macula ( detected with optical coherence tomography ) were included in this study . eyes with vascular retinal disorders other than diabetic retinopathy ( eg , choroidal neovascularization ) , eyes that received previous intravitreal injection of any agents , eyes with previous intraocular surgery or with previous laser treatment were excluded from the study . eyes with bcva < 0.1 or > 0.25 were excluded ; also eyes with edema not affecting the central 1 mm were excluded from the study . complete ophthalmic examination was done for all patients including measurement of bcva , iop measurement , slit lamp examination , and dilated fundus examination . macular examination with heidelberg spectralis - oct ( heidelberg engineering , heidelberg , germany ) was done for all eyes . macular thickness map with follow - up software was used to detect and measure the changes in central macular thickness ( cmt ) . the anti - vegf agents were injected into the eyes in both the groups at baseline and then every 1 month for three intravitreal injections as a loading dose . anti - vegf was re - injected if the macular edema was persistent or if the visual acuity or the cmt worsened . all eyes in group i received an injection of 2 mg/0.05 ml aflibercept ( eylea ; regeneron pharmaceuticals , ny , usa ) and those in group ii received an injection of 0.5 mg/0.1 ml ranibizumab ( lucentis ; genentech , usa , inc . after disinfection , draping , and proper topical anesthesia , the anti - vegf agent was injected into the vitreous cavity . a 27-gauge needle was used in intravitreal injection , which was done at 4 mm from the limbs if the patient was phakic and at 3.5 mm if the patient was pseudophakic . evaluation of vision was done at the end of the maneuver to ensure that the intraocular pressure was not excessively high . all eyes were examined monthly for 12 months after the last injection of the loading dose . the number of re - injections ( not including the loading dose injections ) was calculated and reported in each group . the collected data were analyzed statistically using non - paired t - test in statistical package for the social sciences ( spss ) ( ibm spss statistics 14.0 ; chicago , il , usa ) . the 1st , 3rd , 6th , and 12th month follow - up visits were the benchmark visits , and the results of examination at these visits were used in the tables and figures to make results easier to comprehend . patients with type i or ii diabetes , dme in eyes as diagnosed clinically and with oct , patients with best corrected visual acuity ( bcva ) ranged from 0.1 to 0.25 ( moderate visual loss ) and with edema affecting the central 1 mm of the macula ( detected with optical coherence tomography ) were included in this study . eyes with vascular retinal disorders other than diabetic retinopathy ( eg , choroidal neovascularization ) , eyes that received previous intravitreal injection of any agents , eyes with previous intraocular surgery or with previous laser treatment were excluded from the study . eyes with bcva < 0.1 or > 0.25 were excluded ; also eyes with edema not affecting the central 1 mm were excluded from the study . complete ophthalmic examination was done for all patients including measurement of bcva , iop measurement , slit lamp examination , and dilated fundus examination . macular examination with heidelberg spectralis - oct ( heidelberg engineering , heidelberg , germany ) was done for all eyes . macular thickness map with follow - up software was used to detect and measure the changes in central macular thickness ( cmt ) . the anti - vegf agents were injected into the eyes in both the groups at baseline and then every 1 month for three intravitreal injections as a loading dose . anti - vegf was re - injected if the macular edema was persistent or if the visual acuity or the cmt worsened . all eyes in group i received an injection of 2 mg/0.05 ml aflibercept ( eylea ; regeneron pharmaceuticals , ny , usa ) and those in group ii received an injection of 0.5 mg/0.1 ml ranibizumab ( lucentis ; genentech , usa , inc . after disinfection , draping , and proper topical anesthesia , the anti - vegf agent was injected into the vitreous cavity . a 27-gauge needle was used in intravitreal injection , which was done at 4 mm from the limbs if the patient was phakic and at 3.5 mm if the patient was pseudophakic . evaluation of vision was done at the end of the maneuver to ensure that the intraocular pressure was not excessively high . all eyes were examined monthly for 12 months after the last injection of the loading dose . the number of re - injections ( not including the loading dose injections ) was calculated and reported in each group . the collected data were analyzed statistically using non - paired t - test in statistical package for the social sciences ( spss ) ( ibm spss statistics 14.0 ; chicago , il , usa ) . p<0.05 was considered statistically significant . the 1st , 3rd , 6th , and 12th month follow - up visits were the benchmark visits , and the results of examination at these visits were used in the tables and figures to make results easier to comprehend . a total of 70 eyes of 42 patients received intraocular injections of aflibercept ( group i , 35 eyes ) or intraocular injections of ranibizumab ( group ii , 35 eyes ) . the mean age of the patients was 55.054.7 years ( range : 4565 years ) in aflibercept group and 56.645.8 years ( range : 4268 years ) in ranibizumab group . the mean baseline bcva and cmt of eyes treated with aflibercept were 0.170.05 m and 465.2933.7 m and of eyes treated with ranibizumab were 0.180.04 m and 471.534.4 m , respectively . the bcva was recorded monthly for 1 year after the last loading injection . at the end of the follow - up period , the mean bcva in eyes treated with aflibercept improved to 0.420.28 and that in eyes treated with ranibizumab improved to 0.370.23 with no significant difference between the two groups ( p=0.27 ) ( table 1 and figure 1 ) . at the end of follow - up period , eyes in aflibercept group ( 35 eyes ) showed improvement of vision in 22 eyes ( 62.9% ) , vision was worsened in 7 eyes ( 20% ) , and remained stationary in 6 eyes ( 17.1% ) . eyes in ranibizumab group ( 35 eyes ) showed vision improvement in 20 eyes ( 57.1% ) , worsening vision in 7 eyes ( 20% ) , and stationary vision in 8 eyes ( 22.9% ) ( figure 2 ) . the cmt was measured at every visit during the follow - up period . at the end of the follow - up period , the mean cmt in eyes of group i ( aflibercept ) decreased to 360.885.7 m , whereas in eyes of group ii ( ranibizumab ) , the mean cmt decreased to 387.387.8 m with no significant difference between the two groups ( p=0.2 ) ( table 2 and figure 3 ) . the number of dry eyes ( with no macular edema ) at the end of the study was 25 ( 71% ) in the aflibercept group and 23 ( 66% ) in the ranibizumab group . the drugs were injected into the study eyes at baseline and then every 1 month until the 3rd month ( loading dose of three injections ) . during the follow - up period , the drug re - injection was considered on monthly basis if the macular edema persisted or worsened in comparison with the preceding visit and if visual acuity worsened in comparison with the preceding visit . the treatment was withheld if there was no change of macular thickness or visual acuity for two successive visits but was reinstated once vision or macular edema worsened again . improvement or worsening of macular edema was defined as a 10% change of cmt in comparison with last visit while 0.1 change of visual acuity in comparison with last visit was considered a significant change . the number of injections in group i ( aflibercept ) ranged from 2 to 5 times with an average of 2.620.68 , whereas for eyes in group ii , the number of ranibizumab re - injections ranged from 2 to 6 times with an average of 3.030.95 . no serious complications such as retinal detachment or endophthalmitis were detected in any patient of both the groups during the follow - up period . dme is caused by excessive retinal vascular permeability , resulting in increase in retinal thickness with leakage of fluid and plasma constituents.16 vegf is associated with the breakdown of the blood retinal barrier , which may be the cause of increased retinal vascular permeability that results in retinal and macular edema . many studies have found elevated levels of vegf in ocular fluids of patients with diabetic retinopathy and in eyes with dme.17,18 the development of vegf inhibitors ( anti - vegf ) has revolutionized dme treatment with an improvement in visual acuity.19 although ranibizumab is a fab fragment of a monoclonal anti - vegf , aflibercept is a recent anti - vegf agent made of a recombinant fusion protein that binds both vegf isoforms a and b , and placental growth factor.20 this study compares intravitreal aflibercept and ranibizumab in the treatment of sme in eyes with moderate visual loss . aflibercept has a longer half - life in eye than ranibizumab , and it binds vegf and placental growth factor as well . this may explain the lesser treatment burden of intravitreal aflibercept recorded in this study . in this study , only one eye in the aflibercept group and none in the ranibizumab group had no injections in the first 3 months after the initial three loading doses . this may suggest that a loading dose of three intravitreal injections may not be enough in those eyes . in protocol t,15 there was no loading doses , and the treatment was given monthly as needed from the start , but the number of injections in the first 6 months was comparable . many studies proved the efficacy and safety of ranibizumab intravitreal injection in the treatment of dme.21,22 wells et al15 studied the effect of aflibercept , bevacizumab , and ranibizumab for dme . when the initial visual acuity loss was mild , there were no apparent differences , on average , among the study groups . at worse levels of initial visual acuity , the baseline visual acuity was between 0.25 and 0.1 , which is a common presenting visual acuity in parts of the world without a tight diabetic retinopathy screening program . in those eyes , other studies15,23 showed that aflibercept was superior to bevacizumab and ranibizumab regrading visual gain . at the end of the follow - up period , this study failed to show significant difference in bcva between aflibercept and ranibizumab ( p=0.27 ) but showed less treatment burden with aflibercept . the baseline cmt in both the groups did not differ significantly , and the results indicated that both aflibercept and ranibizumab showed the same degree of cmt decrease at the end of the follow - up time ( p=0.2 ) . all eyes in this study were injected with anti - vegf agents with the loading dose of three injections ( with 1 month interval ) , then during the follow - up period , the anti - vegf was re - injected when the macular edema was still present . the mean number of aflibercept was 2.620.68 and that of ranibizumab was 3.030.95 with significant difference between the two drugs ( p=0.02 ) . both aflibercept and ranibizumab improve visual acuity and decrease cmt in eyes with dme and moderate visual loss with no difference between the two drugs , whereas aflibercept needs a number of re - injections less than that of ranibizumab ( 2.620.68 versus 3.030.95 ) . all procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 helsinki declaration and its later amendments or comparable ethical standards .

purposethe purpose of this study was to compare the efficacy of intravitreal aflibercept and ranibizumab in the treatment of diabetic macular edema ( dme ) in eyes with moderate visual loss.patients and methodsthis study is a randomized prospective study . seventy eyes with dme were divided into two groups ( each containing 35 eyes ) . eyes in group i were treated with intravitreal injection of 2 mg/0.05 ml aflibercept and eyes in group ii were treated with intravitreal injection of 0.5 mg/0.1 ml ranibizumab . all the eyes had three successive injections as a loading dose ( with 1 month interval ) , and then the patients were followed up monthly for 12 months . the outcomes of the study were visual acuity , central macular thickness ( cmt ) , and the number of re - injections of the drug.resultsmean age of the patients in group i was 55.054.7 years and in group ii was 56.645.8 years ( p=0.17 ) . the mean baseline best corrected visual acuity ( bcva ) of eyes treated with aflibercept was 0.170.05 and with ranibizumab was 0.180.04 ( p=0.9 ) . bcva was improved in both the groups at the end of the follow - up period and was found to be 0.420.28 and 0.370.23 , respectively ( p=0.27 ) . the mean baseline cmt of eyes in group i was 465.2933.7 m and in group ii was 471.534.4 m ( p=0.65 ) . cmt decreased in both the groups to 360.885.7 m and 387.387.8 m , respectively ( p=0.2 ) . the mean number of drug re - injection was 2.620.68 and 3.030.95 in both the groups , respectively ( p=0.02).conclusionaflibercept and ranibizumab have the same efficacy in the treatment of dme in eyes with moderate visual loss but with less number of drug re - injection and less treatment burden with aflibercept ( 2.620.68 versus 3.030.95 ) .

the avermectins and milbemycins , which are part of the macrocyclic lactones , are potent pesticides for cow , sheep , pigs , and horses . ivermectin ( ivm ) , doramectin ( dor ) , abamectin ( aba ) , and eprinomectin ( epm ) belong to the avermectin group , whereas mox is one of the milbemycins and derived from the fungus streptomyces cyanogriseus . ivermectin ( ivm ) and moxidectin ( mox ) are most commonly used for animal husbandry , whereas dor and aba are not allowed in lactating animals [ 2 , 3 ] . mox is a potent and persistent anthelmintic , whilst oral formulations of ivm for sheep are generally less potent and persistent anthelmintics . mox is marketed as topical ( pour - on ) formulations for use in beef and dairy cattle . some studies have shown that cattle treated with mox topical pour - on achieved an advantage over the other endectocide compounds in attaining a high and persistent level of nematode control . the use of mox during the lactation period has been correlated with a significant enhancement on the volume of milk produced . topical formulations of mox are currently approved for use in dairy cattle without requiring milk withdrawal time in some countries . moxidectin is active at very low doses ( 0.2 mgkg ) against a wide variety of nematode and arthropod parasites in cow and sheep . in the european union regulations , the maximum residue limit ( mrl ) value for moxidectin was 40 gkg in bovine and ovine milk , whereas for ivm no mrl in milk the fat tissues act as a reservoir , contributing to the long term persistence in the body . moxidectin is substantially excreted also in milk , with residues reaching up to 5% of the dose administered . however , the residue of mox in foodstuff samples could lead to undesirable risks of public health . it therefore becomes essential that the residue is strictly regulated from food safety point of view . the results showed that , after administration of mox ( 200 gkg ) in goats , 5.7% of the administered dose was excreted in milk for more than 35 days after dosing . thus , it is important to develop a rapid , simple , and effective method for the determination of mox in milk samples . since mox is very potent in the antiparasitic activity and the regulated effective dose is very small , the concentration of mox in milk is very low and simultaneously accompanied by a large number of interfering substances . thus , sample enrichment and purification techniques are highly required prior to chromatography or mass spectrometry analysis . protein precipitation using an organic solvent followed by solid - phase extraction ( spe ) with c18 sorbents was the strategy most commonly applied for mox analysis in foodstuff samples . however , all of these reported spe processes are time - costing because spe consists of four steps : column preparation , sample loading , column postwash , and sample desorption . in contrast , magnetic solid - phase extraction ( mspe ) has received considerable attention in recent years due to its potential applications in separation science . a unique and attractive property of this technology is that magnetic adsorbents can be isolated from sample solutions by an external magnetic field . thus , suspended magnetic particles tagged with the analytes can be removed from large volume samples in a short time . the extraction procedure can be directly performed in crude samples to avoid blockage of the traditional spe column caused by sample matrix . -cyclodextrins ( -cd ) as a novel functional monomer has attracted sufficient interest in recent years . due to the rigidity of the hydrophobic cavity , -cd unit can form a complex with the target molecules through various kinds of intermolecular interactions , such as van der waals force and hydrophobic interaction , which are helpful to obtain binding sites with high affinity . zhang 's group successfully prepared imprinted polymers with high affinity and selectivity for erythromycin as a structural analogue of mox using -cd as the monomer . in this study , magnetic / gelatin composites were prepared and modified using -cyclodextrins and acrylic acid as binary functional monomers . the extraction procedure could be carried out within 5 min without extraction instrument and centrifugation . direct contact of the composites with mox causes direct extraction . the magnetic / gelatin composites with mox are then separated from the solution by using an external magnetic field ( magnet ) . coupling with liquid chromatography - fluorescence detection ( lc - fld ) , a rapid , simple , and effective method was established for determination of mox in milk samples . gelatin ( from bovine skin , average molecular weight 80,000kd ) , acrylic acid ( aa ) , mox , sodium hydride ( nah ) , -cyclodextrin ( -cd ) , and glycidilmethacrylate ( gma ) were obtained from sigma ( st . louis , mo , usa ) . anhydrous ethanol , iron chloride tetrahydrate , ammonia solution , and n , n - dimethylformamide were purchased from kemiou chemical reagent company ( tianjin , china ) . ultrapure water was obtained from a milli - r04 purification system ( millipore , germany ) . all measurements were performed using an agilent 1200 series hplc ( palo alto , ca , usa ) equipped with a quaternary pump , an autosampler , a thermostatic cartridge compartment , and a fluorescence detector ( fld ) . -cd ( 3.0 g ) was dissolved in 100 ml of anhydrous dmf to which nah ( 0.3 g ) was added . the mixture was stirred at room temperature until no gas was released , and the excessive nah was removed by filtration . thereafter , gma ( 1.0 g ) was added and formed complexes with -cd through the coupling reaction accompanied with ring - opening process . then the mixture was magnetically stirred at 90c for 5.0 h under nitrogen protection . the complexes were obtained by washing with acetic ether several times and finally dried in a vacuum oven . briefly , gelatin ( 3 g ) was soaked in deionized water and then dissolved at 55c . after 6 h , the product was magnetically collected , washed with distilled water and ethanol several times , and finally dried in a vacuum oven . subsequently , 200 mg of magnetic gelatin was dispersed into aqueous solution and 400 l of acrylic acid ( aa ) was added subsequently . then the mixture was incubated at room temperature for 12 h. finally , magnetic gelatin , 200 mg of acryloyl--cd , and 10 mg of ammonium persulfate were gradually added and the mixture was incubated at 37c for 12 h. the magnetic composites were washed with ethanol and deionized water , respectively , and then dried at 60c for 24 h. real milk samples were collected from a local market ( wuhan , china ) . an aliquot of 5 g of sample was placed into a 50 ml polypropylene tube and homogenized after adding 2.5 ml of ultrapure water . two aliquots of 2.5 ml and one of 5 ml of acetonitrile were successively added to the samples with 10 s of mixing on a vortex between each solvent addition . after shaking for 20 min and centrifugation for 5000 rpm , subsequently , 10 mg magnetic composites were added into the extraction solution and incubated for 3 min . after magnetic separation , acetonitrile / water ( 9 : 1 , v / v ) ( 1.0 ml ) was used to remove the sample matrix . finally , magnetic composites were eluted with 0.5 ml of methanol / acetic acid ( 9 : 1 , v / v ) to remove mox from the materials . the eluting fraction was collected and evaporated to dryness under a stream of n2 . the residue was dissolved with 1 ml of acetonitrile and analyzed by lc - fld . for lc - fld direct detection , extraction solution ( 800 l ) was previously derivatized with acetic acid ( 40 l ) , trifluoroacetic anhydride ( 80 l ) , and 1-methylimidazole ( 80 l ) , at 64c in a heating block for 20 min . after this , the derivatized solution was left at room temperature for 10 min and then analyzed by lc - fld . all separations were performed on a sunfiretm c18 column ( 150 mm 4.6 mm i d , particle size 5 m , waters , milford , usa ) at a flow rate of 1.0 mlmin at room temperature . the mobile phase was acetonitrile / water solution ( 85 : 15 , v / v ) . fluorescence detector was operated at 365 nm ( excitation ) and 470 nm ( emission ) . the synthesis of magnetic composites involved preparation of magnetic gelatin , synthesis of acryloyl--cd , and modification of acryloyl--cd and acrylic acid on magnetic gelatin ( figure 1 ) . first of all , gelatin acts as an efficient adsorbent of ionic species through polar or ionic interactions because its functional groups such as -oh , -nh2 , and cooh act as binding sites . at thus , iron oxide nanoparticles were well distributed in the gelatin and then separated from a suspension system under an external magnetic field . subsequently , magnetic gelatin was dispersed into an acid solution adjusted by acrylic acid ( aa ) . at low ph solution , -nh2 groups of gelatin get protonated and the high adsorption capacity of acrylic acid can be expected by the electrostatic attraction between the carboxyl groups and the amino groups . thus , double bond was introduced onto the surface of magnetic gelatin to ensure further modification of -cd . finally , acryloyl--cd and aa ( 1/1 , w / v ) were modified on the surface of magnetic gelatin using ammonium persulfate as initiator . the obtained film was beneficial for the high adsorption of mox and then avoiding the swelling of gelatin resulted in the disintegration of magnetic gelatin . zeta potential measurements were performed to verify the modification on the surface of magnetic gelatin . it is shown that the zeta potential of magnetic gelatin in acid solution ( ph = 5.0 ) was + 10.14 mv , since the gelatin had a net positive charge on the surface at low ph . however , the zeta potential of aa modified magnetic gelatin was 0.78 mv due to the crosslinking of amino / carboxyl groups and decreasing of amino groups . the zeta potential of -cd / aa modified magnetic gelatin was 9.21 mv , due to the carboxyl groups and hydroxyl groups of functional monomers . it was shown that the -cd / aa modified films were created on the surface of magnetic gelatin . to determine the adsorption capacity of the magnetic composites for mox , the adsorption isotherm and the corresponding scatchard analysis were acquired . figure 2 shows that the amount of mox bound to the magnetic adsorbent increased when the initial concentration of mox increased . compared with magnetic gelatin , the adsorption capacity of mox on aa modified composites was lower , because the amino groups of gelatin interact with carboxyl groups of aa , which decrease the amount of functional groups . however , the adsorption capacity of mox was the highest on the -cd / aa modified magnetic composites , because of the presence of hydrophobic cavity and carboxyl groups . in the scatchard analysis , the results indicated that kd and qmax values were calculated to be 0.003 lmg and 98.7 mgg , respectively . it was shown that magnetic sorbents could be used to determine mox in real milk samples . to evaluate the applicability of magnetic composites in the separation and enrichment of mox in milk samples , firstly , different amounts of magnetic composites ranging from 1 mg to 15 mg were applied to extract mox from milk samples . the results indicated that 10 mg of composites enabled almost complete recovery of mox , and increasing the amount did not produce any significant improvement . thus , different loading solvents , including water , methanol , acetonitrile , methanol / water ( 1 : 1 , v / v ) , methanol / acetonitrile ( 1 : 1 , v / v ) , and methanol / acetic acid ( 9 : 1 , v / v ) , were used to extract 100 ngml of mox . the results are shown in figure 3 . when acetonitrile was used as the loading solvent , magnetic composites had an excellent adsorption performance for the target compound . it was also observed that methanol / acetic acid ( 9 : 1 , v / v ) was not available for extraction of the target molecule . thus , it can be used as an eluting solution in this study and the ratio of methanol / acetic acid was optimized . the results indicated that a good eluting effect was obtained when 10% of acetic acid was mixed with the methanol solution . it was shown that 0.5 ml was enough to elute mox completely from the magnetic composites . finally , extraction time profiles were conducted by increasing the vortex time from 0.5 to 5 min . it can be seen that extraction of mox could reach equilibrium when the time is 3 min . the adsorption rapidly reached equilibrium probably because of the surface modification . under the optimal protocol , milk samples spiked with mox at 1 , 10 , 50 , 100 , and 500 ngml were extracted with the magnetic composites . figure 4 shows the chromatograms obtained by the spiked sample with extraction with c18 spe , extraction with mspe , and standard solution . the results indicated that peak area of mox from the spiked samples without extraction was lower than that from the standard solution , because of matrix interference in the derivatization reaction . however , efficient cleaning and high recoveries of mox were received by mspe procedure . a simple , rapid , and low - cost analytical method based on mspe coupled with lc - fld 5 ml sample solutions and 0.5 ml eluting solution were used in the extraction procedure to obtain the low limits of detection . the results indicated that linear calibration curves were obtained in the range of 1500 ngml and the linear coefficient was 0.998 . it was performed for the evaluation of accuracy and precision . spiked milk sample in five replicates at different concentrations was analyzed in one day or in five different days . the lods , defined as the concentrations that yielded s / n ratios greater than or equal to 3 , and the loqs , defined as the concentrations that yielded s / n ratios greater than or equal to 10 , were determined through mspe coupled with lc - fld . in this study , the results showed that the lod was 0.1 ngml and the loq was 0.5 ngml . a comparative study of our developed method to other reported sample preparation procedures was performed , and the results are presented in table 1 . it can be seen that the proposed method was very simple and sensitive for the extraction of complex samples with high fat content . compared with other studies , mspe takes 3 min to extract mox ( 100 ngml ) in milk . then magnetic composites with mox are isolated with milk by using an external magnetic field ( magnet ) , without the need for centrifugation or filtration . after that , just 1.5 ml washing / eluting solvent can completely elute mox from the magnetic composites . thus , this method exhibited the advantages of easy operation , shorter extraction time , low solvent consumption , and high extraction efficiency . real milk samples collected from a local region were used to validate the feasibility and reliability of the proposed method . mox of spiked milk samples were extracted and determined using mspe coupled with lc - fld . the recoveries ( n = 5 ) were in the range of 93.8%112.5% , with rsd being not higher than 6.4% . therefore , it was shown that the proposed method was suitable for analyzing mox in real milk samples . in summary , our approach provided a simple , rapid , and economical protocol for the determination of mox in milk samples based on mspe combined with lc - fld . in this paper , -cd / aa modified magnetic composites were firstly synthesized and exhibited the advantages of easy operation , shorter extraction time , low solvent consumption , and high extraction efficiency . mox was extracted with the magnetic composites in only 5 min with only 1.5 ml eluting solvent . , a large number of real milk samples were analyzed to confirm the reliability and practicality of the proposed method , and the results revealed that the method may be a promising alternative to conventional techniques for mox in dairy products .

-cyclodextrins / acrylic acid modified magnetic gelatin was prepared and then employed as the magnetic solid - phase extraction ( mspe ) sorbent for extraction of moxidectin in milk samples . due to the rigidity of hydrophobic cavity of -cyclodextrins and carboxyl groups of acrylic acid , magnetic composites are prepared to form a complex with target molecules through various kinds of chemical reactions and then showed excellent extraction performance . this method exhibits the advantages of simplicity of implementation , short extraction time ( 5 min ) , low solvent consumption , and high extraction efficiency . a rapid , simple , and effective method for the analysis of moxidectin in milk samples was established by mspe coupled with liquid chromatography - fluorescence detection . the limit of detection was 0.1 ngml1 and the recoveries from milk samples were in the range of 93.8%112.5% . the relative standard deviation was not higher than 6.4% . in conclusion , magnetic solid - phase extraction is a simple and robust preconcentration technique that can be coupled to other analytical methods for the quantitative determination of target molecules in complex samples .

the elements of lateral movement in the sitting position are included in many sitting activities of daily living ( adl ) . lateral movement in the sitting position requires that the load - side buttock holds pelvic inclination and that the opposite - side buttock is raised from the bearing surface . these movements involve attitude adjustment of the trunk through activity of the trunk and hip girdle muscles . therefore , lateral movement in the sitting position is impaired by weakness of the trunk and hip girdle muscles1,2,3 . for example , after total hip arthroplasty or femoral neck fracture , the hip girdle muscles become weak . in addition , post - cerebral infarction hemiplegic patients have low muscle tone of the trunk and hip girdle muscles on one side . in such cases , it is observed that these patients have difficulty in improvement of adl because lateral movement from a sitting position at the time of practice does not occur . therefore , the previous study has examined the activity of the hip girdle muscles at the time of lateral movement in the sitting position using electromyography ( emg)4 , 5 . as a result , features of the hip girdle muscle and pelvic tilt on lateral movement in the sitting position suggested the following . 1 ) when the subject initiates lateral movement in the sitting position , the pelvis tilts backward by hip extension with body weight transfer to the sciatic portion of the moving side , and this movement controls the hip flexion action of the rectus femoris ( rf ) muscle on the moving side . 2 ) on the moving side , hip internal rotation of the tensor fascia latae ( tfl ) , gluteus medius ( gm ) , and gluteus maximus ( upper fibers ) muscles is aggressively involved in lateral pelvic tilt on lateral movement in the sitting position . the above mentioned movement mechanisms are related to the control of weight shift at the time of execution of lateral movement in the sitting position . however , the muscle activity on initiation of lateral movement from the start position is though not clear . as a driving force at the start of lateral movement from the rest sitting position , reverse reaction of center of pressure ( cop ) reverse reaction of cop begins when the cop is displaced to the opposite side from a state that is commensurate with the floor reaction force of the seat surface and the center of gravity . the direction of the floor reaction force changes to the moving side through this phenomenon , and the center of gravity in response to this is the driving force that causes movement toward the moving side . further , when reverse reaction of cop occurs on lateral movement in the sitting position , it reportedly allows lateral pelvic tilt on the opposite side by upper elevation of the pelvis on the movement side from the starting position8 . however , reports on emg at the time that reverse reaction of cop occurs are scattered . also , anticipatory postural adjustments ( apa ) to control trunk lateral tilt before initiating the movement have been reported9 . therefore , the kinematics before and after the start of movement , including reverse reaction of cop on lateral movement in the sitting position , remain largely unknown . in this study , cop and surface emg of the hip girdle muscles on lateral movement in the sitting position were measured , and video analysis of displacement of the trunk and pelvis during the task was also evaluated . further the study has examined the kinematic features of lateral movement in the sitting position before and after the start of motion . the study subjects consisted of 11 healthy male ( mean age : 24.8 3.7 years , height : 170.3 2.1 cm ) . sufficient explanation was provided to the subjects regarding overview and management of personal information in this study . the experiment was carried out on the subjects who agreed to participate in the study . this study was approved by the ethical review board at hachimine clinic ( no.0015 - 01 ) and the experiments were conducted in accordance with the declaration of helsinki . the electrodes , electromyography transmitter ( telemetry muscle electrocardiograph mq - air , kisseicom ) , and markers were attached to the subjects before the start of the experiment . the electrodes were attached to both sides of the tfl , gm , and rf muscles ( fig . 1fig . 1.recording muscle of surface emgshowing the electrode pasting position of each muscle ( : electrode of tfl , : electrode of gm , : electrode of rf ) ) . the negative electrode of the tfl was placed on the midpoint of the line connecting the anterior superior iliac spine and the leading edge of the greater trochanter , and the positive electrode was placed 2 cm proximal to the negative electrode . the negative electrode of the gm was placed on the proximal one - third of the line connecting the iliac crest and greater trochanter , and the positive electrode was placed 2 cm proximal to the negative electrode . the negative electrode of the rf was placed on the proximal one - third of the line connecting the anterior inferior iliac spine and the top edge of the patella , and the positive electrode was placed 2 cm proximal to the negative electrode . furthermore , electrodes were placed on both sides of the middle finger to clarify the timing of the start and end of the motor task . the markers were attached to th1 , th7 , th12 , and l4 of the spine and both upper posterior superior iliac spines ( psis ) . the measurement plate of the body sway meter ( gravity balance system jk-101 ii , unimec ) was then placed on the table , and the video camera ( handycam , sony ) for video analysis was placed on the rear side at 175 cm from the center of the measurement plate and at a height of 110 cm from the floor . the subject sat on the measurement plate with both shoulders abducted to 90 degrees ( fig . 2.schematic diagram of the experimental environmentvideo was recorded from the rear and included the head , trunk , and pelvis of the subjects . ) . recording muscle of surface emg showing the electrode pasting position of each muscle ( : electrode of tfl , : electrode of gm , : electrode of rf ) schematic diagram of the experimental environment video was recorded from the rear and included the head , trunk , and pelvis of the subjects . the provision of the motor task was for the subject to reach 15 cm to the lateral side on the goal plate , maintain the position for 1 second , and return to the rest sitting position for 1 second . the thigh and lower leg of the moving side were moved without tilting during the task , and the bottoms of both feet were kept on the ground . however , tilting of the opposite lower leg was allowed . the subjects performed the lateral reach task on both sides for a total of six times . measurement of the emg recordings was performed with vital recorder 2 software ( kisseicom ) . cop data were measured using simple measurement software of the body sway meter ( unimec ) at a sampling frequency of 20 hz . the video camera was synchronized with the emg measurement software and video data was recorded along with the surface emg . the selected analysis data were the times closest to 1 second of the lateral reach task in three measurements . emg and cop data of 22 cases were subjected to synchronization processing using the bimtas - video analysis software ( fig . 3.example of surface emg and copresults showing reach movement to the right , 500 ms before and 1,500 ms after the start of the movement . ) . the analysis interval of this study was set at 100% , from 300 ms before the start of the task to the opposite side maximum cop displacement by the reverse reaction . muscle activity start time , cop displacement , and marker displacement were examined in the analysis interval . example of surface emg and cop results showing reach movement to the right , 500 ms before and 1,500 ms after the start of the movement . the start time of muscle activity was measured from the recorded emg waveform . in this study , the start time of muscle activity referred to a report on the start reference of muscle activity10 . the measured surface emg was changed to a full - wave rectified waveform and the start time of muscle activity was the emg amplitude value exceeding the maximum amplitude value of the start position . cop analysis examined the amount of displacement from the mean values of the x - axis and y - axis respective coordinates . the mean value of the respective coordinates was calculated from the data with the 5-second hold at the start position . in the present study , the definition of cop displacement referred to the report of kudo et al6 . displacement in the constant direction of the cop occurs when the displacement of the cop has exceeded twice the maximum displacement at the start position . video analysis examined the amount of marker displacement of the second and subsequent sheet images from the marker of the first still image . in this study , for the analysis the amount of marker displacement , 1 pixel of the image was 1.3 mm . furthermore , marker displacement in a certain direction was defined as marker displacement exceeding 5 mm . the marker displacement analysis used the image - processing software image - j ( free software ) . the motor task started from between the 60% and 70% intervals in all subjects . however , each data changed from before this interval ( tables 13 ) . the values in each table represent the data at the time of arrival of each analysis interval . the emg waveform amplitude at the start of the opposite side tfl and gm muscle activity was between the 40% and 50% intervals in all subjects . in 13 cases , the opposite side tfl had continued muscle activity at the 90% interval from after the start of the task . in 15 cases , the opposite side gm had continued muscle activity at the 100% interval from after the start of the task . rf on both sides began muscle activity from just before the start of the task or after the start of the task . in 5 cases , the opposite side rf and in 6 cases , the moving side rf started muscle activity before the start of the task . in 15 cases , the opposite side rf and in 13 cases , cop of the x - coordinate was the opposite side displacement from the time that was more than the 20% interval . cop of the y - coordinate was the anterior displacement from the time that was more than the 30% interval in all subjects ( table 1table 1.amount of cop displacement [ mm]10%20%30%40%50%x coordinate0.39 0.660.6 0.660.88 0.741.39 0.982.19 1.31y coordinate0.2 1.381.09 2.831.16 2.911.3 3.091.52 3.4360%70%80%90%100%x coordinate3.26 1.674.49 2.035.7 2.246.58 2.416.84 2.5y coordinate1.82 4.032.18 4.772.63 5.433.09 5.883.62 a positive value of the x - coordinate indicates moving - side displacement and a positive value of the y - coordinate indicates anterior - side displacement . a positive value of the x - coordinate indicates moving - side displacement and a positive value of the y - coordinate indicates anterior - side displacement . the y - coordinate shows anterior displacement from the 30% interval . th1 and th7 marker displacement on the moving side was between the 50% and 60% intervals in all subjects . th12 marker displacement on the moving side was from the time that exceeded the 60% interval . l4 and bilateral psis marker was displacement to moving side from the time that exceeded the 80% interval ( table 2table 2.marker displacement of x - coordinates [ mm]10%20%30%40%50%th100001.73 2.22th70000.13 0.311.6 1.7th120000.1 0.250.47 0.95l400000moving side psis000.2 0.080.33 0.530.2 0.69opposite side psis000.000.2 0.20.2 0.3360%70%80%90%100%th18.02 4.7716.98 7.6230.87 10.1845.6 11.863.1 13.02th76.67 4.4215.07 7.2227.67 10.1541.27 12.0957.6 13.97th123.2 3.378.47 5.3517.27 8.428.1 10.6642.2 12.76l40.4 1.371.73 5.1714.53 6.1opposite side psis0.13 1.331.2 3.144.2 3.2110.2 5.1514.93 7.17values are mean standard deviation . the thoracic spine showed moving - side displacement from the time that was more than the 50% interval . the lumbar spine and pelvis showed moving - side displacement from the 80% interval . ) . moreover , psis marker was displacement to upward from the 80% interval ( table 3table 3.marker displacement of y - coordinates [ mm]10%20%30%40%50%th100000.13 0.33th700000.07 0.24th1200000l400000moving side psis00000opposite side psis000.000.0318960.07 0.2460%70%80%90%100%th10.07 0.610.13 0.940.13 1.160.4 1.320.8 1.51th70.07 0.430.13 0.60.27 0.660.93 1.171.13 1.58th120.33 0.580.73 0.680.8 1.011.4 1.361.67 1.8l40.07 0.940.2 1.730.93 2.10.53 2.260.47 4.12moving side psis0.07 0.240.1 0.490.2 0.880.21 1.120.64 1.69opposite side psis0.6 1.221.47 2.123.47 2.776.27 3.379.73 3.9values are mean standard deviation . the psis of the opposite side from when it exceeded the 80% interval showed upward displacement . ) . the thoracic spine showed moving - side displacement from the time that was more than the 50% interval . the lumbar spine and pelvis showed moving - side displacement from the 80% interval . the psis of the opposite side from when it exceeded the 80% interval showed upward displacement . opposite - side tfl and gme and bilateral rf confirmed the start of muscle activity in the analysis section . the study discusses the action of each muscle before or after the start of lateral movement in the sitting position as follows . opposite side tfl and gme showed muscle activity starting from when it exceeded the 40% interval . this result is consistent with cop being largely displaced to the opposite side ( table 1 ) . in addition , in more than half of the cases , there was continuity in muscle activity even after the start of the task . this also is consistent with continued cop displacement to the opposite side after the start of the task . from these results , it is considered that the tfl and gm on the opposite side were related to the occurrence of the reverse reaction of cop . in a previous study , it was reported that tfl and gme in the sitting position had actions of medial rotation of the hip joint4 , 5 , 11 . in other words , the study considered that the pelvis was displaced to the opposite side by medial hip rotation of the tfl and gm on the opposite side . this is in agreement with report of fujisawa et al8 . who stated that the pelvis was tilted to the opposite side in the reverse reaction of cop . while opposite side displacement of the pelvis could not be confirmed within the provisions of this present study , coordinate values of the pelvic markers showed a slight tendency toward opposite side displacement ( table 2 ) . therefore , this study considered that the pelvis was displaced to the opposite side by medial hip rotation of the tfl and gme on the opposite side in the reverse reaction of cop . forward pelvic tilt is reportedly decreased in the sitting position compared to the standing position12 , 13 . also in our previous studies , cop was confirmed to be displaced to the posterior side in accordance with displacement of the movement side during lateral movement in the sitting position5 . when the ischial tuberosity of one side supports the load in the sitting position , the backward pelvic tilt is considered to increase . therefore , it was speculated that holding the pelvis in a forward tilt direction is necessary for smooth lateral movement in the sitting position . therefore , the present study considered that the hip flexion action of rf on both sides was related to control of backward pelvic tilt by hip extension in accordance with lateral movement in the sitting position . the start time of muscle activity of rf on both sides was more often after rather than before the start of the task . furthermore , the anterior displacement of cop showed a tendency toward increased displacement after the start of the task . therefore , it was considered that hip flexion was involved in the anterior displacement of cop . cop of the x - coordinate confirmed opposite side displacement from the time of more than the 20% interval . cop of the y - coordinate confirmed anterior displacement from the time of more than the 30% interval . the study discusses the cop displacement direction of each coordinate as follows . from the results of the cop displacement of the x - coordinate , it was considered that the subjects transferred the pelvic load of the opposite side from the sitting position just before the start of the task . furthermore , cop displacement of the opposite side of the x - coordinate increased even after the start of the task . this indicated that subjects held the pelvic load of the opposite side even after the start of the task . this result of the reverse reaction of cop on lateral movement in the sitting position was similar to other studies6,7,8 . the reverse reaction of cop occurred on lateral movement in the sitting position , the reason for which will be described later together with consideration of marker displacement in the next section . as previously mentioned , it is easy to tilt the pelvis backward in the sitting position , and our previous study confirmed posterior side displacement of cop on lateral movement in the sitting position . the study considered that anterior displacement of cop in the sitting position was due to forward pelvic tilt by aggressive hip flexion motion , when transferring load to the opposite side of the pelvis , it was also considered that the subject avoided backward displacement of cop with backward pelvic tilting . it is thought that this phenomenon is to facilitate load transfer from the opposite side to the moving side . on checking each marker s displacement time , the th1 , th7 , and th12 markers were displaced to the moving side in the 60% interval from the 50% interval just before the start of the task . the l4 and bilateral psis markers were displaced to the moving side when exceeding the 80% interval . furthermore , the psis marker of the opposite side confirmed upward displacement when it exceeded the 80% interval . the marker displacement of the thoracic portion indicated that lateral movement in the sitting position was initiated by lateral flexion of the thoracic spine . in addition , marker displacement of l4 and the pelvis indicated that these markers began moving - side displacement by lateral pelvic tilt when it exceeded the 80% interval . the results of the lumbar and pelvic marker displacement are similar to that for cop of the x - coordinate with continued opposite displacement . in other words , it was considered that the lumbar and pelvic parts maintained the start position even after the start of the task . from the results of the cop displacement of the x - coordinate and the displacement of each marker , further the role of the reverse reaction of cop is as follows . there are two hypotheses for the role of the reverse reaction of cop on lateral movement in the sitting position . one is the theory of a mechanism to stabilize the postural upset caused by the start of lateral movement in the sitting position6 , 7 . the other is the theory of driving the pelvis and trunk by the ground reaction force for starting lateral movement in the sitting position9 . however , in this present study , the lumbar and pelvic markers were displaced to the moving side later than the thoracic markers . furthermore , cop maintained opposite side and anterior displacement even after the start of the task . if the reverse reaction of cop drives the pelvis and trunk in order to start the task , it was thought that the pelvis and trunk displace to the moving side by the ground reaction force generated through the pelvis almost simultaneously . for this reason , it is considered that the reverse reaction of cop is a mechanism to adjust the attitude at the start of the task . lateral movement in the sitting position begins by the thoracic portion succumbing to the moving side . the lumbar portion and pelvis hold the load to the opposite side in order to control for trunk tilt to the moving side at that time . therefore , moving side displacement of the lumbar spine and pelvis is slower than the thoracic portion . from the results of the present study , to encourage the lateral tilt of the pelvis for causing the lateral movement in the sitting position , it is necessary to consider the following points . when the before or after the start of lateral movement in the sitting position , there is a need for the opposite side tilt of the pelvis to control the movement side slope of the trunk . this phenomenon is involved the medial rotation action of hip girdle muscles of opposite . the kinematic interpretation of lateral movement in the sitting position must be considered the motion of the hip joint with the flexed position and the understanding of the shape of the pelvis . therefore , it is necessary to repeated measure the more data in the future .

[ purpose ] this study aimed to evaluate the kinematic characteristics at the start of lateral movement in the sitting position , for application in physical therapy . [ subjects and methods ] eleven healthy male subjects ( mean age , 24.8 3.7 years ) were included in the study after they provided informed consent . the electromyographic activities of the tensor fascia lata , gluteus medius , and rectus femoris , and the center of pressure ( cop ) displacement during lateral reach in the sitting position were measured . the task was recorded on video for analysis . [ results ] in almost all subjects , before the beginning of the task , the electromyographic activity in the opposite side of each studied muscle was recorded , and the opposite and anterior displacement of the cop was observed . the video analysis revealed that all subjects showed lateral displacement of the thoracic part of the trunk after the start of the task . however , the lumbar region and pelvis maintained their starting positions . [ conclusion ] cop displacement occurred in the reverse reaction before the task , and this involved the hip girdle muscles of the opposite side . a reverse reaction displaced the pelvis to the opposite side to ensure instability of posture through side tilting of the trunk at the beginning of the task .

keratoconus is a bilateral , progressive , noninflammatory disease of the cornea which often leads to high myopia and astigmatism with an estimated prevalence of approximately 1 in 2000 . in the general population , the incidence of keratoconus is estimated to be between 50 and 230 per 100,000 [ 24 ] . it seems to be a multifactorial disease with an unknown exact etiology which impairs the quantity and quality of vision secondary to thinning in and protrusion of the cornea . this results in an irregular astigmatism with or without myopia [ 57 ] . despite the fact that only one eye may be affected initially the conservative management of keratoconus in early stages consists of spectacle correction or rigid contact lenses . in more advanced stages with severe corneal irregular astigmatism and stromal opacities , surgical treatment with deep lamellar keratoplasty and penetrating keratoplasty ( pk ) intrastromal corneal ring segments ( icrss ) represent a substantial evolution in the management of keratoconus . moreover , long - term data on icrs procedures demonstrated promising results in topographic regularity and uncorrected visual acuity ( ucva ) , indicating the possibility of putting back or even replacing keratoplasty in keratoconus patients [ 14 , 15 ] . different brands of icrss are currently on the market , including intacs ( addition technology , inc . ) , ferrara ( ferrara ophthalmics ltd . ) , and keraring ( mediphacos ltd . ) . kerarings are made of medical grade polymethyl methacrylate ( pmma ) with a uv blocker . they are characterized by a triangular cross section with variable thickness and an arc length that induces a flattening effect on the cornea . keraring 355 intrastromal corneal ring ( icr ; mediphacos , minas gerais , brazil ) is a new unique intracorneal ring design especially developed for a nipple type keratoconus . it is available in a diameter of 5.7 mm and a thickness range of 200 and 300 m . to our knowledge , there are no reports on the effect of insertion or implantation of keraring 355 on the postoperative outcome . to investigate the short - term visual and refractive outcomes after implantation of keraring 355 , we conducted the current study in which all eyes had a 6-month follow - up . this prospective , consecutive , interventional study included 15 eyes from 15 patients ( 12 men , 3 women ) with a mean age of 28.87 6.94 years ( range 21 to 49 years ) with keratoconus . it was approved by the institutional review board of the eye research center , bina eye hospital , and followed the tenets of the declaration of helsinki . after fully explaining the purpose and procedures of the study , all patients were asked to sign an informed consent form before treatment . inclusion criteria were nipple type keratoconic eyes with clear central cornea , age between 21 and 49 years , minimum corneal thickness of 360 microns , mean keratometry between 45 and 52 d , contact lens intolerance , an uncorrected visual acuity ( ucva ) not better than 20/50 , and no visual dysfunctions other than keratoconus . exclusion criteria were positive pregnancy test , breast - feeding , history of vernal and atopic keratoconjunctivitis , history of keratorefractive surgery on the operative eye , patients with dry eye , history of corneal stromal disorders , nystagmus , immunosuppressive drugs users , hyperopia , advanced keratoconus with inferior corneal thinning less than 360 m , and patients with severe ocular and systemic pathologies ( e.g. , history of herpes keratitis , diagnosed autoimmune disease , systemic connective tissue disease , glaucoma , cataract , diabetic retinopathy , and age - related macular degeneration ) . a complete ophthalmic examination was performed preoperatively and postoperatively , including uncorrected visual acuity ( ucva ) , best spectacle - corrected visual acuity ( bscva ) , manifest refraction , spherical equivalent ( se ) , keratometry ( k ) readings , and ultrasound pachymetry . corneal topography was measured using the orbscan ii slit scanning corneal topography / pachymetry system ( orbscan ii , bausch & lomb ) . visual acuity was measured using snellen notation and then converted to logmar for statistical analysis . diagnosis of keratoconus was established by the combination of computerized video keratography of the anterior and posterior corneal surfaces ( orbscan iiz ) , k readings , and corneal pachymetry [ 16 , 17 ] . the safety of implantation of keraring 355 in patients with keratoconus was assessed using a refractive surgery safety index ( safety index = postoperative best - corrected visual acuity preoperative best - corrected visual acuity ) . efficacy was assessed using a refractive surgery efficacy index ( efficacy index = postoperative uncorrected visual acuity preoperative best - corrected visual acuity ) [ 18 , 19 ] . we asked every patient about their overall satisfaction with icrs implantation after three years based on a six - point likert scale ( ( 0 ) no satisfaction ; ( 1 ) very little satisfaction ; ( 2 ) little satisfaction ; ( 3 ) moderate satisfaction ; ( 4 ) high satisfaction ; ( 5 ) very high satisfaction ) . all surgical procedures were performed by the same experienced surgeon ( khosrow jadidi ) in an operating room under topical anesthesia with proparacaine hydrochloride 0.5% ( alcaine , alcon ) drops . in order to mark the central point of intrastromal corneal ring implantation , the surgical procedure included creation of a pocket within the corneal stroma of 8.5 mm in diameter at 300-micron depth using a pocketmaker microkeratome ( dioptex gmbh ) as described elsewhere [ 28 , 38 , 39 ] with a minor modification : when correct position of the blade was determined , the microvibrating diamond blade was set at 300 m of the measured corneal thickness and a single 2 mm radial incision was made at the steepest meridian . the suction ring was removed from the eye after creating a closed intrastromal pocket of 8.5 mm diameter and 300 m depth through the small incision tunnel . the keraring 355 segment was inserted at the steepest meridian into the circular channel via the notch , using implantation forceps . the appropriate keraring 355 segment thickness was selected and then implanted in the eye according to the new nomogram designed based on the author 's experiences ( table 1 and figure 1 ) . subsequently , a silicone - hydrogel bandage contact lens ( bausch & lomb ) was placed on the cornea . postoperatively , patients were prescribed betamethasone drops ( sina darou ) four times a day , chloramphenicol drops ( sina darou ) four times a day , and nonpreserved artificial tears ( artelac , bausch & lomb , france ) six times per day . chloramphenicol drops were discontinued one week postoperatively , but betamethasone drops were tapered after four to six weeks . patients were then scheduled for postoperative clinical examinations at one month and three and six months . continuous variables with normal distribution are presented as mean sd . the paired t - test was used to compare preoperative and postoperative values of ucva , bscva , se , kmax , kmin , and kmean . the difference as a function of time was analyzed using paired two - tailed t - tests ( at time intervals before operation to three months , before operation to six months , and three to six months of the follow - up period ) . the mean age of the patients was 28.87 6.94 years ( range 21 to 49 years ) , and the male / female ratios were 4 : 1 ( table 2 ) . the mean ucva improved significantly from 0.79 0.48 logmar preoperatively to 0.28 0.15 logmar ( p = 0.001 ) six months after implantation ( figure 2 ) . the mean bscva improved to 0.18 0.9 logmar ( p = 0.009 ) at six months after implantation ( figure 2 ) . there was a significant improvement in spherical equivalent refractive error from 5.46 1.52 diopters ( d ) preoperatively to 2.01 1.63 ( p the mean k readings improved in the same period , from 48.11 1.95 d to 43.31 2.31 ( p < 0.001 ) ( figure 3 ) . our results present a significant flattening effect postoperatively , since the mean topographic k values showed decreases in kmean , kmax , and kmin at six months postoperatively ( p < 0.001 ) . the means ( standard deviation ) of all data are shown in tables 3 and 4 . postoperatively , ucva and bscva showed a significant improvement and sphere , cylinder , se , and keratometry readings were significantly reduced . likewise , on a scale of 0 to 5 for current overall satisfaction , 86.7% of patients noted that they were moderately to very happy with the results ( scores 35 ) ( table 5 ) . the purpose of the present study was to determine the effects of keraring 355 on uncorrected visual acuity ( ucva ) , best spectacle - corrected visual acuity ( bscva ) , refraction , topography , and the safety and efficacy indices in keratoconic eyes . several possible alternatives to manage keratoconus have been reported in the literature , including scleral - fitted gas - permeable contact lenses , inferior eccentric penetrating grafts , deep lamellar keratoplasty , penetrating keratoplasty , and a recently developed therapeutic tool : intrastromal corneal ring segments ( icrss ) . icrss were designed with the goal of delaying or avoiding corneal grafts in keratoconus patients . it represents a prominent evolution in the management of keratoconus via flattening the central corneal curvature to achieve a refractive adjustment due to the removable and tissue saving nature of the technique . the high efficiency of intacs in correcting keratoconic eyes has been reported by several authors [ 914 , 2330 ] . icrs implantation in post - lasik ectasia appears to be safe and effective in decreasing myopia , corneal steepness , and decentration of the corneal apex and offers potential improvement of ucva and bscva in keratoconus patients [ 9 , 20 , 22 , 24 , 25 , 3040 ] . keraring 355 is a new intracorneal ring design made of polymethyl methacrylate ( pmma ) and is especially developed for a nipple type keratoconus . mechanical dissection and femtosecond laser are the two main techniques generally used for tunnel creation during icrs implantation . the traditional mechanical technique for tunnel creation can cause complications such as epithelial defects at the keratotomy site , extension of the incision , anterior and posterior perforations , infectious keratitis , shallow placement of intrastromal corneal ring segments , decentration , asymmetric placement , persistent incisional gaping , corneal stromal edema around the incision , and stromal thinning [ 21 , 22 , 32 ] . a study by hellstedt et al demonstrated a 35% rate of postoperative complications such as corneal melt , segment movement , and exposure with the mechanical tunnel dissection method . these complications could be reduced with femtosecond laser due to the more precise localization , dimensions , diameter , depth , and width of the channel . despite this , ferrer et al . found no significant difference between the use of femtosecond laser and mechanical dissection . also , a significant improvement in cdva , udva , and k readings after icrs implantation was reported with a mechanical and femtosecond laser tunnel creation in other studies [ 4345 ] . reported improvement of cdva in 15.68% of 50 eyes , a decrease in the mean keratometry from 50.6 d to 47.5 d and the mean se from 5.6 d to 2.4 d after icrs implantation at 1 year . similarly , kubaloglu et al . compared the outcomes of keraring icrs implantation with mechanical and femtosecond laser tunnel creation and demonstrated an improvement of the udva and cdva in 86% and 88% of eyes , respectively . in addition , a decrease in the mean maximum k value from 53.5 d to 48.9 d and an improvement in the mean se from 5.05 to 1.87 d at 1 year in femtosecond laser group were found . the mean maximum k value decreased from 54.1 d to 43.8 d , and the mean se went from 5.75 to 0.75 d at 1 year in the mechanical group . in our study , we used the mechanical technique to create a pocket within the corneal stroma with the pocketmaker microkeratome ( dioptex gmbh ) . according to our experiences , thirdly , with the mechanical technique , reshaping and remodeling of the cornea are more feasible than with the femtosecond laser technique . in our cases , postoperative results revealed a significant reduction in the magnitude of corneal steepening , an increase in topographical regularity , and an improvement in the ucva and bscva when the keraring 355 was implanted at 300 m thickness ( see table 3 ) . furthermore , all operations were uneventful , and no extrusions of the rings were found . additionally , the integrity of the cornea was well preserved in all eyes . on one hand our results are in contrast to the study by kwitko and severo that demonstrated a higher rate of extrusion using standard mechanical stromal dissection for keraring implantation . on the other hand , despite the small sample of eyes ( 15 eyes ) in our study , our finding is similar to the study by shabayek and ali using femtosecond laser for keratoconus correction . they found a significantly increased ucva from 0.06 to 0.3 and bscva from 0.54 to 0.7 , and the spherical equivalent and the average keratometric values ( k value ) decreased by 2.28 diopters ( d ) and 2.24 d , respectively . the major changes in refraction and topographic findings in our series seem to take place during the early postoperative period ( the first three postoperative months ) for ucva but bscva improved after this period , contradicting the results of shabayek and ali which showed no significant difference between the 3- and 6-month follow - ups . these results were similar to the results of intacs implantation in low myopia patients [ 4648 ] , patients with keratoconus [ 9 , 2327 ] , and patients with post - lasik ectasia [ 14 , 24 , 29 ] where stability in refraction and visual acuity after the sixth month were observed . additionally , in our study , the efficacy index and the safety index were more than 1.00 at six months postoperatively , which showed the visual outcomes were satisfactory . additionally , all patients were satisfied with keraring 355 implantation and the majority of cases agreed to have an implant inserted in the other eye ( data not shown ) . we believe that this new technique with the unique and specialized characteristics of the keraring 355 may explain the reliable results in our study . our study has potential limitations , including the small sample of treated eyes , the lack of higher - order aberration analysis , and the lack of a control group . however , the results in our study are similar to those in a keratoconus study in which icrss were used for treatment [ 4345 ] . in conclusion , we have shown that icrs ( keraring 355 ) implantation using a mechanical dissection with the pocketmaker microkeratome is a unique , safe , efficient , and minimally invasive procedure in treating nipple type keratoconic eyes , and it reduces the risk of operative and postoperative complications . further studies with a longer follow - up period and a larger number of patients are recommended to draw final conclusions about the efficacy and safety of icrss ( keraring 355 ) and their role in controlling the progression of keratoconus .

we evaluate the efficacy and safety of keraring 355 intrastromal corneal ring segment ( icrs ) implantation aided by pocketmaker microkeratome for the correction of keratoconus . patients underwent icrs insertion using mechanical dissection with pocketmaker microkeratome and completed 6 months of follow - up . uncorrected visual acuity ( ucva ) , best spectacle - corrected visual acuity ( bscva ) , refraction , topographic findings , safety , efficacy index , and adverse events were reported for six months postoperatively . we evaluated 15 eyes of 15 patients ( 12 men ) with a mean age of 28.87 6.94 years ( range 2149 years ) . at final postoperative examination , there was a statistically significant reduction in the spherical equivalent refractive error compared to preoperative measurements ( 5.46 1.52 to 2.01 1.63 d , p < 0.001 ) . mean preoperative ucva ( logmar ) before implantation was 0.79 0.48 , and postoperative ucva was 0.28 0.15 , p = 0.001 . mean preoperative bscva ( logmar ) before implantation was 0.36 0.21 ; at final follow - up examination bscva was 0.18 0.9 , p = 0.009 . mean k decreased from 48.33 to 43.31 d , p < 0.001 . all patients were satisfied with icrs implantation ; 86.7% were moderately to very happy with the results . no intraoperative or postoperative complications were demonstrated . this preliminary study shows that icrs ( keraring 355 ) implantation is an efficient , cost - effective , and minimally invasive procedure for improving visual acuity in nipple type keratoconic corneas .

the term cerebral palsy ( cp ) refers to permanent , mutable motor development disorders stemming from a primary brain lesion that causes secondary musculoskeletal disorders which limitations regarding activities of daily living1 . the prevalence of cp ranges from 1.5 to 2.5 among every 1,000 live births , with little or no difference among western countries2 . motor impairment is the main manifestation of this condition and causes difficulties for cp subjects with in the biomechanics of the body3 , 4 . thus , functional mobility ( how an individual moves in the surrounding environment in interactions with society)5 is an important physiotherapeutic goal for children with cp , as walking with or without assistance favors physical development and allows such children with cp to participate in activities6 . approximately 90% of children with cp have compromised gait performance due to excessive muscle weakness , altered joint kinematics and reduced postural reactions7 . consequently , such children have a reduced capacity to participate in games and sports with sufficient intensity to develop adequate cardiorespiratory fitness8 , 9 . indeed , exercise programs that include aerobic and muscle strengthening components have often been contraindicated for individuals with cp , as greater exertion was believed to result in an increase in muscle tone and reductions in the both the gamut of movements and overall function10 , 11 . while there is current evidence of the physiological benefits of aerobic exercise for children with cp , the influence of these benefits on functional capacity remains unkown11 . continual , intensive physical therapy is considered the gold standard in the treatment of individuals with cp , but achieves varying results . different approaches have been employed to favor selective motor control , coordinated muscle action during gait7 , 12 and physical fitness10 , 11 . recent such approaches that are currently being studied are gait training on a treadmill as and as mobility and balance training using a virtual reality system . postural control involves coordinating balance ( stability and orientation of the body ) among different body segments13 , and requiries interactions between motor control and the visual , vestibular and somatosensory systems14 . the maintenance of postural control without alteration of the support base is determined by the ability to maintain the center of body mass within the limits of stabilty15 . many patients with neurological disorders have considerable difficulty maintaining the visual , vestibular and somatosensory systems in harmony for adequate postural stability16 . a body is in equilibrium when at rest ( static balance ) or during stable movements ( dynamic balance ) . in a stable system , movements do not significantly deviate from the desired trajectory even when submitted to perturbations17 . motor impairment in children with cp exerts a negative impact on their performance of activities of daily living , which is more evident in the standing position due to their greater demand for postural control18 , 19 . the development of novel therapeutic modalities for use in combination with conventional physical rehabilitation is important for the optimization of functional capacity20 . in this context , , significant improvements have been reported following short periods of cerebral stimulation in individuals with brain lesions20 , 21 . transcranial direct current stimulation ( tdcs ) is a promising low - cost technique , that is easy to administer and well tolerated , which has minimal adverse effects22 . it is therefore important to determine the effects of novel techniques , such as tdcs and virtual reality , on the static and dynamic balance of patients with neurological disorders . the aim of the present study was to investigate the effects of a single session of tdcs over the primary motor cortex combined with virtual reality training on the balance of children with cp . a cross - sectional , randomized , placebo - controlled , double - blind clinical trial was carried out . this study received approval from the human research ethics committee of university nove de julho ( brazil ) under process number 69803/2012 and was conducted in accordance with the ethical principles established by the declaration of helsinki . the study is registered with the brazilian registry of clinical trials under process number rbr-9b5dh7 . all parents or guardians agreed to the participation of their children by signing a statement of informed consent . the study took place at the movement analysis lab , university nove de julho , sao paulo , brazil , from march 2013 to july 2014 . twenty children with cp were recruited from specialized outpatient clinics and the physical therapy clinics of the university . the following were the inclusion criteria : levels i , ii or iii of the gross motor function classification system ( gmfcs ) ; independent gait for at least 12 months ; age between four and twelve years ; and degree of comprehension compatible with the execution of the procedures . the following were the exclusion criteria : history of surgery or neurolytic block in the previous 12 months ; orthopedic deformities ; epilepsy ; metal implants in the skull or hearing aids . all children who met the eligibility criteria ( n = 12 ) were submitted to an initial evaluation and randomly allocated to an experimental group ( virtual reality training combined with active tdcs ) and control group ( virtual reality training combined with placebo tdcs ) . block randomization was used and stratified based on gmfcs level ( levels i ii or level iii ) . stabilometric analysis was performed for the evaluation of static balance . for this , a force plate ( kistler model 9286ba ) was used . this force plate , which allows the recording of oscillations in the center of pressure ( cop ) . the acquisition frequency was 100 hz , captured by four piezoelectric sensors positioned at the extremities of the force plate , at a sampling frequency of 100 hz ; the force plate s size is 400 600 mm . the data were recorded using the sway software program ( bts engineering ) and integrated and synchronized by the smart - d 140 system . the children were instructed to remain in a stand barefoot position on the force plate , with their arms alongside the body , with an unrestricted foot base , heels aligned , and to look at fixed on a point marked at a distance of one meter at the height of the glabellum ( adjusted for each child ) . children classified on level iii of the gmfcs used their normal gait - assistance device , which was positioned off the force plate . a foam mat23 measuring 40 60 5 cm was used as a proprioceptive stimulus . measurements were taken under four conditions : feet on the force plate with the eyes open ; feet on the force plate with the eyes closed ; feet on the foam mat with the eyes open ; and feet on the foam mat with the eyes closed . three 45-second measurements were taken under each condition , and the mean was used in the analysis . the order of the different conditions was randomized to avoid the possible effects of motor learning . between measurements , the participants were given a one - minute rest period in the sitting position . stabilometric evaluations were conducted in a single session prior to and immediately following the training protocol . the children first received an explanation of the procedures , then remained at rest for 20 minutes . two raters were in charge of the procedures to ensure blinding and the reliability of the results . rater 1 was in charge of placing the electrodes and the administration of tdcs ( active or placebo ) . both the children and rater 2 were blinded to the allocation to the different groups . the intervention consisted of a single session of tdcs using two sponge ( non - metallic ) electrodes ( 5 5 cm ) moistened with saline solution . the anodel electrode was positioned over the primary motor cortex , following the 1020 international electroencephalogram system , and the cathode was positioned over the supra - orbital region on the contralateral side24 . in the experimental group , a 1-ma current was applied to the primary motor cortex region for 20 minutes while the children performed the virtual reality mobilty training . the device has a knob that allows the operator to control the intensity of the current . in the first ten seconds , the stimulation was gradually increased until it reached 1 ma , and it was gradually diminished in the last ten seconds of the session . in the control group , the electrodes were positioned at the same sites and the device was switched on for 30 seconds , giving the children the initial sensation of the 1 ma current , but no stimulation was administered during the rest of the virtual reality training . mobility training with virtual reality was performed for 20 minutes with simultaneous tdcs ( active or placebo ) . the children used their habitual braces and gait - assistance devices , when necessary . the braces were placed by the physiotherapist and an assessment of the gait - assistance device was performed and adjustments were made when necessary to achieve the proper size . mobility training with virtual reality was conducted using an xbox 360 with a kinect ( motion sensor ) was used for mobility training . the your shape : fitness evolved 2012 game was selected for aerobic exercises ( walking and walking with obstacles ) . the children were instructed to stand at a distance of 2 to 3 meters in front of the motion sensor for the estimation of height and the calculation of the body mass index . training was performed in a specific room of the human movement analysis laboratory of the university , measuring 250 400 cm . a screen measuring 200 150 cm was projected on the wall and stereo speakers were used to provide adequate visual and auditory stimuli . the displacement of the center of pressure ( cop ) of the feet on the force plate in the anteroposterior ( ap ) and mediolateral ( ml ) directions was used to analyze body sway . prior to the analysis , the signals were filtered using a low - pass butterworth filter with a cutoff frequency of 10 hz . body sway was determined based on the oscillation area of the cop , displacement , mean sway velocity and oscillation frequency in the ap and ml directions . for the oscillation area ( cm ) , was estimated using principal component analysis , was used to calculate the area of the ellipse from the cop - ap and cop - ml data considered for 95% of the data25 . mean displacement ( cm ) was calculated as the sum of the distances of all the consecutive points of the cop trajectory divided by the number of points26 . sway velocity ( cm / s ) was calculated as on the total distance divided by the signal capture time27 . the displacement frequency ( hz ) was determined as the frequency of 80% of the spectral power of the cop28 . for this purpose , repeated measures manovas with the bonferroni post hoc tests was used to determine differences between the experimental and control groups considering the following factors : group ( experimental and control ) , treatment ( pre - and post - treatment ) vision ( eyes open and eyes closed ) , foot support ( ground and foam mat ) and direction of cop oscillation ( ap and ml ) . levene s test was used to determine the evenness of variance between the groups . all statistical tests were performed with using of the spss 20.0 program ( spss inc . , the analysis of the immediate effect of tdcs on oscillation area , displacement , oscillation frequency and body sway velocity in the ap and ml directions revealed a statistically significant interaction only for sway velocity ( table 1table 1.sway velocity before and after treatment in the ap and ml directions of the control and experimental groups with the eyes open ( eo ) and the eyes closed ( ec ) ( units : cm / s)foam matgroundprepostprepostcontrol groupcop - apeo12.522.5613.261.8715.973.8314.482.46ec10.831.7312.071.4511.011.6312.621.97cop - mleo10.872.4112.912.1111.674.0712.263.24ec10.473.0810.641.7911.674.0711.922.31experimental groupcop - apeo10.781.4612.911.5510.801.6613.661.99ec9.851.3113.131.589.520.4713.211.93cop - mleo8.681.3012.902.099.582.0311.962.05ec9.171.1810.931.569.582.0312.452.21mean values ( sd ) and significant differences between pre - and post - treatment are reported.p < 0.05 ; p < 0.001 : bonferroni post hoc tests ) . mean values ( sd ) and significant differences between pre - and post - treatment are reported . p < 0.05 ; p < 0.001 : bonferroni post hoc tests in the comparison of the pretreatment and post - treatment evaluations of sway velocity in the control group , significant differences were only found in the ml direction under both visual conditions ( eyes open and eyes closed ) with the foam mat as the support base and only with the eyes open when the ground was the support base . in the experimental group , significant differences in sway velocity were found only in the ml direction under both visual conditions ( eyes open and eyes closed ) when the foam mat was used and in both the ap and ml directions under both visual conditions when the floor was the support base . the present study describes the effects of a single session of tdcs combined with virtual reality mobility training on the static balance of children with cp . the literature offers studies on the effects of tdcs combined with other physiotherapy modalities for adults with neurological disorders stemming specifically from a stroke and parkinson s disease29 , as well as some studies involving patients with cp . the findings of these studies indicate the considerable potential of this form of stimulation in the treatment of neurological disorders and the investigation of changes in cortex excitability29,30,31 . short - term tdcs is reported to have short - term effects , whereas long - term administration is reported to have lasting effects on neuroplasticity30 . tdcs is a way of to modulating cortex activity to enhance and prolong the functional gains achieved in physical therapy . stimulation causes a change in dysfunctional cortex excitability allowing that physical therapy to mold the functional pattern of cortex activity through the activation of neural networks specific to the activity in question29 . according to minhas et al.32 , a current of 1 ma is adequate for children and was therefore used in the present study . the positioning of the electrodes was based on the description given by fregni et al.24 , with the anode placed over the primary motor cortex and the cathode placed over the supraorbital region . this configuration was also used in previous studies involving the same cp population conducted by grecco et al.33 , 34 and duarte et al.35 . in the present investigation , a statistically significant difference between groups was only found with regard to body sway velocity . comparing children with typical development to those with cp , gatica et al.36 found a greater body sway velocity only with the eyes open in the patients with cp , and a greater oscillation area only with the eyes closed . in the present study , sway velocity was significantly greater in the ml direction in both groups only with the eyes open when the foam mat was used as the support base . the control group exhibited greater ml oscillation on the ground only with the eyes open , and the experimental group exhibited greater ap and ml oscillations under both visual conditions ( eyes open and eyes closed ) when the ground was the support base . cherng et al.14 conducted a study involving children with typical development and those with cp using a foam mat for the proprioceptive stimulus of the feet . the children were evaluated three times under each condition ( eyes open , eyes closed and the use of a headset to alter the sensory environment ) . no statistically significant differences between the groups were found in static balance on the floor with the eyes open and with the closed . however , when the sensory environment was altered , the children with cp exhibited a reduction in static balance , with an increase in the oscillation area in comparison to the control group . in a study involving children with cp and those with typical development , donker et al.37 evaluated static balance with the eyes open , the eyes closed and real - time visual feedback of the cop . they authors found less postural control in the group with cp , and body sway was more irregular when visual feedback was provided , which confirmed the expected imbalance when one s attention is drawn to another function . rose et al.38 found that children with cp exhibited greater body sway in comparison to children with typical development with the eyes open , but found no significant differences between groups with the eyes closed . damiano et al.39 conducted a study with three groups of children : one with hemiparetic cp , one with diparetic cp , and one with typical development . they authors found greater oscillation area in the ap and ml directions in both groups of children with cp in comparison to the group with typical development , while sway velocity was only greater in the group with hemiparesis . duarte et al.35 conducted a longitudinal study of tdcs ( active and placebo ) combined with treadmill training for children with cp over 10 consecutive days , with evaluations performed before , immediately after and one month after the intervention . statistically significant reductions in ap and ml cop area were found with the eyes open and with the eyes closed in the group submitted to active tdcs , which was maintained until to the follow - up evaluation . salem et al.40 found an improvement in balance in the one - leg stance test in a pilot study involving children with delayed motor development . in another pilot study , 41 found a 7% improvement in the gross motor function measure among children with cp . tdcs studies generally have involved adults patients and have analyzed its of the effect on the balance among stroke survivors42,43,44 or sedentary individuals with obesity45 . games facilitate improvements in static and dynamic balance during functional tasks by stimulating the displacement of the center of body mass and changes in the support base46 , 47 . moreover , games are a viable option for aerobic exercise and therefore promoting physical fitness , which in line with the recommendations of the american college of sports medicine48 , 49 . practical guidelines for the use of virtual reality in the treatment of children with cp were published in february 201250 . although there are few studies involving this population , the findings demonstrate improvements in postural control , balance , upper limb function , selective motor control and gait50 . the effects of tdcs combined with virtual reality mobility training are promising with regard to improvements in balance , as demonstrated in longitudinal studies with the techniques applied separately . we believe that the results of the immediate effect of the combination of these two methods are of considerable importance to future investigations in this line of research . based on the findings of the present study , a single session of anodal tdcs over the primary motor cortex combined with virtual reality mobility training appears elicits to lead to an increase in body sway velocity in the anteroposterior and mediolateral directions on stable ground with the eyes open and the eyes closed , however , an the increase in sway velocity only occurs in the mediolateral direction with the eyes open and with the eyes closed only occurred when proprioceptive information ( foam mat foot support ) was also provided .

[ purpose ] the aim of the present study was to investigate the effects of a single session of transcranial direct current stimulation combined with virtual reality training on the balance of children with cerebral palsy . [ subjetcs and methods ] children with cerebral palsy between four and 12 years of age were randomly allocated to two groups : an experimental group which performed a single session of mobility training with virtual reality combined with active transcranial direct current stimulation ; and a control group which performed a single session of mobility training with virtual reality combined with placebo transcranial direct current stimulation . the children were evaluated before and after the training protocols . static balance ( sway area , displacement , velocity and frequency of oscillations of the center of pressure on the anteroposterior and mediolateral axes ) was evaluated using a force plate under four conditions ( 30-second measurements for each condition ) : feet on the force plate with the eyes open , and with the eyes closed ; feet on a foam mat with the eyes open , and with the eyes closed . [ results ] an increase in sway velocity was the only significant difference found . [ conclusion ] a single session of anodal transcranial direct current stimulation combined with mobility training elicited to lead to an increase in the body sway velocity of children with cerebral palsy .

most studies on the prevalence of oral mucosal lesions have been conducted in the developed world such the american , european and asian continents . since there are vast cultural , ethnic and geographical differences between the indian subcontinent and the countries on these continents , it would not be realistic to apply the results of those studies to the indian population . as stated previously , tobacco and its related products are notable risk factors for the development of pre - cancerous lesions and conditions , which may eventually lead to oral cancer . the prevalence of melanin pigmentation , various oral lesions and white lesions is higher among smokers . however , some studies do not identify smoking as a risk factor for oral lesions . the use of dentures in the elderly has also been known to act as a potential risk factor for oral lesions and the development of oral cancer as well as other characteristic lesions such as denture stomatitis and hyperplasia . the present study was carried out to verify the association between the use of tobacco and dentures and the development of oral mucosal lesions and to find the prevalence of oral lesions in a north indian population . for the study , 3,749 patients attending the department of oral medicine and radiology , jodhpur dental college general hospital , between september 2008 and december 2012 for the treatment of dental problems other than oral mucosal lesions , were selected . ethical clearance was obtained from the institutional ethical committee , jodhpur dental college general hospital and jodhpur national university . the study was designed as per the world health organization ( who ) guidelines for the conduct of oral health epidemiological studies and all diseases were diagnosed and recorded according to the who classification . the oral examination was conducted with appropriate instruments under suitable lighting . a patient who has smoked > 8 cigarettes a patient who uses either partially removable or complete dentures was included as a wearer of dentures for the study . if there was no improvement in the lesion after 15 days , a biopsy of the lesion for histopathological examination was advised . the results were evaluated using the pearson chi - square test , with yate s correction and anova tests . the study sample comprised 2035 ( 54.3% ) male and 1714 ( 45.7% ) female patients . the ages of the patients ranged from 28 years to 83 years with a mean age of 49.7 years . most of the patients ( n = 1049 ; 58.34% ) in the 28 - 37 years age group were smokers and the number of patients with either partial or complete dentures ( n = 553 ) ( non - denture wearers , n = 318 ) increased after the fourth decade [ table 2 ] . a total of 2,318 patients ( 61.8% ) were diagnosed with lesions associated with tobacco use and the wearing of dentures . the most frequently seen lesion was melanin pigmentation ( n = 1121 ; 29.9% ) followed by linea alba ( n = 835 ; 22.2% ) and frictional keratosis ( n = 746 ; 19.9% ) [ table 3 ] . melanin pigmentation was more commonly seen in males ( n = 632 ; 40.8% ) while females showed a higher prevalence of linea alba ( n = 423 ; 24.6% ) and frictional keratosis ( n = 418 ; 24.4% ) . ulcers ( n = 232 ; 6.1% ) were the most commonly seen oral lesion in patients who wore dentures , followed by hyperplasia ( n = 181 ; 4.8% ) . it was noticed that a large number of the lesions occurred on the buccal mucosa and the lips . most of the patients examined were of middle and lower socio - economic status ( > 80% ) . distribution of the sample according to age and gender distribution of the sample according to age and smoking habits and age and denture wearing status distribution of oral pathology lesion according to gender the distribution of the oral mucosal lesions according to the habit of smoking and the use of dentures is shown in table 4 . the statistical analysis showed a statistically significant relation ( p < 0.05 ) between the tobacco habit and melanin pigmentation ( p = 0.0001 ) , frictional keratosis ( p = 0.0016 ) , traumatic ulcerations ( p = 0.0342 ) and leukoplakia . similarly , in wearers of dentures a statistically significant relation ( p < 0.05 ) was found between candidiasis ( p = 0.0004 ) , traumatic ulceration ( 0.0073 ) , stomatitis ( p = 0.0116 ) and frictional keratosis ( p = 0.0000 ) . no significant relation was established between gender and melanin pigmentation ( p = 0.0012 ) and melanin pigmentation and the socio - economic status of patients ( p > 0.05 ) . however , a statistically significant relation was found between frictional keratosis and gender ( p = 0.0024 ) as well as in patients of low socio - economic status ( p < 0.05 ) . the prevalence of the various oral mucosal lesions in the present study ( 61.8% ) was similar to other studies mentioned in the literature . however , these results are much higher than the global prevalence , which is reported to be < 10% . the number of patients who have the habit of smoking is much higher ( 55.0% ) , than in similar studies with the reported number of smokers as approximately 34 - 37% . some studies had a higher percentage of patients who wore dentures , but the present study had 969 patients who wore dentures ( 25.8% ) . in certain countries , the pigmentation of the oral mucosa ranges from 70.5% to 88.4% . the current study showed melanin pigmentation in 29.9% of the patients , a much higher finding than those of salonen et al . who reported prevalence of 9.9% in 1976 and 6.6% in the 1990 study . similar to the findings of axll et al . and hedin and axll melanin pigmentation was more common in males ( 40.8% ) . however , a few other studies have reported a higher prevalence in females taking contraceptives . hedin and axll reported a statistically significant relationship between melanin pigmentation and the tobacco habit , but no statistically significant relation was established in the present study . the prevalence of leukoplakia in the current study was 6.6% , similar to the findings of other studies . this was , however , much higher than the 0.5% prevalence reported by martnez daz - canel and garca - pola vallejo . some studies link tobacco habits to leukoplakia , as in the study , which showed 13.6% of smokers afflicted with leukoplakia . while others have shown no relation between leukoplakia and smoking . lichen planus represents approximately 20% of the diagnoses in routine practice and is the most frequent non - infectious disease of the oral mucosa . the present study reported prevalence of 1.5% , a figure which is much higher than that of the previous study done in an indian population . other similar studies done in thailand and in sweden showed a higher prevalence of 3.8% and 2.4% respectively . lichen planus was more commonly seen in males in the current study , similar to the findings of bouquot and gorlin and salem . on the other hand , a few studies have shown it to be more common in females . according to the symposium on white lesions held in uppsala in 1994 , the origin of a white lesion of the oral mucosa is unlikely to be trauma . the theory of cheek biting in anxiety and stress is shown by the difference in prevalence depending on where the study is conducted . in a study in denmark , 0.5% of the patients presented with cheek or lip biting while in another study carried out in south africa the prevalence was 4.6% . the prevalence in the present study was estimated as 16.9% , a much higher figure than the studies done in american and spanish populations showed . the prevalence of frictional keratosis observed in the current study was 19.9% , which was much higher than was reported by salonen et al . . the greater frequency of keratosis seen in patients younger than 50 years , was thought to have originated from caries and fractured teeth rather than from dentures . the prevalence of pathologies of the tongue in the present study ( 11.1% ) was similar to that observed axll et al.(12,6% ) . the prevalence of geographical tongue or benign migratory tongue in patients of the present study was 3.3% , which is closer to what was seen in the swedish population . however , a much lower prevalence was seen in the united states ( 0.3% ) and in the spanish population ( 0.8% ) . the prevalence of hairy tongue , was 7.8% , a much higher figure than has been reported in similar studies . no statistically significant relation was established between this lesion and the habit of smoking or the wearing of dentures as proposed by beaven and brooks . the prevalence of traumatic ulcerations found in the present study ( 11.5% ) was higher than was estimated by salonen et al . who reported it as 8% . the prevalence of recurrent aphthous stomatitis also shows great variations in different populations owing to the differences in the methodology used for the study , socio - economic status and the professional level . the result of the present study ( 8.2% ) is higher than the 2% found in the swedish study . however , there was no indication in the present study that this entity is related to a determined socioeconomic - professional level . the prevalence is higher in females , which is in accord with other similar studies . indeed some similar studies have reported a relationship between non - denture wearers and non - smokers and the presence of aphthous . these variations may be seen as possibly due to social and ethnic differences , as seen in other asian countries . the differences in the results of the present study and the study of salonen et al . may be because in these studies , the lesion was verified when it was present and not from recurrence in the clinical history . it has been suggested , that recurrent herpes is more frequent in low social classes than in higher social classes . the values for the prevalence of candidiasis in previous studies ( < 0.5% ) were much lower than the present study ( 5.5% ) . these differences may be due to the high percentage of wearers of dentures in the present study sample . this was statistically significant , as in other similar studies . in the present study , the percentage of hyperplasia caused by the suction effect or negative pressure diagnosed in those who do not wear dentures is very similar to the findings of salonen et al . and axll et al . as indicated in similar studies , our study shows that wearers of dentures more frequently suffer from traumatic ulceration . while a few other studies have shown that frictional keratosis , denture stomatitis and hyperplasia were more commonly seen in wearers of dentures . due the presence of fibrous hyperplasia and candidiasis in a high number of denture wearers , it is essential to carry out periodic examination of the oral mucosa in these patients to establish a preventive protocol to eradicate the pathology caused by the use of dentures . the results of the present study indicate a strong association of frictional keratosis with tobacco users and wearers of dentures with the etiology of oral cancer . this should be of interest to dental clinicians since it would help in quick diagnosis and planning the treatment of oral cancer . wearers of dentures should also be recalled periodically for a check - up , so that optimum preventive measures could be taken .

aim : the aim of the present study was to determine the prevalence and the relationship of oral mucosal lesions in tobacco users and denture wearers in a north indian population.materials and methods : the study comprised 3,749 patients attending the department of oral medicine and radiology , jodhpur dental college general hospital between september 2008 and december 2012 , for the treatment of dental problems other than oral mucosal lesions . the ages ranged from 28 years to 83 years , with a mean age of 49.7 years . the results were evaluated using the pearson chi - square test , with yate s correction and anova tests.results:a total of 2,318 patients were diagnosed with lesions associated with the use tobacco and the wearing of dentures . the most frequently seen lesion was melanin pigmentation ( 29.9% ) followed by linea alba ( 22.2% ) and frictional keratosis ( 19.9% ) . ulcers ( 6.1% ) were the most common oral lesion seen in patients who wore dentures , followed by hyperplasia ( 4.8% ) . a statistically significant relation ( p < 0.05 ) was established between tobacco and melanin pigmentation , frictional keratosis , traumatic ulcerations and leukoplakia . in those who wore dentures a statistically significant relation ( p < 0.05 ) was found between candidiasis , traumatic ulceration and frictional keratosis.conclusion:the results of the present study indicate that the association of frictional keratosis with tobacco users and wearers of dentures can help in determining the diagnosis and treatment plan for oral cancer . wearers of dentures should also be recalled for periodic checks at regular intervals and optimum preventive measures implemented .

the ponto - caspian amphipod dikerogammarus villosus ( sowinsky , 1894 ) , also known as the killer shrimp , is recognized as one of the 100 worst alien species in europe . this invader has colonized most of the european main inland water bodies in less than 20 years [ 24 ] . the killer shrimp is an efficient , high trophic level predator , feeding on other amphipods and on almost all other available benthic invertebrates [ 7 , 8 ] . in addition , this species is characterised by wide ecophysiological tolerance to a number of environmental factors including water temperature , salinity and oxygen concentrations [ 912 ] as well as by very high fecundity [ 1315 ] . initial expansion of d. villosus in continental europe followed the two so - called invasion corridors for ponto - caspian fauna , associated with major rivers ( i.e. the southern corridor via danube / rhine and the central corridor via dnieper / vistula ) often referred to as invasion highways . the populations migrating via the two invasion corridors originating in different ponto - caspian watersheds are about to come into contact in poland and possibly hybridize . it has recently colonized many lakes in the alpine region and was even accidentally introduced overseas to the uk . finally , the risk of its future introduction to the north american great lakes is not negligible . the microsatellite markers will be a valuable tool in assessing the demographic processes associated with invasion of the killer shrimp from a genetic point of view . for example , they will help to identify the origin of populations in the uk and in alpine lakes as well as to assess the dynamics of the invasion process ( e.g. via the associated bottleneck or founder effect ) . such marker will also help to estimate the differentiation between invasion corridors and chances for putative hybridization in case the two populations originating in different areas of the native range ( danube vs. dnieper ) meet in poland . the three already known loci proved to be useful but additional loci are needed to answer more detailed questions . enrichment for eight microsatellite motifs [ i.e. ( ag)10 , ( ac)10 , ( aac)8 , ( agg)8 , ( acg)8 , ( aag)8 , ( acat)6 , ( atct)6 ] was based on a biotin protocol adapted from kijas et al . . the sequences were produced by pyrosequencing on a 454 gs - flx titanium apparatus ( roche diagnostics ) . both , the enrichment and the pyrosequencing were as described by malausa et al . . using the open access qdd program , the resulting 32,084 sequences were first screened for microsatellite ( minimum of five repeats ) and flanking sequences presence and then pcr primers were designed for selected sequences . from a total of 4,206 candidate sequences including microsatellites , the primer design was effective for 102 putative loci . all the steps from enrichment down to primer design were performed at genoscreen ( lille , france ) . the pcrs were carried out in a 10 l volume including 20 ng dna template , 200 nm each primer ( table 1 ) , 0.025 m of 5 labeled m13 primer ( either 700 or 800 dye ) , 5 l dreamtaq master mix ( 2x ) dna polymerase ( thermo scientific ) . the reactions were run in a biorad thermocycler with an initial denaturation step at 95 c for 3 min , followed by 35 cycles consisting of 20 s at 95 c , 45 s at 50 c and 1 min at 72 c , and a final extension step at 72 c for 2 min . the polymorphism was tested on seven individuals from five locations in europe : liman duru golu , turkey ( 41.316n ; 28.621e ) ; danube delta , ukraine ( 45.337n ; 28.955e ) ; dnieper mouth , ukraine ( 47.792n ; 35.126e ) ; grafham water , uk ( 52.292n ; 0.324w ) ; constance lake , germany ( 47.748n ; 9.137e ) . from the 33 microsatellite loci chosen for amplification , ten failed to produce readable patterns , fifteen loci were monomorphic and eight primer pairs revealed polymorphism further , the allelic diversity of the eight candidate loci was tested on 64 individuals , from the danube delta in ukraine ( dan ; n = 32 ) and from the dnieper mouth in ukraine ( dni ; n = 32 ) . these two populations may be considered as representatives of the two distinct watersheds areas in the ponto - caspian region providing starting points for the killer shrimp invasion . the allelic diversity , observed ( ho ) and expected ( he ) heterozygosities , deviations from hardy weinberg proportions as well as gametic disequilibrium and differentiation between dan and dni ( fst as estimated by weir and cockerham theta ) were estimated using the software fstat version 2.9.3.2 . presence and possible source of genotyping errors ( null allele , stuttering , short allele dominance , were checked with micro - checker version 2.2.3 . .table 1characterization of 8 polymorphic microsatellite loci for dikerogammarus villosus locusrepeat motifprimer sequence ( 5-3)genbank accession#size range ( bp ) pop n k1 k2 h o/ h e fis null dv1-f842 k(ac)7 f : caatgggtgacacatcgagagf112174170178dan:25320.120/0.2460.5170.097r : gctcggctgcttgttttatt dni:2720.185/0.3720.5080.132dv6-gql0 m(ca)7 f : acactgcctatgtttccccagf112181150190dan:20660.400/0.6050.3450.119r : aggaagcaaggatttagggc dni:3140.419/0.6540.3620.136dv11-cons108(tg)7 f : atatgtctgagagcattttgccgf112175190194dan:26330.538/0.6640.1930.068r : gtcggtaaatcgacgcat pop sampled populations ; dan danube delta in ukraine ; dni dnieper mouth in ukraine ; n number of successfully genotyped individuals ; k1 and k2 number of alleles for both populations combined and in each population respectively ; h o and h e observed and expected heterozygotes ; fis standarised genetic variance within populations at each locus ; null frequency of null allele as measured by brookfield1 method in micro - checker characterization of 8 polymorphic microsatellite loci for dikerogammarus villosus pop sampled populations ; dan danube delta in ukraine ; dni dnieper mouth in ukraine ; n number of successfully genotyped individuals ; k1 and k2 number of alleles for both populations combined and in each population respectively ; h o and h e observed and expected heterozygotes ; fis standarised genetic variance within populations at each locus ; null frequency of null allele as measured by brookfield1 method in micro - checker out of the 33 microsatellite loci chosen for testing , ten did not amplify at all , 15 were monomorphic and eight amplified successfully and revealed polymorphism . based on the 64 genotyped individuals from the danube ( dan ) and the dnieper ( dni ) populations , we obtained a mean diversity of 4.87 alleles per locus , ranging from 2 to 13 ( table 1 ) . the mean observed and expected heterozygosities were , respectively , 0.439 ( 0.0910.844 ) and 0.468 ( 0.0890.843 ) . the fstat software detected neither the gametic disequilibrium for any pair of loci , nor a deviation from the hardy weinberg proportions in any locus in any of the two populations . however , micro - checker detected sign of a null allele at dv1 in both dan and dni and at dv6 for dni only . dan and dni populations were differentiated with a significant fst value of 0.17 . although the invasion dynamics of the killer shrimp along the danube and in french rivers was assessed by wattier et al . based on the three microsatellite loci available at that time , additional loci are needed for further assessment of its expansion all over europe . the eight new loci will be highly valuable in identifying sources of introduction for the alpine lakes and for the uk , that are not directly connected to any of the invasion highways ( fig . 1 ) . the differentiation between dan and dni populations illustrates that such source populations could be relatively easily identified with a higher number of loci . moreover , these markers could help to detect possible hybridization and/or introgression between the two populations of d. villosus which may become in contact in poland .fig . 1allele frequency distribution for each locus for the dan ( black ) and dni ( grey ) populations . axis x allele size in bp , axis y frequency of alleles allele frequency distribution for each locus for the dan ( black ) and dni ( grey ) populations . axis x allele size in bp , axis y frequency of alleles finally , it is known that microsatellite markers characterized for one species may often reveal polymorphism in other closely related taxa . thus we suggest that the loci described here have potential to be amplified in species closely related to the killer shrimp such as dikerogammarus haemobaphes ( eichwald , 1841 ) and dikerogammarus bispinosus martynov , 1925 which are also invasive in european inland waters and , in case of the latter , also in the uk . .

dikerogammarus villosus is a freshwater amphipod of the ponto - caspian origin recognized as one of the 100 worst alien species in europe , having negative impact on biodiversity and functioning of the invaded aquatic ecosystems . the species has a wide ecophysiological tolerance and during the last 20 years it has rapidly spread throughout european inland waters . in consequence , it presents a major conservation management problem . we describe eight polymorphic microsatellite loci developed for d. villosus by combining a biotin - enrichment protocol and new generation 454gs - flx titanium pyrosequencing technology . when genotyped in 64 individuals from two locations , the loci exhibited a mean diversity of 4.87 alleles per locus ( 213 ) . the mean observed and expected heterozygosities were , respectively , 0.439 ( 0.0910.844 ) and 0.468 ( 0.0890.843 ) . gametic disequilibrium was not detected for any pair of loci . the microsatellite markers will be a valuable tool in assessing the demographic processes associated with invasion of the killer shrimp from a genetic point of view .

antibody deficiency may be either primary or secondary , and the most common severe primary antibody deficiency ( pad ) is common variable immunodeficiency ( cvid ) , which accounts for the majority ( 57% ) of all symptomatic primary immunodeficiency ( pid ) cases on the european society for immunodeficiencies ( esid ) registry ( http://www.esid.org ) . cvid is an important diagnosis not only because of its prevalence ( one in 25 00050 000 ) 1 , but also because of the frequent requirement for medical attention and intervention . the morbidity and need for health resources is exacerbated by a diagnostic delay of , on average , between 6 and 7 years before appropriate treatment is started 2 . in contrast to many other pid diseases , patients with cvid may be diagnosed at almost any age 2 , which has implications for screening approaches . the misconception that pid always presents in infancy in combination with the protean manifestations of cvid , the number of specialities to which patients may present and the wide range of non - infectious complications all contribute to the delay in recognition of the underlying diagnosis . this may result in recurrent infections leading to irreversible end organ damage , such as bronchiectasis . clinical strategies to reduce diagnostic delay include improved education and awareness of antibody deficiency in both primary and secondary care , patient - centred screening 3 , the use of the 10 warning signs of pid ( http://www.info4pi.org ) and using computer sorting of diagnostic codes 4 . a study into the clinical features which identify children with immunodeficiency found that of the 10 warning signs the strongest predictors were family history , intravenous antibiotics for sepsis ( neutrophil defects ) and failure to thrive ( t cell defects ) , while for b cell defects the only predictor was family history of immune deficiency 5 . laboratory approaches have involved opportunistic detection cases of antibody deficiency by cascade testing for immunoglobulins when there is a very low background in enzyme - linked immunosorbent assays ( elisas ) used for coeliac disease testing , such as the immunoglobulin ( ig)a anti - tissue transglutaminase ( ttg ) assay 6 , and also when there is a very low ige these opportunistic methods lack specificity and depend upon the clinical details on the request form , which are often incomplete or lacking . furthermore , the very successful newborn screening for severe combined immunodeficiency ( scid ) looking for t cell receptor excision circles ( trecs ) in dna extracted from guthrie spots 8 , when extended to b cell kappa - deleting recombination excision circles ( krecs ) 9 , will not detect cvid due to the adult onset and presence of b cells in the majority of patients . guthrie spots are also unsuitable for the detection of iga deficiency in neonates , as the iga at that age is of both fetal and maternal origin 10 . there still remains a major challenge in the early detection of b cell immunodeficiency and thus antibody deficiency . calculated globulin ( cg ) is derived from the difference between total protein and albumin results , and forms part of the liver function test ( lft ) profile . cg is used widely in primary and secondary care to detect high levels that may indicate haematological malignancy , such as multiple myeloma . the globulin fraction consists of alpha 1 , alpha 2 , beta 1 , beta 2 and gamma globulin fractions , and as proteins within the alpha and beta fractions can act as acute - phase proteins ; high levels of these could potentially mask a low gamma globulin ( antibody ) level . cg levels have been shown to be low in patients with antibody deficiency 11 , and as they reflect antibody levels there is an opportunity to aid clinicians in the diagnosis of a range of conditions with both high and low levels ( table 1 ) . causes of a high or low calculated globulin ebv = epstein barr virus ; hbc = hepatitis b core antigen ; cvid = common variable immunodeficiency ; tb = tuberculosis ; itu = intensive care unit . in preparation for this study , a survey of practice in biochemistry laboratories across wales and a pilot study into the interpretation of calculated globulin were undertaken . lfts are performed in very high numbers ( 19 million / year for a population of 306 million in wales ) with total protein already included . even if not tested as standard within the lft profile the inclusion of total protein is inexpensive , costing 007 , us$011 or 008 per test . cg also fulfils the accepted criteria for a screening test based on those developed originally by the world health organization ( who ) , which encompass the condition , the test and the treatment 12 . these state that the condition should be an important health problem , the natural history should be understood and there should be a detectable risk factor , disease marker , recognizable latent or early symptomatic stage . the test should be simple , safe , precise and validated with a defined cut - off level . the test should be acceptable to the population and there should be an agreed policy on the further investigation of individuals with a positive result . there should be an effective treatment or intervention for patients identified through early detection , with evidence of early , rather than late , treatment leading to better outcomes . in the setting of pid , such as cvid , immunoglobulin replacement remains the mainstay of treatment , reduces infection frequency and severity and increases life expectancy . late diagnosis and delayed institution of immunoglobulin replacement therapy results in increased morbidity and mortality 15,16 . there is now a large range of immunoglobulin treatment options for patients comprising intravenous ( ivig ) , subcutaneous ( scig ) weekly or biweekly , facilitated subcutaneous ( fscig ) and rapid push 13,1719 made possible by improvements in immunoglobulin manufacturing , resulting in more concentrated products , faster infusion rates using ivig and for fscig the development of recombinant human hyaluronidase ( rhuph20 ) 19,20 . a survey was carried out via the wales biochemistry audit group using a questionnaire to determine current practice with respect to the use of calculated globulin ( cg ) across the 13 biochemistry laboratories in wales . an analysis of anonymized adult ( > 16 years ) samples with a range of cg levels from 15 to 22 g / l was undertaken for 50 samples at each level of cg ( abbott diagnostics , maidenhead , uk ) using the colorimetric bromocresol green ( bcg ) method for albumin and the architect biuret method ( 16200 abbott analyzer ; abbott diagnostics ) for total protein ( tp ) . immunoglobulin levels were then measured by nephelometry ( siemens bn2 nephelometer ; siemens , camberley , uk ) to confirm the relationship between cg and igg and to identify an appropriate cut - off value for subsequent study . based on the results of the pilot study , ethical and research and development committee approval was obtained for a more detailed study of samples having cg levels of < 18 g / l from nine participating hospitals in wales [ university hospital of wales ( uhw ) , university hospital llandough , velindre hospital , royal gwent hospital , neville hall hospital , caerphilly miners hospital , bronglais hospital , west wales general hospital and prince charles hospital ] . samples collected from adults ( age > 16 years ) during a period of 1 year were anonymized and sent to uhw for assay of immunoglobulin levels ( igg , iga and igm ) ( siemens bn2 nephelometer ; siemens ) , serum electrophoresis ( sebia capillarys 2 ; sebia , norcross , ga , usa ) and , where appropriate , serum immunofixation ( sebia hydrasys ; sebia ) . the laboratory and clinical data were analysed using microsoft excel and graphpad prism version 6 . duplicate samples were identified through the patient initials , sex , age , source or location of request and clinical details , and removed from further analysis . the initial presence of duplicate samples , however , resulted in improved capture of clinical details . the results were presented to the biochemistry department at uhw and reporting practice audited 1 year later . following a subsequent change in the measurement method of albumin from bcg ( abbott architect ) to bromocresol purple ( bcp ) ( abbott architect ) in our centre , a further study was performed to identify the method - specific cg cut - off using 25 anonymized samples for each cg level from 19 g / l to 26 g / l , as there was predicted to be a change in the albumin value between the methods . results were analysed using the analyse it method validation edition in microsoft excel and receiver operating characteristic ( roc ) curves used to identify the most appropriate cut - off for the bcp method . the survey of practice with respect to use and interpretation of cg results was performed with responses from all 13 biochemistry laboratories in wales ( population 306 million ) , and the findings showed that approximately 19 million lfts were performed across wales , with 082 million of these in primary care . although analytical platforms were different between the laboratories , all were using the bcg method for measuring serum albumin and the biuret method for total protein measurement . conversely , there was no consistent reference range for cg , varying at the upper end of the range from 35 to 45 g / l and at the lower end from no lower limit to 2028 seven of the 10 laboratories reporting cg either took no action or did not report low levels of cg , while two flagged low results and one reviewed results if < 25 g / l ( three laboratories did not report cg ) . the pilot study showed a linear relationship between values of igg and cg with a wide range of igg levels for each level of cg ( fig . 1 ) , with a cg of 18 g / l corresponding with an igg of 6 g / l ) . the cut - off value of cg < 18 g / l was chosen which corresponded to a sensitivity of 082 and a specificity of 071 for an igg < 3 g / l ( table 2 ) . following the presentation of these data to the biochemistry department a number of samples with low cg were referred to immunology for immunoglobulin levels , and at least one patient was diagnosed with pid and commenced on replacement immunoglobulin therapy . however , audit 1 year later of 100 random samples with a cg of < 18 fifty samples obtained anonymously for each level of calculated globulin ( cg ) at 15 , 16 , 17 , 18 , 19 , 20 , 21 and 22 g / l were tested for immunoglobulin levels and the correlation with igg is shown . g / l and this was selected as the cut - off value for the bromocresol green ( bcg ) method . comparison of sensitivity and specificity using bcg and bcp methods sensitivity and specificity of variable cut - off for immunoglobulin ( ig ) g below set value when using bromocresol green ( bcg ) and bromocresol purple ( bcp ) methods for albumin and biuret method for total protein . in the study of serum samples with cg < 18 g / l from nine hospitals in wales , after exclusion of duplicates ( on the same patient ) , 826 samples were tested for immunoglobulin levels and serum electrophoresis with the addition of immunofixation to confirm and type any paraproteins . the results demonstrated that 89% of samples had an igg of < 6 g / g / l ) and 56% had an igg < 4 g / l with the distribution shown in fig . the bcp method , using a cut - off of < 23 g / l for cg , had a sensitivity of 076 and specificity of 054 at an igg level of < 3 g / l ( table 2 ) . the numbers of samples at each level of igg are shown for samples with a calculated globulin ( cg ) of < 18 g / l with only 11% of samples recorded with an igg level in the normal range ( 616 g / l ) ; 56% of samples had an igg of < 4 g / l , which is 4 standard deviations below the mean for adults [ bromocresol green ( bcg ) method ] . g / l are shown in fig . 3 , with a wide range of clinical specialities and primary care represented . it is noteworthy that when the stringency in terms of significant antibody deficiency is increased by using an igg level of < 4 g / l there is a major fall in samples from surgery of 122 to 20 , perhaps suggesting that some of the changes associated with intravenous replacement of fluid or blood result in more limited reductions in antibody levels . a wide range of specialities is represented with a high number from surgery due probably to the use of intravenous fluids and blood products which may temporarily alter a calculated globulin ( cg ) [ intensive care unit ( itu ) , general practice ( gp ) , outpatients department ( opd ) , teenage cancer trust unit ( tctu ) , accident and emergency ( a&e ) , medical assessment unit ( mau ] . the speciality with the highest number of samples with low igg apart from surgery was haematology , and the associated disorders with an igg < 4 g / l are shown in fig . unexpectedly , the largest diagnostic group was lymphoma , at 34% , rather than cll at 20% ; however , it is possible that some of the cll patients may already be receiving immunoglobulin , which may lower the numbers of cll patients with antibody deficiency detected by cg . the results identify substantial numbers of undiagnosed secondary antibody - deficient patients , including some with neutropenic sepsis or pneumonia stated in the clinical information on the request form . the largest category of antibody - deficient patients from haematology is from requests labelled lymphoma [ lymphoma , non - hodgkin lymphoma , diffuse large b cell lymphoma ( dlbcl ) , follicular lymphoma , hodgkin lymphoma , renal lymphoma , mantle cell lymphoma , marginal zone lymphoma ( mzl ) , burkitt 's lymphoma and central nervous system ( cns ) lymphoma ] and a range of other haematological diagnoses reflecting either secondary antibody deficiency due to the disorder or subsequent to its treatment . in addition to the antibody deficiency , in 12% of samples ( 10 of 826 ) a new , previously undetected small paraprotein with immune - paresis was identified , with igg levels ranging from 19348 the associated immune - paresis suggests that these patients may no longer fall into the category of monoclonal gammopathy of undetermined significance ( mgus ) and would merit referral to haematology for further assessment and potential intervention . these highlight antibody deficiency in patients being monitored for immunosuppressive therapy and with a known diagnosis of cll , but also in patients with diabetes , which is more difficult to explain , and with a range of non - specific symptoms such as tiredness and bloating . diagnoses with immunoglobulin ( ig)g < 4 g / l from primary care . diagnoses from 32 request forms from patients in general practice with igg levels less than 4 there are a significant number where drug monitoring is taking place and a number with a range of more non - specific symptoms . congestive cardiac failure ( ccf ) , ischaemic heart disease ( ihd ) , chronic lymphocytic leukaemia ( cll ) and information not available ( na ) . the impact of reduction in diagnostic delay on wellbeing , schooling , end - organ damage and the use of resources is demonstrated by the patient histories of three cases identified initially by low cg levels who were seen following the study . a 19-year - old male was referred to immunology on the basis of low cg . his tongue was too big for his mouth. he had tonsillectomy and adenoidectomy aged 4 years and three insertions of grommets and a hearing aid . he had a chronic cough from the age of 4 years and subsequently sinusitis and post - nasal drip . he was sent home from school as his coughing was disrupting the class , and recalled falling asleep in a geography examination as he had been coughing all night . when asked when he last felt well he said in the past year he had six courses of antibiotics , two admissions with pneumonia and had investigations for hiv and cystic fibrosis . during the previous 15 years it was estimated he would have visited his general practitioner ( gp ) on 180 occasions , and in 2012 he was found to have pan - hypogammaglobulinaemia with absent vaccine responses and was diagnosed as having cvid . since commencing replacement immunoglobulin 18 months ago he has required no further admissions to hospital . a 23-year - old mechanic was referred to gastroenterology with blood in his stools aged 21 years and was investigated with endoscopies and diagnosed as having a distal colitis of indeterminate type . at this time his cg was 14 , and treatment for colitis was commenced ( asacol ) with little benefit . he was investigated for coeliac disease when a duodenal biopsy showed normal villous architecture with a lack of plasma cells , his cg was 15 and results showed iga deficiency . in 2011 , on the basis of the low cg , immunoglobulin levels were tested ( igg < 134 g / l , iga < 006 he had had watery or semi - formed motions eight times per day for 34 years , suffered from joint pains , was coughing an eggcup of light green sputum per day for the past year , with sinusitis and a post - nasal drip , and had lost , on average , 40 days of work per year . he was found to have low numbers of class - switched memory b cells , did not respond to test vaccination with pneumovax ii and so was diagnosed with cvid . immunoglobulin replacement therapy with privigen 50 g intravenously every 3 weeks was commenced , and at his most recent follow - up his colitis had resolved with two formed motions per day and his previous colitis medication was discontinued . he no longer had a cough and his sinusitis , post - nasal drip and joint pains had settled . prior to replacement immunoglobulin he rated his wellbeing as three out of 10 on a visual analogue scale , and in the year following commencement of immunoglobulin this improved to eight out of 10 , and he missed no days from work . a 26-year - old female working for the university had a 9-year history of recurrent infections , including pneumonias requiring chest x - rays , intravenous antibiotics and admissions to hospital on three occasions . she felt she has a constant chest infection for 6 months of the year and had been diagnosed as having irritable bowel syndrome . in the previous year she had lost 21 days from work , needed six courses of antibiotics and rated her wellbeing as 6 out of 10 on a visual analogue scale . in the previous 9 years she had had 72 visits to her gp and had been told she needed to explore psychological causes for her symptoms . g / l , with pan - hypogammaglobulinaemia ( igg < 134 g / l , iga < 024 she was diagnosed with cvid and commenced on subgam 675 g per week subcutaneously , and had a single visit to the gp in the year following the start of treatment , had no infections , required no antibiotics and felt the treatment had had a brilliant effect on her quality of life. a 19-year - old male was referred to immunology on the basis of low cg . his tongue was too big for his mouth. he had tonsillectomy and adenoidectomy aged 4 years and three insertions of grommets and a hearing aid . he had a chronic cough from the age of 4 years and subsequently sinusitis and post - nasal drip . he was sent home from school as his coughing was disrupting the class , and recalled falling asleep in a geography examination as he had been coughing all night . when asked when he last felt well he said never , and rated his wellbeing as 3 out of 10 . in the past year he had six courses of antibiotics , two admissions with pneumonia and had investigations for hiv and cystic fibrosis . during the previous 15 years it was estimated he would have visited his general practitioner ( gp ) on 180 occasions , and in 2012 he was found to have pan - hypogammaglobulinaemia with absent vaccine responses and was diagnosed as having cvid . since commencing replacement immunoglobulin 18 months ago he has required no further admissions to hospital . a 23-year - old mechanic was referred to gastroenterology with blood in his stools aged 21 years and was investigated with endoscopies and diagnosed as having a distal colitis of indeterminate type . at this time his cg was 14 , and treatment for colitis was commenced ( asacol ) with little benefit . he was investigated for coeliac disease when a duodenal biopsy showed normal villous architecture with a lack of plasma cells , his cg was 15 and results showed iga deficiency . in 2011 , on the basis of the low cg , immunoglobulin levels were tested ( igg < 134 g / l , iga < 006 g / l , igm < 017 he had had watery or semi - formed motions eight times per day for 34 years , suffered from joint pains , was coughing an eggcup of light green sputum per day for the past year , with sinusitis and a post - nasal drip , and had lost , on average , 40 days of work per year . he was found to have low numbers of class - switched memory b cells , did not respond to test vaccination with pneumovax ii and so was diagnosed with cvid . immunoglobulin replacement therapy with privigen 50 g intravenously every 3 weeks was commenced , and at his most recent follow - up his colitis had resolved with two formed motions per day and his previous colitis medication was discontinued . he no longer had a cough and his sinusitis , post - nasal drip and joint pains had settled . prior to replacement immunoglobulin he rated his wellbeing as three out of 10 on a visual analogue scale , and in the year following commencement of immunoglobulin this improved to eight out of 10 , and he missed no days from work . a 26-year - old female working for the university had a 9-year history of recurrent infections , including pneumonias requiring chest x - rays , intravenous antibiotics and admissions to hospital on three occasions . she felt she has a constant chest infection for 6 months of the year and had been diagnosed as having irritable bowel syndrome . in the previous year she had lost 21 days from work , needed six courses of antibiotics and rated her wellbeing as 6 out of 10 on a visual analogue scale . in the previous 9 years she had had 72 visits to her gp and had been told she needed to explore psychological causes for her symptoms . g / l , with pan - hypogammaglobulinaemia ( igg < 134 g / l , iga < 024 g / l , igm < 017 she was diagnosed with cvid and commenced on subgam 675 g per week subcutaneously , and had a single visit to the gp in the year following the start of treatment , had no infections , required no antibiotics and felt the treatment had had a brilliant effect on her quality of life. there are no published studies of the utility of cg to screen for antibody deficiency , and the principal findings of the current study are that using appropriately established method - dependent cg cut - off values it is possible to detect primary and secondary antibody deficiencies , as well as small new paraproteins associated with immune - paresis . this approach fulfils the criteria of a screening test 12 , and as a proof - of - principle , patients detected with low cg have been diagnosed with cvid and commenced on immunoglobulin replacement . using the bcg methodology for albumin measurement and the biuret method for total protein 21 , a cut - off value of < 18 g / l for calculated globulin defines a population in which 89% have an igg level of < 6 g / l . a limitation of this study is that it did not evaluate the utility of cg in children , as the normal ranges for immunoglobulins vary with age ; however , levels approximating the adult ranges are achieved by the age of 910 years 22 and potentially further studies could address whether screening using cg could be used from 10 years of age . clinically , children below this age have a higher frequency of infection which declines with age . the most straightforward would be on samples from primary care , as the use of intravenous fluids ( which may temporarily perturb cg levels ) in this setting would be unlikely , and the patients in the case histories all reported frequent contact with their gp . several comments were developed for a low cg in primary care and the gp laboratory management committee liaison group selected the following comment on the basis that it interprets the underlying abnormality and suggests action which may be considered in the appropriate clinical setting : low calculated globulin may represent antibody deficiency . consider immunoglobulin measurement if there is a history of infections. in secondary care a similar comment could be used , with an added caveat that intravenous fluid administration in surgical , medical or intensive care may cause a temporarily low cg . some laboratories could also choose to cascade directly on discovery of a low cg , adding immunoglobulins and sep as deemed appropriate ; however , this may be hampered by the frequent lack of clinical data on the request form and the requesting clinician may be in the best position to make the most informed decision , potentially also limiting costs . with increasing pressure on health resources , the removal of total protein has been advocated on cost grounds . as part of the process of test profile harmonization and demand management 23 it has been suggested that its inclusion is more as an opportunistic screening test for monoclonal gammopathy , but no specific intervention exists other than watchful waiting 24 . data from the current study , however , argue that the range of disorders for which additional information is gained is wider than monoclonal gammopathy ( table 1 ) ; in particular , when defined cut - offs are in place , there are clear treatment advances for multiple myeloma , clinically important monoclonal bands are detected with a low cg and screening criteria are fulfilled for both primary and secondary antibody deficiency . in addition , testing costs must be evaluated in the context of the complete patient pathway , the utility losses associated with delay in the diagnosis of patients , the benefits associated with accurate immunological testing and the potentially increased use of health resources if the test is not performed . where the test is already being performed it would seem reasonable to obtain best value for money by education regarding its use at both ends of the reference range . there may be circumstances when an antibody deficiency does not require immunoglobulin replacement , but further investigation , monitoring and treatment may still be altered in the light of the result and the clinician and patient are able to proceed in a more informed manner . the increasing use of b cell ablation therapy across a range of specialities ( in particular haematology ) and the growth of maintenance therapy with rituximab , alongside the development of a range of agents targeting b cells for malignant , autoimmune and inflammatory indications , is likely to result in an increase in secondary antibody deficiency in a subset of these patients , further increasing the potential utility of cg . in chronic lymphocytic leukaemia ( cll ) , 3050% of deaths are due to infection caused by secondary antibody deficiency , t cell defects and the effects of therapy 25 . variations in detection rate may be expected , as the populations being tested will be different for different laboratories and health settings . the presence of a large haematology department and oncology centre will have influenced the results in terms of secondary antibody deficiency in this study . the ability of cg to be used as a screening test should be widely applicable , regardless of the presence of major hospitals or specialist services . given the low costs of testing , use of cg may also be relevant in resource - poor settings . the information presented by this study could be used to inform health policy regarding the most appropriate test profiles to use . there is the potential to reduce diagnostic delay , optimize the use of health resources and reduce the burden of disease not only for patients with antibody deficiency but also a much wider range of conditions . cg is an underutilized test which is cheap ( 007 , us$011 or 008 ) , widely available and fulfils the need for a screening approach for antibody deficiency . t. e. has received payment from biotest and csl behring for attendance at advisory board meetings . s. j. has received support from csl behring , baxter , biotest , bpl and octapharma for projects , advisory boards , meetings and clinical trials .

summarycalculated globulin ( total protein albumin ) is usually tested as part of a liver function test profile in both primary and secondary care and determines the serum globulin concentration , of which immunoglobulins are a major component . the main use hitherto of calculated globulin is to detect paraproteins when the level is high . this study investigated the potential to use low levels of calculated globulin to detect antibody deficiency . serum samples with calculated globulin cut - off < 18 g / l based on results of a pilot study were collected from nine hospitals in wales over a 12-month period . anonymized request information was obtained and the samples tested for immunoglobulin levels , serum electrophoresis and , if appropriate , immunofixation . a method comparison for albumin measurement using bromocresol green and bromocresol purple was undertaken . eighty - nine per cent ( 737 of 826 ) samples had an immunoglobulin ( ig)g level of < 6 g / l using the bromocresol green methodology with a cut - off of < 18 g / l , and 56% ( 459 ) had an igg of < 4 g / l . patients with both secondary and primary antibody deficiency were discovered and serum electrophoresis and immunofixation showed that 12% ( 10 ) had previously undetected small paraproteins associated with immune - paresis . using bromocresol purple , 74% of samples had an igg of < 6 g / l using a cut - off of < 23 g / l . screening using calculated globulin with defined cut - off values detects both primary and secondary antibody deficiency and new paraproteins associated with immune - paresis . it is cheap , widely available and under - utilized . antibody - deficient patients have been discovered using information from calculated globulin values , shortening diagnostic delay and time to treatment with immunoglobulin replacement therapy .

a total of 284 poultry workers at 110 poultry farms throughout the campania region of italy were recruited into the study from december 2005 through march 2006 . the workers were interviewed confidentially in their workplace regarding demographics , work activity , knowledge of transmission and prevention of avian influenza , attitudes toward this disease , compliance with precautions at work , and sources of information ( figure a1 ) . multiple logistic regression analysis with adjusted odds ratios and 95% confidence intervals and multiple linear regression analysis with adjusted coefficients were performed with stata software ( 6 ) . average age was 43 years ( range 1975 years ) , average duration of work activity was 18 years , and median number of daily exposures to breeder animals was 18,500 . of the 257 workers , 63.8% correctly defined avian influenza as a contagious infection caused by a virus that can affect all species of birds ( table 1 ) , and 21.8%81.7% knew that avian influenza can be transmitted by touching uncooked eggs or infected animals . most knew that poultry workers had a high risk of being infected and that butchers and veterinarians had a lower risk . only 22.6% provided a correct definition of this disease and knew routes and vectors of transmission . knowledge was greater in persons with more education , those who worked a longer time , those who believed they were at high risk of contracting avian influenza , and those who needed information ( table 2 ) . with respect to identifying measures that protect poultry workers from exposure to avian influenza , correct responses ranged from 34.2% for all protective measures to 43.2% for eye protection and 68.5% for face masks . greater knowledge was observed in those who received information from health professionals and employers , those who believed they were at high risk , and those who worked only with poultry ( table 2 ) . * or , odds ratio ; ci , confidence interval ; df , degrees of freedom . reference category . most poultry workers believed that avian influenza was a serious ( 69.7% ) but preventable ( 70.8% ) disease . mean total scores ( scale of 1 to 10 ) for perceived risk of contracting avian influenza during work activity and for co - workers and family members were 3.2 and 3.1 , respectively , which indicated low - risk perception . respondents who were more likely to believe that they were at high risk worked fewer hours , knew protective measures for exposure to avian influenza , had received information from the mass media , and needed information . workers who were exposed only to poultry were less likely to perceive risk ( table 2 ) . a total of 23.7% reported that in the past 3 months they had modified their work habits because of fear of contracting avian influenza . those more likely to modify their behavior were younger , married , had more knowledge of avian influenza , believed that it was a serious but preventable disease , and received information from sources other than health professionals and employers ( table 2 ) . regarding compliance with precautions to avoid spreading virus through food while working , 59.9% routinely washed their hands and disinfected surfaces and utensils that had been in contact with raw meat . wearing personal protective equipment was not a routine practice because 82.9% always wore outer garments , 82.9% wore boots or protective boot covers , 59.9% wore gloves , 59.9% wore face masks , 24.5% wore eye protection , and 87.9% washed their hands . a total of 24.1% always wore protective clothing and washed their hands ; these practices were more common in poultry workers who knew that these measures were protective and less common by workers who handled only eggs ( table 2 ) . the most common sources were mass media ( 91.8% ) , health professionals ( 47.5% ) , and employers ( 6.2% ) ; 62.3% wanted more information . knowledge of avian influenza can be improved , as shown in a study of consumers ( g. di giuseppe et al . we expected to find more knowledge in educated poultry workers , especially in how to identify potential animal cases and minimize risk for transmission . however , our observations indicate that information is not correctly disseminated because those who receive information from the mass media and who need information were more likely to have a perception of high risk . therefore , tailored educational programs , including booklets and seminars , could be beneficial in improving self - risk assessment of poultry workers . two thirds of poultry workers believed that avian influenza is a serious and preventable disease , but the study showed a perception of low risk of contracting this disease at work because only 4.3% indicated strong concern , although the largest percentage correctly recognized that they are a risk group . workers who handled only poultry were less likely to perceive a risk than those who handled other products . studies have identified direct exposure to infected poultry as the primary risk factor in transmission of avian influenza virus to humans . a cohort study of poultry workers in hong kong showed that greater exposure to poultry was associated with antibody to h5 hemagglutinin ( 4 ) . in thailand , a case - control study showed that activities involving exposure to poultry were associated with influenza caused by h5n1 virus ( 7 ) . in vietnam , a population study in a rural area with outbreaks of highly pathogenic avian influenza showed a dose - response relationship between poultry exposure and illness ( 8) . low adherence to the recommendations of the world health organization to avoid spread of avian influenza through food while working has been reported ; use of protective clothing and handwashing is inadequate ( 9 ) . in our study , there was a subset of workers who routinely followed guidelines because lower compliance was observed in those working only with eggs . moreover , those who did not know precautions had a 6-fold greater risk for inconsistent adherence to preventive guidelines compared with those who identify them . improving knowledge of transmission and application of preventive measures is a useful public health strategy for reducing the effects of avian influenza in poultry workers . employers and health professionals should work together to provide effective and coordinated information to these workers .

we asked italian poultry workers about knowledge , attitudes , and practices regarding avian influenza . it was perceived to be a low occupational hazard , and wearing protective equipment and handwashing were not routine practices . knowledge of transmission and preventive measures should be improved . employers and health professionals should provide more effective information .

interest in developing new drugs for the treatment of acute heart failure ( ahf ) remains high and the past , present and future of diuretics , vasodilators and inotropes is source of debate and expectations . among these three drug therapies , while waiting for new promising drugs to be confirmed as serious players , the present manuscript aims to review the latest addition to the available repertoire of inotropes and inodilators , levosimendan ( figure 1 ) . levosimendan is an inodilator developed for intravenous use in hospitalised patients with acutely decompensated heart failure ( adhf ) . the pharmacological effects of levosimendan are : a ) increased cardiac contractility mediated by calcium sensitisation of troponin c ; b ) vasodilation through the opening of potassium channels on the sarcolemma of smooth muscle cells in the vasculature ; c ) cardioprotection through the opening of mitochondrial potassium channels in the cardiomyocytes . clinical data from heart failure patients show that levosimendan improves haemodynamics without a significant increase in oxygen consumption [ 20 , 21 ] , reduces symptoms of ahf [ 17 , 18 , 22 , 23 ] , has a beneficial effect on neurohormone levels , has a sustained efficacy due to formation of an active metabolite [ 24 , 25 ] , and suffers no loss of effect in patients under beta - blockade [ 17 , 26 ] . levosimendan offers a predictable safety profile [ 17,18,19 , 23 ] , no impairment of diastolic function [ 27 , 28 ] , with no development of tolerance . the most common adverse events are hypotension , headache , atrial fibrillation , hypokalemia and tachycardia [ 22 , 23 , 29 ] . levosimendan is indicated for the short - term treatment of acutely decompensated severe chronic heart failure in situations where conventional therapy is not sufficient , and in cases where inotropic support is considered appropriate . in the latest decade , however , levosimendan has been studied in numerous clinical trials outside the field of acute heart failure . primarily , the drug has been tested in the cardiac surgery settings , field in which the drug has shown beneficial haemodynamic and cardioprotective effects and a favorable outcome effect . in addition , several studies with repetitive levosimendan dosing in patients suffering from advanced chronic heart failure have shown beneficial effects on haemodynamics , neurohormone levels and symptoms . finally , levosimendan has also shown preliminary positive effects - mainly in small - scale studies - in right ventricular failure , cardiogenic shock , septic shock , takotsubo cardiomyopathy , and in other states requiring inotropic support [ 32 , 33 ] . our aim is to review the current data , clinical use and future development of levosimendan in all therapy settings envisioning the future development for this drug , the first in class of the cardioprotective inodilators . clinical development most of the studies in the regulatory clinical development of levosimendan ( table 1 ) were performed in patients with acute worsening of chronic heart failure . the regulatory clinical development program included about 3,500 patients . the therapeutic dose range of levosimendan administered over a 24-h period was identified in a , double - blind , placebo - controlled study including 151 patients with stable ( mainly nyha class iii ) heart failure of ischaemic origin . patients were treated with a bolus dose of levosimendan of 3 - 36 g / kg in 10 min followed by a 24-h intravenous infusion at doses ranging from 0.05 to 0.6 g / kg / min . dose escalation study up - titration , maintenance and withdrawal of levosimendan were studied in a randomised , double - blind , placebo - controlled study in 146 patients hospitalised for decompensated heart failure ( nyha class iii or iv ) due to coronary artery disease ( 60% ) or dilated cardiomyopathy ( 40% ) . patients received an intravenous infusion of levosimendan at doses ranging from 0.1 to 0.4 g / kg / min . escalated doses of levosimendan ( n=98 ) were compared with placebo ( n=48 ) . from 6 to 24 h , patients in the levosimendan group continued to receive the study medication as an open - label infusion . at 24 h , the patients remaining in the study were randomised to continue on levosimendan ( levosimendan continuation group ) or placebo ( levosimendan withdrawal group ) , administered double - blind up to 48 h [ 18 , 25 ] . levosimendan was compared with dobutamine in the lido ( levosimendan infusion versus dobutamine ) study , a double - blind , randomised study in 203 patients with low - output heart failure , who required right heart catheterisation and treatment with an intravenous inotropic drug . patients randomised to levosimendan were treated with an initial intravenous bolus of 24 g / kg in 10 min , followed by a 24-h intravenous infusion of levosimendan at doses from 0.1 to 0.2 g / kg / min . the safety of levosimendan in patients with left ventricular failure complicating an acute myocardial infarction was studied in russlan ( randomised study on safety and effectiveness of levosimendan in patients with left ventricular failure after an acute myocardial infarct ) , a placebo - controlled , double - blind , parallel - group , randomised study in 504 patients enrolled within 5 days of an index infarction . patients randomised to levosimendan were treated with a bolus dose of 6 - 24 g / kg in 10 min , followed by a 6-h intravenous infusion at rates ranging from 0.1 to 0.4 g / kg / min . the revive ( randomised multicenter evaluation of intravenous levosimendan efficacy ) studies ( reviev i and reviev ii ) evaluated the efficacy of levosimendan on symptoms of heart failure by means of a composite endpoint comprising patients subjective symptom assessments ( at 6 h , 24 h and 5 days ) and signs of worsening symptoms ( including death ) during the 5 days after starting a 24-h trial drug infusion . these two randomised , double - blind , placebo - controlled , parallel - group studies in patients with adhf were conducted mainly in the u.s.a . revive i ( n=100 ) was a pilot study designed to evaluate the suitability of the chosen composite ; revive ii ( n=600 ) was a phase iii study . patients randomised to levosimendan ( given in addition to current medications ) received an initial bolus of 6 - 12 g / kg in 10 min , followed by a 24-h intravenous infusion at rates of 0.1 to 0.2 g / kg / min . the survive ( survival of patients with acute heart failure in need of intravenous inotropic support ) study was a double - blind , randomised study in 1327 patients with severe systolic heart failure comparing the effects of levosimendan with dobutamine on mortality . patients in the levosimendan arm were treated ( on top of current ahf medications ) with a bolus of 12 g / kg in 10 min , followed by a 24-h intravenous infusion of 0.1 to 0.2 g / kg / min . main therapeutic efficacy and safety data levosimendan has repeatedly been shown to produce significant dose - dependent increases in cardiac output , stroke volume and heart rate , and decreases in pulmonary capillary wedge pressure , mean blood pressure , mean pulmonary artery pressure , mean right atrial pressure and total peripheral resistance . the effect of levosimendan on haemodynamic variables ( cardiac output , stroke volume , heart rate and pulmonary capillary wedge pressure ) are clearly evident already at the end of a 5-min bolus infusion . there is no sign of development of tolerance even with a prolonged infusion up to 48 h . haemodynamic effects are maintained up to 7 - 9 days after stopping levosimendan infusion , due to the formation of an active metabolite , designated or-1896 . compared with dobutamine , levosimendan produces a slightly greater increase in cardiac output and a profoundly greater decrease in pulmonary capillary wedge pressure [ 17 , 37 ] . in contrast to dobutamine , the haemodynamic effects are not attenuated with concomitant beta - blocker use . at 48 h after the start of infusion in beta - blocked patients with severe adhf , a 24-h infusion of levosimendan achieves better haemodynamic effects than a 48-h dobutamine infusion . accordingly , the current summary of product characteristics ( spc ) labelling suggests levosimendan be used with caution in patients with low baseline systolic or diastolic blood pressure or those at risk for a hypotensive episode . post - hoc analyses of the revive studies identified systolic blood pressure < 100 mmhg or diastolic blood pressure < 60 mmhg at baseline as a factor increasing mortality risk [ 22 , 38 ] . conversely , in patients with higher blood pressure at baseline ( systolic 100 mmhg and diastolic 60 mmhg ) , mortality was 8% with levosimendan and 9% with placebo ( p = 0.81 ) . of importance is the finding that the primary composite endpoint was still positive and statistically significant in this subgroup . dyspnoea improved in significantly more patients treated with levosimendan compared with placebo at 6 hours after the start of treatmentin one early controlled trial in severe heart failure . on the other hand , in the lido study , symptoms of dyspnoea and fatigue improved equally well in levosimendan - or dobutamine - treated patients at 24 h after the start of infusion . in the revive ii study , symptoms over the 5-day assessment period improved significantly more with levosimendan than with placebo . it should be noted that in revive levosimendan ( or placebo ) was administered on top of the standard of care and that in the placebo group , the majority of the patients also improved vs baseline . in the revive ii study , the primary endpoint was a composite consisting of patients subjective symptom assessments ( at 6 hours , 24 hours , and 5 days ) and signs of worsening symptoms ( including death ) during the 5 days after starting a 24-h trial drug infusion . improvement was observed more frequently ( 19% vs 15% ) and worsening less frequently ( 19% vs 27% ) in levosimendan treated patients compared with placebo ( p = 0.015 ) . the improvement in the composite endpoint was accompanied by a lower need for rescue medication in the levosimendan group [ 22 , 38 ] . . found that a 24-h levosimendan infusion induced a 40% decrease in plasma n - terminal atrial natriuretic peptide and n - terminal pro - bnp ( nt - probnp ) levels and the treatment effect was estimated to last up to 16 and 12 days , respectively . in the survive study , a similar decrease in bnp was seen but duration of the effect could not be determined as the last time - point for measuring bnp was 5 days . in the revive ii study the significant effect on bnp was also evident until day 5 . in the lido study , 8% of patients assigned to levosimendan died , compared with 17% assigned to dobutamine ( p = 0.049 ) . the follow - up was retrospectively extended to 180 days , at which point the respective figures were 26% for levosimendan and 38% for dobutamine mortality in the russlan study was prospectively followed for 14 days after starting treatment and shown to be significantly lower with levosimendan than with placebo ( 12% vs 20% ; p = 0.031 ) . there was a trend for this positive effect to persist up to 180 days in a retrospective analysis ( 23% vs 31% ; p = 0.053 ) . in the revive ii study , mortality was numerically , but not statistically significantly , higher in the levosimendan group , with 45 ( 15% ) deaths in the levosimendan group and 35 ( 12% ) in the placebo group during the 90-day study period ( p = 0.21 ) . in the survive study there was no significant difference in survival between levosimendan and dobutamine ( 26% vs 28% , hazard ratio 0.91 ; 95% ci , 0.74 - 1.13 ; p = 0.40 ) . a majority ( 88% ) of patients in this study in these patients , mortality at 31 days was significantly lower with levosimendan than with dobutamine ( p = 0.046 ) . similarly , mortality was significantly lower with levosimendan than with dobutamine in the subset of patients receiving concomitant beta - blockers . several meta - analyses on the effect of levosimendan on mortality have been recently published . among these , the investigation by landoni et al . levosimendan significantly reduced mortality both in cardiology and cardiac surgery settings , and both against dobutamine and placebo . a registry study , alarm - hf ( acute heart failure global survey of standard treatment ) , reviewed in - hospital treatments in eight countries .unadjusted analysis showed a significantly higher in - hospital mortality rate in patients receiving intravenous inotropes ( 25.9% ) compared to those who did not ( 5.2% ) ( p < 0.0001 ) . a propensity - based analysis was performed to compare in - hospital mortality in patients treated only with intravenous levosimendan versus those treated only with catecholamines within 24 h of therapy initiation . propensity score matching produced 104 matched pairs and showed that the use of levosimendan resulted in a significant reduction in the risk of in - hospital mortality ( hazard ratio 0.25 , 95% ci 0.07 - 0.85 ) . a way to evaluate the effect of a medication on both mortality and morbidity is to assess the number of days a patient is both alive and out of hospital during the follow - up period . in the lido study , patients in the levosimendan group spent significantly more days alive and out of hospital than dobutamine - treated patients in a retrospective 180-day follow - up analysis ( median 157 vs. 133 days for levosimendan and dobutamine , respectively ; p = 0.027 ) . in the revive ii study , the mean duration of the initial hospitalisation was almost 2 days shorter in the levosimendan group ( 7.0 days ) than in the placebo group ( 8.9 days ) [ 22 , 38 ] . significantly more patients treated with levosimendan were released within 5 days and fewer had extended hospitalisations ( p = 0.008 ) . in a recent single center study that compared levosimendan with standard of care in a population of ahf patients , the mean length of hospitalization was 12.1 and 13.6 days in the levosimendan ( n=147 ) and control groups ( n=147 ) , respectively ( p < 0.05 ) . re - hospitalization rates were lower in the levosimendan group at 12 months ( 7.6 vs 14.3% ; p < 0.05 ) , and mortality rates at 1 month were 2.1% vs 6.9% , respectively ( p < 0.05 ) . in conformity with these observations from individual studies , in the earlier - mentioned meta - analysis by landoni et al . the mean length of stay in hospital was 1.59 ( 95% ci 0.85 - 2.33 ) days shorter in levosimendan - treated patients in the cardiology setting ( p < 0.0001 ) . renal function often worsens in patients with ahf , and this deterioration is associated with adverse outcome . studies show improved serum creatinine levels in levosimendan - treated patients [ 47 , 48 ] . estimated glomerular filtration rate ( egfr ) improved in levosimendan compared to dobutamine treated patients with heart failure who required inotropic therapy . a placebo - controlled study in patients hospitalized for decompensated heart failure and renal dysfunction , showed a statistically significant improvement in egfr in levosimendan treated patients . peak effect was seen at 3 days after a 24-h infusion , with discernible effects persisting up to 14 days . in addition to beneficial effects on central hemodynamics , levosimendan has direct effects on renal circulation and induces preglomerular vasodilation , leading to improved renal blood flow and glomerular filtration rate . the renal oxygen demand / supply relationship is not affected by levosimendan . in a meta - analysis of 4 randomized studies on the effect of levosimendan in cardiac surgery , a reduction in the rate of acute renal failure was seen in favor of levosimendan treated patients ( odds ratio 0.26 [ 96% ci 0.12 - 0.60 ] , p = 0.002 , with 228 patients included ) . interestingly , a study was just started on the effect of levosimendan in acute kidney injury ( lakis , nct01720030 ) . severe renal failure is a contraindication for levosimendan use as no formal pharmacokinetic studies in heart failure patients with severe renal failure have been conducted . however , many heart failure patients in large regulatory studies such as revive and survive had severe renal failure [ 22 , 23 , 26 ] . according to pharmacokinetic study in severe renal impairment , the elimination of the levosimendan metabolites was prolonged . the results suggest that if levosimendan was given to heart failure patients with severe renal impairment , the dose should be reduced . effects on other organs some reports on a positive effect of levosimendan on splanchnic , liver [ 56 , 57 ] , and diaphragm functions have been published . it must be highlighted , however , that these data were not collected in an ahf population , but in other settings , such as septic shock , cardiac surgery , or chronic obstructive pulmonary disease ( copd ) . levosimendan infusion has generally been well tolerated in the ahf population , despite the high - risk nature of these patients . hypotension was seen more frequently with levosimendan than with placebo , but not when levosimendan was compared with dobutamine . levosimendan has been associated with a higher incidence of atrial fibrillation compared both with placebo and with dobutamine . however , conflicting results have been presented with regard to ventricular arrhythmias . in revive a higher incidence of ventricular tachycardia was observed with levosimendan compared with placebo . in survive , ventricular tachycardia was observed with similar frequency in the levosimendan and dobutamine groups . in both studies , cardiac failure as an adverse event was less frequent in levosimendan arm , although the result was statistically significant only in survive . safety laboratory values the changes in safety laboratory variables have been modest in levosimendan studies . a decrease in potassium levels has been seen with levosimendan more often than with comparators . clinical trials settings other than adhf during the past years , various independent research groups have described the use of levosimendan in a range of clinical situations other than adhf , e.g. advanced chronic heart failure , cardiogenic- and septic - shock , cardiac- and non - cardiac surgery , etc . advanced chronic heart failure patients with refractory heart failure are hospitalised frequently for clinical deterioration . during such admissions , they often receive infusions of positive inotropes ( dobutamine , dopamine , or milrinone ) and vasodilators in an effort to improve cardiac performance , facilitate diuresis and promote clinical stability . despite favourable haemodynamic and symptomatic improvement in small clinical studies , both dobutamine and milrinone increase the myocardial oxygen demand and intracellular calcium concentration , thus increasing the risk of arrhythmic events and , possibly , death [ 60 , 61 ] . a number of small - scale investigator - initiated studies , in which levosimendan has been administered repeatedly to patients with advanced chronic heart failure , have been reported and are summarized below . in general , the results suggest that levosimendan improves haemodynamics , neurohormones and clinical outcomes . exposed 36 consecutive patients with nyha class iv heart failure , resistant to a 24 h continuous infusion of dobutamine : a group was exposed to continuous infusion of dobutamine 10 mg / kg / min for at least 48 h , followed by weekly ( or more often if needed ) intermittent dobutamine infusions of 8 h ( n=18 ) while the second group , after the initial 24 h infusion of dobutamine , to a 24 h infusion of levosimendan ( 0.2 mg / kg / min ) followed by further biweekly 24 h infusions at the same dose . the addition of intermittent levosimendan infusions was associated with a prolonged survival ( 45-day survival of 6% vs 61% ; p=0.0002 ) . mavrogeni et al . performed an open - label prospective study in 50 patients with advanced heart failure ( nyha iii or iv ) . half of the patients received 24 h infusions of levosimendan ( 0.1 - 0.2 mg / kg / min after a 6 mg / kg loading dose ) given monthly for 6 months in addition to standard of care and half of the patients were treated with standard of care . at the end of the study , the proportion of patients reporting improvement in symptoms of heart failure was larger in the levosimendan group than in the control group ( 65% vs 20% ; p < 0.01 ) . after 6 months , the levosimendan group had a significant increase in left ventricular ejection fraction ( lvef ) ( 287% vs controls 214% ; p=0.003 ) . twenty - four hours holter - recordings revealed no significant changes in the occurrence of atrial or ventricular arrhythmias between the two groups . two patients in the levosimendan group died during the 6-month follow - up period , compared with 8 patients in the control group ( p < 0.05 ) . . reported on 44 consecutive heart failure patients with systolic impairment , who received between 2 and 26 repeated infusions of levosimendan . the bolus dose was omitted in 65% of administrations and the maximum maintenance infusion was 0.2 mg / kg / min in 60% of patients . the interpretation of efficacy and safety is hampered by the absence of a control group . however , a significant drop in bnp levels and nyha class was observed and overall the infusions were judged to be well - tolerated . performed an open - label , randomised , placebo - controlled study in 25 patients with decompensated chronic heart failure . five 24 h infusions of levosimendan ( a bolus of 6 mg / kg/10 min followed by an infusion of 0.1 mg / kg / min ) were given to 17 patients in a 3 weeks interval . levosimendan treatment was accompanied by significant reductions in cardiac end - systolic and end - diastolic dimensions and volume indices ( p < 0.01 vs baseline ) . the effects of long - term , intermittent treatment of levosimendan , dobutamine , and the combination of levosimendan with dobutamine on outcomes have recently been studied in 63 patients with decompensated end - stage heart failure . three groups , each of 21 patients , were assigned to dobutamine 10 mg / kg / min , to levosimendan 0.3 mg / kg / min , or to dobutamine 10 mg / kg / min and concomitant levosimendan 0.2 mg / kg / min . the durations of the infusions were 6 h , and the drugs were given weekly for 6 months . the 6-month survival was 80% in the levosimendan - only group , 48% in the dobutamine - only group and 43% in the combined group . cardiac index was significantly increased and pulmonary capillary wedge pressure significantly decreased only in the levosimendan group . moreover , a study was conducted in 28 patients with pulmonary hypertension of various etiologies ( including secondary to left - sided heart failure ) . patients were randomised 2:1 to levosimendan and placebo and they received in total five infusions . the initial 24 h levosimendan infusion ( 12 mg / kg bolus followed by the maintenance infusion of 0.1 - 0.2 mg / kg / min ) produced a significant decrease in pulmonary vascular resistance . thereafter , levosimendan was administered every 2 weeks as a 6-h infusion with the infusion rate of 0.2 mg / kg / min . these repeated administrations were found to be safe and the effect on pulmonary vascular resistance was maintained throughout the course of treatment . finally , the largest study with repetitive dosing with levosimendan is the levorep ( randomised trial investigating the efficacy and safety of pulsed infusions of levosimendan in outpatients with advanced heart failure ) study , a randomised , double - blind , placebo - controlled study in advanced chronic heart failure . levosimendan was administered as 6-h infusions ( 0.2 mg / kg / min ) every 2 weeks for 8 weeks ( four infusions per patient ) . meanwhile , three similar studies has been started : the intermittent intravenous levosimendan in ambulatory advanced chronic heart failure patients study ( lion - heart , nct01536132 ) , the randomized , double - blind , placebo controlled , multicenter trial to study efficacy , security , and long term effects of intermittent repeated levosimendan administration in patients with advanced heart failure ( laica , nct00988806 ) , and the early levosimendanvs usual care in advanced chronic heartfailure ( elevate , nct01290146 ) . right ventricular failure right ventricular failure is most commonly related to left ventricular heart failure . in isolated right ventricular failure there is low output syndrome in the absence of pulmonary congestion , with increased jugular venous pressure , with or without hepatic congestion , and a low left ventricular filling pressure . right ventricular failure can be caused by myocardial ischemia , volume overload and/or pressure overload . investigator initiated studies have been performed in patients with right ventricular failure . in these studies , levosimendan has been shown to : 1 ) reduce the increased right ventricular afterload ; 2 ) improve right ventricular contractility ; 3 ) improve diastolic function of the right ventricle . reported that , in a placebo - controlled study in 54 patients with advanced right ventricular heart failure ( nyha iii - iv , lvef < 35% ) , levosimendan ( 0.1 - 0.2 g / kg / min for 24 h ) improved doppler echocardiographic markers of systolic and diastolic right ventricular function . . administered open - label levosimendan ( bolus in the range of 6 - 12 g / kg and infusion in the range of 0.075 - 0.2 g / kg / min for 24 h ) to 18 patients with ahf ( characterized by lvef 30% , cardiac index 2.5 l / min / m , right atrial pressure 10 mmhg , pulmonary capillary wedge pressure 15 mmhg ) and observed improved right ventricular contractility but not any change in right ventricular afterload . russ et al . evaluated right ventricular function in 25 consecutive acute myocardial infarction patients with cardiogenic shock not responding sufficiently to conventional treatment . a 24 h levosimendan infusion ( 12 g / kg bolus in 10 min , followed by a dose of 0.1 - 0.2 g / kg/ min ) decreased pulmonary vascular resistance and improved cardiac power index ( both right and left ventricles ) , indicating decreased right ventricular afterload and improved right ventricular contractility . studied 35 mechanically ventilated patients with acute respiratory distress syndrome ( ards ) related to septic shock . patients were treated with a 24 h infusion of levosimendan ( 0.2 g / kg / min , n=18 ) or placebo ( n=17 ) . reductions in pulmonary vascular resistance and mean pulmonary arterial pressure , and improvements in cardiac index , right ventricular ejection fraction and mixed venous oxygen saturation were observed only in the levosimendan group . the underlying pathophysiology is profound depression of myocardial contractility and the state can be defined as decreased cardiac output and evidence of tissue hypoxia in the presence of adequate intravascular volume . the standard of care consists of primary percutaneous coronary intervention for st - elevated myocardial infarction , fluid therapy , vasopressors and inotropes . the role of intra - aortic balloon pump ( iabp ) counter - pulsation has recently been challenged . nevertheless , the drug appears to be safe and to improve some haemodynamic and ventricular indices [ 69 , 76,77,78 ] . the improved survival reported by fuhrmann et al . is notable ( 30 day survival 69% with levosimendan vs 37% with enoximone ; p=0.023 , n=32 ) , but the current dataset is too small to draw definitive conclusions . sepsis and septic shock are among the leading causes of death in intensive care units ( icus ) with associated mortality rates up to ~50% . sepsis frequently causes myocardial depression of both ventricles via an impaired response of beta - receptors to endogenous and exogenous catecholamines and via diminished sensitivity of contractile myofilaments to calcium . the results of the few investigator - initiated studies with levosimendan in septic shock support the possibility that levosimendan may have some beneficial effects in this highly vulnerable patient population . randomly exposed 28 septic patients with persisting left ventricular dysfunction after 48 h of conventional treatment to receive a 24 h infusion of either levosimendan ( 0.2 mg / kg / min , n=15 ) or dobutamine ( 5 mg / kg / min , n=13 ) . in addition to improved haemodynamics , levosimendan increased gastric mucosal flow , creatinine clearance and urinary output and decreased lactate levels , without negatively affecting mean arterial pressure . reports by the same author of favourable effects of levosimendan in patients with sepsis - related ards have already been noted in this review ( see section on a 500-patient study sponsored by the nihr - eme program is shortly to commence in the uk ( led by dr . gordon in conjunction with the imperial clinical trials unit ) assessing the role of levosimendan for the prevention of acute organ dysfunction in sepsis ( leopards , isrctn12776039 ) . this double - blind randomised placebo - controlled trial will test if levosimendan , when added to standard of care , reduces multiple organ failure and improve patient outcome . weaning from ventilator acquired diaphragm muscle weakness is a key feature in several chronic conditions , including copd and congestive heart failure , and patients who are difficult to wean from mechanical ventilation . about 10 - 20% of intubated patients in icus are difficult to wean from mechanical ventilation , resulting in increased morbidity , mortality and health care costs . the pathophysiology of muscle weakness in these patients is complex , but includes muscle fiber atrophy and reduced calcium sensitivity of the contractile proteins . as respiratory muscle troponin resembles cardiac troponin , it is plausible that levosimendan may enhance contractility in the same way it enhances cardiac contractility.this supposition has support from in vitro data , experimental observations , and a healthy volunteer study . positive effects were seen both in slow and rapid diaphragm muscle fibers [ 58 , 85 ] . in addition , shifting from mechanical ventilation to spontaneous ventilation may dramatically increase left ventricular filling pressure and pulmonary artery pressure , especially in patients with pre - existing cardiac and or pulmonary comorbidities . levosimendan resulted in significantly greater inhibition of spontaneous ventilation induced increase in pulmonary artery occlusion pressure . similarly , mean pulmonary artery pressure increased to a lesser extent with levosimendan than with dobutamine . in a prospective observational study in ventilator - dependent difficult - to - wean icu - patients with diminished left ventricular function ( lvef < 40% ) , levosimendan improved cardiac contractility and oxygenation variables and increased the likelihood of separation from mechanical ventilation . a study on the effects of levosimendan on diaphragm function in mechanically ventilated patients ( nct01721434 ) run at the university medical center nijmegen is currently recruiting . peri - operative acute cardiovascular dysfunction occurs in more than 20% of patients undergoing cardiac surgery ; yet current ahf classification is not applicable to this period . indicators of major perioperative risk include unstable coronary syndromes , decompensated heart failure , significant arrhythmias and valvular disease . clinical risk factors include history of heart disease , including heart failure , cerebrovascular disease , presence of diabetes mellitus , renal insufficiency and high - risk surgery . neither is the use of an iabp or left ventricular assist device ( lvad ) risk - free [ 89 , 90 ] . the comparative data on levosimendan in this situation suggest that it has the potential to become a drug of choice among the agents with inotropic properties , possibly due to its cardioprotective qualities . several studies have demonstrated that levosimendan protects the myocardium and improves tissue perfusion , while minimising tissue damage during the cardiac surgery and reperfusion periods [ 20 , 33 , 91 ] . current data from individual studies and meta - analyses suggest that levosimendan is superior to traditional inotropes ( dobutamine , phosphodiesterase - inhibitors ) , delivering sustained haemodynamic improvement , diminished myocardial injury , and better outcomes [ 92 , 93 ] . performed a randomised , double - blind , placebo - controlled study in 106 patients undergoing elective multivesselcoronary artery bypass grafting ( cabg ) . levosimendan ( bolus only , 24 g / kg over 10 min ) , or placebo was given before initiation of cardiopulmonary bypass ( cpb ) . mean tracheal intubation time and length of icu stay were significantly shorter in the levosimendan group ( both p<0.01 ) and the number of patients needing inotropic support for > 12 h was significantly lower with levosimendan ( 18.0% vs 3.8% ; p = 0.021 ) . significantly higher postoperative values of mean arterial pressure , cardiac index and cardiac power index , and a lower systemic vascular resistance index were observed with levosimendan , while troponin i increases were significantly smaller ( all p < 0.005 or less ) . in a placebo - controlled study in 60 patients undergoing cabg eriksson et al . showed that levosimendan ( 0.2 g / kg / min , 24 h ) increases the success of primary weaning from cpb ( 73% vs 33% , p=0.002 ) . reported a randomised , double - blind , placebo - controlled study in 200 patients assigned to undergo heart valve or combined heart valve and cabg surgery . levosimendan was given as a 24-h infusion started at the induction of anaesthesia with a 24 g / kg bolus over 30 min and thereafter at a dose of 0.2 g / kg / min . the primary outcome measure was heart failure , defined as cardiac index 2 or failure to wean from cpb necessitating inotrope administration for at least 2 hours postoperatively after cpb . heart failure was less frequent in the levosimendan group than in the placebo group ( 15% vs 58% ; p<0.001 ) . need of inotrope use for rescue was also lower in the levosimendan group ( risk ratio 0.11 ; 95% ci 0.01 - 0.89 ) , and iabp was utilised in one patient ( 1% ) in the levosimendan versus nine ( 9% ) in the placebo group ( risk ratio 0.11 ; 95% ci 0.01 - 0.87 ) . creatine kinase was lower in the levosimendan group on the first post - operative day ( p=0.011 ) . the levosimendan group had more hypotension and needed norepinephrine more often ( 83 vs 52 , p<0.001 ) . there was no difference in in - hospital or 6-month mortality ( overall , 12% of the patients died in both groups ) . the meta - analysis by landoni et al . on mortality with intravenous levosimendan identified 17 studies in cardiac surgery , including 1233 patients . levosimendan reduced mortality significantly in cardiac surgery patients compared with the control arms ( 5.8% vs 12.9% ; risk ratio 0.52 , 95% ci 0.35 - 0.76 ) . in another independent meta - analysis , maharaj and metaxa reported a favourable effects of levosimendan on cardiac index ( mean difference 1.63 , 95% ci 1.43 - 1.83 , p<0.00001 ) , length of icu stay ( mean difference -26 hours 95% ci -46 to -6 , p=0.01 ) , rate of atrial fibrillation ( odds ratio 0.54 , 95% ci 0.36 - 0.82 , p=0.004 ) , and troponin i levels ( mean difference -1.59 , 95% ci 1.78 - 1.40 , p<0.00001 ) as well as a mortality reduction after coronary revascularisation ( odds ratio 0.40 , 95% ci 0.21 - 0.76 , p=0.005 ) . these data amplified the findings of previous meta - analyses . recently , a group of experts published a consensus report on how to use levosimendan in cardiac surgery and the study levosimendan in high risk patients undergoing cardiac surgery ( hsr - levo , nct00994825 ) was initiated and is currently recruiting . of interest also two recent papers on reduction of perioperative mortality [ 96 , 97 ] in which levosimendan is cited as a drug with potential to reduce mortality in surgical settings . congestive heart failure is a non - uncommon co - morbidity in patients undergoing non - cardiac surgery , and is strongly associated with a doubling of the in - hospital mortality . some authors have suggested a role for levosimendan in the preoperative optimization of cardiac function in patient undergoing major elective non - cardiac surgery . reported on the use of levosimendan in high risk patients undergoing abdominal surgery , while ponschab et al . described how levosimendan infusion improves haemodynamics in elderly heart failure patients undergoing urgent hip fracture repair both groups demonstrated that the administration of levosimendan was safe and observed improvements in ejection fraction , echochardiographic parameters as well as a range of haemodynamic indices both intra- and postoperatively . therefore , morelli et al . suggested that the prophylactic administration of levosimendan in patients with compromised myocardial physiologic reserve , undergoing anesthesia and major non - cardiac surgery , is safe and advisable for preoperative cardiac optimization . in the paediatric cardiac settings , inotropic support is often employed on the basis of extrapolations from adult studies , the underlying pathophysiology , the pharmacodynamics of inotropes , and anecdotal experience . there are currently no official indications for levosimendan in patients under 18 years of age ( see levosimendan spc ) , but the drug has been indeed studied and used as a rescue drug in the paediatric icu ( picu ) and in the operating room . the published paediatric experience in levosimendan comprises mainly observational studies [ see recent reviews by hoffman and by angadi et al . ] or analyses of patient registers , but also four randomised and blinded trials have been reported . the pharmacokinetic profile of levosimendan in children with congenital heart disease is similar to that in adult patients with congestive heart failure . ten hours after the initiation of 48 hours levosimendan infusion in neonates undergoing cardiac surgery , the active metabolite or-1896 was detectable in plasma and remained measurable up to 14 days . this observation suggests that levosimendan will exert prolonged hemodynamic effects in neonates after cessation of infusion similar to those noted in adults . as it regards the doses , levosimendan has been administered in children without or with a bolus dose of 6 - 24 mcg / kg , and by infusion of 0.05 to 0.2 mcg / kg / min similarly to adults . levosimendan infusions have been well tolerated in children with acute heart failure or children who are undergoing cardiac surgery ; only transient hypotension or tachycardia has been reported in the beginning of the infusion . the largest published data in paediatrics included retrospectively - gathered data on 484 levosimendan infusions delivered to 293 patients at a single picu . most of the physicians surveyed ( 89% ) thought that levosimendan postponed or reduced the need for mechanical cardiac support in children with cardiomyopathy or who were undergoing cardiac surgery . levosimendan was shown to be as efficacious as milrinone with comparable hemodynamic data in two randomised and double - blind studies in children and in neonates undergoing cardiac surgery [ 107 , 110 ] . in another comparison of milrinone and levosimendan in neonates undergoing cardiac surgery , levosimendan group had higher ph , lower blood glucose level and lower inotrope score in the picu . in a randomised double - blind study in children younger than 4 years of age undergoing cardiac surgery , patients receiving levosimendan had significantly higher cardiac index and lower pulmonary artery pressure than children receiving dobutamine . of interest is a report describing a strategy for rotating inotropes in pediatric decompensated heart failure . other clinical settings case reports , uncontrolled small series or small - scale comparative studies with levosimendan have been published e.g. in calcium - channels - blockers intoxication and takotsubo cardiomyopathy [ 113 , 114 ] . guidance for clinical use , future developments its pharmacologic and pharmacodynamic properties differentiate levosimendan from other inotropes . the infusion of levosimendan has consistently been shown to enhance left ventricular performance and to decrease left ventricular filling pressure and plasma bnp concentrations without increasing myocardial oxygen consumption ( table 2 ) . neither age nor gender influence the responses to levosimendan . following a 24-h infusion of levosimendan , the active metabolite reaches pharmacologically active plasma levels , resulting in a prolonged haemodynamic effect , wich persists for at least 7 days . for at least 7 days . the haemodynamic and neurohumoral improvement is associated with a symptomatic benefit that is sustained and superior to that of placebo . in contrast to dobutamine , the effects of levosimendan are not attenuated with concomitant beta - blocker use . in two early phase iii studies , a significant mortality benefit with levosimendan was observed in comparison with both placebo ( russlan ) and dobutamine ( lido ) . these favourable results were not , however , confirmed in two subsequent studies where levosimendan was compared with placebo ( revive ) and dobutamine ( survive ) . subgroup analyses from the survive study indicate that levosimendan outperforms dobutamine in beta - blocked patients and in patients with acute decompensation of an existing chronic failure . recent independent meta - analyses on the effect of levosimendan on mortality suggest a survival benefit of levosimendan both compared to placebo and dobutamine ; a trend towards a more favourable outcome effect was noted with lower levosimendan doses ( 0.1 g / kg / min ) . dosing guidance for levosimendan in adhf ( table 3 ) may be proposed from the experience of controlled trials . data from the revive and survive studies - the two largest studies conducted to date- indicate that hypotension was more frequent with levosimendan than with placebo , though not dobutamine . levosimendan was also associated with higher incidence of atrial fibrillation relative to both those comparators . it should be recalled that , in addition to contractility increasing effects , levosimendan has profound vasodilatory effects . clinical studies have indicated that levosimendan should be given cautiously to patients with low blood pressure , especially in case of hypovolaemia . use of lower infusion rates without the loading bolus should be considered for such patients . in case of unintended overdose , pronounced haemodynamic effects arrhythmias may be treated with intravenous beta - blockade or amiodarone ( if blood pressure allows ) . due to the formation of the active metabolite , the follow - up may need to be prolonged , if the total dose of parent drug is substantial . applications of this drug in fields such as cardiac and non - cardiac surgery , cardiogenic- and septic - shock , and others have been proposed . the effects of levosimendan in these settings have been described in many independent studies , and there is a strong rationale for suitably powered studies to corroborate those reports

levosimendan is an inodilator indicated for the short - term treatment of acutely decompensated severe chronic heart failure , and in situations where conventional therapy is not considered adequate . the principal pharmacological effects of levosimendan are ( a ) increased cardiac contractility by calcium sensitisation of troponin c , ( b ) vasodilation , and ( c ) cardioprotection . these last two effects are related to the opening of sarcolemmal and mitochondrial potassium - atp channels , respectively . data from clinical trials indicate that levosimendan improves haemodynamics with no attendant significant increase in cardiac oxygen consumption and relieves symptoms of acute heart failure ; these effects are not impaired or attenuated by the concomitant use of beta - blockers . levosimendan also has favourable effects on neurohormone levels in heart failure patients . levosimendan is generally well tolerated in acute heart failure patients : the most common adverse events encountered in this setting are hypotension , headache , atrial fibrillation , hypokalaemia and tachycardia . levosimendan has also been studied in other therapeutic applications , particularly cardiac surgery - in which it has shown a range of beneficial haemodynamic and cardioprotective effects , and a favourable influence on clinical outcomes - and has been evaluated in repetitive dosing protocols in patients with advanced chronic heart failure . levosimendan has shown preliminary positive effects in a range of conditions requiring inotropic support , including right ventricular failure , cardiogenic shock , septic shock , and takotsubo cardiomyopathy .

tinnitus is an unpleasant and often agonizing condition marked by the phantom perception of sound . according to recent epidemiological estimates , 25% of the us general populations are affected with one in three subjects reporting daily tinnitus ( shargorodsky et al . , 2010 ) . while there is evidence of a genetic susceptibility to tinnitus ( sand , 2011 ) , a complex mode of inheritance suggests the presence of multiple risk genes , and comparatively small effect sizes for any single risk allele . heritability estimates vary from 0.11 to 0.39 ( petersen et al . , 2002 ; kvestad et al . , 2010 ) owing to the lack of linkage studies , the search for candidate genes in primary tinnitus is hypothesis - driven . a well - established theoretical framework for tinnitus has been provided by the disruption of gabab receptor signaling in animal models ( szczepaniak and mller , 1995 , 1996 ) that may explain altered cortical inhibition in patients ( eichhammer et al . , 2004 ) . more recently , a key role for gabab receptors has been confirmed by the effects of receptor agonists on tinnitus symptomatology ( zheng et al . , 2012 ) , renewing the interest in controlled clinical trials ( westerberg et al . , 1996 ) . in the light of these developments , further characterization of the gabab receptor complex is advocated , including the genes encoding the respective receptor structures . an auxiliary subunit that associates tightly with the carboxy terminus of gabab2 receptors is kctd12 ( also known as pfet1 or btb / poz domain - containing protein ) , a potassium channel tetramerization domain - containing protein ( bartoi et al . , 2010 ) . coassembly of kctd12 and gabab2 changes the properties of the gabab(1,2 ) core receptor by increasing agonist potency , by altering g - protein signaling , and by promoting desensitization ( schwenk et al . , effects on the pharmacology and the kinetics of gabab receptors occur in various cochlear cell classes , e.g. , in type i fibrocytes of the spiral ligament and in type i vestibular hair cells ( resendes et al . , 2004 ) . knockdown of the kctd12 ortholog right on leads to improper neuronal differentiation in the zebrafish auditory pathway ( kuo , 2005 ) . unlike other kctd proteins , however , kctd12 is also widely expressed in the adult mammalian brain ( metz et al . , 2011 ) and kctd12 is encoded by an intronless gene on human chr13q21 for which only limited data are presently available in hearing disorders . to determine the impact of this candidate gene on chronic tinnitus , we systematically screened the entire open reading frame for genetic variants , and compared observed allele frequencies to published reference data . in 95 german outpatients ( 67 men and 28 women , age 50.6 12.1 years , mean sd ) consulting for chronic tinnitus , the diagnosis was confirmed by a detailed neurootological examination including otoscopy , stapedius reflexes , middle ear pressure measurements , and pure tone audiometry . for the present study tinnitus severity was assessed by the tinnitus questionnnaire ( tq ) ( goebel and hiller , 1994 ) . an additional 50 subjects with chronic tinnitus ( 40 men and 10 women , age 49.3 11.3 years , mean sd ) formed an extension sample and underwent the same diagnostic workup as outlined above . all participants were cauacasians and a majority originated from the upper palatinate region of bavaria . all provided informed consent and the study was approved by the local ethics committee at the university of regensburg . genomic dna was extracted from lymphocytes using standard procedures prior to amplification of the kctd12 open reading frame and adjacent 3 sequence by pcr . briefly , two overlapping amplicons of 438 bp ( a ) and 819 bp ( b ) were generated using the following primer pairs : 5-cgg ttg cag ctc ctg agt-3 ( forward , a ) , 5-agc tct ggc agc tcg aag ta-3 ( reverse , a ) , 5-ctc gtg ctg ccc gac tac tt-3 ( forward , b ) and 5-gac agg tct cac cca gct ac-3 ( reverse , b ) . pcr products were purified with exosap - it ( ge healthcare , freiburg , germany ) for sanger sequencing , and for the identification of variants against the human genome reference ( genome reference consortium build 37 , february 2009 release ) . in the extension sample , multiple sequence alignments were conducted with dna dynamo 1.0 ( blue tractor software , uk ) . linkage disequilibrium and conformity with hardy weinberg equilibrium was measured with haploview 4.2 ( barrett et al . , 2005 ) . kctd12 allele frequencies from a large reference population of european ancestry ( nhlbi go exome sequencing project , esp ) were retrieved with the exome variant server ( url : http://evs.gs.washington.edu/evs/ ) . esp allele frequency data were compared to the frequencies observed in tinnitus using fisher 's exact tests . t - tests were employed to compare self - reported tinnitus severity in carriers and non - carriers of the minor kctd12 alleles . stata 8.0 ( stata corporation , college station , tx , usa ) was used for descriptive statistics . the shapiro wilk statistic served to test the null hypothesis of normally distributed tq scores . all p - values are uncorrected for multiple testing . for estimating the functionality of confirmed sequence variants , evolutionary conservation in primates was assessed with a phylogenetic hidden markov model - based method , phastcons , that describes the process of dna substitution at each site in a genome and the way this process changes from one site to the next ( siepel et al . , 2005 ) . computational annotations of snp function ( xu and taylor , 2009 ) were obtained from the snpinfo webserver ( url : http://snpinfo.niehs.nih.gov/snpfunc.htm , accessed dec . 2011 ) . in silico predictions of structural effects at the amino acid level were based on information from homologous proteins using metaprdos at default parameters ( ishida and kinoshita , 2008 ) . we confirmed the existence of two coding variants , f87f ( rs73237446 ) and t178 t ( rs34544607 ) , plus one previously described , non - coding variant in the gene 's 3 utr ( rs41287030 ) at heterozygosities of 0.01 , 0.10 , and 0.02 , respectively . all genotype distributions conformed to the hardy weinberg equilibrium ( p > 0.75 ) . no novel sequence variants emerged and 11 kctd12 variants listed in dbsnp were absent from our sample ( rs141180437 , rs116710456 , rs143013358 , rs694997 , rs146434030 , rs141477426 , rs144225285 , rs139291676 , rs151278314 , rs142368706 , and rs140689403 , table 1 ) . when allele frequencies in subjects with chronic tinnitus were compared to reference frequencies from a large control population of european ancestry , an increased prevalence of the minor allele was noted for t178 t ( 0.0494 vs. 0.0263 , p = 0.04 ) . to put this finding into perspective , the original screening sample was augmented by 100 chromosomes from a second set of patients , whereupon the maf in cases dropped to 0.0458 for rs34544607 , weakening the association with tinnitus ( p = 0.07 ) . power simulations , based on the entire sample of patients diagnosed with chronic tinnitus and on esp control data , indicated that we should expect a statistical power of > 80% to detect a susceptibility factor with an allelic relative risk of > 1.77 for the t178 t variant . allele frequencies for the kctd12 sequence screened in subjects with chronic tinnitus as compared to frequencies in a large control population . call rates of 85% were achieved in the first round of screening amplicon b. reference population of european ancestry from the nhlbi go exome sequencing project . data retrieved with the exome variant server ( url : http://evs.gs.washington.edu/evs/ ) , accessed december 2011 . overall , tq scores followed a gaussian distribution ( figure 1 ) and averaged 37.1 16.3 ( mean sd ) out of 84 points ( n = 144 ) . by this measure , tinnitus was rated mild ( 030 points ) in 55 subjects ( 38.2% ) , moderate ( 3146 points ) in 46 subjects ( 31.9% ) , severe ( 4759 points ) in 29 subjects ( 20.1% ) , and extreme ( 6084 points ) in 14 subjects ( 9.7% ) . there was no significant difference in mean tq scores or in the degree of concomitant hearing loss between carriers and non - carriers of the minor allele at rs34544607 ( p = 0.52 and p = 0.48 , respectively , t - test , figure 2 ) . a positive family history of tinnitus in first - degree relatives did not predict rs34544607 genotype ( p = 0.67 , fisher 's exact test ) . as we encountered only one carrier of rs73237446 , and only two carriers of rs41287030 , the interplay of these substitutions with tinnitus severity , hearing loss , or with a family history of tinnitus could not be fully judged . distribution of tq scores in 144 subjects with chronic tinnitus does not deviate from the expected gaussian curve ( p = 0.22 ) . open circles indicate t178 t carriers , filled circles indicate homozygous carriers of wildtype alleles . the degree of hearing loss is expressed as the binaural pure tone average involving air conduction across seven test frequencies ( 0.125 , 0.25 , 0.5 , 1 , 2 , 4 , and 8 khz ) . using the degree of evolutionary conservation as a surrogate parameter of functionality , both rs73237446 and rs34544607 scored high on the comparative genomics scale ( figure 3 ) . further in silico analyses confirmed that rs73237446 maps to the potassium channel tetramerization domain ( figure 3 ) whereas residue 178 , encoded by rs34544607 , maps to a disordered region of kctd12 ( figure 4 ) which may affect the molecular recognition of proteins and dna . the non - coding variant rs41287030 is only poorly conserved among primates but could have acquired a functional role in the recent past . thus , rs41287030 would appear to alter a micro rna binding site and may thereby inhibit protein translation ( see the corresponding snpinfo entry for prediction results ) . comparative genomic analysis of the kctd12 sequence f87f ( rs73237446 ) and t178 t ( rs34544607 ) map to regions ( x - axis ) highly conserved in primates . conservation scores ( y - axis ) for the potassium channel tetramerization domain ( delimited by residues 36 and 125 ) are plotted in red . in silico eight prediction scores on the y - axis ( see legend ) are combined using metaprdos to locate disordered regions based on homologies with other proteins ( blue line ) . t178 t ( rs34544607 ) maps to a relative maximum , screening of the kctd12 orf in chronic tinnitus extends preliminary results on genomic variation as obtained from 88 subjects with congenital deafness ( kuo , 2005 ) . as in the earlier study , no novel sequence variants were identified . however , a trend was observed for association of chronic tinnitus with a highly conserved , synonymous substitution , rs34544607 . the relevance of this finding is unclear in view of the moderately sized sample and the use of an external reference population . it is conceivable that some control subjects from the esp may have experienced mild , subclinical forms of tinnitus , increasing the likelihood of a type ii error . pending replication of this association trend at a larger scale , the mechanism by which rs34544607 can affect hearing also remains to be elucidated . possible explanations for synonymous mutations ' functionality are offered by interference with rna processing , or by changes in translation kinetics that affect protein folding ( sauna and kimchi - sarfaty , 2011 ) . phenotypically , rs34544607 carriers may be indistinguishable from other subjects unless treated with baclofen or another gabab receptor agonist . if rs34544607 truly impacts on gabab signaling , we should expect electrophysiological measures of cortical inhibition to discriminate between carriers and non - carriers . electrophysiological data ( motor threshold , short - interval intracortical inhibition , intracortical facilitation , and cortical silent period ) were available only in a subset of our sample and did not suggest a major effect . the degree of hearing loss did not predict rs34544607 carrier status but further stratification by etiology ( noise - induced vs. congenital ) is recommended in future studies . with regard to rs41287030 , the current lack of publicly available control data and a maf < 0.01 in tinnitus subjects call for a re - examination in a larger population of affecteds and controls in order to test for a possible association with the phenotype . taken together , the present results implicate genetic variation in a gabab receptor auxiliary subunit as a possible risk modifier in chronic tinnitus . more research is also invited to address kctd12 promoter variants , and to explore the interaction with variants in genes encoding other elements of the receptor complex , e.g. , gabab1 and gabab2 proteins . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

tinnitus is a common and often incapacitating hearing disorder marked by the perception of phantom sounds . susceptibility factors remain largely unknown but gabab receptor signaling has long been implicated in the response to treatment and , putatively , in the etiology of the disorder . we hypothesized that variation in kctd12 , the gene encoding an auxiliary subunit of gabab receptors , could help to predict the risk of developing tinnitus . ninety - five caucasian outpatients with a diagnosis of chronic tinnitus were systematically screened for mutations in the kctd12 open reading frame and the adjacent 3 untranslated region by sanger sequencing . allele frequencies were determined for 14 known variants of which three ( rs73237446 , rs34544607 , and rs41287030 ) were polymorphic . when allele frequencies were compared to data from a large reference population of european ancestry , rs34544607 was associated with tinnitus ( p = 0.04 ) . however , kctd12 genotype did not predict tinnitus severity ( p = 0.52 ) and the association with rs34544607 was weakened after screening 50 additional cases ( p = 0.07 ) . pending replication in a larger cohort , kctd12 may act as a risk modifier in chronic tinnitus . issues that are yet to be addressed include the effects of neighboring variants , e.g. , in the kctd12 gene regulatory region , plus interactions with variants of gabab1 and gabab2 .

kidney cancer is the third most common genitourinary cancer.13 in europe , the standardized incidence ( to the world health organization world standard population ) is 8.8 per 100,000 , with a standardized incidence of 5.1 per 100,000 in women and 10.5 per 100,000 in men.3 in denmark , the number of new cases reported in 2009 was 669 ; of these , 420 cases were seen in men and 249 in women.2 a study on survival of patients with kidney cancer in the nordic countries in the period 19642003 found that throughout the study period kidney cancer patients in denmark had the lowest 5-year relative survival ratios for both men and women compared with the other scandinavian countries.4 for patients diagnosed between 1999 and 2003 , the relative survival was 40% ( 95% confidence interval [ ci ] 3843 ) for danish women and 41% ( 95% ci 3843 ) for danish men , while survival in the other nordic countries ranged between 52% and 62% for women and between 51% and 69% for men.4 in 1998 , engeland et al published a study that compared relative survival of cancer patients between denmark and the other nordic countries and found that for cancers of the stomach , colon , rectum , breast ( female ) , and prostate the danish patients had a markedly lower relative survival than the patients in the other countries.5 as a response to such poorer prognosis , the danish national board of health launched the national cancer treatment plans i and ii in 2000 and 2005 , focusing on improvement of cancer treatment in denmark.6 the cancer treatment plans focused on centralization of treatment , national clinical guidelines , shorter diagnostic delay , and establishment of multidisciplinary cancer teams . hypothesizing that improvement of treatment of kidney cancer would lead to better survival and lower mortality from kidney cancer in denmark , we used data from the population - based danish national registry of patients ( dnrp ) from 1998 to 2009 to examine potential changes in the survival of kidney cancer patients we conducted this study in the central and the northern denmark regions , with a combined population of 1.8 million . the study period was divided into the following four consecutive time periods : 19982000 , 20012003 , 20042006 , and 20072009 . the national health service provides tax - supported health care for all inhabitants in denmark , guaranteeing free access to hospitals . virtually no kidney cancer patients were treated in private hospitals during the study period . through the dnrp , we identified all patients who had a first - time hospitalization with kidney cancer in the period january 1 , 1998 through december 31 , 2009 . the dnrp contains information about all admissions from nonpsychiatric hospitals in denmark since 1977.7 outpatient and emergency room visits at hospitals have been included since 1995 . this registry includes information on civil registration number , dates of admission and discharge , surgical procedure(s ) performed , and up to 20 diagnoses from each hospital contact . diagnoses have been classified according to the international classification of diseases ( icd ) 8th edition until the end of 1993 and 10th edition ( icd-10 ) thereafter . we used the dnpr to identify nephrectomy procedures ( kac.00 [ nephrectomy ] , kac.01 [ percutaneous endoscopic nephrectomy ] , kac.20 [ nephroureterectomy ] , and kac.21 [ percutaneous endoscopic nephroureterectomy ] ) conducted within 90 days of kidney cancer diagnosis . since 1968 , the central office of civil registration has assigned a unique 10-digit personal identification number to all danish citizens.8 this number , unique to each danish resident , is used in all danish registries , allowing unambiguous individual - level data linkage . from the civil registration system we also obtained information on vital status ( dead or alive ) , date of death , and residence for all cancer patients . we followed each patient from date of cancer diagnosis until emigration , death , or june 25 , 2010 , whichever came first . to visualize crude survival , we constructed kaplan meier curves stratified according to period of diagnosis ( 19982000 , 20012003 , 20042006 , and 20072009 ) . we estimated 1- , 3- , and 5-year survival . in the latter periods we estimated 3- and 5-year survival using a hybrid analysis in which we included the actual survival for as long as possible and then estimated the conditional probability of surviving thereafter based on the corresponding survival experience of patients in the previous period ( ie , using a period analysis technique).9 to compare mortality over time we used cox proportional hazards regression analysis with 19982000 as the reference period to estimate 1- , 3- , and 5-year mortality rate ratios ( mrrs ) and corresponding 95% cis adjusting for age group ( 1559 years , 6074 years , 75 years ) and gender . in separate models we included age as a continuous variable and as a cubic spline with three knots , respectively . for the patients who underwent nephrectomy within 90 days of kidney cancer diagnosis , we likewise computed 30-day mortality rates after surgery for the four time periods . we used cox proportional hazards regression analysis and estimated 30-day mrrs with 19982000 as the reference period , adjusting for age group and gender . analyses were performed using sas version 9.2 ( sas institute inc , cary , nc ) . through the dnrp , we identified all patients who had a first - time hospitalization with kidney cancer in the period january 1 , 1998 through december 31 , 2009 . the dnrp contains information about all admissions from nonpsychiatric hospitals in denmark since 1977.7 outpatient and emergency room visits at hospitals have been included since 1995 . this registry includes information on civil registration number , dates of admission and discharge , surgical procedure(s ) performed , and up to 20 diagnoses from each hospital contact . diagnoses have been classified according to the international classification of diseases ( icd ) 8th edition until the end of 1993 and 10th edition ( icd-10 ) thereafter . we used the dnpr to identify nephrectomy procedures ( kac.00 [ nephrectomy ] , kac.01 [ percutaneous endoscopic nephrectomy ] , kac.20 [ nephroureterectomy ] , and kac.21 [ percutaneous endoscopic nephroureterectomy ] ) conducted within 90 days of kidney cancer diagnosis . since 1968 , the central office of civil registration has assigned a unique 10-digit personal identification number to all danish citizens.8 this number , unique to each danish resident , is used in all danish registries , allowing unambiguous individual - level data linkage . from the civil registration system we also obtained information on vital status ( dead or alive ) , date of death , and residence for all cancer patients . we followed each patient from date of cancer diagnosis until emigration , death , or june 25 , 2010 , whichever came first . to visualize crude survival , we constructed kaplan meier curves stratified according to period of diagnosis ( 19982000 , 20012003 , 20042006 , and 20072009 ) . we estimated 1- , 3- , and 5-year survival . in the latter periods we estimated 3- and 5-year survival using a hybrid analysis in which we included the actual survival for as long as possible and then estimated the conditional probability of surviving thereafter based on the corresponding survival experience of patients in the previous period ( ie , using a period analysis technique).9 to compare mortality over time we used cox proportional hazards regression analysis with 19982000 as the reference period to estimate 1- , 3- , and 5-year mortality rate ratios ( mrrs ) and corresponding 95% cis adjusting for age group ( 1559 years , 6074 years , 75 years ) and gender . in separate models we included age as a continuous variable and as a cubic spline with three knots , respectively . for the patients who underwent nephrectomy within 90 days of kidney cancer diagnosis , we likewise computed 30-day mortality rates after surgery for the four time periods . we used cox proportional hazards regression analysis and estimated 30-day mrrs with 19982000 as the reference period , adjusting for age group and gender . analyses were performed using sas version 9.2 ( sas institute inc , cary , nc ) . a total of 2659 patients was diagnosed with kidney cancer in the period 19982009 . of these patients , the annual number of patients diagnosed with kidney cancer increased from 583 in 19982000 to 853 in 20072009 . overall , the survival improved over the study period ( figure 2 ) ; the 1-year survival improved from 56% in 19982000 to 63% in 20072009 , corresponding to an age and gender adjusted mrr of 0.78 ( 95% ci 0.660.93 ) ( table 1 ) . it is expected that the 3-year survival will increase from 40% in 19982000 to 51% in 20072009 , and the 5-year survival from 33% to 42% , corresponding to predicted age and gender adjusted mrrs of 0.76 ( 95% ci 0.660.87 ) and 0.77 ( 95% ci 0.680.89 ) respectively ( table 1 ) . including age as either a continuous variable or as a spline in the model did not change these estimates . tables 2 and 3 show 1- , 3- , and 5-year survival for each gender , stratified by age . for women , an improvement in survival was observed throughout the study period . for men , an improvement was seen above the age of 60 years , whereas men younger than 60 years seemed not to have experienced the same improved survival . from 1998 to 2009 , the annual number of patients who received a nephrectomy within 90 days of kidney cancer diagnosis increased from 282 patients in 19982000 ( corresponding to 48.4% of patients diagnosed in this period ) to 411 ( 48.2% ) in 20072009 . during the study period the 30-day mortality varied between 3.5% and 2.2% , corresponding to an age- and gender - adjusted 30-day mrr of 0.62 ( 95% ci 0.251.52 ) in 20072009 compared with 19982000 ( table 4 ) . in the study population of more than 2500 patients with kidney cancer , we observed an increase in the survival over the study period ; although not for men below 60 years of age . at the same time , the annual number of patients diagnosed with kidney cancer also increased . our use of data collected through a uniformly organized health care system with free access for all patients allowed for a population - based design with complete follow - up . furthermore , although no validation studies of the coding of kidney cancer in the dnpr exists , a previous study comparing ovarian cancer data from the dnpr with data from the danish cancer registry found a completeness of 96% and a positive predictive value of 87%.10 we have no reason to believe that the coding quality of kidney cancer is inferior to that of ovarian cancer , and thus , we do not expect misclassification of the kidney cancer diagnosis in the dnpr to have major impact on our estimates . when interpreting our findings we also need to consider weaknesses of our study . since we did not include information about the general population in our region we could not compute incidence rates . we therefore could not confirm that the increase in the annual number of kidney cancer cases also reflected an increasing incidence of kidney cancer in northern denmark . another weakness was our lack of information on clinical characteristics such as cancer stage at diagnosis and cancer - directed treatment , other than nephrectomy . we were therefore unable to examine whether changes in stage or treatment could explain our findings of an improved survival . a previous study in patients diagnosed with rectal cancer in the nordic countries and scotland found that part of the differences in survival could be explained by differences in stage distribution between denmark and the other countries.11 therefore , a high prevalence of advanced stage at diagnosis could be a possible explanation of the lower survival for danish kidney cancer patients . because we lacked stage information , we could not compare the stage distribution with that of kidney cancer patients in other countries . moreover , we could not examine whether our increasing annual number of kidney cancer patients was a result of increasing use of abdominal imaging giving rise to a higher proportion of early stage cancers and incidentalomas.1215 if so , such tumors could be slow - growing lesions ( not yet causing symptoms ) with a better prognosis and thereby falsely improving the observed survival in the later study periods as compared with the early study period ( length time bias ) . another weakness is our lack of information regarding comorbidity and lifestyle factors , and we are therefore unable to examine whether changes in these factors may influence survival over time . a number of other factors may explain the improved survival observed in our study . following the national cancer treatment plans , a more aggressive diagnostic strategy has been implemented resulting in earlier diagnosis and thus an increased likelihood of curative treatment . if more patients are being diagnosed at an earlier stage in the most recent periods , they will be living longer with the kidney cancer diagnosis even without any improvements in time of death ( lead - time bias ) . improved surgical technique , extensive use of minimally invasive surgical procedures , and the introduction of new medical treatment modalities such as tyrosine kinase and mtor ( mammalian target of rapamycin ) inhibitors also have the potential to improve survival.1618 however , several of these different initiatives have only recently been implemented , and therefore the full effect on overall cancer survival still remains to be seen . in this 12-year period in northern denmark we found an increasing annual number of kidney cancer patients . this increase was followed by a limited improvement in survival and relative mortality , although not in men younger than 60 years .

objectivefor decades , kidney cancer patients in denmark have had lower survival than patients in the other scandinavian countries . our aim was to study possible changes in survival of patients with kidney cancer after implementation of two national danish cancer plans.study design and settingfrom 1998 through 2009 we included all patients ( n = 2659 ) with an incident diagnosis of kidney cancer in two danish regions ( population 1.8 million ) . data were retrieved from the danish national registry of patients . we computed survival after 1 , 3 , and 5 years , stratified by age , and estimated mortality rate ratios ( mrrs ) using cox regression to assess changes over time , controlling for age and gender . we lacked data on stage distribution . among patients who had a nephrectomy we also computed 30-day mortality and 30-day mrrs.resultsduring the study period , we identified 2659 patients with kidney cancer . the annual number of patients increased from 583 in the period 19982000 to 853 in the period 20072009 . the median age at diagnosis was 69 years throughout the study period . the overall 1-year survival improved from 56% ( 19982000 ) to 63% ( 20072009 ) , corresponding to an adjusted mrr of 0.78 ( 95% confidence interval [ ci ] 0.660.93 ) . we predicted the 3-year survival to increase from 40% to 51% and the 5-year survival to increase from 33% to 42% , corresponding to predicted mrrs of 0.76 ( 95% ci 0.660.87 ) and 0.77 ( 95% ci 0.680.89 ) , respectively . survival increased in all age groups ( 1559 years , 6074 years , 75 + years ) and in both genders , except for men below 60 years , for whom the 1-year survival declined from 76% to 69% . the 30-day mortality after nephrectomy declined from 4% to 2% during the study period.conclusionwe observed an improvement in the survival and relative mortality in kidney cancer patients , although not in men younger than 60 years .

unless otherwise indicated , chemicals were purchased from sigma - aldrich , while media and supplement for cell culture were from gibco - life technologies . wild - type human osteosarcoma u2os cells were obtained from the american type culture collection ( atcc ) and maintained in dulbecco modified essential medium ( dmem ) supplemented with 10% fetal bovine serum ( fbs ) , 10 mm hepes buffer , 100 units / ml penicillin sodium , 100 g / ml streptomycin sulfate , and 1x non - essential amino acids . the generation of u2os cells stably expressing h2b - rfp or green fluorescence protein ( gfp)-lc3 chimeras has previously been described . genetically modified u2os cells were propagated in the presence of 5 g / ml blasticidine . five thousand h2b - rfp- or gfp - lc3-expressing cells were seeded in 96-well bd falcon black / clear imaging plates ( bd biosciences ) and maintained under standard culture conditions ( 200 l medium / well , 37 c , 5% co2 ) for 24 h. thereafter , 160 l medium were removed from each well and replaced with 150 l of fresh medium that had previously been saturated with pre - determined gas mixtures by bubbling ( flow rate = 1 l / min , 10 min ) . ten l of drug - free culture medium or medium containing a 20 concentration of the chemicals employed in this study were employed to restore a final culture volume of 200 l , and plates were transferred into system duetz gas - tight chambers ( enzyscreen ) . upon verification of the tightness of the chamber , gas mixtures were washed in ( flow rate = 10 l / min ) , until co2 concentrations at the outlet and inlet ports of the chamber , measured by a g100 gas analyzer ( geotech ) , were equivalent . chambers were incubated at 37 c in the dark for 6 or 16 h , after which cells were fixed with 3.7% paraformaldehyde ( w : v in pbs ) , optionally supplemented with 1 g / ml hoechst 33342 ( molecular probes - life technologies ) for 20 min . eventually , the fixative was replaced with pbs , and plates were either destined to automated imaging as such ( for the assessment of cell number ) or further processed for immunostaining ( to determine cytochrome c release and caspase-3 activation ) . gas mixtures were kept at room temperature for at least 48 h before the experiment to avoid liquefaction - related changes in composition . cytochrome c release and caspase-3 activation were monitored by automated imaging upon immunostaining with specific antibodies , as previously described to this aim , cells fixed and stained with hoechst 33342 as described above were washed once in pbs and permeabilized with 0.1% triton x100 ( v : v in pbs ) , followed by the blocking of unspecific binding sites with 2% bovine serum albumin ( w : v in pbs ) . thereafter , cells were sequentially immunostained with primary antibodies , specific for cytochrome c ( rabbit monoclonal igg1 # 556432 , bd biosciences ) and active caspase-3 ( casp3a , rabbit monoclonal igg # 9661 , cell signaling technology inc ) , and appropriate alexa fluor conjugates ( molecular probes - life technologies ) . upon 2 washes in pbs , a robot - assisted bd pathway 855 high - content bioimager ( bd biosciences ) equipped with photofluor light sources ( chroma ) , multiple excitation and emission filters ( semrock ) and an orca - ag deep cooled digital camera ( hamamatsu photonics ) was used to acquire at least 4 viewfields per sample ( 20 magnification ) . images were automatically processed by means of the bd attovision software version 1.7 ( bd biosciences ) . in particular , the following regions of interests ( rois ) were defined : nuclei , identified using the h2b - rfp or hoechst 33342 signals ; and cells , defined by the nuclear area plus a perinuclear extension of 10 pixels . objects were equalized and contrasted from the background by means of shading and rolling - ball pre - processing filters , followed by automatic signal - to - noise thresholding . to exclude events corresponding to more than one nucleus from the analysis , successful segregation of each nucleus as a unique roi was conducted using an open pre - processing filter combined with a watershed erosion algorithm . the putative bias coming from cellular debris of overlapping clusters of nuclei was eluded by applying a scrap algorithm set to minimal and maximal size limits of 50 and 2500 pixels , respectively . to monitor apoptosis - associated parameters , cells were co - stained with 1 g / ml propidium iodide ( pi ) ( sigma - aldrich ) , which identifies cells with ruptured plasma membranes , and 40 nm 3,3dihexiloxalocarbocyanine iodide ( dioc6(3 ) ; molecular probes - life technologies ) , which measures m , as previously reported . cytofluorometric acquisitions were performed on a facscalibur cytometer ( bd biosciences ) , and first - line statistical analyses were with the cellquest software ( bd biosciences ) upon gating on the events characterized by normal forward and side scatter . unless otherwise specified , experiments were performed in triplicate instances . data were analyzed with excel ( microsoft co ) and prism 5 ( graphpad software inc ) . raw data on cell number and caspase-3 activation were obtained from a population of at least 100 cells per experimental condition , in duplicate parallel instances , and normalized to appropriate control values . unless otherwise indicated , chemicals were purchased from sigma - aldrich , while media and supplement for cell culture were from gibco - life technologies . wild - type human osteosarcoma u2os cells were obtained from the american type culture collection ( atcc ) and maintained in dulbecco modified essential medium ( dmem ) supplemented with 10% fetal bovine serum ( fbs ) , 10 mm hepes buffer , 100 units / ml penicillin sodium , 100 g / ml streptomycin sulfate , and 1x non - essential amino acids . the generation of u2os cells stably expressing h2b - rfp or green fluorescence protein ( gfp)-lc3 chimeras has previously been described . genetically modified u2os cells were propagated in the presence of 5 g / ml blasticidine . five thousand h2b - rfp- or gfp - lc3-expressing cells were seeded in 96-well bd falcon black / clear imaging plates ( bd biosciences ) and maintained under standard culture conditions ( 200 l medium / well , 37 c , 5% co2 ) for 24 h. thereafter , 160 l medium were removed from each well and replaced with 150 l of fresh medium that had previously been saturated with pre - determined gas mixtures by bubbling ( flow rate = 1 l / min , 10 min ) . ten l of drug - free culture medium or medium containing a 20 concentration of the chemicals employed in this study were employed to restore a final culture volume of 200 l , and plates were transferred into system duetz gas - tight chambers ( enzyscreen ) . upon verification of the tightness of the chamber , gas mixtures were washed in ( flow rate = 10 l / min ) , until co2 concentrations at the outlet and inlet ports of the chamber , measured by a g100 gas analyzer ( geotech ) , were equivalent . chambers were incubated at 37 c in the dark for 6 or 16 h , after which cells were fixed with 3.7% paraformaldehyde ( w : v in pbs ) , optionally supplemented with 1 g / ml hoechst 33342 ( molecular probes - life technologies ) for 20 min . eventually , the fixative was replaced with pbs , and plates were either destined to automated imaging as such ( for the assessment of cell number ) or further processed for immunostaining ( to determine cytochrome c release and caspase-3 activation ) . gas mixtures were kept at room temperature for at least 48 h before the experiment to avoid liquefaction - related changes in composition . cytochrome c release and caspase-3 activation were monitored by automated imaging upon immunostaining with specific antibodies , as previously described to this aim , cells fixed and stained with hoechst 33342 as described above were washed once in pbs and permeabilized with 0.1% triton x100 ( v : v in pbs ) , followed by the blocking of unspecific binding sites with 2% bovine serum albumin ( w : v in pbs ) . thereafter , cells were sequentially immunostained with primary antibodies , specific for cytochrome c ( rabbit monoclonal igg1 # 556432 , bd biosciences ) and active caspase-3 ( casp3a , rabbit monoclonal igg # 9661 , cell signaling technology inc ) , and appropriate alexa fluor conjugates ( molecular probes - life technologies ) . for automated ( immuno)fluorescence microscopy , a robot - assisted bd pathway 855 high - content bioimager ( bd biosciences ) equipped with photofluor light sources ( chroma ) , multiple excitation and emission filters ( semrock ) and an orca - ag deep cooled digital camera ( hamamatsu photonics ) was used to acquire at least 4 viewfields per sample ( 20 magnification ) . images were automatically processed by means of the bd attovision software version 1.7 ( bd biosciences ) . in particular , the following regions of interests ( rois ) were defined : nuclei , identified using the h2b - rfp or hoechst 33342 signals ; and cells , defined by the nuclear area plus a perinuclear extension of 10 pixels . objects were equalized and contrasted from the background by means of shading and rolling - ball pre - processing filters , followed by automatic signal - to - noise thresholding . to exclude events corresponding to more than one nucleus from the analysis , successful segregation of each nucleus as a unique roi was conducted using an open pre - processing filter combined with a watershed erosion algorithm . the putative bias coming from cellular debris of overlapping clusters of nuclei was eluded by applying a scrap algorithm set to minimal and maximal size limits of 50 and 2500 pixels , respectively . to monitor apoptosis - associated parameters , cells were co - stained with 1 g / ml propidium iodide ( pi ) ( sigma - aldrich ) , which identifies cells with ruptured plasma membranes , and 40 nm 3,3dihexiloxalocarbocyanine iodide ( dioc6(3 ) ; molecular probes - life technologies ) , which measures m , as previously reported . cytofluorometric acquisitions were performed on a facscalibur cytometer ( bd biosciences ) , and first - line statistical analyses were with the cellquest software ( bd biosciences ) upon gating on the events characterized by normal forward and side scatter . data were analyzed with excel ( microsoft co ) and prism 5 ( graphpad software inc ) . raw data on cell number and caspase-3 activation were obtained from a population of at least 100 cells per experimental condition , in duplicate parallel instances , and normalized to appropriate control values .

although chemically non - reactive , inert noble gases may influence multiple physiological and pathological processes via hitherto uncharacterized physical effects . here we report a cell - based detection system for assessing the effects of pre - defined gas mixtures on the induction of apoptotic cell death . in this setting , the conventional atmosphere for cell culture was substituted with gas combinations , including the same amount of oxygen ( 20% ) and carbon dioxide ( 5% ) but 75% helium , neon , argon , krypton , or xenon instead of nitrogen . the replacement of nitrogen with noble gases per se had no effects on the viability of cultured human osteosarcoma cells in vitro . conversely , argon and xenon ( but not helium , neon , and krypton ) significantly limited cell loss induced by the broad - spectrum tyrosine kinase inhibitor staurosporine , the dna - damaging agent mitoxantrone and several mitochondrial toxins . such cytoprotective effects were coupled to the maintenance of mitochondrial integrity , as demonstrated by means of a mitochondrial transmembrane potential - sensitive dye and by assessing the release of cytochrome c into the cytosol . in line with this notion , argon and xenon inhibited the apoptotic activation of caspase-3 , as determined by immunofluorescence microscopy coupled to automated image analysis . the antiapoptotic activity of argon and xenon may explain their clinically relevant cytoprotective effects .

clinicians should have a good basic knowledge before performing any surgical procedure on a patient . it is difficult for practising gynaecologists and last year trainees to gain adequate experience in new laparoscopic surgical procedures . the traditional training programme is based on simple black box modules , computer models , animal models followed by learning on patients under supervision of a trainer . an additional possibility and extra - step is training on fresh or embalmed human cadavers . performing surgical procedures in these circumstances is close to real life without the stress of operating on a living patient . in the present paper , we present the objectives , structure , outcome and acceptance of postgraduates course of the vvog , specifically aimed to improve the surgical skills and performance of gynaecologists . the course was started in june 2010 as an initiative of the big ge ( bijzondere interessegroep gynaecologische endoscopie or the particular interest group on gynaecological endoscopy ) of the vvog and had as initial goal to improve the experience with more advanced laparoscopic gynaecological procedures . all cadavers were prepared according to the thiel technique ( thiel , 2002 ) and procedures were performed at the laboratory of human anatomy ledganck , ghent university , gent , belgium . all participants were gynaecologists . before entering this course they had to attend two other types of courses : the basic course with exercises in the black box ( half day ) and the advanced course with live surgery on pigs ( 2 day course ) . the cadaveric course started with interactive lectures on the embalming technique and the preparation of the corpses , the pelvic anatomy , the laparoscopic positioning , followed by videos on laparoscopic hysterectomy , pelvic sidewall exploration and pelvic and para - aortic lymphadenectomy . the anatomy of the anterior abdominal wall was reviewed from a surgical point of view with positioning of the trocars ( fig . 1 ) and the laparoscopic view of the pelvis ( fig . 2 ) . the practical approach of opening the pelvic side wall , with identification of the ureter and its relation to the uterine artery was discussed . the participant should be able to dissect the ureter from the bifurcation of the common iliac artery to the cross of the uterine artery and if possible the bladder , open the pararectal and paravesical spaces ( fig . 3 ) , clamp the uterine artery , dissect the bladder from the isthmus and perform a hysterectomy with ultrascission . furthermore the pelvic sidewall vessels , and the obturator nerve should be dissected , with the removal of all lymphatic structures surrounding them ( fig . 4 ) . at the end of the procedure an anatomical view of the pelvic sidewall , with clear identification of all structures should be obtained . during the hands - on procedure the trainer s role is to facilitate the surgical procedures performed by the trainees in a controlled , nonthreatening and interactive way . this approach allows for interactive discussion on the used surgical techniques and points out the possible complications . the faculty included 3 to 4 experienced laparoscopic surgeons in order to train simultaneously on three cadavers . per cadaver the vvog endoscopic training course was organized for the first time in june 2010 and until now 5 courses have been done with a total of 27 participants ( 15 female and 12 male gynaecologists ) . the postgraduate course was initiated as an extension of the existing laparoscopic training programme for trainees of the flemish society of obstetrics and gynaecology . the program can be found at the website of the vvog ( www.vvog.be/endogent ) or at www.endogent.be . each participant had to fill in an evaluation questionnaire at the end of the course . in table 1 the questionnaire also included the possibility to give free remarks on the course ( table 2 ) . the average age of the participant was 47 years ( range 33 - 57 ) and the average lapse of time since certification as a gynaecologist was 17 years ( range 3 - 30 ) . the cadaver surgery course was found to be very valuable and all participants would recommend the workshop to their colleagues and trainees . the course not only allowed for a refreshment of anatomy knowledge and surgical procedures , but it also gave the possibility to have an interaction with colleagues and peers regarding daily surgical practice . laparoscopic gynaecological surgical techniques have increased considerably the last decades . since laparoscopic surgery tends to be more stressful than open surgery , it is therefore mandatory that laparoscopic surgeons have the opportunity to gain good technical skills ( berguer et al 2001 ) . oncological staging operations in cervical and ovarian cancers have risks but provide considerable opportunities in tailoring the treatment ( tjalma , 2003 ; tjalma 2005 ) . theoretical and practice based knowledge is the keystone in the medical management . for the general gynaecologist it is difficult to keep up with all these new techniques . the principle of see one , do one and teach one can not ethically be justified and from a medical legal point of view it is dangerous to perform operations in which one never has been trained . the aim was to learn laparoscopic procedures in a relaxed and safe surrounding in order to improve surgical skills . our anatomical knowledge principally depends on lectures and on training in the dissection room during medical school . to learn anatomy by heart is considered old - fashioned and nowadays in medical school the anatomical training programmes and dissection courses are reduced in favour of simulation models . however , up till now computer models havent shown to be effective in clinical teaching ( sutherland et al . , 2006 ) , while the evidence regarding cadaver dissection as the best way to learn topographical anatomy is growing ( raferty , 1996 ; willan et al . , 1996 ; gordinier et al . in the training program for specialists in belgium there is no formal anatomical surgical teaching and examination in contrast to other countries . during our training as gynaecologist we gradually increase our knowledge by seeing from our tutors and performing operations under guidance . the last decades the field of obstetrics and gynaecology has expanded enormously ; as a consequence a training of 5 or 6 years is insufficient to gain experience in all fields of expertise . the opportunities to gain practical experience in common surgical procedures like for instance hysterectomies are reduced , also due to the reduction in numbers . indeed , almost ten years ago it was already established that newly qualified gynaecologists had less surgical skills than their predecessors ( eisenkop and spirtos , 2004 ) . furthermore there is a reduction in working hours as a result of the european working time directives , which leads to lesser time to perform surgery . like colleagues from other disciplines of surgery , we feel that the graduate and postgraduate surgical training is compromised ( monkhouse , 1996 ; ger , 1996 ) . these diminishing opportunities to perform common operative laparoscopic procedures during the training period therefore an alternative approach to learn laparoscopic surgical skills based on training in animal models was introduced . these surgical model training have proven to enhance the surgical skills and increase the competence ( taylor and hammond , 2004 ; lentz et al . , 2005 ; winkelmann , 2007 ; goff , 2008 barton et al . , 2009 the rabbit and pig models in our training programs allow the resident to develop confidence and experience in surgical handling . however , these models do not reproduce the anatomical configuration of the human female pelvic viscera . gynaecologic surgery involves operating near the bowel , bladder , ureter and the major vessels in a well - established fashion . human cadaver surgery allowed operating in a real - life setting . the surgical approach of dissecting and cutting of the different structures in the pelvis can be practised with the bowel , bladder , ureter , and major blood vessels in the immediate vicinity . laparoscopic cadaver dissection sessions are known to improve surgical skills ( levine et al . , 2009 ) . despite the fact that a benefit of integrating cadaver dissection into a resident training program in obstetrics and gynaecology has been proven , cadaver surgery is not yet integrated in our training programme ( corton et al . , 2003 ) . however classical enbalming of the cadavers does not allow for laparoscopic surgery : the traditionally formalin - fixed cadavers are less useful because the fixation causes ( 1 ) tissue rigidity ( 2 ) loss of tissue texture , colour and consistency ( 3 ) limited preservation of surgical planes , and spaces , and ( 4 ) difficulty in identifying small structures such as autonomic nerves ( barton et al . , 2009 ) . the embalmed bodies according to thiel have a more or less elastic tissue structure , which is completely different from the traditional formalin - fixed cadavers . this results in well preserved organs and tissues with regard to colour , consistency , flexibility and plasticity . the articular joints remain freely movable and the peritoneal cavity can be inflated for laparoscopic procedures . moreover , in the embalmed bodies it is possible to dissect the thin tissue layers from the underlying structures ( barton 2009 , kerckaert , 2008 ) . teaching in a small group , one cadaver per two trainees and an experienced teacher , was regarded as essential in the training session . for instance opening the pelvic sidewall and identifying the ureter is very realistic in these circumstances . the participant hesitated in the beginning but once started , motivated and guided by their private teacher , they were eager to extend their surgical explorations . furthermore , the direct response and interaction gave the opportunity to learn from each other and practice standard techniques . it is only by training that one develops learning points , allowing you to avoid complications . the direct feedback at the surgical table of participants revealed that cadaver dissection enhanced their understanding of pelvic surgical anatomy and gave confidence in performing standard and new procedures . the structured evaluation afterwards confirmed that all participants found it a valuable learning experience , which improved their surgical capabilities . participants would encourage their colleagues and trainees to attend this postgraduate surgical workshop . since no validated tools were available to measure the surgical performance before and after the course , it is impossible to assess what the long - term impact would be on the surgical development of the attendees . hammond et al reported that the participants of a surgical workshop , being contacted 6 months after the workshop , all reported an increased confidence in their surgical ability in less common surgical situations , which they ascribed to attending the workshop ( hammond et al . , 2004 ) . however live surgery does not provide the possibility for the trainees to get hands - on experience . from a medical legal point of view it would be a major step forward if we could learn new techniques on embalmed bodies . regardless how one looks at surgical training the last two decades several complex new techniques like radical trachelectomy or sentinel node procedures have been introduced . without shame we have to acknowledge that the majority of us learned these new techniques by performing them ( under supervision ) on patients . the next step in this course could be the availability of internet databases with video presentations demonstrating standard procedures as well as atypical situations , including the management of complications ( cibula and kesic , 2009 ; hoffman and bodurka , 2009 ) . moreover we should consider to organize , as the attendees mentioned , live surgical teaching sessions in our hospital . human cadaver surgery in small groups , consisting of two trainees and one teacher , is a useful model for gynaecologists . this model provides the opportunity to gain more knowledge in anatomy and get acquainted with laparoscopic procedures in a close to real life situation , this without the stress of operating on a living patient . it allows the participants to perform a surgical procedure in a controlled , nonthreatening and interactive way under the guidance of an experienced trainer . short - term evaluation of the course yielded appreciation scores of more than 90 % . what the long - term impact will be on managing patients is difficult to measure . maintaining surgical skills the flemish society of obstetrics and gynecology will offer this workshop twice yearly to their members in order to improve and extend laparoscopic procedures .

objective : to describe the postgraduate surgical skills training programme of the flemish society of obstetrics and gynaecology ( vvog * ) . laparoscopic surgical techniques and indications have increased substantially during the past two decades . from surgeons it is expected that they keep up with all techniques and should be able to perform all relevant procedures . learning new procedures in daily practice is hazardous and difficult to achieve . a training experience with cadaver surgery could improve the course and outcome of surgery on patients . we present the objective , structure , and outcome of the endoscopic postgraduate training course.structure : the overall objective of the endoscopic postgraduate training course is to refresh anatomical knowledge and improve general gynaecological laparoscopic surgical skills . the vvog endoscopic training programme is based on black box training , followed by pig surgery . new is the possibility to perform surgical procedures on specifically prepared human cadavers . the course consists of an anatomical teaching session followed by lectures with videotaped procedures on the anatomical exploration of the pelvis , laparoscopic hysterectomy and pelvic lymphadenectomy . during the hands - on session the participant performs the surgical procedures in a controlled , nonthreatening and interactive way under the guidance of an experienced trainer.conclusions : all participants provided feedback on their experiences . the evaluation of the workshop revealed that this course is an opportunity to practise and improve clinical laparoscopic skills of gynaecological procedures and anatomy . attending the course was regarded as of genuine additional value for surgical practice .

ion - selective electrode ( ise ) is the most routinely used method for electrolyte estimation in clinical laboratories . direct ise uses an undiluted sample to interact with ise membrane , and the devices based on indirect ise use preanalytical dilution . direct ise may use whole blood as in the case of an arterial blood gas ( abg ) analyzer , or may use serum as in the case of benchtop auto - analyzers ( aas ) . it is , therefore , important to determine the concordance of electrolyte values obtained by various methods . moreover , there are a lot of studies comparing the results of electrolytes in an arterial sample processed by an abg analyzer , and serum sample processed by a benchtop aa , both of which use the direct ise method without any need for pre - dilution of the sample . the present study was done with the objective to investigate whether the results from abg analyzers and aas are equal when used to assess electrolyte levels ( sodium and potassium ) , in the institute of medicine ( iom ) , kathmandu . a cross - sectional observational study was conducted for a period of 2 months , ie , 10 october to 10 december 2016 , in the central biochemistry laboratory and intensive care unit ( icu ) of iom . ethical clearance to conduct the study was obtained from the institutional research board of iom reference number 293(6 - 11-e)/073/074 . oral consent was taken from patients to be part of this research . the sample from patients admitted to we maintained a record of electrolyte results from abg analyzers and benchtop aa for those cases from whom simultaneous arterial and venous blood was drawn . the serum sample was obtained by withdrawing 3 ml of venous blood in a plain vacutainer through venipuncture under aseptic conditions . the arterial sample was collected in a 2 ml plastic syringe in a sterile environment . the syringe was flushed thoroughly with a solution of liquid heparin ( 1:10,000 ) , which was removed completely before drawing the sample . quality control was ensured by having the blood samples collected by trained staff of the icu and analyzed on a benchtop aa located in the central laboratory , and abg machine located in the icu under similar environmental conditions , and calibrated using standard protocol . abg samples received in heparinized syringes were processed immediately for electrolytes , and the samples received in vacutainers for serum were centrifuged within 30 minutes after collection . electrolytes were measured with a benchtop aa ( fully automated electrolyte analyzer ex - d , jokoh co. ltd , japan ) and with an abg analyzer ( phox ultra , nova biomedical , waltham , ma , usa ) . the reference ranges for sodium and potassium were taken as 135145 mmol / l and 3.55.2 mmol / l , respectively . agreement between the two analyzers was assessed using the bland - altman approach.1,2 limit of agreement was defined as mean bias 2 standard deviation ( sd ) . we used a mountain plot to compare different distributions between methods as a complementary plot to bland - altman findings.3 the mountain plot provides information about the distribution of the differences between methods.1 if two assays are unbiased with respect to each other , the mountain will be centered over zero and long tails in the plot reflect large differences between the methods.4 passing and bablok plot was used to estimate agreement between two methods and to check for linear model validity.57 the mean sodium level measured by abg analyzer and benchtop aa was 140.1 ( 7.58 ) mmol / l and 144.6 ( 7.63 ) mmol / l , respectively , as shown in table 1.1 a bland - altman comparison of aa and abg analyzer for the sodium measurement results revealed that the limits of agreement were between 4.45 to 13.1 and the mean difference was 4.3 mmol / l ( figure 1).8 similarly , for potassium the limit of agreement ranged from 1.15 to 1.24 and the mean difference was 0.04 the mountain plot is shown in figure 2 as a complementary plot to bland - altman plot.1,5 likewise , passing and bablok regression with 95% confidence interval ( ci ) has an intercept of zero , showing no systematic differences and slope one , showing no proportional differences in both sodium and potassium measurements by the two instruments.57 the 95% ci is 6.32 to 6.32 for sodium and 0.84 to 0.84 for potassium . the cumulative sum test for linearity shows a p - value 0.10 for sodium and 0.85 for potassium , signifying no significant deviation from linearity ( figure 3 ) . the united states clinical laboratory improvement amendments ( us clia ) accepts a 0.5 mmol / l difference in measured potassium levels and a 4 mmol / l difference in measured sodium levels , in the gold standard measure of the standard calibration solution . in this scenario , even though the methods of measurement of the two instruments are comparable , the mean difference of sodium between aa and abg analyzer was 4.3 mmol / l , which is not within the acceptable limit for sodium as defined by the us clia . from the dawn of civilization , human beings have been developing new technologies for the benefit of society . the discovery of new techniques has provided options to choose various instruments for analyzing data of the analytes . thus , we need to analyze variations among different methods used in hospitals for the benefit of the patient . electrolyte abnormalities are one of the common reversible causes of morbidity and mortality in patients admitted to the icu . the levels of electrolytes need to be monitored on a regular basis in these patients , which are ordered in abg or serum sample , as per the convenience of sampling and requirement . the results of both types of measurements are used in an interchangeable manner with the assumption that they are equal . on average , 150160 samples for electrolyte estimation are ordered every day in the biochemistry lab , and 4050 samples are ordered every day for abg analysis in icu in the iom . we included only those patients from whom simultaneous arterial and venous blood could be drawn . various types of instruments are available for measurement of electrolytes , and every institution should establish their own protocol for acceptance of the results obtained by these instruments . the use of different techniques is not a limitation in the present study , as we have compared results of two instruments using the direct ise method . the magnitude of difference in our study was consistent with the literature data , as reported earlier.9 the us clia accepts a 0.5 mmol / l difference in measured potassium levels , and a 4 mmol / l difference in measured sodium levels , in the gold standard measure of the standard calibration solution . the value for potassium was within the acceptable limits of the us clia.10 furthermore , there was a strong correlation between the results of the two instruments , concomitant with the acceptable 95% limits of agreement in the bland - altman analysis . the mean difference in the measurement of sodium by the two instruments is 4.3 mmol / l , which is not within the acceptable limits as per the us clia . the use of different heparin volumes for flushing abg sampling syringes dilute the whole blood and lower the levels of measured electrolytes in abg testing , which could provide a meaningful explanation for lower values of electrolytes measured by the abg machine . since the normal range for sodium is higher than potassium , sodium values might be largely affected by dilution , giving lower values in abg machine than aa . the study agrees with previous studies that sodium values obtained from abg machines are not reliable for making clinical decisions.11 the small sample size and use of different volumes of heparin for abg samples , are limitations of the study . it can be concluded that benchtop aas and abg analyzers may be used interchangeably for measurement of potassium in the iom , while the same can not be concluded for the measurement of sodium because of the significant difference in sodium measurements by the two instruments .

objectiveto investigate whether benchtop auto - analyzers ( aas ) and arterial blood gas ( abg ) analyzers , for measuring electrolyte levels of patients admitted to intensive care units ( icu ) , are equal and whether they can be used interchangeably.materials and methodthis study was conducted on 98 patients admitted to the icu of the institute of medicine , kathmandu , nepal between 15 october and 15 december 2016 . the sample for aa was collected from the peripheral vein through venipuncture , and that for abg analyzer was collected from radial artery simultaneously . electrolyte levels were measured with abg analyzer in the icu itself , and with benchtop aa in the central clinical biochemistry laboratory.resultsthe mean value for sodium by aa was 144.6 ( standard deviation [ sd ] 7.63 ) and by abg analyzer 140.1 ( sd 7.58 ) , which was significant ( p - value < 0.001 ) . the mean value for potassium by aa was 3.6 ( sd 0.52 ) and by abg analyzer 3.58 ( sd 0.66 ) . the bland - altman analysis with the 95% limit of agreement between methods were 4.45 to 13.11 mmol / l for sodium and the mean difference was 4.3 mmol / l and 1.15 to 1.24 mmol / l for potassium and the mean difference was 0.04 mmol / l . the united states clinical laboratory improvement amendments accepts a 0.5 mmol / l difference in measured potassium levels and a 4 mmol / l difference in measured sodium levels , in the gold standard measure of the standard calibration solution . the passing and bablok regression with 95% confidence interval has an intercept of zero and slope one for both sodium and potassium , and the 95% of random difference is 6.32 to 6.32 for sodium and 0.84 to 0.84 for potassium , showing no significant deviation from linearity.conclusionit can be concluded that aa and abg analyzers may be used interchangeably for measurement of potassium in the institute of medicine , while the same can not be concluded for the measurement of sodium , because of the significant difference in sodium measurement by the two instruments .

blepharospasm is a localized form of dystonia consisting of involuntary tonic and spasmodic contractions of the orbicularis oculi , corrugator supercilii , and procerus muscles , leading to partial or complete closure of the eyelids . these contractions may be intense and last from several seconds to a few minutes.1 it is a bilateral condition with tonic rather than clonic spasms , which develops typically between 50 and 70 years of age . the exact cause remains unknown , but may result from damage to areas of the basal ganglia including the superior colliculus , pars reticularis of the substantia nigra , and nucleus raphe magnus . the most common form is called benign essential blepharospasm ( beb ) , which is limited to the orbitopalpebral area.1 botulinum toxin ( bont ) injections were first used to treat strabismus in 1977 by alan scott , a pediatric ophthalmologist,2 and subsequently used to treat blepharospasm in the early 1980s by frueh et al3 and scott et al.4 bont is highly effective and well tolerated in the symptomatic treatment of a very broad range of conditions involving either muscle hyperactivity such as blepharospasm or cervical dystonia , or cholinergic hyperactivity such as hyperhidrosis or hypersalivation.5,6 recently , bont has been approved for the treatment of glabellar rhytids and migraine headaches.7 bonts act on the peripheral nervous system where they inhibit acetylcholine exocytosis at the motor endplate within the neuromuscular junction by inhibition of proteolytic cleavage of different proteins of the acetylcholine transport protein cascade ( soluble n - ethylmaleimide - sensitive fusion attachment protein receptor ( snare ) proteins ) . botulinum toxin type a ( bont / a ) hydrolyses snap-25 ( synaptosomal - associated protein 25 ) which is located on the presynaptic cell membrane whereas type b ( bont / b ) acts on synaptobrevin or vamp ( vesicle - associated membrane protein ) which is embedded in the acetylcholine vesicle membrane . by cleaving these target proteins , bont prevents the fusion of the synaptic vesicle with the presynaptic membrane , thereby blocking the release of acetylcholine in the synaptic cleft . between a heavy chain ( 100 kda ) and a light chain ( 50 kda ) of neurotoxin , only the light chain is responsible for the pharmacological action of bont.1,8 the various strains of the anaerobic bacterium clostridium botulinum produce 7 distinct serotypes of botulinum toxin , of which 5 are pharmacologically active in man ( a , b , e , f , and g ) and 2 are inactive ( c and d).9 today , 2 serotypes are used in therapeutics , botulinum toxin type a and type b. in august , 2010 the us food and drug administration ( fda ) approved xeomin ( incobotulinumtoxina10 ; merz pharmaceuticals gmbh , frankfurt , germany ) for the treatment of cervical dystonia or blepharospasm in adults ( figure 1 ) . now xeomin is the fourth bont product licensed for the us market , following botox ( onabotulinumtoxina ; allergan inc , irvine , ca ) , dysport ( abobotulinumtoxina ; ipsen ltd , slough , berks , uk ) , and myobloc ( rimabotulinumtoxinb ; solstice neurosciences inc , malvern , pa).11 with this recent entry , 3 type a and 1 type b brands of botulinum neurotoxins are available in the us10 ( table 1 ) . the main long - term side effect of bont use is the development of an immunologic resistance due to the production of neutralizing antibody for the neurotoxin after repeated injections . the frequency of this sensitization reported by several authors is around 3% to 10%.12 the newly approved bont / a drug may overcome this limitation of the other 3 previous products , since xeomin contains only the pure neurotoxin ( 150 kda ) through a manufacturing process that separates it from complexing proteins such as hemagglutinins ( ha ) and other proteins in the neurotoxin complex produced by fermentation of c. botulinum.11 xeomin was first introduced in germany in july , 2005.13,14 it received approval for the treatment of blepharospasm and cervical dystonia in a number of european countries , and then argentina and canada , for the treatment of blepharospasm , cervical dystonia , and poststroke upper - limb spasticity,15 and now in the us for cervical dystonia and blepharospasm . the fda s approval of xeomin for the treatment of blepharospasm restricts the drug to adult patients previously treated with botox . therapy with the newer bont / a product should be based on the previous dose , number , and location of botox injections . regardless of whether the patient has cervical dystonia or blepharospasm xeomin is supplied in single - use vials containing 50 or 100 units of lyophilized incobotulinumtoxina , which do not require refrigeration before use.11 in all naturally occurring serotypes of botulinum toxin ( types a g ) , the active neurotoxin ( 150 kda ; 100 kda of a heavy chain and 50 kda of a light chain ) is noncovalently associated with a set of nontoxic and inactive complexing proteins ( hemagglutinins [ ha ] and non - hemagglutinins [ nha ] ) and thus forms high molecular toxin complexes.6,1618 the molecular weight of the toxin complex ranges between 230 and 900 kda , depending on the serotype.1 all previous bont drugs , including botox and dysport , contain these nontoxic and inactive complexing proteins in addition to the neurotoxin ( figure 2).6 this new preparation , xeomin , is derived from a wild - type strain of c. botulinum type a ( atcc 3502 ) which is the same strain from which botox is derived and thus has similar biologic activity to botox.17,19 unlike botox it contains only the active neurotoxin moiety with complexing proteins removed through a manufacturing process for removal all clostridial contaminants.6,11 the function of the nontoxic portions of the protein complexes in bont / a preparations has been studied . it was initially thought that these proteins protected the native neurotoxin from destruction in the gastrointestinal tract with oral ingestion ( its natural route of entry).18,20 this was subsequently confirmed in biochemical analyses ( protease resistance ) of different toxin serotypes.21 others have suggested that complexing proteins may have a role in the uptake and transcytosis of botulinum toxin through the intestinal epithelium to reach and affect muscle.2224 xeomin does not have complexing proteins to protect it from the low ph and gastric enzymes and therefore shows poor oral bioavailability and toxicity.25 however , in the therapeutic setting these proteins are not relevant to clinical efficacy . another consideration is that complexing proteins may in fact limit botulinum toxin diffusion from the injection site and thereby minimize adverse events , due to the large size of the toxin complex.2628 the smaller size of xeomin might more rapidly and easily diffuse away from the target tissue into adjacent tissues and produce an adverse effect profile different from other bont / a drugs . however , an in vivo study using botox , dysport , and a purified preparation of bont / a ( 150 kda ) showed that diffusion from the injection site does not differ between the 3 preparations.29 another study using i - radiolabeled botulinum toxin type a showed no difference in the diffusion of the free or complexed form of bont / a after injection into the muscle , even when using high doses.30 comparing the adverse effect profiles of conventional bont / a drugs and complexing protein - free bont / a drug , indeed , did not reveal any of those differences.5,13,14,31,32 these findings can be explained by a dissociation of the complex consisting of neurotoxin and complexing proteins immediately after injection.33 at physiologic ph values , the active 150 kda neurotoxin is efficiently released in less than 1 minute from the 900 kda complex.34 this is in contrast with the onset time of its therapeutic effect , which is measured in days . therefore , complexing proteins do not seem to be essential for either the stability of the 900 kda toxin complex at physiologic ph or for limiting diffusion of the 150 kda neurotoxin after injection of conventional bont / a preparation.19 another possible function of complexing proteins in a therapeutic setting could be to enhance the stability of the botulinum toxin drug during storage before use . however , a series of stability studies ( stress experiments ) of xeomin do not support this hypothesis . the stability of xeomin was evaluated in both long - term storage studies and in short - term temperature - stress studies . the studies evaluated the active ingredient ( neurotoxin ) and inactive ingredients ( sucrose and human serum albumin [ hsa ] ) of vials containing xeomin stored at 5c or 25c , as well as the biologic activity of the neurotoxin ( mouse median lethal dose [ ld50 ] ) . after 48 months of storage in a refrigerator ( 5c ) or at room temperature ( 25c ) , no significant changes in neurotoxin , sucrose , or hsa content or , more importantly , biologic activity , were observed in the xeomin . furthermore , storage studies showed that xeomin was stable for at least 18 months at 30c and for at least 6 months at 40c . also in short - term temperature - stress studies , there was neither loss of activity nor degradation detectable after 30 days at 60c . even at 80c , the reduction of biologic activity occurred within 5 days , although proteolytic activity had not fallen to below one - third of the initial value after 10 days , with a decline over time considerably slower than for biologic activity.35 overall , these results demonstrate that complexing proteins are not required to maintain the stability of bont / a preparations during storage.19 the manufacturer reports that xeomin is stable at room temperature for 3 years and new data demonstrate stability for 4 years at room temperature.11,36 complexing proteins may have the disadvantage of immunostimulation , since ha , which belong to the complexing proteins , are lectins known as potent stimulators of immune cells.11 lee et al37 reported vaccination experiments in mice with formalin - treated botulinum toxin type b ( toxoid ) . the amount of neutralizing antibodies increased when the neurotoxoid was complexed with ha , compared with the neurotoxoid complexed with nha , or when the neurotoxoid was administered alone . further analysis showed that among the 4 subcomponents of ha designated ha1 , ha2 , ha3a , and ha3b , ha1 and ha3b subcomponents of ha accounted for the adjuvant activity . the mechanism of increased immune response to ha1 and ha3b appeared to be mediated by an increase in interleukin-6 , leading to increase in cd19 + cells . further experiments showed that in vitro enzyme - linked immunosorbent assay analysis of antibody binding to bont / a large toxin complex showed that ha1 was responsible for most of the immunogenic response.37 some have commented that this study was flawed and does not reflect a therapeutic situation . the dose of toxin used was about 1000 times greater than in typical clinical use . the antigen immunized into the mice was actually inactivated toxin ( toxoid ) that was treated with formaldehyde , which in itself causes the molecule to be more antigenic than native proteins.38 however , these and other data confirmed that ha1 and ha3b appear to increase the immunogenic potential.37 hence the presence of complexing proteins in commercially available bont / a preparations may facilitate an immunogenic reaction and the development of neutralizing antibodies against the active neurotoxin leading to partial or complete clinical unresponsiveness ( treatment failure ) to bont / a.19 indeed , incidence of antibody development following treatment with botox had been significantly reduced since its original formulation was changed to reduce complexing proteins and inactive neurotoxin.39,40 furthermore , removing the complexing proteins in the manufacture of xeomin may reduce this risk markedly . preliminary experiments with xeomin also suggest that the absence of complexing proteins is indeed associated with reduced immunogenicity . in a preclinical animal study with cynomolgus monkeys , repeated injections with 4 , 8 , or 16 u / kg xeomin or control were not associated with the development of neutralizing antibodies in each group , despite clear evidence of biologic activity of the neurotoxin , particularly in the highest - dose group.41 the immunogenicity of xeomin was compared with that of botox and dysport in new zealand white rabbits . after repeated injection , xeomin did not induce the formation of neutralizing antibodies , unlike the other preparations.42 similar results were shown in a human study of up to 89 weeks in patients with upper limb spasticity who received multiple injections of xeomin ; no patient developed neutralizing antibodies throughout the study.43 although in the clinical development program of xeomin in the us , 12 of 1080 subjects developed antibodies against the neurotoxin , each of these patients was previously treated with a conventional bont / a product which contained complexing proteins . they may have already been primed by the previous treatment.44 several risk factors of this sensitization have been identified:12,45 injection of over 100 units of botox or 300 units of dysport per session , interval of less than 3 months between 2 injections , booster technique where another dose is injected 2 to 3 weeks after the first injection , and use of a bont drug with a low intrinsic activity . cumulative dose , treatment time , and patient age have been excluded as risk factors . antibody - induced therapy failure usually develops within the first 2 to 3 years of bont therapy.46 among the above risk factors , the intrinsic activity of bont drugs is defined as the number of toxin units per the amount ( nanogram ) of clostridial proteins ( ie , toxin complex ) . at each injection of toxin , the administered protein mass will be greater when using a toxin with a low intrinsic activity . the toxin s antigenic potential is probably related to the total protein concentration injected ( protein load ) , and according to borodic et al,47 this may be a much more relevant parameter in the development of a resistance than the number of units . indeed , in patients with cervical dystonia , the original formulation of botox ( 100 u/25 ng protein ) was 6 times more likely to elicit the production of neutralizing antibodies than the newer formulation of botox ( 100 u/5 ng protein ) which contains fewer complexing proteins and reduced inactive neurotoxin . the authors conclude that the low risk of antibody formation after newer botox treatment is related to lower protein load.39 xeomin contains only 0.6 ng of clostridial proteins per vial ( 100 u/0.6 ng protein ) , whereas the other products contain much more protein : 55 ng in myobloc , 5 ng in botox ( 100 u/5 ng protein ) , and 12.5 ng in dysport ( 500 u/12.5 ng protein).11 based on a conversion factor of 1 botox or xeomin unit to 3 dysport units,6,4850 100 units of botox or xeomin are bioequivalent to 300 units of dysport . if this same equivalent dose of the preparations is injected , in the case of xeomin , the patient is treated with 0.6 ng of clostridial protein ( neurotoxin alone ) . in contrast , when treated with botox or dysport , the patient receives a much higher amount of clostridial proteins including complexing proteins , 5 ng or 7.5 ng respectively ( figure 3 ) . in other words thus it has the highest specific potency , and with xeomin the patients gets the lowest amount of foreign protein,11 so that the risk of development of any immunogenicity may be reduced . but long - term , trials in nave patients comparing xeomin with conventional bont / a drugs are required to confirm the low immunogenicity of xeomin . based on the fact that xeomin is obtained from the same strain of c. botulinum as botox and on preclinical studies , the manufacturer first assumed xeomin had the same potency as botox . subsequently , the registration studies using a cross - over design were based on identical potency labeling . the result of equivalent experiments in which ld50 of 5 different batches of xeomin and botox was compared in a blinded fashion demonstrates that there was no difference in the potency between the 2 preparations.51 results from both registration studies13,14 together with subsequent head - to - head potency testing using a mouse lethality ( ld50 ) assay17 confirmed the equivalence rate between xeomin and botox . the conclusion of a focal dystonia study also showed that xeomin had the same efficacy as botox , which means that 1 unit of xeomin is equipotent to 1 unit of botox.6 dressler50 further confirmed the identical potency labeling by his study converting botox in a blinded fashion to xeomin using a 1:1 conversion ratio . therefore , clinically xeomin and botox can be exchanged easily using a straightforward 1:1 conversion ratio.50 the efficacy and safety of xeomin in the treatment of blepharospasm has been confirmed by a prospective , double - blind , placebo - controlled , randomized , multicenter study.31,32 in this study involving 109 patients ( mean total dose of xeomin per treatment visit was 64.8 units ) , the jankovic rating scale ( jrs ) severity score was significantly reduced compared with placebo ( p < 0.001 ) . and the most commonly reported adverse effects related to xeomin vs placebo were eyelid ptosis ( 18.9 vs 8.8% ) and dry eye ( 16.2 vs 11.8% ) . several large clinical studies to compare the efficacy of botox and xeomin have been reported . in comparative clinical trials , the efficacy and tolerability of xeomin were noninferior to that of conventional bont / a drugs.13,14 the efficacy of xeomin was compared with that of botox in a 16-week randomized , double - blind , noninferiority trial in 463 patients with cervical dystonia . both treatments significantly improved the toronto western spasmodic torticollis rating scale severity score compared with baseline , and noninferiority of xeomin vs botox was demonstrated.14 similarly , a randomized , double - blind study of xeomin and botox in 300 patients with blepharospasm found that both treatments significantly reduced jrs score from baseline , indicating noninferiority of xeomin.5,13 there was also no difference in the time course of the 2 treatments demonstrated in the kaplan there were no clinically relevant differences between xeomin and botox in safety parameters , 40 of 148 patients ( 27.0% ) treated with xeomin reporting adverse events vs 45 of 155 patients ( 29.0% ) treated with botox . the most common adverse event was ptosis ( 6.1% xeomin and 4.5% botox).5 in conclusion , the clinical evidence to date suggests that xeomin is an effective treatment for blepharospasm , which does not differ from botox in terms of its potency , duration of effect , or adverse reaction profile.5 the same efficacy and safety profiles could be explained by the immediate dissociation , which would lead to the generation of the same active agent , ie , the 150 kda active neurotoxin , eliciting the same diffusion characteristics and therapeutic effects . if the previous information is not available , the recommended starting dose is 2.5 to 5.0 units per each injection site in blepharospasm.52 there is some evidence of a dose response relationship for efficacy and its duration , in which the greatest benefits for xeomin were observed with the highest dose , and dressler50 reported his experience of off - label indications and maximum therapeutic dose up to 840 units of xeomin as well as botox in a variety of muscle hyperactivity disorders without producing clinically detectable systemic adverse effects . however , few patients with blepharospasm received a total dose of greater than 75 units in the controlled trials and less than 70 units ( 35 u / eye ) is recommended for initial total dose so far.52 based on stability data , xeomin is the only preparation that remains active for up to 3 or 4 years at room temperature before reconstitution . in contrast , other botulinum toxin products require refrigerated storage.19,36,53,54 after reconstitution , xeomin should be stored for only 24 hours at 4c , because otherwise sterility problems , such as bacterial contamination , could occur.11 xeomin was originally developed to reduce drug antigenicity , which can lead to partial or complete treatment failure . many studies have also shown that xeomin has the same efficacy and safety profile as complexing protein - containing products such as botox and is exchangeable with botox using a simple 1:1 conversion ratio . xeomin represents a new treatment option for the repeated treatment of patients with blepharospasm in that it may reduce antibody - induced therapy failure . but , long - term , trials in nave patients comparing xeomin with conventional bont / a drugs are required to confirm the low immunogenicity of xeomin .

even though conventional botulinum neurotoxin ( bont ) products have shown successful treatment results in patients with benign blepharospasm ( beb ) , the main , potential long - term side effect of bont use is the development of immunologic resistance due to the production of neutralizing antibody to the neurotoxin after repeated injections . xeomin ( incobotulinumtoxina ) , a unique botulinum neurotoxin type a ( bont / a ) drug free of complexing proteins otherwise contained in all conventional bont / a drugs , was recently approved by us food and drug administration for the treatment of cervical dystonia or blepharospasm in adults . the newly approved bont / a drug may overcome this limitation of previous conventional products , since it contains pure neurotoxin ( 150 kda ) through a manufacturing process that separates it from complexing proteins such as hemagglutinins produced by fermentation of clostridium botulinum . many studies have also shown that xeomin has the same efficacy and safety profile as complexing protein - containing products such as botox and is exchangeable with botox using a simple 1:1 conversion ratio . xeomin represents a new treatment option for the repeated treatment of patients with blepharospasm in that it may reduce antibody - induced therapy failure . but , long - term comparative trials in nave patients between xeomin and conventional bont / a drugs are required to confirm the low immunogenicity of xeomin.

the importance of dental implantology has influenced that many oro - maxillofacial surgeons get involved in this issue . this influence has made that today we have many methods and manufacturers in this field . the aim of our study is to evaluate the advances and weaknesses in patient treatment with tiolox - dentaurum dental implants in cases of partial and total lack of teeth , compared to classic prosthesis / dentures , whether static or mobile . to do case analysis before and after placing the implants , at what age they have undergone the surgical intervention and in which cases the placing of implants can not be done . the prospective method of research has been used , analyzing cases that have undergone surgical treatment at the policlinic shoshi in prishtina , in a two year period , from 2010 to 2011 . patients were divided in two groups : research group with 23 patients in which the implants were placed in a shorter time periodcontrol group with 6 patients research group with 23 patients in which the implants were placed in a shorter time period control group with 6 patients with distribution of cases based on gender , out of 23 cases in the first group , 12 or 52.2% were females and 11 or 47.8% were male , while in the control group we had three females and three males , but without differences with significant statistical importance . the patients involved in the study were of age 30 - 59 years old . in structure higher than 52.2% were in group age of 30 - 39 years , then 40 - 49 years were 30.4% and 50 - 59 years were 17.4% . with distribution of cases based on group ages and gender , we got statistically important difference ( chi test = 12,5 , p= 0,0078 ) . we had more females in the group age 30 - 39 years old ( 58.3% vs. 45.5% ) , while men in the group age 40 - 49 years old ( 25.0% vs. 36.4% ) and 50 - 59 years ( 16.7% vs. 18.2% ) . all the tiolox implants used were of 3.5 mm thickness , and 10 - 16 mm long . in our patients , mostly were used implants 16 mm long in 58.1% , 14 mm long in 17.1% , and 10 mm long in 16.2% . in the research group , the average time of placing the dental bridges in the lower jaw was 2 months ( sd 0,1 month ) , and in control group 4.2 months(sd 0,5 months ) . in the upper jaw , the average time of placing the dental bridges at the experimental group was 3 months ( sd 0,15 months ) , and in control group was 5,4 months ( sd 0,6 months ) . at the end of this scientific study , at the cases we have worked , we can conclude that the period after the placement of implants until the placements of prosthesis , can be shortened for the upper jaw from 4.6 months to 3 months , and for the lower jaw from 3 months to 2 months , based on co - existence of the bone and primary stability of the implant . oral implantology is a part of stomatology that works with placement of implants aiming to improve the function of chewing , esthetics and phonetics in cases of total or partial lack of teeth . a very important factor which determines the success of the implantation is the selection of the patient who would be ready to cooperate with the doctor at every stage of the work . presenting of the implant placement of cover caps over implants placement of mucoperiostal flap and stitching final phase of placing the implants the anamnesis and the examination of the mouth cavity should be done in all patients who are willing to undergo implantation . anamnesis will show us , what cause led to the loss of teeth and the level of the oral hygiene . the overall and the local health condition of the patient and also the characteristics of the bone tissue in which the implant will be placed , are crucial for the selection of the patient . implanting is not limited to age , having in mind that all the other factors are fulfilled . it is also necessary to do a radiography and as needed , to make the study models and photographs , biochemical analysis of blood , blood screen , sedimentation and leukocyte formula ( 22,23 ) . the importance of dental implantology has influenced many oro - maxilofacial surgeons to get involved in this field of maxillofacial - surgery . to do evaluation of advantages and disadvantages of the treatment with tiolox - dentaurum dental implants in cases of partial and total lack of teeth , compared to classic dentures . to analyze the cases before and after the placement of implants , at what age they undergo surgical treatment and in which cases the implantation can not be applied . to conduct this scientific research we have used the prospective method of research , analyzing cases of dental implantology which undergo the surgical treatment at the policlinic shoshi in prishtina , in a two year period , 2010 - 2011 . comparison will be done for the period from the placement of the implants until the placement of dental dentures in the policlinic shoshi , with dental implants tiolox - dentaurum , with the data from other authors . for verification of statistical importance of differences between the parameters , we have used statistical tests , verification of which will be done with the confidence scale with error for p<0.05 and p<0.01 . 29 patients that are included in the study , have been divided in two groups 1 . control group with 6 patients patients were divided into two groups : research group in which were included 23 patients in which the placement of the superstructure was done two months after the placement of the implants in the lower jaw and after three months in the upper jaw.control group this included 6 patients in which the superstructure was placed four months after the placement of the implants in the lower jaw and five months in the upper jaw . research group in which were included 23 patients in which the placement of the superstructure was done two months after the placement of the implants in the lower jaw and after three months in the upper jaw . control group this included 6 patients in which the superstructure was placed four months after the placement of the implants in the lower jaw and five months in the upper jaw . the patients were divided into two groups : research group in which were included 23 patients in which we placed tiolox - dentaurum implants , and the placement of the superstructure was done after two months in the lower jaw and after three months in the upper jaw.control group this included 6 patients in which the same type of implants was used , but the superstructure was placed after four months in the lower jaw and five months in the upper jaw . research group in which were included 23 patients in which we placed tiolox - dentaurum implants , and the placement of the superstructure was done after two months in the lower jaw and after three months in the upper jaw . control group this included 6 patients in which the same type of implants was used , but the superstructure was placed after four months in the lower jaw and five months in the upper jaw . with case distribution based on gender , out of 23 cases in the first group , or 52.2% were female , and 11 of 47.8% were male , while in the control group we had 3 females and 3 males , but without important statistical significance ( chi test= 1.734 , p=0.1879 ) . . a higher structure of 52.2% was of age 30 - 39 years , then 40 - 49 years were 30.4% and 50 - 59 years were 17.4% . with case distribution based on the group - age and gender , we have gained difference with statistically important significance ( chi test= 12.5 , p=0.0078 ) there were more females in the group - age 30 - 39years ( 58.3% vs. 45.5% ) , while men in the group - age 40 - 49 years ( 25.0% vs. 36.4% ) and 50 - 59 years ( 16.7% vs.18.2% ) ( diagram 1 ) . structure of patients based on group - age and gender . all the implants used were 3.5 mm thick , and 10 - 16 mm long . in our patients , we mostly used implants with 16 mm length , 58.1% , than 14 mm or 17.1% and 10 mm or 16.2% ( diagram 2 ) . the structure of implants used based on length . at the research group , the average time of placement of the bridges in the lower jaw was 2 months , and at the control group 4.2 months . in the upper jaw , the average time of placement of the bridges , at the research group was 3 moths and at the control group 5.4 months . rebuilding over implants in both jaws . finishing the case placing of porcelain bridges in both jaws . based on the analyzed materials , we came to the following conclusions : with the case distribution based on gender , out of 23 cases in the first group , 12 or 52.2% were females and 11 or 47.8% were male , while in the control group we had three females and three males , but without significantly important statistical differences . patients involved in the research were the age 30 - 59 years old . in a higher structure of 52.2% were of group age 30 - 39 years , then 40 - 49 years were 30.4% , and 50 - 59 years were 17.4% . with the distribution of cases based on group - age and gender , we gained significant statistically important difference ( chi test = 12,5 , p= 0,0078 ) . we had more females in the group age 30 - 39 years ( 58.3% vs.45.5% ) , while men in the group = age 40 - 49 years ( 25 , 0% vs. 36,4% ) and 50 - 59 years ( 16,7% vs. 18,2% ) . all the implants used were tiolox , 3.5 mm wide and 10 - 16 mm long . in our patients , mostly were used implants of 16 mm length , 58.1% , then 14 mm 17.1% , and 10 mm 16.2% implants based on regions , mostly were placed in the inter - canine region 58.1% and 41.9 % in the trans - canine region . at the research group , the average time of placement of bridges in the lower jaw was 2 months , ( sd 0,1 month ) , and at the control group was 4.2 months ( sd 0,5 months ) . in the upper jaw , the average time of placing the bridge at the research group was 3 months ( sd 0,1 month ) and at the control group 5.4 months ( sd 0,6 ) . at the end of this scientific study , from the cases we have worked on , we can conclude that the period after placing the implants until the placement of the prosthetics could be shortened from 4 - 6 months for the upper jaw to 3 months , and from 3 months for the lower jaw it could be shortened to 2 months , based on the coexistence of the bone and the primary stability of the implant .

introduction : the importance of the dental implantology has influenced the development of many treatment methods in this field . the aim of our study is to evaluate the advantages and disadvantages of the treatment with tiolox - dentaurum dental implants in cases of partial and total lack of teeth , compared to classic dentures . to conduct our research we have used the prospective method.material and methods : the analyzed cases have undergone surgical treatment at the policlinic shoshi in prishtina during 20102011 . this study included 29 patients , 23 in the research group and 6 in the control group . the patients involved in the study were 30 - 59 years old . 52,2% of our patients were 30 - 39 years old , while 17,4% were 50 - 59 years old.results:in the research group , the average time of placing the dental bridges in the lower jaw was 2 months and 4.2 months in the control group . in the upper jaw , the average time was 3 months for the experimental group and 5,4 months for the control group.conclusion:the period after the placement of implants until the placement of dentures , can be shortened from 4 - 6 months to 3 months for the upper jaw and from 3 to 2 months for the lower jaw .

the ever - changing safety regulations related to the drug use demands the development of dosage forms , which would be able to deliver drugs at specific sites , preferably through non - invasive routes in a rate - controlled manner . the biotechnologically produced protein drugs also need parallel delivery devices because of their inherent instability in gastrointestinal tract . presently , the pharmaceutical research is focused on the development of improved delivery routes for the existing drugs . in this context , 40% of the drug delivery candidates under clinical evaluation in united states belonged to the transdermal category . however , the route was thought to be of questionable worth for the hydrophilic drugs , as they resulted in inadequate skin permeation . stratum corneum , the outermost skin layer , a thin matrix of dehydrated dead keratinocytes ( 10 - 15 m ) , is designed by nature to minimize the trans - epidermal water loss . only small molecules , neutral and lipophilic , can pass through them in limited quantity . the permeants experience high resistive force while traversing the complicated lipophilic hydrophilic serial pathways of the skin . for most of the molecules , the skin resistance is too high to be overcome by the concentration gradient , which is the driving force of passive permeation . enhancer molecules can increase the permeation rate , but are prone to cause irreversible skin damage and irritation . physical propulsion of the drug molecules into the deeper layers of the skin was thought to be a safer alternative . the first reports of iontophoresis had appeared in the turn of the century when leduc demonstrated the technique by delivering strychnine and cyanide into rabbits in 1908 . but reports of burns and skin damages associated with the use of electric current created dubious repercussions about the utility of the process for routine administration of drugs . the interest was revived only after some 60 years by the pioneering work of a group of researchers who had shown that drug permeation could be safe and successfully enhanced by using low - intensity controlled current , bringing the electrically assisted drug delivery back into the limelight . against intravenous infusions , current - induced controlled delivery is projected to have higher patient compliance , as it is associated with minimum of histological damage and spares the recipient of psychological trauma of needle . last two decades have seen biotechnologically produced proteins , added to the repertoire of drugs , which due to their macromolecular nature are unstable and unable to pass the gastro - intestinal barrier . but , the problem looked less complicated when it was discovered that they could slip through the aqua - filled pores when repelled by an electric force . data obtained from the studies of smaller molecules also projected iontophoresis , an electrically driven delivery device , as a promising tool for routine non - invasive drug administration . needing an additional energy source , the iontophoretic devices are bound to be expensive compared to the passive patches . hence , to be commercially viable , iontophoretic delivery must have certain advantages over their passive counter parts . analysis is normally done on this aspect before selecting a candidate for the iontophoretic system development . but , fewer attempts have been undertaken to compare and evaluate the data obtained from the processes . in this review , we attempt to examine and analyze the prospects of iontophoretic research as well as potential benefits of the technique above and over that of passive permeation , with special reference to cardiovascular drugs . the source once activated generates the energy in programmed and controlled manner . since the patches are to be worn on the body , the battery that provides current should be small , convenient to wear , and inexpensive , making the challenge formidable for the system developers . fortunately , giant leaps in microelectronics have bridged that gap and made the theoretical approach a practicability . the technique works by principle of electrostatic repulsion of charges ; hence , to be of use , the drug must be in the ionic form . electric current creates a potential gradient across the skin tissue and from the drug reservoir placed below the active electrode , charged drug gets repelled . positive ions were delivered by anode and negative ions by cathode [ figure 1 ] . skin has a net negative charge at physiological ph and cations cross the skin barrier better than the anions . for anions belonging to the molecular range of 35 - 500 dalton , since the permeability of stratum corneum in the presence of electric current is altered , uncharged water - soluble molecules can also permeate the skin by electro - osmosis . as carriers of electricity , hydrophilic drug that ionizes at physiological ph gets priority . these drugs permeate through the water - filled natural pores of the skin that takes them to the root of the pores , from where systemic uptake is very rapid . since the pore diameter of human skin ( 22 - 54 ) are much wider and linear than the tortuous irregular path that run through the lipophilic matrix of the unbroken skin , the permeation is speedy and enhanced . as the iontophoretic propulsion is directly proportional to the intensity of the current , the technique is likely to ensure the programmed and reproducible delivery of drug substances , to fulfill the demand of modern therapy . schematic representation of a typical transdermal iontophoretic system showing the electromigration transport of charged cationic drug molecules ( c ) into skin commercially available passive patches carry small molecules and have evaded the interest of the iontophoretic researchers so far . the above molecules by virtue of their low molecular weight and good lipophilicity can permeate through the lipophilic skin matrix at an adequate rate without the assistance of active energy supply . the follicular area of the skin is only 0.1% , and small molecules can utilize both unbroken skin as well as the shunt pathway of the follicular route . here , the use of electricity can result in no or minimal benefit . in iontophoresis , the follicular route because of its aqueous environment is predominantly utilized , though there is a nominal contribution from the passive flux too . hence , the high - molecular - weight drug that can not accommodate into the narrow slits of the unbroken skin gets a decided advantage in the iontophoretic process . in electrically repelled drug delivery , the total permeation is the sum of iontophoresis , electro - osmosis , and passive flux ; the electrostatic repulsion being the predominant one . moreover , for the biotechnologically produced peptide drugs , iontophoresis seems to be the ideal route for non - invasive , rate controlled , and reproducible drug delivery . iontophoretic system development is a multidisciplinary research activity and can be broadly classified into several areas . while the energy supply device is a specialized microelectronic research zone , formulation development needs to address certain key issues ; the pathways of percutaneous penetration be characterized , rate limiting steps in transdermal absorption be identified ; the specific drug structure - penetration relation be understood ; and the safety factors of the process on skin be evaluated . laser scanning confocal microscopy and vibrating probe electronic technique are being utilized to ascertain the penetration mechanism of molecules . investigations on pindolol had indicated that radial mass transport is the dominant mechanism of iontophoresis . identifying the preference of skin for specific ions and structures can also optimize a formulation . measurement of skin impedance and reversibility are usually carried out to evaluate the safety factors , optimizing drug delivery . however , the key factor on which the pharmaceutical research is focused on is the enhancement of permeation , which can result in beneficial therapeutic efficacy of drugs . being a promising approach , researchers are assessing the feasibility of this technique to extract the drug molecule from the subdermal region for a noninvasive therapeutic drug monitoring . a recent market survey by cleary and beskar had shown that in terms of sales , the analgesic patch , fentanyl has reached the status of a blockbuster product with an annual turn over of 1.59 billion us dollars in the year 2002 . however , the patches belonging to the cardiovascular category were rated to be of higher clinical benefit . the major reasons could be the requirement of steady - state concentration throughout the treatment and ensure safe therapy . the only commercially available antihypertensive patch of clonidine and two antianginal patches of nitroglycerine and isosorbide dinitrate have made significant contribution in therapy in terms of reduction of cardiovascular emergencies . american heart association has endorsed the utility of transdermal nitroglycerine in acute myocardial infarction , irrespective of involvement of left ventricular failure . a retrospective analysis of the medicaid claims in two american states had shown that though the prescription expenditure of the patients using the clonidine patch was significantly higher , it saved them from hospitalization and reduced the overall health expenditure . drugs belonging to these groups are getting extensively investigated for transdermal development and both passive and energy - assisted permeation are being attempted . table 1 compares the flux values of both the processes and the benefits associated with use of electrical energy . transdermal permeation data of certain cardiovascular agents by passive and iontophoresis the ultimate challenge in passive delivery is the reproducibility of drug flux . usually , experiments are designed to achieve a target flux , calculated from the reported pharmacokinetic data using in vivo animal models . flux values obtained from passive permeation are subjected to variations by species , skin type , and skin area . similarly , electrical impedance of skin also varies widely amongst the species , individuals , as well as the location of the body parts . iontophoresis uses trans - follicular pathway and the follicle densities of animals and humans were different . tailor - made artificial skin offering resistance comparable to that of human skin is the ideal choice for the comparative study . for in vivo studies , the comparison becomes more complicated because of the different volume of distributions . exactly matching experiments were rare and permeation efficiencies of passive and active processes were compared in table 1 , by using data obtained from grossly similar experiments . flux values obtained depend upon the concentration as well as the characteristics of the donor vehicles . increase in drug supply in donor usually results in higher flux value in passive permeation , whereas iontophoretic flux is proportional to the energy supply as long as a sufficient donor concentration is maintained . since the donor concentrations used by different researchers were different , the quantitative comparison of flux values becomes meaningless and flux enhancement becomes a more appropriate parameter . in table 2 , the comparisons of the flux enhancement with that of physicochemical parameters reveal some puzzling pattern . metoprolol tartrate , the drug with highest water solubility , showed a moderate benefit of 1.8 times enhancement in iontophoresis as compared to the passive flux . this may result from its high partition coefficient that makes it accommodative into the lipophilic stratum corneum that constitutes 99.9% of the skin surface . in such cases , the contribution of the passive process in the total iontophoretic flux turns significant and the net benefit of electrical energy is moderate . in case of propranolol hydrochloride and nadolol , the drugs were delivered from special vehicles containing ion - exchange resins . for propranolol hydrochloride , , higher flux was reported through the mice skin , whereas in human the passive permeation was very low compared to the iontophoretic flux . since the drug has moderate water solubility and freebase shows high lipophilicity , enhancement effect of iontophoresis can be attributed to the assistance of the ion - exchange resins . for nadolol , the free base is much less lipophilic than propranolol and the contribution of passive permeation is likely to be less . timolol maleate has somewhat similar parameters to that of propranolol hydrochloride , but the benefits of iontophoresis were somewhat lower . however , the donor used in this experiment is plain water and there was no assistance of the ion - exchange resins . comparison of the iontophoretic flux with physicochemical parameters of certain cardiovascular agents being low - molecular - weight and water - soluble drug , atenolol qualifies as a good candidate for iontophoresis theoretically . however , the iontophoretic permeation conducted by dennet et al . showed widely variable pattern . in twenty - hour long current application , the initial permeation rate was low , and then the rate increased drastically . though a discount can be given for species variation , the average iontophoretic flux was 30% lower compared to that of a passive experiment . the results indicate that theoretically ideal candidates may not always be a best bet for iontophoretic system development . since the drug gets extensively ionized at the physiological ph and has a low octanol - water partition coefficient , the results match the theoretical expectation . optimization of the iontophoretic delivery needs standardization of various parameters , the condition of the skin , the formulation excipients , but the most important and investigated area is the mode and duration of energy supply . mode of energy supply influences the flux and hence investigation is carried out for the desired current density as well as the pattern of energy supply . switching iontophoresis , i.e. , switching of the polarity of electrodes on transdermal absorption was one of the approaches adopted . it was seen that verapamil permeation was greater at certain switching interval and permeability coefficient and cumulative amount both improved significantly . increasing the current density usually increases mass transport , but a limiting value of 0.5 ma / cm is thought to be safe and satisfactory in most of the cases . for the formulation parameters , optimization is usually done with ionic strength , ph , buffer types , special adjuvants , and the membranes incorporated in the formulation . in case of captopril , iontophoretic flux was significantly affected by factors like ph , ionic strength , drug concentration , etc . use of special adjuvants like ion exchange resins in the device is also found to be beneficial . in certain cases , manipulation of the association state and formation of the charged analogues may turn out to be a useful approach to enhance the iontophoretic delivery . formation of the insulin analogues with extra negative charges had shown beneficial results in terms of 50 to 100 times flux enhancement . skin hydration was found to be an important parameter that can enhance the permeation rate . so , efforts are also on to optimize the permeation rate by this basal treatment . use of permeation enhancers in the donor vehicle to enhance the iontophoretic flux is another approach investigated by some researchers . as electroporation and iontophoresis both have same common denominator of electro - repulsion , attempt is also being taken to enhance iontophoretic flux by decreasing the skin impedance by an initial electroporation . the source once activated generates the energy in programmed and controlled manner . since the patches are to be worn on the body , the battery that provides current should be small , convenient to wear , and inexpensive , making the challenge formidable for the system developers . fortunately , giant leaps in microelectronics have bridged that gap and made the theoretical approach a practicability . the technique works by principle of electrostatic repulsion of charges ; hence , to be of use , the drug must be in the ionic form . electric current creates a potential gradient across the skin tissue and from the drug reservoir placed below the active electrode , charged drug gets repelled . positive ions were delivered by anode and negative ions by cathode [ figure 1 ] . skin has a net negative charge at physiological ph and cations cross the skin barrier better than the anions . for anions belonging to the molecular range of 35 - 500 dalton , since the permeability of stratum corneum in the presence of electric current is altered , uncharged water - soluble molecules can also permeate the skin by electro - osmosis . as carriers of electricity , hydrophilic drug that ionizes at physiological ph these drugs permeate through the water - filled natural pores of the skin that takes them to the root of the pores , from where systemic uptake is very rapid . since the pore diameter of human skin ( 22 - 54 ) are much wider and linear than the tortuous irregular path that run through the lipophilic matrix of the unbroken skin , the permeation is speedy and enhanced . as the iontophoretic propulsion is directly proportional to the intensity of the current , the technique is likely to ensure the programmed and reproducible delivery of drug substances , to fulfill the demand of modern therapy . schematic representation of a typical transdermal iontophoretic system showing the electromigration transport of charged cationic drug molecules ( c ) into skin commercially available passive patches carry small molecules and have evaded the interest of the iontophoretic researchers so far . the above molecules by virtue of their low molecular weight and good lipophilicity can permeate through the lipophilic skin matrix at an adequate rate without the assistance of active energy supply . the follicular area of the skin is only 0.1% , and small molecules can utilize both unbroken skin as well as the shunt pathway of the follicular route . in iontophoresis , the follicular route because of its aqueous environment is predominantly utilized , though there is a nominal contribution from the passive flux too . hence , the high - molecular - weight drug that can not accommodate into the narrow slits of the unbroken skin gets a decided advantage in the iontophoretic process . in electrically repelled drug delivery , the total permeation is the sum of iontophoresis , electro - osmosis , and passive flux ; the electrostatic repulsion being the predominant one . moreover , for the biotechnologically produced peptide drugs , iontophoresis seems to be the ideal route for non - invasive , rate controlled , and reproducible drug delivery . iontophoretic system development is a multidisciplinary research activity and can be broadly classified into several areas . while the energy supply device is a specialized microelectronic research zone , formulation development needs to address certain key issues ; the pathways of percutaneous penetration be characterized , rate limiting steps in transdermal absorption be identified ; the specific drug structure - penetration relation be understood ; and the safety factors of the process on skin be evaluated . laser scanning confocal microscopy and vibrating probe electronic technique are being utilized to ascertain the penetration mechanism of molecules . investigations on pindolol had indicated that radial mass transport is the dominant mechanism of iontophoresis . identifying the preference of skin for specific ions and structures can also optimize a formulation . measurement of skin impedance and reversibility are usually carried out to evaluate the safety factors , optimizing drug delivery . however , the key factor on which the pharmaceutical research is focused on is the enhancement of permeation , which can result in beneficial therapeutic efficacy of drugs . being a promising approach , researchers are assessing the feasibility of this technique to extract the drug molecule from the subdermal region for a noninvasive therapeutic drug monitoring . a recent market survey by cleary and beskar had shown that in terms of sales , the analgesic patch , fentanyl has reached the status of a blockbuster product with an annual turn over of 1.59 billion us dollars in the year 2002 . however , the patches belonging to the cardiovascular category were rated to be of higher clinical benefit . the major reasons could be the requirement of steady - state concentration throughout the treatment and ensure safe therapy . the only commercially available antihypertensive patch of clonidine and two antianginal patches of nitroglycerine and isosorbide dinitrate have made significant contribution in therapy in terms of reduction of cardiovascular emergencies . american heart association has endorsed the utility of transdermal nitroglycerine in acute myocardial infarction , irrespective of involvement of left ventricular failure . a retrospective analysis of the medicaid claims in two american states had shown that though the prescription expenditure of the patients using the clonidine patch was significantly higher , it saved them from hospitalization and reduced the overall health expenditure . drugs belonging to these groups are getting extensively investigated for transdermal development and both passive and energy - assisted permeation are being attempted . table 1 compares the flux values of both the processes and the benefits associated with use of electrical energy . transdermal permeation data of certain cardiovascular agents by passive and iontophoresis the ultimate challenge in passive delivery is the reproducibility of drug flux . usually , experiments are designed to achieve a target flux , calculated from the reported pharmacokinetic data using in vivo animal models . flux values obtained from passive permeation are subjected to variations by species , skin type , and skin area . similarly , electrical impedance of skin also varies widely amongst the species , individuals , as well as the location of the body parts . iontophoresis uses trans - follicular pathway and the follicle densities of animals and humans were different . tailor - made artificial skin offering resistance comparable to that of human skin is the ideal choice for the comparative study . for in vivo studies , the comparison becomes more complicated because of the different volume of distributions . exactly matching experiments were rare and permeation efficiencies of passive and active processes flux values obtained depend upon the concentration as well as the characteristics of the donor vehicles . increase in drug supply in donor usually results in higher flux value in passive permeation , whereas iontophoretic flux is proportional to the energy supply as long as a sufficient donor concentration is maintained . since the donor concentrations used by different researchers were different , the quantitative comparison of flux values becomes meaningless and flux enhancement becomes a more appropriate parameter . in table 2 , the comparisons of the flux enhancement with that of physicochemical parameters reveal some puzzling pattern . metoprolol tartrate , the drug with highest water solubility , showed a moderate benefit of 1.8 times enhancement in iontophoresis as compared to the passive flux . this may result from its high partition coefficient that makes it accommodative into the lipophilic stratum corneum that constitutes 99.9% of the skin surface . in such cases , the contribution of the passive process in the total iontophoretic flux turns significant and the net benefit of electrical energy is moderate . in case of propranolol hydrochloride and nadolol , the drugs were delivered from special vehicles containing ion - exchange resins . for propranolol hydrochloride , two passive permeation data vary widely indicating the effect of species variation . as usual , higher flux was reported through the mice skin , whereas in human the passive permeation was very low compared to the iontophoretic flux . since the drug has moderate water solubility and freebase shows high lipophilicity , enhancement effect of iontophoresis can be attributed to the assistance of the ion - exchange resins . for nadolol , the free base is much less lipophilic than propranolol and the contribution of passive permeation is likely to be less . timolol maleate has somewhat similar parameters to that of propranolol hydrochloride , but the benefits of iontophoresis were somewhat lower . however , the donor used in this experiment is plain water and there was no assistance of the ion - exchange resins . comparison of the iontophoretic flux with physicochemical parameters of certain cardiovascular agents being low - molecular - weight and water - soluble drug , atenolol qualifies as a good candidate for iontophoresis theoretically . however , the iontophoretic permeation conducted by dennet et al . showed widely variable pattern . in twenty - hour long current application , the initial permeation rate was low , and then the rate increased drastically . though a discount can be given for species variation , the average iontophoretic flux was 30% lower compared to that of a passive experiment . the results indicate that theoretically ideal candidates may not always be a best bet for iontophoretic system development . since the drug gets extensively ionized at the physiological ph and has a low octanol - water partition coefficient , the results match the theoretical expectation . optimization of the iontophoretic delivery needs standardization of various parameters , the condition of the skin , the formulation excipients , but the most important and investigated area is the mode and duration of energy supply . mode of energy supply influences the flux and hence investigation is carried out for the desired current density as well as the pattern of energy supply . switching iontophoresis , i.e. , switching of the polarity of electrodes on transdermal absorption was one of the approaches adopted . it was seen that verapamil permeation was greater at certain switching interval and permeability coefficient and cumulative amount both improved significantly . increasing the current density usually increases mass transport , but a limiting value of 0.5 ma / cm is thought to be safe and satisfactory in most of the cases . for the formulation parameters , optimization is usually done with ionic strength , ph , buffer types , special adjuvants , and the membranes incorporated in the formulation . in case of captopril , iontophoretic flux was significantly affected by factors like ph , ionic strength , drug concentration , etc . use of special adjuvants like ion exchange resins in the device is also found to be beneficial . in certain cases , manipulation of the association state and formation of the charged analogues may turn out to be a useful approach to enhance the iontophoretic delivery . formation of the insulin analogues with extra negative charges had shown beneficial results in terms of 50 to 100 times flux enhancement . skin hydration was found to be an important parameter that can enhance the permeation rate . so , efforts are also on to optimize the permeation rate by this basal treatment . use of permeation enhancers in the donor vehicle to enhance the iontophoretic flux is another approach investigated by some researchers . as electroporation and iontophoresis both have same common denominator of electro - repulsion , attempt is also being taken to enhance iontophoretic flux by decreasing the skin impedance by an initial electroporation . drug delivery is not the only area where iontophoresis is thought to be of practical use . reverse iontophoresis is used in the non - invasive monitoring of the glucose level for diabetics . it is getting investigated for the development of a diagnostic test for the determination of phenylketonuria , a potentially fatal metabolic disease in infants . though the opponents of iontophoresis argue that electroporation can be a better substitute for active drug delivery rather than iontophoresis , the technique fares better in terms of both safety and efficacy . it also shows more promise than the micro - conduit technology , which uses the targeted impact of high - energy micro - particles on specified areas on skin surface to create pores for non - invasive sampling of analyte as well as drug administration . the electroporation technique requiring much higher voltage , i.e. , 100 - 1000v , can give rise to hazardous side effects and is likely to be a component of specialized healthcare zone , while the hi - tech micro - conduits technology is still in its infancy . on the other hand , iontophoretic systems involving low level of electricity can be constructed with a 9-volt button cell and can be easily commercialized . cardiovascular agents because of their demand for chronic administration have already had an assured market for transdermal products . the advanced state of research in this field indicates that iontophoretic administration of such drugs is likely to be a reality in the near future .

clinical benefits , industry interest , regulatory precedence , and strong market potential have made transdermal research the fastest growth area in drug delivery . as most drugs permeate poorly through skin , a major challenge is achieving the therapeutic level by enhancement of permeation rate . iontophoresis , utilizing a minimal amount of current , is found to affect the skin permeation process drastically . ideally suited for protein drugs , attempts have been made to utilize the technology for accelerating the low - molecular - weight drugs for chronic administration . however , because of the difficulty associated with the energy supply , commercialization was not feasible until recent times . fortunately , the unprecedented growth of microelectronics has bridged this lacuna and brought the technology right into limelight . this article analyses the advantages of electrically assisted drug delivery in relation to passive permeation , with special reference to some cardiovascular drugs , for which there is already a demand in the market .

as a result of recent , revolutionary developments in endovascular treatment , self - expanding stent - assisted coiling ( sac ) is now widely accepted for wide - neck or complex intracranial aneurysms , which are technically challenging for conventional coil embolization.5)9 ) sac has various advantages . for example , it creates a barrier which can prevent coil herniation into the parent vessel lumen , facilitates increased overall coil packing density,16 ) and provides flow diversion . currently , two self - expanding closed - cell stents , the enterprise vrd stent ( codman , miami , fl , usa ) and the solitaire ab stent ( covidien , irvine , ca , usa ) , are widely used for sac of wide - neck or complex intracranial aneurysms . several studies have reported results relevant to each stent , including clinical and angiographic outcomes , procedural feasibility , and complications.4)5)12)16)18 ) however , few studies have compared the results of enterprise and solitaire sac for wide - neck or complex intracranial aneurysms . the purpose of this study was to compare enterprise and solitaire sac in terms of several clinical findings and the related follow - up results . between january 2012 and march 2014 , 94 patients with 98 aneurysms underwent sac of intracranial aneurysms at our institutions . nine of the aneurysms were treated with neuroform sac and were therefore excluded from this study . the remaining 86 patients ( male : female ratio , 19:67 ; age range , 41 - 89 years ; mean age , 60.3 years ) harbored 89 aneurysms , which were treated with enterprise ( 57 aneurysms ) or solitaire ( 32 aneurysms ) sac . sac was performed for aneurysms with a neck width of 4 mm or a dome - to - neck ratio of < 2 , as well as for broad - based aneurysms that were not approachable via conventional coiling . the stent device employed for sac was determined according to the endovascular neurosurgeon 's preferences , with attention to the easiness of manipulation , delivery , and deployment . this study was approved by our institution 's review board and informed consent was obtained from all patients included in the study . in addition to clinical presentation and patient demographics , data were collected on the aneurysm dimensions , location , morphology ( saccular , blister - like , or dissecting aneurysms ) , parent artery diameter and tortuosity ( graded qualitatively by the treating endovascular neurosurgeon : mild , moderate , or severe ) , description of the stent , delivery and deployment failure of the stent , presence of coil protrusion into the parent artery , thromboembolic events , periprocedural morbidity and mortality , and immediate postoperative and follow - up angiographic results from digital subtraction angiography with 3-dimensional rotational imaging . immediate postoperative and follow - up angiographic results , including degree of aneurysm occlusion and presence of in - stent stenosis or in - stent thrombosis , were assessed according to the raymond - roy classification as follows : class i ( no filling of the aneurysm or dome ) , class ii ( residual filling of the neck , but not the dome ) , and class iii ( residual filling of the neck and dome ) . clinical outcomes at discharge and follow - up were determined using the modified rankin scale ( mrs).17 ) all data were reviewed retrospectively . the modality of treatment was decided by a neurosurgeon and an endovascular neurosurgeon after considering the patient 's co - morbidities , clinical status , and surgical feasibility in an interdisciplinary discussion . patients were pre - medicated with dual antiplatelet agents : 75 mg of clopidogrel and 100 mg of aspirin daily for seven days before sac . , patients were not preloaded with antiplatelet agents and heparin coagulation was delayed until placement of the framing coil . the activated clotting time sac was performed using the " jailing " or " semi - jailing " technique in all cases.11 ) during stent deployment , we most often performed microcatheter - pullback and microwire - push techniques sequentially in order to optimize stent deployment and minimize stent malposition.6 ) in cases involving coil loop herniation into the parent artery , the position of the coil loop between the parent artery and the stent was confirmed using 3-dimensional digital subtraction angiography . after treatment , all patients were prescribed a dual antiplatelet regimen including 75 mg / day of clopidogrel and 100 mg / day of aspirin for 6 - 12 months , after which they were prescribed only 100 mg / day of aspirin for the rest of their lives . during follow - up , plain images of the skull with the same working projection images of sac were examined every three months . at 12 months after sac , cerebral angiography and magnetic - resonance angiography were performed to evaluate the degree of aneurysm occlusion , in - stent stenosis , stent migration , and any need for retreatment . the pearson chi - square and fisher exact tests were used for assessment of between - group differences for demographic and clinical characteristics . the student t - test was performed for evaluation of the statistical significance of differences in continuous parameters . statistical significance was set at a p - value of less than 0.05 using spss ver . between january 2012 and march 2014 , 94 patients with 98 aneurysms underwent sac of intracranial aneurysms at our institutions . nine of the aneurysms were treated with neuroform sac and were therefore excluded from this study . the remaining 86 patients ( male : female ratio , 19:67 ; age range , 41 - 89 years ; mean age , 60.3 years ) harbored 89 aneurysms , which were treated with enterprise ( 57 aneurysms ) or solitaire ( 32 aneurysms ) sac . sac was performed for aneurysms with a neck width of 4 mm or a dome - to - neck ratio of < 2 , as well as for broad - based aneurysms that were not approachable via conventional coiling . the stent device employed for sac was determined according to the endovascular neurosurgeon 's preferences , with attention to the easiness of manipulation , delivery , and deployment . this study was approved by our institution 's review board and informed consent was obtained from all patients included in the study . in addition to clinical presentation and patient demographics , data were collected on the aneurysm dimensions , location , morphology ( saccular , blister - like , or dissecting aneurysms ) , parent artery diameter and tortuosity ( graded qualitatively by the treating endovascular neurosurgeon : mild , moderate , or severe ) , description of the stent , delivery and deployment failure of the stent , presence of coil protrusion into the parent artery , thromboembolic events , periprocedural morbidity and mortality , and immediate postoperative and follow - up angiographic results from digital subtraction angiography with 3-dimensional rotational imaging . immediate postoperative and follow - up angiographic results , including degree of aneurysm occlusion and presence of in - stent stenosis or in - stent thrombosis , were assessed according to the raymond - roy classification as follows : class i ( no filling of the aneurysm or dome ) , class ii ( residual filling of the neck , but not the dome ) , and class iii ( residual filling of the neck and dome ) . clinical outcomes at discharge and follow - up were determined using the modified rankin scale ( mrs).17 ) all data were reviewed retrospectively . the modality of treatment was decided by a neurosurgeon and an endovascular neurosurgeon after considering the patient 's co - morbidities , clinical status , and surgical feasibility in an interdisciplinary discussion . patients were pre - medicated with dual antiplatelet agents : 75 mg of clopidogrel and 100 mg of aspirin daily for seven days before sac . , patients were not preloaded with antiplatelet agents and heparin coagulation was delayed until placement of the framing coil . the activated clotting time sac was performed using the " jailing " or " semi - jailing " technique in all cases.11 ) during stent deployment , we most often performed microcatheter - pullback and microwire - push techniques sequentially in order to optimize stent deployment and minimize stent malposition.6 ) in cases involving coil loop herniation into the parent artery , the position of the coil loop between the parent artery and the stent was confirmed using 3-dimensional digital subtraction angiography . after treatment , all patients were prescribed a dual antiplatelet regimen including 75 mg / day of clopidogrel and 100 mg / day of aspirin for 6 - 12 months , after which they were prescribed only 100 mg / day of aspirin for the rest of their lives . during follow - up , plain images of the skull with the same working projection images of sac were examined every three months . at 12 months after sac , cerebral angiography and magnetic - resonance angiography were performed to evaluate the degree of aneurysm occlusion , in - stent stenosis , stent migration , and any need for retreatment . the pearson chi - square and fisher exact tests were used for assessment of between - group differences for demographic and clinical characteristics . the student t - test was performed for evaluation of the statistical significance of differences in continuous parameters . statistical significance was set at a p - value of less than 0.05 using spss ver . eighty - nine aneurysms in 86 patients qualified for treatment with sac ( 57 in the enterprise stent group and 32 in the solitaire stent group ) . the mean age of patients was 60.3 years ( range , 41 - 89 years ) . headache was the most common symptom of aneurysms at presentation . in the enterprise group , the locations of aneurysms were as follows : cavernous segment ( n = 4 ) , paraclinoid ( n = 32 ) , supraclinoid ( n = 10 ) , posterior communicating artery ( n = 3 ) , distal internal carotid artery ( n = 1 ) , anterior cerebral artery ( n = 1 ) , and vertebral artery ( n = 6 ) . in the solitaire group , aneurysms were found in the cavernous segment ( n = 1 ) , paraclinoid ( n = 25 ) , supraclinoid ( n = 1 ) , and vertebral artery ( n = 5 ) . seventy - eight ( 87.6% ) of the aneurysms were located in the anterior circulation and 11 ( 12.4% ) were located in the posterior circulation . the mean aneurysm size was 5.69 mm ( range , 2.4 - 20 mm ) in the enterprise group and 5.95 mm ( range , 2.9 - 17 mm ) in the solitaire group . in the enterprise group , one aneurysm was fusiform , two had a blister - like shape , and 54 were saccular . in the solitaire group , six aneurysms were fusiform , one had a blister - like shape , and 25 were saccular . of the sac - treated aneurysms , nine were ruptured ( 10.1% ) , six in the enterprise group and three in the solitaire group . the mean diameter of the parent artery was 3.41 mm ( range , 2.5 - 4.63 mm ) in the enterprise group and 3.81 mm ( range , 2.8 - 5.52 mm ) in the solitaire group . in the enterprise group , 47 aneurysms had mild or moderate parent artery tortuosity and 12 had severe tortuosity . in the solitaire group , 25 aneurysms had mild or moderate and seven had severe tortuosity . there were no cases of stent navigation or deployment failure in either stent group , and there was no occurrence of arterial dissection and intraoperative aneurysmal rupture . we observed three cases ( 3.37% ) of thromboembolic events , all of which occurred in ruptured aneurysms ( enterprise group , n = 2 ; solitaire group , n = 1 ) . however , each case had reverted to its baseline by the last follow - up visit . coil loop or tail protrusion into the parent artery was observed in 17 cases ( 29.8% ) in the enterprise group and seven cases ( 21.9% ) in the solitaire group . no difference in terms of the in - stent thrombosis or stenosis rate , thromboembolic event rate , or overall clinical outcomes was observed between the two stent groups ( table 2 ) . on immediate postoperative angiograms , the occlusion grades in the enterprise group were as follows : class i in 21 ( 36.8% ) aneurysms , class ii in 23 ( 40.4% ) aneurysms , and class iii in 13 ( 22.8% ) aneurysms . in the solitaire group , class i was observed in 16 ( 50% ) aneurysms , class ii in 14 ( 43.8% ) aneurysms , and class iii in two ( 6.2% ) aneurysms . angiographic follow - up imaging was available for 86 aneurysms ( 96.6% ; 55 in the enterprise group and 31 in the solitaire group ) with a mean follow - up period of 12.8 months . on follow - up angiograms , the occlusion grade in the enterprise group was as follows : class i in 32 ( 58.2% ) aneurysms , class ii in 19 ( 34.5% ) aneurysms , and class iii in four ( 7.3% ) aneurysms . in the solitaire group , class i occlusion was found in 25 ( 80.7% ) aneurysms and class ii occlusion in six ( 19.3% ) aneurysms . there were no cases of in - stent stenosis or stent migration , and there was no need for retreatment in either stent group . regarding clinical results , most patients achieved a mrs score of 0 within a mean follow - up period of 20.8 months ( enterprise group , 21 months ; solitaire group , 20.5 months ) however , the two ruptured cases in the enterprise group had mild disability with mrs scores of 2 . no statistically significant difference in terms of clinical ( p = 1.0 ) or angiographic results ( p = 0.184 ) was observed between the two stent groups ( table 3 , 4 ) . eighty - nine aneurysms in 86 patients qualified for treatment with sac ( 57 in the enterprise stent group and 32 in the solitaire stent group ) . the mean age of patients was 60.3 years ( range , 41 - 89 years ) . headache was the most common symptom of aneurysms at presentation . in the enterprise group , the locations of aneurysms were as follows : cavernous segment ( n = 4 ) , paraclinoid ( n = 32 ) , supraclinoid ( n = 10 ) , posterior communicating artery ( n = 3 ) , distal internal carotid artery ( n = 1 ) , anterior cerebral artery ( n = 1 ) , and vertebral artery ( n = 6 ) . in the solitaire group , aneurysms were found in the cavernous segment ( n = 1 ) , paraclinoid ( n = 25 ) , supraclinoid ( n = 1 ) , and vertebral artery ( n = 5 ) . seventy - eight ( 87.6% ) of the aneurysms were located in the anterior circulation and 11 ( 12.4% ) were located in the posterior circulation . the mean aneurysm size was 5.69 mm ( range , 2.4 - 20 mm ) in the enterprise group and 5.95 mm ( range , 2.9 - 17 mm ) in the solitaire group . in the enterprise group , one aneurysm was fusiform , two had a blister - like shape , and 54 were saccular . in the solitaire group , six aneurysms were fusiform , one had a blister - like shape , and 25 were saccular . of the sac - treated aneurysms , nine were ruptured ( 10.1% ) , six in the enterprise group and three in the solitaire group . the mean diameter of the parent artery was 3.41 mm ( range , 2.5 - 4.63 mm ) in the enterprise group and 3.81 mm ( range , 2.8 - 5.52 mm ) in the solitaire group . in the enterprise group , 47 aneurysms had mild or moderate parent artery tortuosity and 12 had severe tortuosity . in the solitaire group , there were no cases of stent navigation or deployment failure in either stent group , and there was no occurrence of arterial dissection and intraoperative aneurysmal rupture . we observed three cases ( 3.37% ) of thromboembolic events , all of which occurred in ruptured aneurysms ( enterprise group , n = 2 ; solitaire group , n = 1 ) . however , each case had reverted to its baseline by the last follow - up visit . coil loop or tail protrusion into the parent artery was observed in 17 cases ( 29.8% ) in the enterprise group and seven cases ( 21.9% ) in the solitaire group . no difference in terms of the in - stent thrombosis or stenosis rate , thromboembolic event rate , or overall clinical outcomes was observed between the two stent groups ( table 2 ) . on immediate postoperative angiograms , the occlusion grades in the enterprise group were as follows : class i in 21 ( 36.8% ) aneurysms , class ii in 23 ( 40.4% ) aneurysms , and class iii in 13 ( 22.8% ) aneurysms . in the solitaire group , class i was observed in 16 ( 50% ) aneurysms , class ii in 14 ( 43.8% ) aneurysms , and class iii in two ( 6.2% ) aneurysms . angiographic follow - up imaging was available for 86 aneurysms ( 96.6% ; 55 in the enterprise group and 31 in the solitaire group ) with a mean follow - up period of 12.8 months . on follow - up angiograms , the occlusion grade in the enterprise group was as follows : class i in 32 ( 58.2% ) aneurysms , class ii in 19 ( 34.5% ) aneurysms , and class iii in four ( 7.3% ) aneurysms . in the solitaire group , class i occlusion was found in 25 ( 80.7% ) aneurysms and class ii occlusion in six ( 19.3% ) aneurysms . there were no cases of in - stent stenosis or stent migration , and there was no need for retreatment in either stent group . regarding clinical results , most patients achieved a mrs score of 0 within a mean follow - up period of 20.8 months ( enterprise group , 21 months ; solitaire group , 20.5 months ) however , the two ruptured cases in the enterprise group had mild disability with mrs scores of 2 . no statistically significant difference in terms of clinical ( p = 1.0 ) or angiographic results ( p = 0.184 ) was observed between the two stent groups ( table 3 , 4 ) . self - expandable closed - cell stents , such as the enterprise and solitaire , have been used with increasing frequency because of their excellent navigability , flexibility , and high rates of successful deployment.2)4)7)18 ) they are retrievable and can be repositioned even after full deployment , particularly when using the solitaire stent.7)9 ) previous studies reported excellent results of stent navigation and deployment in both stent groups ( success rate : 97.1% for enterprise stents and 98.1% for solitaire stents).1)2)9)10)13)15)16)17)18)19 ) among our cases , there were no failures or difficulties in stent navigation or deployment . clinical and angiographic outcomes did not show significant correlation with patient age , patient sex , aneurysm size , or aneurysm type . several studies have reported good initial and follow - up angiographic results in both stent groups . in long - term angiographic results , fargen et al . reported a > 90% rate of occlusion in 81% of 229 patients who underwent enterprise sac.4 ) examining postoperative angiographic results , klisch et al . observed a > 90% rate of occlusion in 94% of 53 patients harboring anterior and posterior circulation aneurysms treated with solitaire sac.12)13 ) in our study , the immediate postoperative and follow - up angiographic results showed no flow or minimal flow into the neck in 83% ( enterprise group , 77.2% ; solitaire group , 93.8% ) and 95.3% ( enterprise group , 92.7% ; solitaire group , 100% ) of aneurysms treated with sac . reported that 36.1% of 84 aneurysms followed in a sac group showed progressive occlusion on angiographic follow - up imaging.10 ) this observation further supports the advantages of sac , as it facilitates increased packing density and thrombosis , as well as providing flow - diversion and a framework for endothelialization . at first , the occlusion grades of aneurysms in the solitaire group appeared to be superior to those in the enterprise group . however , this observation may have been associated with our initial clinical inexperience when primarily using the enterprise stent , and the associated learning curve . alternatively , this may reflect the relatively poorer vessel wall apposition of the enterprise stent , as compared with the solitaire stent.3)11)14 ) however , there were no significant differences in immediate postoperative ( p = 0.054 ) or follow - up angiographic results ( p = 0.184 ) , and there were no cases of in - stent thrombosis / stenosis or stent migration . in our study , both stent groups showed good immediate postoperative and follow - up clinical outcomes , a finding consistent with previous reports.17 ) only 2 patients did not achieve a mrs score of 0 . both of these patients presented with subarachnoid hemorrhage in the enterprise group and achieved a mrs score of 2 . reported a permanent morbidity rate of 2.8% and a mortality rate of 2% in a study of 141 cases treated with enterprise sac.19 ) heller and malek reported a 0% 6-month follow - up permanent morbidity rate and a 0% mortality rate in their study of 62 patients treated with solitaire sac.6 ) in the current study , there were no significant differences between the immediate postoperative ( p = 0.213 ) or follow - up clinical outcomes ( p = 1.000 ) in the 2 stent groups . coil loop or tail protrusion into the parent artery ( 24 cases , 26.9% ) was observed more frequently in our study cohort than in the series by mocco et al . ( 9.9% ) , probably because our study included a very small partial coil loop and tail herniation into the parent artery.18 ) of 24 aneurysms with coil protrusion ( enterprise group , n = 17 ; solitaire group , n = 7 ) , 21 were located in the paraclinoid internal carotid artery and 3 were located in the vertebral artery . although these events presented more frequently when the parent artery had a relatively larger diameter or severe vessel tortuosity , the differences were not statistically significant ( enterprise group , p = 0.052 ; solitaire group , p = 0.540 ) ( table 5 , 6 ) . in an in vitro study of curved vascular models with simulated aneurysm necks , ebrahimi et al . pointed out that enterprise stents showed an increasing trend to flatten and kink with vascular curvatures that were more acute.3 ) this phenomenon may contribute to adverse effects , such as in - stent thrombosis / stenosis or insufficient buttress resulting in coil herniation into the parent artery . as such , we can assume that this phenomenon caused the relatively lower initial occlusion grade of aneurysms in the enterprise group , as compared with those in the solitaire group . heller and malek , who noted the important role of the delivery technique when employing a enterprise stent,6 ) argued that incomplete stent apposition could be minimized in both inner and outer vascular curves by deploying the stent using a dynamic push - pull technique designed to keep the delivery microcatheter centered during deployment . we also used this technique in most cases . according to data on the functional and physical properties of self - expanding intracranial stents , the solitaire stent ( 0.0106 n / mm ) has approximately 30% more radial strength than the enterprise stent ( 0.0082 n / mm).12)14 ) in evaluations with 3/4-mm tubes having a 3.9/4.4-mm radius , krischek et al . reported that the vessel wall apposition of the solitaire stent is similar to that of the neuroform stent , but better than that of the enterprise stent.8)14 ) these observations may support the idea that the solitaire stent is superior to the enterprise considering the role of the buttress in preventing coil herniation , particularly for paraclinoid aneurysms with tortuous vessel curvature or a relatively large parent artery . krischek et al.14 ) also mentioned that the area of the solitaire stent cell is larger than that of the enterprise stent . although an increased cell area may cause coil protrusion into the parent artery , this did not occur in our series . regardless , we observed no statistically significant correlation between coil herniation into the parent artery and clinical or angiographic outcomes in the stent groups . nonetheless , due to the limitations of our study , including a retrospective design , the limited number of patients , and results of long - term follow - up were lacking , some caution is warranted when interpreting our results . the results of our study suggest that patients receiving enterprise and solitaire sac for wide - neck intracranial aneurysms show excellent and comparable clinical and angiographic outcomes . because of its higher radial strength and better vessel wall apposition , we cautiously propose that the solitaire stent may be more effective than the enterprise stent for sac of aneurysms harboring a large or severe tortuous parent artery . more extensive follow - up and a larger patient cohort would be necessary in order to establish any definite conclusions .

objectivethe purpose of this study was to compare clinical findings and outcomes of enterprise and solitaire stent - assisted coiling ( sac).materials and methodsbetween january 2012 and march 2014 , 86 patients ( mean age , 60.3 years ) harboring 89 aneurysms were treated with enterprise ( n = 57 ) or solitaire ( n = 32 ) sac . the patients ' demographics , angiographic results , and clinical outcomes were reviewed retrospectively.resultsthere were no cases of stent navigation , deployment failure , arterial dissection , or intraoperative aneurysmal rupture . angiographic follow - up imaging was available for 86 ( 96.6% ) aneurysms ( enterprise group , n = 55 ; solitaire group , n = 31 ) . immediate postoperative and follow - up angiographic results showed no flow or only minimal flow into the neck in 83% ( enterprise group , 77.2% ; solitaire group , 93.8% ) and 95.3% ( enterprise group , 92.7% ; solitaire group , 100% ) of sac - treated aneurysms , respectively . both stent groups showed good immediate postoperative and follow - up clinical outcomes . excepting 2 cases , all patients achieved modified rankin scale scores of 0 . coil loop or tail protrusion into the parent artery was observed in 17 ( 29.8% ) and 7 ( 21.9% ) cases in the enterprise and solitaire groups , respectively . no statistically significant difference in terms of angiographic results or clinical outcomes was observed between the groups.conclusionexcellent and comparable clinical and angiographic outcomes for wide - neck intracranial aneurysms were achieved using both stents . because of its higher radial strength and better vessel wall apposition , we cautiously propose that the solitaire stent may be more effective for sac of aneurysms harboring a large or severe tortuous parent artery .

arteriovenous fistulas ( avfs ) are anomalous connections between the arterial and the venous systems without an intervening capillary network . renal avfs are often asymptomatic , but can also present with hematuria , pain , hydronephrosis , and heart failure . percutaneous interventions and penetrating trauma cause most renal avfs , although they can rarely be congenital or idiopathic . transcatheter embolization therapy represents a minimally invasive method that can treat these lesions and potentially save the kidney . however , this technique has unique challenges , most notably the risk of embolic material migration to the pulmonary circulation , especially with high - flow fistulas , which necessitates the usage of complex preventative measures and hardware . this article describes a novel endovascular technique for treating high - flow avfs solely using 035 and 018 interlock detachable coils ( idcs ) ( boston scientific corporation , natick , ma , usa ) without the use of flow modulation . idcs are platinum coils with thrombogenic fibers preloaded in a sheath with a coil pusher . a mechanical lock between the junction of the coil and the pusher allows the coil to be retracted as long as the interlock connection is within the delivery sheath . the 035 idc has the same mechanism of deployment but with a larger diameter than the 018 coils , and this results in more stability during the deployment . the denser polyester fabric in the 035 coils promotes rapid thrombosis of the lumen and improved anchorage . a 45-year - old man with a past medical history of refractory hypertension presented with a progressively enlarging right renal avf , which was incidentally discovered a year prior on a computed tomography ( ct ) scan of the abdomen , and demonstrated interval growth on the follow - up ct scan from a maximum diameter of 21 mm to 33 mm . considering the increase in size , however , the poor general medical condition secondary to non - ischemic cardiomyopathy precluded the option of total nephrectomy . ct angiogram ( cta ) showed an enlarged main renal and posterior segment artery which fed a tortuous fistulous tract having many fusiform dilatations ; the maximum diameter of the tract measured 33 mm [ figure 1 ] . following an angiogram performed for selective embolization of the renal avf , access to the right common femoral artery was obtained and a 5-french ( f ) , 45-cm arterial sheath ( ansel sheath , cook , bloomington , in , usa ) was placed with its tip in the infrarenal aorta . the right renal angiogram revealed a large posterior segment branch , measuring up to 8 mm in diameter , supplying a tortuous fistulous tract and arising distal to the origin of the segmental branches [ figure 2a ] . the maximum diameter of the aneurysmal portion of the fistulous tract measured 33 mm and was situated immediately distal to the arteriovenous communication . the posterior segment artery was selected using a cobra catheter over a 035 glide wire . the arterial sheath was further advanced over a super stiff wire into the main right renal artery for better support . a 3-f microcatheter having an inner diameter of 0.021 inches ( renegade , boston scientific corporation ) was advanced coaxially through the cobra catheter over a 018 wire ( transcend wire , boston scientific corporation ) into the fistula . initially , a 018 14 mm 30 cm idc , which was the largest coil available at the time , was advanced through the microcatheter and partially deployed in the feeding artery . however , the coil was unstable in its position and prolapsed into the aneurysmal fistulous tract ; the coil was subsequently withdrawn . the microcatheter was removed and a 035 50 mm 40 cm idc was advanced through the cobra catheter into the feeding artery while maintaining a saline flush . the coil was deployed cautiously over 5 min . once the loops were stable , the idc was detached [ figure 2c ] . the 3-f microcatheter was reinserted coaxially through the cobra catheter over a 018 transcend wire into the feeding artery . then , a 018 10 mm 30 cm fibered idc was advanced through the microcatheter , and loops of the coil were deployed proximally in the feeding artery using the previously deployed coil as an anchor . further coiling of the feeding artery was performed by a 018 10 mm 30 cm and followed by seven 018 10 mm 20 cm . final angiogram demonstrated non - filling of the avf with preservation of the remaining segmental branches . a nephrogram was noted in the final angiogram , which was not present in the initial angiogram , indicating redirected arterial flow from the avf toward the renal parenchyma [ figure 3 ] . follow - up cta obtained 6 months after the embolization demonstrated occlusion of the fistulous tract . focal parenchymal atrophy in the posterior cortex corresponded to the vascular territory of the occluded segment . ( a ) coronal multiplanar reformatted image shows dilated posterior segment artery ( white arrow ) and tortuous fistula tract ( black arrow ) . ( b ) coronal maximal intensity projection image shows enlarged main renal artery , tortuous fistula tract with aneurysmal dilatations ( black arrow ) , and dilated right renal vein ( open white arrow ) ( a ) angiographic image from injection into main renal artery shows the dilated fistulous tract arising from the enlarged posterior segmental artery ( black arrow ) and upper ( open black arrow ) as well as lower ( white arrow ) segmental branches arising proximally . ( b ) selective angiogram of the posterior segmental artery shows an aneurysmal dilatation of the proximal venous outflow tract ( black arrow ) , measuring up to 8.8 mm . ( c ) angiogram from the main renal artery injection after the successful deployment of the first 035 idc in the feeding posterior segmental artery , just proximal to the fistulous track angiographic image after packing several 018 coils proximal to the 035 coil ( black arrow ) in the posterior segmental renal artery shows cessation of distal flow and redirection of flow in the other segmental branches which were not present previously . a nephrogram is apparent with a minimal defect ( white arrow ) at the mid pole corresponding to the territory of the embolized branch the risk of distal embolization into the pulmonary circulation looms large in high - flow avfs and is compounded by the high flow velocity and flow - related ectasia of the fistulous tract along the venous end that prevents proper anchoring of the embolic agents . therefore , while treating these large avfs , various complex techniques involving distal embolization protection , multiple sessions , and combined arterial and venous caliber of the vascular access have been adopted in the past that merit comparison with this new technique . in one such instance , idowu et al . reported treating a large idiopathic renal avf with a tract diameter of 2.2 cm using a 16-mm amplatzer vascular plug ( avp ) ( aga medical , golden valley , mn , usa ) by a combined femoral artery and vein approach . combined deployment occlusion balloons proximal and distal to a fistula have been used in the past in order to achieve flow control in renal avfs . the combination of packing of the tract with guglielmi coils followed by the injection of n - methyl cyanoacrylate glue to occlude any residual flow while deploying the occlusion balloons has been a successful approach . alternatively , using combined venous and arterial access , one such technique makes use of the injection of acrylic glue from the arterial side to block a large avf after the tract was occluded by a balloon from the venous side , without the usage of any coils . notably , this method carries the risk of entrapment of the balloon catheter by the injected glue . yet another combined approach involves stents deployed in the avf in order to downsize the diameter of the fistula ; then the tracks were embolized using coils deployed against the constrained segment of the stent . although ivc filters can be used to capture the embolization materials from an avf embolization procedure , ectasia of the suprarenal ivc and the wide leg strut pattern of the commercially available filters make these filters suboptimal for this purpose . interestingly , opt ease ivc filter ( cordis endovascular , bridgewater , nj , usa ) had been deployed in the fistulous tract through the venous access , which was then used as a scaffold for embolizing a 15-mm arterial feeder using a 22-mm avp - ii and several coils . idc is one such example of a mechanical coil that has been successfully used in treating renal avf , by using a pre - framing technique in which the microcatheter is coiled in the fistulous tract with proximal balloon occlusion . the 018 microcoil is then loaded in the microcatheter and , by the gradual withdrawal of the catheter , the coil is deployed . this technique requires a small side branch in the fistula that can be used to support the tip of the microwire over which the microcatheter can be coiled . very large renal avfs with multilobulated arterial aneurysms have treated using microplex-18 framing microcoils ( terumo , somerset , nj , usa ) followed by azur hydrocoils ( terumo ) in two sessions . these mechanical coils retain the advantages of the electrically detachable coils in terms of safety and control over delivery . compared to electrically detachable coils , idcs are cheaper and do not require galvanic current generating device for detachment . disadvantages of these coils over the electrically detachable coils are decreased pushability , especially when deployed through catheters having sharp angulation , and rigidity of the delivery system . in the procedure described , 035 coils require a maintained saline flush through the catheter in order to improve pushability . rarely , the previously deployed coils could be trapped by the lock of the subsequently deployed coil pusher , which may result in dislodgement of the already deployed coil . in the case presented here , the maximal aneurysmal tract dimension was too large for the available 018 idc . the high - velocity flow in the fistulous tract resulted in instability of a 14-mm , 018 idc , which was oversized with respect to the feeder diameter by 75% , although it had a good circumferential contact with the arterial wall . the 035 coil was then used as a reliable scaffolding against which further embolization was performed without the need of distal embolization protection techniques . avp - ii with 12-mm diameter would have been an alternative for occluding the feeder , which would have necessitated the use of a 5.3-f guiding catheter , requiring upsizing the arterial access to at least 6 f diameter . treatment of high - flow renal avf is challenging and continues to evolve in the wake of new embolization techniques . we present a simple technique to embolize large high - flow renal avf using 035 and 018 interlock coils without the need of flow modulating balloon catheter or additional vascular access for distal protection . this technique provides a potential alternative for the usage of avp in high - flow avf .

transcatheter embolization of renal arteriovenous fistula ( avf ) is a minimally invasive procedure that , in some occasions , can replace surgery and potentially save the kidney . the embolization techniques for the renal avfs have evolved considerably with the availability of newer hardwares . still , the risk of inadvertent migration of the embolization materials to the pulmonary circulation is a concern . this article describes a novel technique of coiling the feeding segmental artery to a large high - flow renal avf using 035 and 018 detachable coils only , and briefly reviews the previously described strategies to safely embolize renal avfs .

malignant peripheral nerve sheath tumor ( mpnst ) is a malignant spindle cell tumor of the soft tissue thought to be derived from the components of nerve sheath such as perineural fibroblasts or schwann cells . these tumors are treated as a subcategory of soft tissue sarcoma , in which they comprise 310% of all such tumors . mpnsts are rare tumors with an incidence of 0.001% in the general population . till date , very few cases of mpnsts of the lateral thoracic nerve have been reported in the medical literature . a 60-year - old male , a case of neurofibromatosis type 1 , presented with swelling in the right axilla , with a rapid growth over 56 months . , there was 1411 cm size swelling over the right lateral aspect of the thorax extending into the axilla , with the presence of dilated veins over it . there was winging of the scapula indicating the involvement of the lateral thoracic nerve [ figure 1a ] . ( d ) postoperative photo of the patient the pa view of the chest x - ray showed a space - occupying lesion on the right side paraspinally extending from d2 to d7 [ figure 2 ] . x - ray of the chest showing meningoceles as a mass lesion a ct scan of the thorax showed a mixed density mass of 1411 cm arising from the neurovascular bundle with a heterogeneous enhancement on the postcontrast study . a cystic lesion of about 85 cm size was also seen in the right intrathoracic paraspinal region [ figure 3a and c ] . ( c ) ct scan showing lateral meningocele with a spinal defect and the tumor . ( d ) mri lateral view showing the meningoceles as the nature of the intrathoracic paraspinal swelling was doubtful , magnetic resonance imaging ( mri ) was done . there was evidence of t2 hyperintense space - occupying lesions in the right paravertebral region from d1 - 2 to d7 - 8 levels which appeared lobulated and septated with well - defined walls , contiguous with each other , and extending into the spinal canal via large neural foramina . there was no internal enhancement on the postcontrast study suggestive of thoracic meningoceles / duralectasia [ figure 3b and d ] . tru - cut biopsy of the swelling indicated malignancy . in view of the presence of the lesions on the right side and history of breathlessness , a decision was taken to operate both the pathologies in the same sitting . under general anesthesia , an elliptical incision was taken on the posterior axillary fold so as to remove the redundant skin along with the mass . subcutaneous flaps were raised and a wide ( 5 cm from all sides ) local excision was done . during dissection , it was found that the tumor was arising from the right lateral thoracic nerve . the nerve along with the mass was excised [ figures 1b and 4a & b ] . ( b ) tumor with the lateral thoracic nerve excised in toto . ( c ) repair of meningocele defects being done with a g - patch ( artificial dural patch ) after the complete removal of the tumor , a right lateral thoracotomy was done to repair the meningoceles in the same position . the meningoceles were dissected till their openings into the defects and then the defects were repaired with a g - patch ( artificial dural graft ; figures 1c and 4c ) . the histopathology of the tumor suggested malignant spindle cell neoplasm , with areas of necrosis , hyalinization , and myxoid change with giant cell reaction to the tumor [ figure 5a and b ] . ( a ) histopathology scanner view ( 4 ) showing tumor with areas of necrosis . ( b ) histopathology photographic view ( 10 ) showing tumor cells arranged in fascicles with nuclear pleomorphism immunohistochemistry ( ihc ) suggested malignant spindle cell neoplasm positive for vimentin with negativity for s100 indicating decreasing differentiation . ihc positive for vimentin in view of the likely residual microscopic disease , the patient was given adjuvant radiotherapy ( rt ) . on follow - up at 6 months , the patient did not have local recurrence [ figure 1d ] . in view of the presence of the lesions on the right side and history of breathlessness , a decision was taken to operate both the pathologies in the same sitting . under general anesthesia , an elliptical incision was taken on the posterior axillary fold so as to remove the redundant skin along with the mass . subcutaneous flaps were raised and a wide ( 5 cm from all sides ) local excision was done . during dissection , it was found that the tumor was arising from the right lateral thoracic nerve . the nerve along with the mass was excised [ figures 1b and 4a & b ] . ( c ) repair of meningocele defects being done with a g - patch ( artificial dural patch ) after the complete removal of the tumor , a right lateral thoracotomy was done to repair the meningoceles in the same position . the meningoceles were dissected till their openings into the defects and then the defects were repaired with a g - patch ( artificial dural graft ; figures 1c and 4c ) . the histopathology of the tumor suggested malignant spindle cell neoplasm , with areas of necrosis , hyalinization , and myxoid change with giant cell reaction to the tumor [ figure 5a and b ] . ( a ) histopathology scanner view ( 4 ) showing tumor with areas of necrosis . ( b ) histopathology photographic view ( 10 ) showing tumor cells arranged in fascicles with nuclear pleomorphism immunohistochemistry ( ihc ) suggested malignant spindle cell neoplasm positive for vimentin with negativity for s100 indicating decreasing differentiation . ihc positive for vimentin in view of the likely residual microscopic disease , the patient was given adjuvant radiotherapy ( rt ) . on follow - up at 6 months , the patient did not have local recurrence [ figure 1d ] . the disease is characterized by a classical triad of skin lesions , mental deficiency , and skeletal deformities . now we distinguish neurofibromatosis as types 1 and 2 ( nf 1 and nf 2 ) , which are the peripheral and the central forms of the disease , respectively . as defined by the who , mpnsts are malignant tumors arising from a peripheral nerve or showing a nerve sheath differentiation with the exception of tumors originating from the epineurium or the peripheral nerve vasculature . approximately , one - third of mpnsts arise de novo , whereas the remainder represent the sarcomatous degeneration of a preexisting plexiform neurofibroma . mpnsts are associated with genetic aberrations such as loss of the neurofibromatosis 1 gene protein product . mpnsts occur in the third to sixth decade of life usually arising from medium and larger nerves . tumor location has been found to be a strong prognostic factor , with those in the thoracic and retroperitoneum having worse outcomes . in our patient , the mpnst was found to be arising from the right lateral thoracic nerve , suggesting a better prognosis with good surgical resection . the international consensus group has recommended that the current management of mpnsts should be identical to that of any other soft tissue sarcomas ( stss ) , . adjuvant rt should be considered for all intermediate- and high - grade lesions as well as low - grade tumors with positive margins . our patient also received rt as the tumor was negative for s100 which indicates decreasing differentiation . mpnsts are more aggressive tumors compared with other stss ; hence , an attempt should be made for near total surgical debulking of the tumor . the supportive literature is generally in the context of sarcomas and is not specific to mpnsts . disease - free survival and overall survival were reported to be approximately 64% and 30% at 5 years , respectively . the changes in neurofibromatosis can be classified as primary , which are related to mesodermal dysplasia ; secondary , which result from the compressive effects of nerve sheath tumors ; and mixed that combine the two previous . ventrolateral meningoceles of the thoracic spine are secondary to congenital mesodermal dysplasia and hypoplastic bone changes . our patient had the meningoceles since a long time , but presented with symptoms like breathlessness along with the mpnst . we postulate that the mpnst arose in a preexisting neurofibroma in this patient of nf 1 who already was harboring lateral thoracic meningoceles . such a case of co - existent meningoceles and mpnst has not been reported yet in any english medical literature . rarity of this tumor along with lateral spinal meningoceles made us to present this case with a multidisciplinary approach to tumor still undefined .

malignant peripheral nerve sheath tumor ( mpnst ) is a malignant spindle cell tumor of the soft tissue thought to be derived from the components of nerve sheath . mpnsts are mainly located in the buttocks , thighs , brachial plexus , and paraspinal region . the objective of this article is to describe a case of neurofibromatosis type 1 who developed neurofibrosarcoma of the right lateral thoracic nerve with thoracic meningoceles , a rare coincidental finding which has not yet been reported in the english medical literature , and how both the conditions were managed in the same sitting .

annular elastolytic giant cell granuloma ( aegcg ) , is a rare granulomatous disorder characterized by annular plaques with elevated borders and central atrophy with a tendency to occur over sun exposed areas , rarely involving covered areas . aegcg falls under noninfectious granulomatous diseases of the skin , in which the granulomatous reactions represent immune reactions to an inciting antigen and may be associated with systemic disease . a case of aegcg associated with hashimoto 's thyroiditis ( ht ) , in a 50-year - old female patient is reported for its rare co - occurrence . a 50-year - old , nondiabetic , nonhypertensive female patient presented with multiple asymptomatic raised skin lesions involving back , upper limbs , and legs for two years . she also had diffuse alopecia , dryness of skin , and thyroid swelling of four years duration . there was no history of photosensitivity , oral ulcers , arthralgia , or drug intake . dermatological examination revealed multiple , skin - colored and erythematous , annular , arciform , and polycyclic plaques of varying sizes involving the back , extensor aspects of forearms , dorsa of both hands , and anterolateral aspects of both legs [ figures 1a and 2 ] . the goiter was diffuse , firm in consistency , with a bosselated surface and was not fixed to underlying structures [ figure 3 ] . ( a ) annular elastolytic giant cell granuloma ( aegcg ) involving back , and ( b ) regressed aegcg lesions in the back annular elastolytic giant cell granuloma involving extensor aspect of elbow diffuse thyroid swelling with bosselated surface routine hematological and biochemical investigations , including blood sugar levels , were within normal limits . however , she had raised tsh levels ( 8 /l ) , with normal t3 ( 8 pmol / l ) and t4 ( 2.5 pmol / l ) levels . antinuclear antibody test , mantoux test , vdrl , and koh examination for fungus were negative . fine - needle aspiration cytology ( fnac ) of the thyroid swelling showed follicular epithelial cells in sheets and clusters , with hurthle cells , lymphocytes , and fibrosis against a hemorrhagic background admixed with colloid , which was suggestive of ht . histopathologial examination ( hpe ) of a lesion from the back revealed diffuse granulomatous infiltrate composed of multinucleated giant cells , histiocytes , and lymphocytes in the dermis . no features of active vasculitis , necrobiosis , or mucin deposition were noted [ figures 4 and 5 ] . van gieson stain showed loss of elastic fibers in upper and mid - dermis and fragmentation of elastic fibers with occasional elastophagocytosis , all features suggestive of aegcg [ figures 6 and 7 ] . diffuse granulomatous infiltration composed of multinucleated giant cells , histiocytes , and lymphocytes in the dermis ( h and e , 200 ) granulomatous dermal infiltrate made up of histiocytes and multinucleated giant cells containing fragments of elastic fibers ( h and e , 400 ) verhoeff van gieson stain showing loss of elastic fibers in upper and mid - dermis ( verhoeff van geison stain , 100 ) fragmentation of elastic fibers with elastophagocytosis by multinucleated giant cells ( verhoeff van geison stain , 400 ) based on hpe findings and fnac study , a clinical diagnosis of aegcg in association with ht was entertained . she was prescribed topical steroids and emollients , but there was no significant improvement after two weeks of treatment . two months later , she presented with much regressed skin lesions [ figure 1b ] and gave a history of having undergone thyroid surgery at a nearby tertiary care hospital . hanke et al . in the year 1979 described a rare granulomatous cutaneous disorder , aegcg , clinically characterized by solitary or grouped papules forming annular plaques with elevated borders and central atrophy . the lesions tend to occur mainly in photoexposed areas and on covered areas rarely , similar to the clinical picture in the present patient , who had asymptomatic plaques in non photo - distributed locations also . the diagnosis of aegcg is mainly based on distinct histopathological findings , which includes granulomatous infiltrate with lymphocytes , histiocytes , and multinucleated giant cells in the upper and mid - dermis . characteristic and unique features of aegcg include fragmentation of elastic fibers , presence of scanty elastic fibers in the areas of the granulomatous infiltrate , and elastophagocytosis by giant cells , all of which were noticed in hpe in the present case . the principal differential diagnosis for aegcg is granuloma annulare ( ga ) , which is considered as an artificial distinction by some authors . although some elastolysis has also been described in ga , the complete loss of elastic tissue in the central zone is used as the basis for the separation of these two conditions . the presence of larger and more numerous giant cells is also a feature in favor of aegcg , as was observed in the present patient . in view of the many giant cells and elastolysis , authors too wish to hold the view that aegcg and ga are two separate entities . also , absence of mucin and collagen necrobiosis helps to establish the distinct identities of the two conditions . regarding the genesis of aegcg lesions , speculations point to ultraviolet radiation , heat , or other unknown factors that might change the antigenicity of elastic fibers , triggering a cellular immune reaction directed to them . aegcg has been associated with acute myelogenous leukemia , cd4 t - cell lymphoma , adult t - cell leukemia , and prostate carcinoma , and this association with malignancy has been interpreted as systemic immunologic host defence against the tumor antigen . in the present patient based on the typical clinical features , positive antithyroid peroxidase antibody , and classical fnac appearances , the diagnosis of ht was established in this case . muller and his coworker considered aegcg as a prodromal stage of mid - dermal elastolysis ( mde ) and also suggested that aegcg and mde might represent different stages in the clinical spectrum of dermal elastolysis . has reported a case of mde associated with ht , suggesting an autoimmune mechanism . in the present case the association of aegcg with ht , which is often of an autoimmune etiology , could be attributed to a similar mechanism . aegcg tends to run a chronic course ; however , cases of spontaneous remission have been recorded . treatment options include topical and intralesional steroids , topical tacrolimus and pimecrolimus , hydroxychloroquine , isotretinoin , dapsone , cyclosporine , and puva therapy with variable response . whether the near complete resolution of the aegcg following thyroidectomy is due to the elimination of underlying contributory immune mechanism or a spontaneous phenomenon , can not be ascertained conclusively in the present patient . alhough there are previous reports of aegcg occurring in isolation or in association with systemic conditions , especially malignancies , the present case is being reported for the rare co - existence of aegcg with ht .

annular elastolytic giant cell granuloma ( aegcg ) is a rare granulomatous skin disease characterized clinically by annular plaques with elevated borders and atrophic centers found mainly on sun - exposed skin and histologically by diffuse granulomatous infiltrates composed of multinucleated giant cells , histiocytes and lymphocytes in the dermis along with phagocytosis of elastic fibers by multinucleated giant cells . we report a case of aegcg in a 50-year - old woman and is highlighted for the classical clinical and histological findings of the disease and its rare co - existence with hashimoto 's thyroiditis .

in august and september 2005 , 29 g. biloba food supplements were sampled in the netherlands . claims for a positive influence on memory and/or concentration or improvement of blood circulation were most frequently mentioned . only four products did n't have any claims on the label ( see table 2 ) . analytical data and claims on products investigated note : na , not analyzed ( method not suitable ) ; & , not present . to determine the degree to which the content of the substances typical for g. biloba were analog to what was declared on the label , the main components of g. biloba , flavone glycosides ( also known as flavonoids ) , and terpene lactones were analyzed . flavone glycosides were analyzed by using high - pressure liquid chromatography ( hplc ) according to the method of mesbah et al . ( 15 ) , but with an extra hydrolysis step added to the pretreatment step . without this hydrolysis step , terpene lactones were analyzed using hplc with refractive index - detection ( ri ) , also based on the method of mesbah et al . ( 15 ) , but with ri - detection instead of the uv - detection , because uv - detection leads to interfering peaks . besluit geneesmiddelen wet ( 18 ) states that the amount of the active substance present in a pharmaceutical product has to be 90110% of the declared amount . to prove the presence of a 24% standardized g. biloba extract in the products , the amount of bilobalide compared to the total amount of terpene lactones was calculated . a ratio of 3959% was used as a reference , corresponding to the ratio used in the european scientific cooperative on phytotherapy ( escop ) monograph ( 19 ) . three commonly encountered claimed health effects for g. biloba were selected : improved blood circulation , effects on symptoms of old age , and improved memory . examples of such claims used are : helps for cold hands and feet ; improves memory ; protects against symptoms of old age ; natural memory booster. per health effect a literature search was conducted in publicly available , objective scientific data : reference and text books and monographs , e.g. escop and who ( 11 , 19);scientific articles , with the emphasis on human studies , searched via pubmed , toxline , cochrane library , and references from articles , in dutch and english . search terms used were : ginkgo ; ginkgo biloba ; li 1370 ; egb761 ; meta - analysis ; review ; randomized controlled trial ; viscosity ; microcirculation ; blood flow ; elderly ; symptoms of old age ; cognition ; cognitive function ; and memory . reference and text books and monographs , e.g. escop and who ( 11 , 19 ) ; scientific articles , with the emphasis on human studies , searched via pubmed , toxline , cochrane library , and references from articles , in dutch and english . search terms used were : ginkgo ; ginkgo biloba ; li 1370 ; egb761 ; meta - analysis ; review ; randomized controlled trial ; viscosity ; microcirculation ; blood flow ; elderly ; symptoms of old age ; cognition ; cognitive function ; and memory . an overview of the available literature per health effect can be found in table 3 , including not only studies in healthy volunteers ( n=24 ) , but also some important patient studies ( n=11 ) . the passclaim criteria were applied to assess if the health claims can be supported by the totality of the data and weighing of the evidence ( 8) . the criteria are that the product needs to be characterized and administered in amounts consistent with its intended consumption . studies should include representative human study groups , have appropriate controls , an adequate study duration , characterized background diets , and monitor compliance of the study group . the target variable should change in a statistically significant way and the change should be biologically meaningful for the target group . dependent on the quality of the data , per health claim a conclusion was drawn as to the substantiation of the claim with reference to the strengths and weaknesses therein . meta - analyses and systematic reviews gci scale by physician cognition , change from baseline mood and emotional function raven 's progressive matrices visual - auditory learning stroop color word test odd - man - out reaction time task ( omo ) inspection time subjective well - being profile of mood states ( poms ) weschler memory scale - revised : immediate and delayed paragraph recall delayed matching - to - sample test long - term episodic memory frontal lobe function two tests selected from cantab sustained attention digit symbol substitution test speed of comprehension test symbol digit modalities test rey auditory verbal learning test cognometer battery of tests ( simple rt and working memory ) digit span backwards ( p<0.05 ) working memory speed ( p<0.05 ) rey auditory verbal learning test , delay list ( p<0.01 ) these sign changes indicate significant egb761 related improvements specifically in memory processes . sternberg memory scanning test vocabulary and digit span subtests wais - r wechsler adult intelligence scale - revised reading span test prose recall test cognitive abilities testing woodcock - johnson psych - educational battery - revised self - ordered pointing odd - man - out reaction time task ( omo ) inspection time subjective well - being profile of mood states ( poms ) information on toxicity of g. biloba was obtained by searching literature databases ( medline , pubmed , toxline ) , international documentation ( commission e monograph , escop monograph , hager 's handbuch , european medicines agency emea information on animal drugs , cbg college ter beoordeling van geneesmiddelen [ dutch medicines evaluation board ] information on drugs , who , herbalgram information , national toxicology program ntp ) , and the internet . in the commission e monograph , escop monograph , and hager 's handbuch , toxicity of the standardized extract egb 761 is described . the toxicity evaluation in the current evaluation is based on those reports as well as recent ntp studies and recent literature , predominantly case reports , and reports on interaction with frequently / commonly used medication . in addition to g. biloba and the gb - ste , data on gas were considered for evaluation . because of their toxic properties , the presence of gas is limited in standardized g. biloba preparations to maximum 5 ppm ( 11 ) . however , in non - standardized g. biloba preparations , gas can be present at higher levels . in august and september 2005 , 29 g. biloba food supplements were sampled in the netherlands . claims for a positive influence on memory and/or concentration or improvement of blood circulation were most frequently mentioned . only four products did n't have any claims on the label ( see table 2 ) . analytical data and claims on products investigated note : na , not analyzed ( method not suitable ) ; & , not present . to determine the degree to which the content of the substances typical for g. biloba were analog to what was declared on the label , the main components of g. biloba , flavone glycosides ( also known as flavonoids ) , and terpene lactones were analyzed . flavone glycosides were analyzed by using high - pressure liquid chromatography ( hplc ) according to the method of mesbah et al . ( 15 ) , but with an extra hydrolysis step added to the pretreatment step . without this hydrolysis step , terpene lactones were analyzed using hplc with refractive index - detection ( ri ) , also based on the method of mesbah et al . ( 15 ) , but with ri - detection instead of the uv - detection , because uv - detection leads to interfering peaks . besluit geneesmiddelen wet ( 18 ) states that the amount of the active substance present in a pharmaceutical product has to be 90110% of the declared amount . to prove the presence of a 24% standardized g. biloba extract in the products , the amount of bilobalide compared to the total amount of terpene lactones was calculated . a ratio of 3959% was used as a reference , corresponding to the ratio used in the european scientific cooperative on phytotherapy ( escop ) monograph ( 19 ) . three commonly encountered claimed health effects for g. biloba were selected : improved blood circulation , effects on symptoms of old age , and improved memory . examples of such claims used are : helps for cold hands and feet ; improves memory ; protects against symptoms of old age ; natural memory booster. per health effect a literature search was conducted in publicly available , objective scientific data : reference and text books and monographs , e.g. escop and who ( 11 , 19);scientific articles , with the emphasis on human studies , searched via pubmed , toxline , cochrane library , and references from articles , in dutch and english . search terms used were : ginkgo ; ginkgo biloba ; li 1370 ; egb761 ; meta - analysis ; review ; randomized controlled trial ; viscosity ; microcirculation ; blood flow ; elderly ; symptoms of old age ; cognition ; cognitive function ; and memory . reference and text books and monographs , e.g. escop and who ( 11 , 19 ) ; scientific articles , with the emphasis on human studies , searched via pubmed , toxline , cochrane library , and references from articles , in dutch and english . search terms used were : ginkgo ; ginkgo biloba ; li 1370 ; egb761 ; meta - analysis ; review ; randomized controlled trial ; viscosity ; microcirculation ; blood flow ; elderly ; symptoms of old age ; cognition ; cognitive function ; and memory . an overview of the available literature per health effect can be found in table 3 , including not only studies in healthy volunteers ( n=24 ) , but also some important patient studies ( n=11 ) . the passclaim criteria were applied to assess if the health claims can be supported by the totality of the data and weighing of the evidence ( 8) . the criteria are that the product needs to be characterized and administered in amounts consistent with its intended consumption . studies should include representative human study groups , have appropriate controls , an adequate study duration , characterized background diets , and monitor compliance of the study group . the target variable should change in a statistically significant way and the change should be biologically meaningful for the target group . dependent on the quality of the data , per health claim a conclusion was drawn as to the substantiation of the claim with reference to the strengths and weaknesses therein . meta - analyses and systematic reviews gci scale by physician cognition , change from baseline mood and emotional function raven 's progressive matrices visual - auditory learning stroop color word test odd - man - out reaction time task ( omo ) inspection time subjective well - being profile of mood states ( poms ) weschler memory scale - revised : immediate and delayed paragraph recall delayed matching - to - sample test long - term episodic memory frontal lobe function two tests selected from cantab sustained attention digit symbol substitution test speed of comprehension test symbol digit modalities test rey auditory verbal learning test cognometer battery of tests ( simple rt and working memory ) digit span backwards ( p<0.05 ) working memory speed ( p<0.05 ) rey auditory verbal learning test , delay list ( p<0.01 ) these sign changes indicate significant egb761 related improvements specifically in memory processes . sternberg memory scanning test vocabulary and digit span subtests wais - r wechsler adult intelligence scale - revised reading span test prose recall test cognitive abilities testing woodcock - johnson psych - educational battery - revised self - ordered pointing odd - man - out reaction time task ( omo ) inspection time subjective well - being profile of mood states ( poms ) information on toxicity of g. biloba was obtained by searching literature databases ( medline , pubmed , toxline ) , international documentation ( commission e monograph , escop monograph , hager 's handbuch , european medicines agency emea information on animal drugs , cbg college ter beoordeling van geneesmiddelen [ dutch medicines evaluation board ] information on drugs , who , herbalgram information , national toxicology program ntp ) , and the internet . in the commission e monograph , escop monograph , and hager 's handbuch , toxicity of the standardized extract egb 761 is described . the toxicity evaluation in the current evaluation is based on those reports as well as recent ntp studies and recent literature , predominantly case reports , and reports on interaction with frequently / commonly used medication . in addition to g. biloba and the gb - ste , data on gas were considered for evaluation . because of their toxic properties , the presence of gas is limited in standardized g. biloba preparations to maximum 5 ppm ( 11 ) . however , in non - standardized g. biloba preparations , gas can be present at higher levels . for 16 products , both the detailed content and the recommended dose were declared on the label ( see table 2 ) . for 13 products , the declaration was unclear , e.g. the recommended dose was stated , but the amount of flavonoids and terpene lactones in this dose was not stated in the declaration . only two of the 29 products met the pharmaceutical guideline ( the amount present in the product is 90110% of the declared amount ) for terpene lactones . for flavonoids , only four products met the criteria of a ratio of 3959% ( the amount of bilobalide compared to the total amount of terpene lactones ) . based on the daily dose and the concentration of terpene lactones and flavonoids found in the products , a dose range of substances typical for g. biloba was calculated ; 16 products did not meet the dose range for terpene lactones and flavonoids for a 24% standardized extract . in summary , when the components of herbal supplements were evaluated according to the guideline for pharmaceuticals , that require the presence of 90110% of the declared amount , one out of 29 g. biloba products meets the guideline.1 the amount of terpene lactones and flavonoids found vary between 27358% for terpene lactones and between 86418% for flavonoids . the ( combined ) passclaim criteria were checked per health effect , for the selected studies ( see table 3 ) . the final step of the assessment , passclaim criterion 6 , weighing of the evidence , is neither worked out in detail in passclaim ( 8) , nor in the efsa guidance document ( 7 ) . in practice , this is done now by efsa on a case - by - case basis ( 2 ) . in our assessment we have used the who criteria ( 20 ) , which categorizes data into convincing evidence ( consistent results from adequately powered , randomized placebo - controlled trials of the claimed substance in human subjects representative of the normal / target population , under normal conditions of use and assessing endpoints relevant to the claim ) , probable evidence ( consistent results from small randomized placebo - controlled trials of the claimed substance or active ingredients contained therein in sufficient numbers of human subjects representative of the normal / target population , under normal conditions of use and assessing endpoints relevant to the claim and/or human epidemiological studies ) , possible ( or supporting ) evidence ( consistent results from animal or in vitro studies of the claimed substance or active ingredients contained therein and assessing endpoints relevant to the claim ) and insufficient evidence ( inconsistent results and/or studies which do not consider the substance which is the subject of the claim and/or the target population and/or endpoints relevant to the claim ) . a detailed description and comments on individual meta - analyses , systematic reviews , and individual studies are given in table 3 . treatment of the disease claudication intermittens is one of the best - known uses of g. biloba . there is a registered medicine on the market containing g. biloba , tavonin , which is used for patients that do n't respond ( enough ) to walking exercise ( 16 ) . in patients with claudicatio intermittens , a positive effect at a dose of 120 mg / day was seen in two meta - analyses ( 21 , 22 ) . however , a recent systematic review that included 739 patients from 14 trials ( with different dosage and duration of treatment ) did n't find a significant effect on walking distance in people with claudication intermittens ( 23 ) . such clinical studies , however , may not be considered pertinent to a claim for the general population . it is an inaccurate expression that is often used to describe a collection of symptoms associated with dementia ( 11 ) or the effects of reduced cerebral blood flow in the elderly ( 24 ) . for the purpose of this evaluation , therefore , cerebrovascular insufficiency has been considered a disease . a systematic review by kleijnen and knipschild including patients with mild to moderate cerebrovascular insufficiency showed that treatment with 112160 mg / day g. biloba for 46 weeks was effective for cerebrovascular insufficiency ( 24 ) . few human studies are available that have investigated specifically the microcirculation and improved blood circulation in healthy subjects consuming g. biloba . santos et al . ( 25 ) did find a lower blood viscosity ( determined with a rotational viscosimeter ) in 48 older men that used 80 mg / day g. biloba extract for a period of 8 months . another study by the same group also described a decreased blood viscosity ( measured using wells - brookfield cone / plate viscometer dv - i ) in 25 adult men taking 80 mg gb - ste a day ( 26 ) . a randomized double blind placebo - controlled trial with 240 mg / day egb761 for 3 weeks in 27 older subjects found a vasoregulation role of g. biloba ( 27 ) . unfortunately , the studies described above included different blood flow parameters , therefore , comparison of the studies is complicated . we concluded there was insufficient evidence that gb - ste use results in improved blood circulation in healthy subjects . to enable scientific evaluation , a claim must include in the wording some reference to the target population and the health effect . symptoms and old age need to be defined and the wording of the claim altered to reflect relevant measurable endpoints . if submitted through article 13.5 , the dossier could be returned to the applicants who in some cases would be afforded the opportunity to re - word the claim to enable evaluation subsequent to re - submission . studies on alzheimer and dementia show inconsistent results for the effect of g. biloba ( 2831 ) . in the latest updated version of a systematic review , looking at cognitive impairment and dementia , it was concluded that the evidence for a clinically significant benefit of g. biloba is unreliable ( 32 ) . a systematic review on the effects of g. biloba on the memory of healthy subjects concluded that g. biloba did n't improve memory ( 33 ) . most studies of g. biloba and aging have considered cognitive endpoints in subjects with mild impairment ( 13 ) or alzheimer and dementia patients ( 2831 ) with inconsistent results for a beneficial effect of g. biloba . in the latest updated version of a systematic review , looking at cognitive impairment and dementia , it was concluded that the evidence for a clinically significant benefit of g. biloba is unreliable ( 32 ) . studies employing clinical samples , however , can not be considered pertinent to a claim for the general population . a large trial including 262 healthy subjects aged 60 years and over , observed improved performance on validated memory tests with gb - ste ( 180 mg / day , 6 weeks ) ( 34 ) . in other trials , g. biloba did not enhance performance on tests of memory or other measures of cognitive function ( 35 , 36 ) . ( 37 ) found some evidence for improved memory function in healthy elderly aged 85 + years ( n=118 ) treated with g. biloba ( 240 mg / day ) compared to placebo after controlling for compliance . the observed effect , however , was weak and disappeared when other variables were controlled . that the subjects were also administered a multi - vitamin , the authors do not appear to have declared the content of the placebo making it difficult to evaluate the report . the most recent trial , the gem study , looking at cognitive decline in healthy older adults , did not find a difference in cognitive functioning between the g. biloba and the placebo group ( 38 ) . in a small ( n=20 ) placebo - controlled study with gb - ste 240 or 360 mg / day , a positive effect was observed in young healthy volunteers in improvement in quality of memory and attention ( 39 ) . however , this result could not be replicated in a similar study in the same study population ( 40 ) . a double blind controlled study showed no effect of gb - ste 120 mg / day on the memory of healthy young males ( n=104 ) ( 41 ) . in conclusion , there is insufficient evidence to substantiate the claim that g. biloba can improve memory in healthy subjects . in the open literature there are no ( animal ) data on acute toxicity , carcinogenicity , reproduction toxicity , teratogenicity , neurotoxicity or immunotoxicity , but information on these toxicological endpoints are available in the commission e monograph , escop monograph , the emea report , and a recent ntp study ( 10 , 19 , 42 ) . however , this should be interpreted with care as g. biloba is an herbal preparation consisting of many components , which may show interactions . indeed , most reports on kinetics focus on known ingredients of g. biloba such as ginkgolides a and b and bilobalide . for gb - ste , low acute toxicity has been reported for rodents with ld50 values ranging from 1,100 ( intravenous , rats and mice ) to 7,725 mg / kg body weight ( bw ) ( oral , mice ) . a 13-week gavage study in mice and rats with gb - ste ( egb761 dose levels 0 , 65.5 [ rats only ] , 125 , 250 , 500 , 1,000 ( rats and mice ) , and 2,000 ( mice only ) mg / kg bw per day ) showed an increase in liver weight at all dose levels tested , and a dose - related increase in hepatocyte hypertrophy in male and female mice at and above 250 mg / kg bw and in male rats at all dose levels . in oral studies with rats and dogs during 6 months , a dose- and time - related mild temporary vasodilatation in cranial blood vessels was observed in dogs dosed at and above 100 mg gb - ste / kg bw from 35 days on ( 19 ) . although gb - ste was reported not mutagenic in ( among other tests ) a ames test by commission e 1994 and escop 2003 , recently ntp ( 43 ) reported a positive ames test . an in vivo micronucleus assay was negative for male mice , whereas for female mice the results were equivocal . escop ( 19 ) reported no carcinogenic effects in a 2-year study in rats at dose levels of 4 , 20 , and 100 mg / kg bw . g / kg bw day ) and rabbits ( up to 0.9 g / kg bw day ) did not show embryotoxic , teratogenic or reproduction toxicity effects ( 19 ) . gb - ste also contains gas , to which immunotoxic , cytotoxic , mutagenic , and carcinogenic properties are ascribed ( 4448 ) . as a consequence , a maximum level for the presence of gas in the gb - ste was set at 5 mg / kg ( 11 ) . however , as not all g. biloba preparations included in the present study contain gb - ste , maximum levels of 5 mg / kg are not guaranteed , which may pose a health risk ( 49 ) . indeed , chiu et al . ( 50 ) report higher levels ( 16733 times ) than 5 ppm in 13 of 14 g. biloba preparations . reportedly , g. biloba is generally well tolerated by human ( 19 , 51 ) , albeit that some mild side effects have been reported [ gastrointestinal complaints , headache , allergic skin reactions , nausea , dizziness , restlessness , heart palpitation , and weakness ( 16 , 22 , 51 , 52 ) ] . in addition to these mild effects , in several case studies side effects have been reported related to blood platelets , hemorrhage , and blood coagulation , which is in agreement with the paf - inhibiting properties of g. biloba ( 5361 ) . in these case reports , reference is made to a ginkgo extract or a ginkgo containing preparation, without further specifications of the g. biloba preparation used . the duration and dose of intake of g. biloba prior to the reported paf - related side effects ranges from 2 weeks to 2 years , respectively , 80160 mg / day , well within the recommended dose range . as for case studies on effects on bleeding , available data on interactions with drugs are also mainly associated with drugs involved in the treatment of blood coagulation and bleeding ( 53 , 6264 ) . the escop monograph ( 19 ) reports that there is no clear evidence for gb - ste on blood coagulation ( alone or in combination with medicines ) . however , various case reports of interactions with drugs have been reported ( 16 , 53 , 54 , 6571 ) . interactions of herbal preparations with drugs are particularly relevant for drugs with a narrow therapeutic margin , it can result in more or stronger side effects or effect on the bioavailability of the drug ( 72 , 73 ) . although a direct causal relationship between g. biloba intake and observed effects in case studies is lacking ( 74 ) , the absence of other risk factors , the paf - inhibiting activity of g. biloba , and the disappearance of effects upon cessation of intake suggest such causality ( 5961 ) . the who has indicated to be careful with the use of g. biloba in combination with drugs that affect blood coagulation or platelet aggregation and to stop g. biloba use before a diagnostic treatment or an operation ( 49 , 75 ) . summarizing , the potential safety issues associated with g. biloba are severalfold . first , in most literature reports on g. biloba , the preparation used is not specified . animal toxicity studies typically were performed with gb - ste , whereas in case studies no specification on the g. biloba preparation is given . second , because of the immunotoxic , cytotoxic , carcinogenic , and genotoxic potential of gas , the maximum level of gas in gb - ste is limited to 5 ppm . however , if a preparation of g. biloba does not contain the gb - ste , a higher level of gas can not be excluded . finally , g. biloba has paf - antagonist activity , which may result in effects on platelet aggregation and blood coagulation . indeed , there are various case studies reporting hemorrhages with intake of g. biloba preparations alone or in combination with drugs affecting platelet aggregation and blood coagulation . moreover , the dose levels of case reports are within the range of recommended dose levels . nevertheless , exact dose levels are unknown . as a result of abovementioned available data , there was insufficient evidence to substantiate the three selected health claims for g. biloba , when applying the passclaim criteria . first , a clear definition of what sort of study sample constitutes a healthy target population is lacking . asp and bryngelsson ( 3 ) concluded that passclaim is useful for applicants for health claims and for the agencies that evaluate the evidence . since herbal food supplements are intended for healthy persons , health claims for herbal food supplements have to be proven in studies performed with healthy persons who are representative of the target population . however , distinction between healthy , complaint and illness is not always clear ( definition of a healthy person is not clear ) . for this analysis we excluded patient studies , which left only a small number of available studies per health effect . secondly , it is unclear to which degree a herbal supplement of an applicant should be identical to the product used in the human studies . should they be exactly the same , or should they contain the same standardized extract ? this study showed that most studies are performed reportedly using standardized g. biloba extract , but also that nearly every product found in the dutch shops was not in conformity with the label declaration . when a claim can be substantiated using those studies , the product that sets the claim should resemble the standardized extract . thirdly , the passclaim criteria 4 and 5 require consideration of specific markers , or endpoints , depending on the type of claim made . finally , the procedure for weighing the evidence was not described in detail in the passclaim project . in contrast to the usa , where qualified health claims are allowed ( 77 ) , the eu regulation 1924/2006 was not designed for supporting qualified health claims . however , qualification of the evidence according to the who criteria has proved useful for scientific purposes . the value of such criteria in consumer communication has not yet been established . indeed , in the usa experience , consumers do not make the anticipated distinction between substantiated and not substantiated ( = qualified ) health claims ( 2 , 7780 ) . hence the need for interdisciplinary groups of expert scientists to monitor and evaluate such claims . firstly , there was no case with product - specific information , delivered by a manufacturer , to evaluate . it is possible that information is missing , e.g. information from negative or null trials or unpublished proprietary or confidential studies . in future , efsa evaluations will endeavor to include all available information , including confidential data , for the substantiation of article 14 and 13.5 claims . secondly , regarding the g. biloba products that were included in this study , the sampling of these products was random from dutch shops . that the search was not exhaustive leaves the possibility that there were products available for purchase which were not analyzed and which contained at least the minimum amount of g. biloba extract . the finding that the standardized 24% of g. biloba extract is often not incorporated into the food supplements , nevertheless , raises doubts as to the efficacy of such products . this analysis has also implied that it is not possible to establish a safe level for intake of g. biloba . review of the science provided insufficient evidence with which to substantiate the three claims for g. biloba . in conclusion , the claim g. biloba and improvement in the symptoms of old age could not be evaluated . a cause and effect relationship between g. biloba and improved circulation or memory has not been established . for 16 products , both the detailed content and the recommended dose were declared on the label ( see table 2 ) . for 13 products , the declaration was unclear , e.g. the recommended dose was stated , but the amount of flavonoids and terpene lactones in this dose was not stated in the declaration . only two of the 29 products met the pharmaceutical guideline ( the amount present in the product is 90110% of the declared amount ) for terpene lactones . for flavonoids , only four products met the criteria of a ratio of 3959% ( the amount of bilobalide compared to the total amount of terpene lactones ) . based on the daily dose and the concentration of terpene lactones and flavonoids found in the products , a dose range of substances typical for g. biloba was calculated ; 16 products did not meet the dose range for terpene lactones and flavonoids for a 24% standardized extract . in summary , when the components of herbal supplements were evaluated according to the guideline for pharmaceuticals , that require the presence of 90110% of the declared amount , one out of 29 g. biloba products meets the guideline.1 the amount of terpene lactones and flavonoids found vary between 27358% for terpene lactones and between 86418% for flavonoids . the ( combined ) passclaim criteria were checked per health effect , for the selected studies ( see table 3 ) . the final step of the assessment , passclaim criterion 6 , weighing of the evidence , is neither worked out in detail in passclaim ( 8) , nor in the efsa guidance document ( 7 ) . in practice , this is done now by efsa on a case - by - case basis ( 2 ) . in our assessment we have used the who criteria ( 20 ) , which categorizes data into convincing evidence ( consistent results from adequately powered , randomized placebo - controlled trials of the claimed substance in human subjects representative of the normal / target population , under normal conditions of use and assessing endpoints relevant to the claim ) , probable evidence ( consistent results from small randomized placebo - controlled trials of the claimed substance or active ingredients contained therein in sufficient numbers of human subjects representative of the normal / target population , under normal conditions of use and assessing endpoints relevant to the claim and/or human epidemiological studies ) , possible ( or supporting ) evidence ( consistent results from animal or in vitro studies of the claimed substance or active ingredients contained therein and assessing endpoints relevant to the claim ) and insufficient evidence ( inconsistent results and/or studies which do not consider the substance which is the subject of the claim and/or the target population and/or endpoints relevant to the claim ) . a detailed description and comments on individual meta - analyses , systematic reviews , and individual studies are given in table 3 . treatment of the disease claudication intermittens is one of the best - known uses of g. biloba . there is a registered medicine on the market containing g. biloba , tavonin , which is used for patients that do n't respond ( enough ) to walking exercise ( 16 ) . in patients with claudicatio intermittens , a positive effect at a dose of 120 mg / day was seen in two meta - analyses ( 21 , 22 ) . however , a recent systematic review that included 739 patients from 14 trials ( with different dosage and duration of treatment ) did n't find a significant effect on walking distance in people with claudication intermittens ( 23 ) . such clinical studies , however , may not be considered pertinent to a claim for the general population . it is an inaccurate expression that is often used to describe a collection of symptoms associated with dementia ( 11 ) or the effects of reduced cerebral blood flow in the elderly ( 24 ) . for the purpose of this evaluation , therefore , cerebrovascular insufficiency has been considered a disease . a systematic review by kleijnen and knipschild including patients with mild to moderate cerebrovascular insufficiency showed that treatment with 112160 mg / day g. biloba for 46 weeks was effective for cerebrovascular insufficiency ( 24 ) . few human studies are available that have investigated specifically the microcirculation and improved blood circulation in healthy subjects consuming g. biloba . santos et al . ( 25 ) did find a lower blood viscosity ( determined with a rotational viscosimeter ) in 48 older men that used 80 mg / day g. biloba extract for a period of 8 months . another study by the same group also described a decreased blood viscosity ( measured using wells - brookfield cone / plate viscometer dv - i ) in 25 adult men taking 80 mg gb - ste a day ( 26 ) . a randomized double blind placebo - controlled trial with 240 mg / day egb761 for 3 weeks in 27 older subjects found a vasoregulation role of g. biloba ( 27 ) . unfortunately , the studies described above included different blood flow parameters , therefore , comparison of the studies is complicated . we concluded there was insufficient evidence that gb - ste use results in improved blood circulation in healthy subjects . to enable scientific evaluation , a claim must include in the wording some reference to the target population and the health effect . symptoms and old age need to be defined and the wording of the claim altered to reflect relevant measurable endpoints . if submitted through article 13.5 , the dossier could be returned to the applicants who in some cases would be afforded the opportunity to re - word the claim to enable evaluation subsequent to re - submission . studies on alzheimer and dementia show inconsistent results for the effect of g. biloba ( 2831 ) . in the latest updated version of a systematic review , looking at cognitive impairment and dementia , it was concluded that the evidence for a clinically significant benefit of g. biloba is unreliable ( 32 ) . a systematic review on the effects of g. biloba on the memory of healthy subjects concluded that g. biloba did n't improve memory ( 33 ) . most studies of g. biloba and aging have considered cognitive endpoints in subjects with mild impairment ( 13 ) or alzheimer and dementia patients ( 2831 ) with inconsistent results for a beneficial effect of g. biloba . in the latest updated version of a systematic review , looking at cognitive impairment and dementia , it was concluded that the evidence for a clinically significant benefit of g. biloba is unreliable ( 32 ) . studies employing clinical samples , however , can not be considered pertinent to a claim for the general population . a large trial including 262 healthy subjects aged 60 years and over , observed improved performance on validated memory tests with gb - ste ( 180 mg / day , 6 weeks ) ( 34 ) . in other trials , g. biloba did not enhance performance on tests of memory or other measures of cognitive function ( 35 , 36 ) . ( 37 ) found some evidence for improved memory function in healthy elderly aged 85 + years ( n=118 ) treated with g. biloba ( 240 mg / day ) compared to placebo after controlling for compliance . the observed effect , however , was weak and disappeared when other variables were controlled . that the subjects were also administered a multi - vitamin , the authors do not appear to have declared the content of the placebo making it difficult to evaluate the report . the most recent trial , the gem study , looking at cognitive decline in healthy older adults , did not find a difference in cognitive functioning between the g. biloba and the placebo group ( 38 ) . in a small ( n=20 ) placebo - controlled study with gb - ste 240 or 360 mg / day , a positive effect was observed in young healthy volunteers in improvement in quality of memory and attention ( 39 ) . however , this result could not be replicated in a similar study in the same study population ( 40 ) . a double blind controlled study showed no effect of gb - ste 120 mg / day on the memory of healthy young males ( n=104 ) ( 41 ) . in conclusion , there is insufficient evidence to substantiate the claim that g. biloba can improve memory in healthy subjects . treatment of the disease claudication intermittens is one of the best - known uses of g. biloba . there is a registered medicine on the market containing g. biloba , tavonin , which is used for patients that do n't respond ( enough ) to walking exercise ( 16 ) . in patients with claudicatio intermittens , a positive effect at a dose of 120 mg / day was seen in two meta - analyses ( 21 , 22 ) . however , a recent systematic review that included 739 patients from 14 trials ( with different dosage and duration of treatment ) did n't find a significant effect on walking distance in people with claudication intermittens ( 23 ) . such clinical studies , however , may not be considered pertinent to a claim for the general population . it is an inaccurate expression that is often used to describe a collection of symptoms associated with dementia ( 11 ) or the effects of reduced cerebral blood flow in the elderly ( 24 ) . for the purpose of this evaluation , therefore , cerebrovascular insufficiency has been considered a disease . a systematic review by kleijnen and knipschild including patients with mild to moderate cerebrovascular insufficiency showed that treatment with 112160 mg / day g. biloba for 46 weeks was effective for cerebrovascular insufficiency ( 24 ) . few human studies are available that have investigated specifically the microcirculation and improved blood circulation in healthy subjects consuming g. biloba . santos et al . ( 25 ) did find a lower blood viscosity ( determined with a rotational viscosimeter ) in 48 older men that used 80 mg / day g. biloba extract for a period of 8 months . another study by the same group also described a decreased blood viscosity ( measured using wells - brookfield cone / plate viscometer dv - i ) in 25 adult men taking 80 mg gb - ste a day ( 26 ) . a randomized double blind placebo - controlled trial with 240 mg / day egb761 for 3 weeks in 27 older subjects found a vasoregulation role of g. biloba ( 27 ) . unfortunately , the studies described above included different blood flow parameters , therefore , comparison of the studies is complicated . we concluded there was insufficient evidence that gb - ste use results in improved blood circulation in healthy subjects . to enable scientific evaluation , a claim must include in the wording some reference to the target population and the health effect . symptoms and old age need to be defined and the wording of the claim altered to reflect relevant measurable endpoints . if submitted through article 13.5 , the dossier could be returned to the applicants who in some cases would be afforded the opportunity to re - word the claim to enable evaluation subsequent to re - submission . studies on alzheimer and dementia show inconsistent results for the effect of g. biloba ( 2831 ) . in the latest updated version of a systematic review , looking at cognitive impairment and dementia , it was concluded that the evidence for a clinically significant benefit of g. biloba is unreliable ( 32 ) . a systematic review on the effects of g. biloba on the memory of healthy subjects concluded that g. biloba did n't improve memory ( 33 ) . most studies of g. biloba and aging have considered cognitive endpoints in subjects with mild impairment ( 13 ) or alzheimer and dementia patients ( 2831 ) with inconsistent results for a beneficial effect of g. biloba . in the latest updated version of a systematic review , looking at cognitive impairment and dementia , it was concluded that the evidence for a clinically significant benefit of g. biloba is unreliable ( 32 ) . studies employing clinical samples , however , can not be considered pertinent to a claim for the general population . a large trial including 262 healthy subjects aged 60 years and over , observed improved performance on validated memory tests with gb - ste ( 180 mg / day , 6 weeks ) ( 34 ) . in other trials , g. biloba did not enhance performance on tests of memory or other measures of cognitive function ( 35 , 36 ) . ( 37 ) found some evidence for improved memory function in healthy elderly aged 85 + years ( n=118 ) treated with g. biloba ( 240 mg / day ) compared to placebo after controlling for compliance . the observed effect , however , was weak and disappeared when other variables were controlled . that the subjects were also administered a multi - vitamin , the authors do not appear to have declared the content of the placebo making it difficult to evaluate the report . the most recent trial , the gem study , looking at cognitive decline in healthy older adults , did not find a difference in cognitive functioning between the g. biloba and the placebo group ( 38 ) . in a small ( n=20 ) placebo - controlled study with gb - ste 240 or 360 mg / day , a positive effect was observed in young healthy volunteers in improvement in quality of memory and attention ( 39 ) . however , this result could not be replicated in a similar study in the same study population ( 40 ) . a double blind controlled study showed no effect of gb - ste 120 mg / day on the memory of healthy young males ( n=104 ) ( 41 ) . in conclusion , there is insufficient evidence to substantiate the claim that g. biloba can improve memory in healthy subjects . in the open literature there are no ( animal ) data on acute toxicity , carcinogenicity , reproduction toxicity , teratogenicity , neurotoxicity or immunotoxicity , but information on these toxicological endpoints are available in the commission e monograph , escop monograph , the emea report , and a recent ntp study ( 10 , 19 , 42 ) . however , this should be interpreted with care as g. biloba is an herbal preparation consisting of many components , which may show interactions . indeed , most reports on kinetics focus on known ingredients of g. biloba such as ginkgolides a and b and bilobalide . for gb - ste , low acute toxicity has been reported for rodents with ld50 values ranging from 1,100 ( intravenous , rats and mice ) to 7,725 mg / kg body weight ( bw ) ( oral , mice ) . a 13-week gavage study in mice and rats with gb - ste ( egb761 dose levels 0 , 65.5 [ rats only ] , 125 , 250 , 500 , 1,000 ( rats and mice ) , and 2,000 ( mice only ) mg / kg bw per day ) showed an increase in liver weight at all dose levels tested , and a dose - related increase in hepatocyte hypertrophy in male and female mice at and above 250 mg / kg bw and in male rats at all dose levels . in oral studies with rats and dogs during 6 months , a dose- and time - related mild temporary vasodilatation in cranial blood vessels was observed in dogs dosed at and above 100 mg gb - ste / kg bw from 35 days on ( 19 ) . although gb - ste was reported not mutagenic in ( among other tests ) a ames test by commission e 1994 and escop 2003 , recently ntp ( 43 ) reported a positive ames test . an in vivo micronucleus assay was negative for male mice , whereas for female mice the results were equivocal . escop ( 19 ) reported no carcinogenic effects in a 2-year study in rats at dose levels of 4 , 20 , and 100 mg / kg bw . g / kg bw day ) and rabbits ( up to 0.9 g / kg bw day ) did not show embryotoxic , teratogenic or reproduction toxicity effects ( 19 ) . gb - ste also contains gas , to which immunotoxic , cytotoxic , mutagenic , and carcinogenic properties are ascribed ( 4448 ) . as a consequence , a maximum level for the presence of gas in the gb - ste was set at 5 mg / kg ( 11 ) . however , as not all g. biloba preparations included in the present study contain gb - ste , maximum levels of 5 mg / kg are not guaranteed , which may pose a health risk ( 49 ) . ( 50 ) report higher levels ( 16733 times ) than 5 ppm in 13 of 14 g. biloba preparations . reportedly , g. biloba is generally well tolerated by human ( 19 , 51 ) , albeit that some mild side effects have been reported [ gastrointestinal complaints , headache , allergic skin reactions , nausea , dizziness , restlessness , heart palpitation , and weakness ( 16 , 22 , 51 , 52 ) ] . in addition to these mild effects , in several case studies side effects have been reported related to blood platelets , hemorrhage , and blood coagulation , which is in agreement with the paf - inhibiting properties of g. biloba ( 5361 ) . in these case reports , reference is made to a ginkgo extract or a ginkgo containing preparation, without further specifications of the g. biloba preparation used . the duration and dose of intake of g. biloba prior to the reported paf - related side effects ranges from 2 weeks to 2 years , respectively , 80160 mg / day , well within the data on interactions with drugs are also mainly associated with drugs involved in the treatment of blood coagulation and bleeding ( 53 , 6264 ) . the escop monograph ( 19 ) reports that there is no clear evidence for gb - ste on blood coagulation ( alone or in combination with medicines ) . however , various case reports of interactions with drugs have been reported ( 16 , 53 , 54 , 6571 ) . interactions of herbal preparations with drugs are particularly relevant for drugs with a narrow therapeutic margin , it can result in more or stronger side effects or effect on the bioavailability of the drug ( 72 , 73 ) . although a direct causal relationship between g. biloba intake and observed effects in case studies is lacking ( 74 ) , the absence of other risk factors , the paf - inhibiting activity of g. biloba , and the disappearance of effects upon cessation of intake suggest such causality ( 5961 ) . the who has indicated to be careful with the use of g. biloba in combination with drugs that affect blood coagulation or platelet aggregation and to stop g. biloba use before a diagnostic treatment or an operation ( 49 , 75 ) . summarizing , the potential safety issues associated with g. biloba are severalfold . first , in most literature reports on g. biloba , the preparation used is not specified . animal toxicity studies typically were performed with gb - ste , whereas in case studies no specification on the g. biloba preparation is given . second , because of the immunotoxic , cytotoxic , carcinogenic , and genotoxic potential of gas , the maximum level of gas in gb - ste is limited to 5 ppm . however , if a preparation of g. biloba does not contain the gb - ste , a higher level of gas can not be excluded . finally , g. biloba has paf - antagonist activity , which may result in effects on platelet aggregation and blood coagulation . indeed , there are various case studies reporting hemorrhages with intake of g. biloba preparations alone or in combination with drugs affecting platelet aggregation and blood coagulation . moreover , the dose levels of case reports are within the range of recommended dose levels . nevertheless , exact dose levels are unknown . as a result of abovementioned available data , there was insufficient evidence to substantiate the three selected health claims for g. biloba , when applying the passclaim criteria first , a clear definition of what sort of study sample constitutes a healthy target population is lacking . asp and bryngelsson ( 3 ) concluded that passclaim is useful for applicants for health claims and for the agencies that evaluate the evidence . since herbal food supplements are intended for healthy persons , health claims for herbal food supplements have to be proven in studies performed with healthy persons who are representative of the target population . however , distinction between healthy , complaint and illness is not always clear ( definition of a healthy person is not clear ) . for this analysis we excluded patient studies , which left only a small number of available studies per health effect . secondly , it is unclear to which degree a herbal supplement of an applicant should be identical to the product used in the human studies . should they be exactly the same , or should they contain the same standardized extract ? this study showed that most studies are performed reportedly using standardized g. biloba extract , but also that nearly every product found in the dutch shops was not in conformity with the label declaration . when a claim can be substantiated using those studies , the product that sets the claim should resemble the standardized extract . thirdly , the passclaim criteria 4 and 5 require consideration of specific markers , or endpoints , depending on the type of claim made . finally , the procedure for weighing the evidence was not described in detail in the passclaim project . in contrast to the usa , where qualified health claims are allowed ( 77 ) , the eu regulation 1924/2006 was not designed for supporting qualified health claims . however , qualification of the evidence according to the who criteria has proved useful for scientific purposes . the value of such criteria in consumer communication has not yet been established . indeed , in the usa experience , consumers do not make the anticipated distinction between substantiated and not substantiated ( = qualified ) health claims ( 2 , 7780 ) . hence the need for interdisciplinary groups of expert scientists to monitor and evaluate such claims . firstly , there was no case with product - specific information , delivered by a manufacturer , to evaluate . it is possible that information is missing , e.g. information from negative or null trials or unpublished proprietary or confidential studies . in future , efsa evaluations will endeavor to include all available information , including confidential data , for the substantiation of article 14 and 13.5 claims . secondly , regarding the g. biloba products that were included in this study , the sampling of these products was random from dutch shops . that the search was not exhaustive leaves the possibility that there were products available for purchase which were not analyzed and which contained at least the minimum amount of g. biloba extract . the finding that the standardized 24% of g. biloba extract is often not incorporated into the food supplements , nevertheless , raises doubts as to the efficacy of such products . this analysis has also implied that it is not possible to establish a safe level for intake of g. biloba . review of the science provided insufficient evidence with which to substantiate the three claims for g. biloba . in conclusion , the claim g. biloba and improvement in the symptoms of old age could not be evaluated . a cause and effect relationship between g. biloba and improved circulation or memory has not been established . the main conclusions in this study were : three selected health claims for g. biloba could not be substantiated.neither safety nor efficacy can be guaranteed at the recommended daily dose.the content of g. biloba containing products often did not conform to what was declared on the label.a multidisciplinary approach is needed to get insight in the relevant issues for health claim evaluation.moreover , the passclaim criteria need to be developed in more detail when used for general application on health claim substantiation for herbal supplements and there is also a need for clarification of the study conditions that are required for claim substantiation , especially with respect to patient studies . a multidisciplinary approach is recommended to assess health claims : analyses of substances typical for g. biloba in the products and toxicological risk assessment are needed for a full assessment . this provides a good insight into the several aspects that are significant for the evaluation of health claims for herbal substances . the content of g. biloba containing products often did not conform to what was declared on the label . a multidisciplinary approach is needed to get insight in the relevant issues for health claim evaluation . the authors have not received any funding or benefits from industry to conduct this study .

backgroundeuropean regulation 1924/2006 states that all health claims made on foods need to be substantiated scientifically.objectiveto apply the passclaim criteria for the scientific substantiation of health claims on foods to herbal supplements containing ginkgo biloba . evaluation of three selected claimed health effects for g. biloba ( improvement of blood circulation , improvement of symptoms of old age , and improvement of memory ) was achieved through review of publicly available scientific data . a total of 35 human intervention studies were evaluated . commercially available products claimed to contain mainly g. biloba ( n=29 ) were randomly sampled in the netherlands and analyzed for their content on ginkgo extract . also , a toxicological risk assessment was performed.resultsthe three selected health claims investigated could not be substantiated . this was mainly because of a lack of data from studies in healthy volunteers . in most studies results performed with a 24% standardized g. biloba extract were described . however , our chemical analysis showed that 25 of the 29 sampled products did not contain the required minimum 24% standardized extract . moreover , in most preparations the content of substances typical for g. biloba did not conform to what was declared on the label . since toxicity data for g. biloba are very limited , a safety limit could not be established.conclusionsevidence is lacking for three health claims of herbal products with g. biloba . neither safety nor efficacy can be guaranteed at the recommended daily dose . the multidisciplinary approach described in this paper provides good insight into issues that are relevant for the evaluation of health claims for herbal food supplements .

malaria is a major public health problem in tropical countries . about 500 million people suffer from malaria , leading to death in 1 to 3 million cases . acute kidney injury ( aki ) is one of the most dreaded complications of severe malaria . the overall prevalence of aki in falciparum malaria varies between < 1 and 60% , with the mortality rate up to 45%.[14 ] aki occurs commonly in plasmodium falciparum ( pf ) malaria and rarely in plasmodium vivax ( pv ) malaria.[57 ] malaria is an important cause of multiple organ failure ; mortality rate is 6.4% when one or fewer organs fail , but increases to 48.8% with failure of two or more organs . prompt and accurate diagnosis of malaria is needed for implementation of appropriate treatment to reduce associated morbidity and mortality . the management of malaria - induced aki includes appropriate antimalarials ( parenteral artesunate or quinine ) , fluid electrolyte management , supportive therapy , avoidance of nephrotoxic drugs , and renal replacement therapy ( rrt ) at the earliest . we carried out prospective single - center study to describe the clinical characteristics , laboratory parameters , prognostic factors , and outcome in patients with malaria with aki who underwent hd . all 50 patients with malaria - associated aki seen at our center in 2010 were included in the study . the diagnosis of malaria was confirmed by direct visualization of the parasite in giemsa - stained peripheral blood smears . in addition , once a diagnosis has been established and treatment has been initiated , serial examinations permit monitoring of the parasitological response . clinical history and assessment were recorded in all the study patients and all other known etiological causes of fever and jaundice were excluded by relevant investigations . other causes of jaundice excluded by absence of evidence of exposure to hepatotoxic drugs and absence of clinical or serological evidence of infective etiology ( like viral hepatitis , leptospirosis ) . serum was tested for sodium , potassium , alkaline phosphatase , alanine aminotransaminase , bilirubin , lactic acid dehydrogenase , and glucose 6-phosphate dehydrogenase . estimation of blood sugar , coagulation profile for disseminated intravascular coagulation ( dic ) , and arterial blood gas analysis were carried out when indicated . chest x - ray and ultrasonography ( usg ) of abdomen were recorded in all the patients . aki was defined as serum creatinine ( scr ) of > 3 mg / dl and urine output < 400 ml/24 hours , with normal kidney size on usg as per world health organization ( who ) definition . jaundice was defined as icteric sclera and/ or total bilirubin levels > 3 mg / dl . the severity of illness was assessed using apache ii , sofa , mods , and gcs scores.[1316 ] the patients were managed using antimalarial drugs , fluid replacement , and rrt . all patients received artesunate 2.4 mg / kg intravenously , followed by 2.4 mg / kg at 12 and 24 hours , followed by 2.4 mg / kg once daily for a total of 7 days and doxycycline ( adults : 100 mg orally twice daily ; children : 2.2 mg / kg [ up to 100 mg ] orally twice daily ) . in absence of parasitological or clinical response to parenteral artesunate , patients received quinine 20 mg salt / kg body weight through intravenous ( iv ) infusion in 5 percent dextrose over 4 hours , then 10 mg / kg body weight every 8 hour for 5 to 7 days . broad - spectrum antibiotic treatment ( iv ceftriaxone 1 g bd ) was given initially until a bacterial infection ( notably salmonella ) was excluded.[1719 ] supportive measures ( e.g. , antipyretics , oxygen , ventilatory support , cardiac monitoring , and pulse oximetry ) were instituted as needed . rrt was initiated for fluid overload , hyperkalemia , clinical evidence of uremia , metabolic acidosis , rapidly increasing scr level , blood urea nitrogen ( bun)>100 mg / dl , and scr > 4 to 5 mg / dl . rrt was initiated prior to the development of overt symptoms and signs of renal failure due to malaria.[131117 ] the hd prescription included a polysulfone membrane dialyzer with surface area 1.3 to 1.8 m , a blood flow rate of 200 to 300 ml/ min , and a dialysate flow rate of 500 ml / min against a 35 meq / l bicarbonate bath . heparin - free ( in most of patients ) intermittent hd was provided on alternate days through temporary femoral / jugular catheter for 4 hours and continuous renal replacement therapies ( crrt ) were done in hemodynamically unstable patients . rrt was usually continued until the patient manifested evidence of recovery of kidney function . in oliguric patients , the primary manifestation of recovery of kidney function was an increase in urine output . in patients who were nonoliguric , recovery of kidney function was manifested by a progressive decline in scr concentration after initial attainment of stable values ( assessed daily during crrt or predialysis in patients managed with intermittent hd ) , despite a constant dose of renal support . they received fluid up to 20 ml / kg of 0.9% saline infused over 60 minutes . to prevent fluid overload , auscultation of the lungs and patients with severe malaria with clinically significant dic were given fresh whole blood transfusions , vitamin k , and fresh frozen plasma . 10 ml / kg of packed red blood cells or 20 ml/ kg of whole blood was transfused during hd . in addition , support of erythropoiesis in the form of replacement of folic acid and iron was instituted since many patients were malnourished . exchange transfusion was done for patients with parasite density of > 10 percent with end organ complications , serum bilirubin > 25 mg/ dl.[3172022 ] renal biopsy was performed when oliguria / heavy proteinuria ( > 3 g / day ) or hematuria persisted for three weeks . the continuous variables were presented as mean standard deviation and assessed by analysis of mann whitney u - test to identify the differences between groups , with statistical significance set at p < 0.05 . analysis of each individual - dependent variable , using bonferroni - adjusted alpha level , shows that there was no contribution of the dependent variables . we considered the values for the wilks lambda and pillai 's trace for robustness of manova . the mean age of the 59 patients was 33.63 14.6 years ( range , 6 - 75 years ) ; 44 ( 74.5 % ) were males and 15(25.5% ) were females . pf malaria was seen in 76.3% ( n = 45 ) , pv in 16.9% ( n = 10 ) , and mixed infection in 6.8% ( n = 4 ) patients . presenting clinical features were fever ( 100% ) , nausea - vomiting ( 85% ) , oliguria ( 61% ) , abdominal pain / tenderness ( 50.8% ) , jaundice ( 74.5 % ) , dyspnea ( 30.5% ) , diarrhea ( 15.2% ) , altered sensorium ( 10% ) , and convulsions ( 8.4%).the time between onset of symptoms and admission to the hospital for aki ranged from 7 to 14 days ( median , 10 days ) . hyponatremia , usually asymptomatic , was observed in 71% ( n = 42 ) of patients . baseline characteristics of the study patients of malaria and aki univariate analysis of factors associated with outcome in survivors and nonsurvivors the mean apache ii score was 18.1 3 , ( 9 - 32 ) ; the mean sofa score was 10.16 3.09 , ( 4 - 17 ) ; the mean mods score was 9.71 2.69 , ( 4 - 16 ) ; and the mean gcs score was 14.15 1.67 , ( 8 - 15 ) , all were higher , indicating more severe abnormalities among the patients who died than among those who survived as shown in tables 1 and 2 . sofa score < 12 were associated with a low mortality rate ( 41 patients , 0 deaths ) , whereas score 12 showed poorer outcome ( 18 patients , 7 deaths , p < 0.0001 ; relative risk , 0.19 ; 95% confidence interval , 0.10 - 0.34 ) . mods score < 12 were associated with a low mortality rate ( 43 patients , 0 deaths ) , whereas score 12 showed a worse outcome ( 16 patients , 7 deaths , p < 0.001 ; relative risk , 0.19 ; 95% confidence interval , 0.100.34 ) . apache ii score < 20 were associated with a low mortality rate ( 42 patients , 0 deaths ) , whereas score 20 showed a worse outcome ( 17 patients , 7 deaths , p < 0.001 ; relative risk , 0.21 ; 95% confidence interval , 0.110.37 ) . on multivariate analysis , statistically significant predictors of outcome were apache ii score ( p = 0.0004 ) , sofa score ( p = 0.0003 ) , mods score ( p = 0.002 ) , gcs score ( p = 0.00002 ) , and lactate dehydrogenase ( ldh ) level ( p = 0.00001 ) . mean number of hd sessions required was 4.59 3.03 ( range , 1 - 15 ) . all patients received artesunate and additional quinine was required in 10 ( 16.9% ) , exchange transfusion was done in 15% ( n = 9 ) patients and 10% ( n = 6 ) of these survived . = 3 ) had patchy cortical necrosis and 40% ( n = 2 ) had acute tubular necrosis ( atn ) with mesangial proliferative glomerulonephritis without any immune deposits . 81.3% ( n = 48 ) of the patients had complete recovery with normal renal function ( scr < 1.4 mg/ dl ) , 5% ( n = 3 ) had incomplete recovery ( scr , 1.5 - 3 mg / dl ) and remained on conservative management , 1.6% ( n = 1 ) ended up as chronic renal failure and continued on maintenance hd , and 11.8% ( n = 7 ) succumbed to malaria due to multiorgan dysfunction syndrome ( mods ) . the most common adverse effects associated with artesunate included nausea , vomiting , anorexia , and dizziness , although these may be due to malaria rather than drug adverse effects . factors related to aki in vivax malaria were heavy parasitemia , hyperbilirubinemia , volume depletion , hypercatabolic state , intravascular hemolysis , and sepsis . our analysis of malaria patients with aki reveals that renal failure was observed in adults and more common in males . this could be explained by more outdoor activities of males in asian countries as compared with females . aki was usually seen in early second week and is oliguric in 61% of cases . the etiology of aki was usually multifactorial due to hyperbilirubinemia , intravascular hemolysis , hyperparasitemia , volume depletion , hypoxia , shock , pigment nephropathy , dic , and sepsis.[192327 ] the cascade of inflammatory cytokine activation leading specifically to renal damage remains enigmatic . the overall mortality rate among those with kidney injury ranges from 15 to 50% in different series . low mortality ( 11.8% ) in our study of malaria with aki despite of having jaundice in 75% is due to prompt diagnosis , timely hd , exchange transfusion , and supportive therapy . in earlier reports , almost all cases of malarial aki were observed in adults and more common in males . the effect of associated aki on mortality in cerebral malaria patients indicated that mortality was as high as 39.5% when associated with aki , whereas it was only 13.9% when unassociated with aki . hyponatremia , usually asymptomatic , is observed in 71% ( n = 42 ) of the patients . hyponatremia in adults with severe malaria is common and associated with preserved consciousness and decreased mortality . it likely reflects continued oral hypotonic fluid intake in the setting of hypovolemia and requires no therapy beyond dehydration . the incidence of jaundice in falciparum malaria has been reported to be between 2 and 57% . jaundice is probably associated with toxicity to tubular cells with more risk of development of acute tubular necrosis . in patients with falciparum malaria with jaundice indirect hyperbilirubinemia from rbc destruction is the most common form of jaundice reported in malaria . some patients with falciparum malaria have direct hyperbilirubinemia and elevated hepatic enzymes . in our study , 75% patients had jaundice as a presenting symptom , which is in accordance to the findings reported in other studies . a number of investigators have recommended that exchange transfusion be considered solely for patients with high - grade parasitemia.[352023 ] the rationale for exchange transfusion therapy is based on rapid reduction in the parasite load by direct removal , decreased risk of severe intravascular hemolysis and its consequences , improved rheology with transfused blood and reduced microcirculatory sludging , and improved oxygen - carrying capacity with transfused erythrocytes . however , the who guidelines indicate that it is not possible to make any recommendations regarding the use of exchange transfusion based on the available evidence . the centers for disease control ( cdc ) recommends consideration of exchange transfusion for patients with parasite density of > 10% with end organ complications . apache ii , sofa , and mods scores have been used in malaria for determining severity of malaria.[84041 ] mods score 16 was associated with significantly longer disease duration . the mods is simple and easy to apply and needs a recording time of less than three minutes . thus , this score might provide a quantitative approach for determining severity in pf malaria . high apache ii score , deep unconsciousness , aki , and acidemia were identified as poor prognostic factors in cerebral malaria patients . with the cut - off point at a score of 24 , the apache ii score stratified the patients mortality outcome with 95.8% accuracy . sofa score < 10 were associated with a low mortality rate , whereas scores > 10 showed a significantly worse outcome . apache ii score 20 , sofa and mods scores 12 were associated with higher mortality in our study . these score can be used for prognosis in malaria with aki . these scores can help us for better stratification of patients for better and intensive managements to improve outcome . in our study , on univariate and multivariate analysis , statistically significant predictors of outcome were apache ii , sofa , mods , gcs scores , and ldh level . there is no direct pathogenic linkage between vivax malaria and aki , but the associated conditions such as heavy parasitemia , hypercatabolic state , volume depletion , hyperbilirubinemia , intravascular hemolysis , sepsis , and dic can contribute to aki . hence , despite the association , cause and effect relationships remain doubtful . hyperbilirubinemia may contribute to reduction in total peripheral vascular resistance and in renal blood flow due to left ventricular dysfunction . acute cortical necrosis ( acn ) has rarely been reported in patients with aki due to malaria.[4447 ] the possible pathogenetic factors in patients with acn were the renal damage through renal hypoperfusion or endothelial injury through release of various circulating substances ( intravascular hemolysis , sepsis , and pancreatitis ) . it should be suspected in patients with aki who have a prolonged phase of oligoanuria.[4448 ] these findings are of importance for clinicians in prognosticating their patients with aki due to malaria . future studies representing a large population with the full spectrum of malaria patients in different geographical areas will determine the usefulness of this scoring method for both clinicians and researchers and recommendation for adjunct exchange transfusion . prompt diagnosis , timely hd , and supportive therapy are associated with improved survival and recovery of kidney functions in malarial with aki . mortality was associated with higher apache ii , sofa , mods , gcs scores , requirement of inotrope , and ventilator support .

acute kidney injury ( aki ) is one of the most dreaded complications of severe malaria . we carried out prospective study in 2010 , to describe clinical characteristics , laboratory parameters , prognostic factors , and outcome in 59 ( 44 males , 15 females ) smear - positive malaria patients with aki . the severity of illness was assessed using acute physiology and chronic health evaluation ( apache ) ii , sequential organ failure assessment ( sofa ) score , multiple organ dysfunction score ( mods ) , and glasgow coma scale ( gcs ) scores . all patients received artesunate and hemodialysis ( hd ) . mean age of patients was 33.63 14 years . plasmodium falciparum malaria was seen in 76.3% ( n = 45 ) , plasmodium vivax in 16.9% ( n = 10 ) , and mixed infection in 6.8% ( n = 4 ) patients . presenting clinical features were fever ( 100% ) , nausea - vomiting ( 85% ) , oliguria ( 61% ) , abdominal pain / tenderness ( 50.8% ) , and jaundice ( 74.5% ) . mean apache ii , sofa , mods , and gcs scores were 18.1 3 , 10.16 3.09 , 9.71 2.69 , and 14.15 1.67 , respectively , all were higher among patients who died than among those who survived . apache ii 20 , sofa and mods scores 12 were associated with higher mortality ( p < 0.05 ) . 34% patients received blood component transfusion and exchange transfusion was done in 15% . mean number of hd sessions required was 4.59 3.03 . renal biopsies were performed in five patients ( three with patchy cortical necrosis and two with acute tubular necrosis ) . 81.3% of patients had complete renal recovery and 11.8% succumbed to malaria . prompt diagnosis , timely hd , and supportive therapy were associated with improved survival and recovery of kidney functions in malarial with aki . mortality was associated with higher apache ii , sofa , mods , gcs scores , requirement of inotrope , and ventilator support .

the diagnosis and management of pancreatic cystic lesions has become an area of developing interest over the past decade . increasing use of advanced abdominal imaging modalities has resulted in the discovery of previously unrecognized pancreatic cystic neoplasms ( pcns ) . as these lesions have become a more common finding , health care providers should be familiar with the different types of cystic pancreatic lesions in order to assess the potential for malignancy within a cyst . this allows providers to effectively risk - stratify patients for surveillance , surgery , or expectant management . the purpose of this review is to provide both general practitioners and specialists with evidence - based data to aid in the management of patients found to have pcns . pancreatic cysts are classified as either a true cyst in which there is a true epithelial lining to the lesion ; or a pseudocyst ( false cyst ) , in which there is a walled - off collection of fluid without a true epithelial lining , usually the result of acute pancreatitis . pcns are true pancreatic cysts , and they represent at least 50% of all pancreatic cystic lesions . pcns are generally divided into four subtypes : mucinous cystic neoplasms ( mcns ) , intraductal papillary mucinous neoplasms ( ipmns ) , serous cystadenomas ( scas ) , and solid pseudopapillary neoplasms ( spns ) ( table 1).13 the mcn and ipmn types are further classified as mucin - producing lesions , while the remainder are non - mucinous.4 differentiating between the different cyst types can be challenging ; however , certain radiographic , histological , and pathological features may help distinguish these cysts from one another , and thus help guide management for the patient . they occur more commonly in women , and their peak incidence is in the seventh decade of life . the cyst lining is composed of a simple , glycogen - rich cuboidal epithelium . on imaging , these cysts appear as honeycomb - like microcysts , often with the presence of a central scar on computed tomography ( ct ) or magnetic resonance imaging ( mri ) . the tiny microcystic spaces may often coalesce , and the lesion can begin to appear as a solid mass - like structure ( figure 1 ) . these lesions are usually benign , with a very low potential for malignant transformation , and thus they are typically managed conservatively unless the patient is symptomatic from the cyst ( eg , abdominal pain).3 however , cases of malignant transformation of a serous cystic lesion into a serous cystadenocarcinoma have been reported . in a retrospective review of 158 resection specimens of serous cystic pancreatic lesions from a single institution , one case of histologically confirmed malignancy was identified.5 also , three of these cases were classified as locally aggressive benign lesions , one of which later developed metachronous metastatic lesions . additionally , a literature review of serous cystic lesions in 2009 indicated that an average lesion size of 10 cm was associated with carcinoma.6 therefore , consideration should be made to treat larger and locally advanced lesions aggressively . a relationship between scas and von hippel lindau ( vhl ) disease has been noted . in one study , a histopathological analysis of pancreatic cysts from nine vhl patients was performed . a total of 21 benign serous lesions , 63 microscopic microcystic ( serous ) adenomas , and 35 macroscopic microcystic ( serous ) adenomas were found.7 all lesions displayed similar histology , and deoxyribonucleic acid ( dna ) extracted from the cysts showed allelic deletions in the vhl gene . another study showed evidence of vhl gene alterations not only in vhl - disease - associated cysts , but also in sporadic microcystic ( serous ) adenomas ; thus implying that changes in the vhl tumor suppressor gene play an important role in the pathogenesis of these types of cysts , regardless of whether or not the individual has vhl disease.8 mcns comprise 10%45% of pcns , occur mostly in the female population , and are typically discovered in the fifth and sixth decades of life . the mcns are histologically very similar to ipmns , as both lesions have a mucin - producing epithelial lining . however , a distinguishing feature between mcns and ipmns is the characteristic histopathological dense mesenchymal ovarian - like stroma seen in mcns . also , mcns do not communicate with the pancreatic ductal system , as they develop out in the periphery of the gland ( figure 2 ) . all mcns have a risk for malignant transformation , and therefore resection is generally considered in individuals who are good surgical candidates depending upon their clinical risk factors.3 the prevalence of invasive carcinoma in mcns at the time of surgical resection varies from 6% to 36%.4 however , some studies have not used ovarian - type stroma as a necessary criterion to distinguish mcns from ipmns , making these data difficult to interpret . in some studies , the prevalence of invasive carcinoma strictly in mcns with ovarian - type stroma ranges from 6% to 27% . to avoid mistakenly classifying ipmns as mcns , which may have different clinical implications for the patient , the diagnosis of mcn should be limited to cysts containing ovarian - type stroma.4 ipmns represent approximately 21%33% of pcns . ipmns occur with equal frequency in both men and women , commonly in the sixth and seventh decades of life , and more often in the head of the pancreas . the cyst lining consists of a mucin - secreting columnar epithelium . all ipmns have malignant potential , and similar to mcn lesions , an algorithm for risk - stratification and management is of paramount importance ( discussed below ) . ipmns with adenomatous or borderline changes have been shown to have an excellent prognosis if resected ; however , the prognosis is less favorable when findings of carcinoma in situ or invasive carcinoma are present.3 ipmns can be divided into three different subtypes : 1 ) main duct ipmn ( md - ipmn ) , involving dilation of the main pancreatic duct ( mpd ) only ; 2 ) branch duct ipmn ( bd - ipmn ) , involving cystic dilation of one of the ductal side - branches ; and 3 ) mixed type , in which both the main duct and side - branch are involved in cystic dilation . segmental or diffuse dilatation of the mpd > 5 mm , in the absence of other secondary causes such as chronic pancreatitis , is suggestive of md - ipmn ( figure 3 ) . mucinous cysts communicating with the pancreatic ductal system without evidence of mpd dilation are categorized as bd - ipmns . radiographically , they often appear as a bunch of grapes growing from the end of a pancreatic ductal side - branch ( figure 4 ) . ipmns which meet criteria for both md- and bd - ipmn are categorized as mixed type , which can have varying degrees of involvement with both the main duct and branch ducts.4,9 definitive diagnosis of an ipmn is made based on the histology of resected cysts . many bd - ipmns exhibit some involvement with the main duct microscopically , and therefore grading these lesions in terms of the extent of main duct involvement may be a preferable approach , in contrast to categorizing all ipmns as strictly md - ipmn or bd - ipmn.4,9 distinguishing between these subtypes is important , as md - ipmns are at increased risk for malignant transformation compared with bd - ipmns . the prevalence of malignancy in resected md - ipmn lesions ranges from 57% to 92% , in contrast with 6%46% for bd - ipmn lesions.4 interestingly , on cyst fluid dna analysis studies of ipmns , the histological grade of dysplasia increases with the frequency of mutations in the k - ras gene.10 these findings suggest that k - ras gene mutations play a significant role in the process of carcinogenesis for these mucinous pcns . spns represent less than 10% of pcns , and they occur predominantly in younger women , with a peak incidence ranging from the second to fourth decades of life.3 they are most commonly located in the body or tail of the pancreas , but the location is variable . solid and cystic components may be present , as well as occasional calcifications within the cyst ( figure 5 ) . histologically , spns contain uniform cells with ovoid nuclei and eosinophilic granules , which are arranged in sheets . spns have a low potential for malignant transformation , but in general are considered to have a much higher risk for cancer compared with benign scas . these lesions have an excellent prognosis when completely resected , as the overall incidence of malignancy is estimated to be less than 15% . metastasis to sites including the liver , peritoneum , and lymph nodes has been reported . even in malignant cases , prolonged survival has been shown in patients with residual disease after surgery , or in patients with unresectable tumors.3,11 one additional less common class of pcns is the cystic neuroendocrine neoplasm . these cysts represent less than 10% of pcns , and they occur with equal distribution among men and women . cytology of these non - mucinous cysts shows small cells with scant cytoplasm and monomorphic nuclei with salt - and - pepper chromatin.3 cystic neuroendocrine neoplasms are typically nonfunctioning tumors that have undergone cystic degeneration and can be difficult to distinguish from other pcns on imaging alone . one study found this cystic degenerative form represented 10% of all pancreatic neuroendocrine tumors.12 their location is most commonly in the body and tail of the pancreas . these lesions are generally indolent in behavior and carry a good prognosis ; however , surgical resection should be considered in appropriate surgical candidates , given the premalignant nature of these lesions , particularly when they are larger than 2 cm in size.12,13 most pancreatic cysts are asymptomatic at the time of diagnosis , and they are often discovered incidentally when abdominal imaging is performed for evaluation of an unrelated problem . when pcns are symptomatic , symptoms are typically the result of pancreaticobiliary duct obstruction . as such , clinical findings may include recurrent pancreatitis , chronic abdominal pain , or jaundice . other nonspecific symptoms which may be present include nausea , vomiting , back pain , weight loss , or anorexia . the symptoms of advanced pcns with malignant transformation may mimic those caused by pancreatic ductal adenocarcinoma ( eg , jaundice , weight loss , and pain ) . obstruction of the mpd ( typically from mucin due to an ipmn , or compression of the duct by mass effect from the lesion ) may present as acute or chronic pancreatitis ( figure 6 ) . development of exocrine and endocrine pancreatic insufficiency is not uncommon , due to atrophy of the distal gland downstream of the obstruction . pcns and small pancreatic pseudocysts are often mistaken for one another given their similar presentations and imaging characteristics . the clinical context is often needed to help differentiate between pcns and pseudocysts , as the latter are more likely to develop in the setting of prior pancreatitis ( either recent or remote past ) , and are typically associated with pain.3,14 one study investigating 212 patients with pancreatic cystic lesions in a surgical practice showed that 36.7% of the patients were asymptomatic.1 these asymptomatic cysts were more common in the elderly , smaller in size than symptomatic cysts , and less likely to be pseudocysts on final surgical pathology . furthermore , greater than half of the asymptomatic cysts were found to be pcns with dysplastic changes or malignant transformation . another recent study followed 105 patients who underwent pre operative endoscopic ultrasound ( eus ) for cyst evaluation . in this study , only 10% of patients were asymptomatic . of the 70 patients with eus cyst size less than 3 cm , 12 patients ( 17% ) had a malignancy as diagnosed from eus.15 the above studies support the role for periodic surveillance in all patients with pcns , regardless of the size of the lesion or presence of symptoms . pancreatic cystic lesions are often initially detected on cross - sectional abdominal imaging such as ct or mri . these modalities can help characterize the morphological features of pancreatic cysts such as cyst wall calcification , the presence of septae or mural nodules , and concurrent changes consistent with pancreatitis . in contrast to ct scan , mri with magnetic resonance cholangiopancreatography ( mrcp ) serves to evaluate the pancreatic ductal system and often establishes the existence of ductal communication with a pancreatic cyst . according to a consensus of surveyed radiologists , the optimal procedure for evaluating pancreatic cystic lesions is a dedicated mri - mrcp due to superior contrast resolution with better visualization of septae , nodules , and ductal communications.3,9,16 in another study evaluating the accuracy of ct versus mri - mrcp in the characterization of ipmn disease , ductal connection was found on 73% of mrcp scans and only 18% of ct scans.17 ct scans overestimated mpd involvement when compared with mrcp and surgical pathology . mrcp identified multifocal disease in 72% of cases versus 50% on ct . additionally , mrcp was superior in visualization of branch duct lesions.17 although pcns often appear morphologically similar on cross - sectional imaging , particular characteristic features of specific pcn types can aid in diagnosis without further invasive testing . for example , the presence of a central scar seen on ct or mri is highly suggestive of a serous lesion ( sca ) , and noted in roughly 20% of these cysts . for mcn lesions , ( small calcifications on the periphery of macrocystic spaces within the lesion ) is rare but strongly predictive of malignancy . newer types of ct imaging , such as high - resolution multi - slice helical imaging , can more accurately identify potentially malignant features of ipmns , such as the presence of mural nodules and segmental or diffuse dilatation of the mpd > 15 mm in diameter . certain characteristics on ct , in combination with clinical history , can distinguish true cysts from pseudocysts . pseudocysts are more likely to be associated with findings of chronic pancreatitis such as gland atrophy , ductal dilatation , parenchymal calcification , and calculi in the pancreatic duct.3 when further evaluation of a pcn beyond routine cross - sectional imaging is required , eus with fine - needle aspiration ( fna ) may be utilized . eus - fna allows for better imaging characterization of cyst morphology ( eg , the presence of a mural nodule or a solid component ) , and it enables aspiration of cyst fluid for further analysis . despite its unique ability to obtain high quality pancreatic imaging from within the lumen of the upper gastrointestinal tract , previous studies have suggested a limited ability of eus alone to distinguish between benign and early dysplastic or malignant lesions , especially if features of frank malignancy are not present.3,16 furthermore , the accurate interpretation of eus findings is operator dependent and often varies from one endoscopist to another . one study investigated the ability of endosonographers to establish a diagnosis of a pcn , and also to determine the presence or absence of malignancy solely based on the eus findings.18 poor to fair agreement was observed between endosonographers in both tasks . another study evaluated the accuracy of preoperative imaging with ct , endoscopic retrograde cholangiopancreatography ( ercp ) , or eus in the detection of invasive versus noninvasive ipmn and mcn lesions.19 the overall accuracy for detecting invasion was less than 80% for all three diagnostic modalities . ercp enables inspection of the duodenal papilla for mucin extrusion , a finding which occurs in 20%50% of main duct ipmns , and is essentially pathognomonic for the disease . in addition , ercp allows for the ability to perform pancreatography to assess communication of the cyst with the mpd . that said , with today s high quality ( noninvasive ) mrcp studies , there is little role for diagnostic ercp in the work - up of a pcn . due to the relatively high risk of post - procedural pancreatitis when performing ercp of the pancreatic duct ( 20%30% ) , this invasive procedure is most often reserved for patients in whom the diagnosis of main duct ipmn is highly suspected , and one is attempting to diagnose frank malignancy within the duct . the combination of intraductal pancreatoscopy with intraductal ultrasound at the time of ercp can demonstrate malignancy with a high level of precision , and allow for accurate sampling of ductal nodules and other areas of concern.3,4,16 these procedures are generally performed only at specialized centers by expert interventional endoscopists . compared with ercp , eus evaluation with fna is a less invasive and safer endoscopic procedure for the diagnosis of a pcn . cyst fluid is obtained to assess for the presence of mucin , cytological atypia , carcinoembryonic antigen ( cea ) , and amylase levels , and dna for molecular analysis . studies have shown varying success in the accurate diagnosis of pcns using cytology alone . however , identifying certain cell types using fna can help narrow the diagnosis in certain instances . for example , the finding of glycogen - rich cuboidal cells suggests a diagnosis of sca in the appropriate clinical setting ; or the aspiration of inflammatory cells , such as macrophages and neutrophils , usually is suggestive of a pseudocyst.3,9,16 for the diagnosis of malignancy within pcns , eus - fna with cytology alone is highly specific ( approximately 90% ) ; yet , the sensitivity of eus - fna for a malignant pcn may be as low as 40%50% , with high false - negative rates . in addition to cytology , the cyst fluid from pcns can be used to measure levels of tumor markers and pancreatic enzymes ( table 2 ) . cea is a marker that has been shown to differentiate mucinous from non - mucinous cysts with 80% accuracy using levels > 192 ng / ml as a threshold suggestive of a mucinous lesion.3 however , a low cea level ( < 192 ng / ml ) does not fully exclude a mucinous cyst . furthermore , cea levels have not been shown to distinguish benign from malignant lesions.9,16 the presence of amylase in cyst fluid may suggest a communication between the pcn and the pancreatic ductal system , typically characteristic of an ipmn . low amylase concentrations in cyst fluid are associated with noncommunicating lesions such as scas and mcns.3 although the tumor marker carbohydrate antigen ( ca ) 19 - 9 has been shown to have an association with pancreatic adenocarcinoma , its presence in cyst fluid has not been helpful in distinguishing between mucinous and non - mucinous pcns.3 the presence of ca 72 - 4 in cyst fluid has , however , been shown to be indicative of a mucinous lesion . one study showed that elevated serum ca 19 - 9 in combination with elevated ca 72 - 4 in cyst fluid is associated with mucinous neoplasms and ductal adenocarcinomas , and these patients should be considered for resection.3,20 recent developments in dna molecular analysis of cyst fluid have identified genes potentially associated with certain cyst types or pcns . this can further help with the diagnosis of pcns when cytological analysis is unrevealing due to scant cellularity in the cyst fluid , especially when a solid component is not present for sampling . a recent study showed that the presence of a k - ras gene mutation is diagnostic of a mucinous cyst.21 furthermore , cyst fluid demonstrating large amounts of dna , high - amplitude mutations , or a mutational sequence of k - ras mutation followed by allelic loss ( loss of heterozygosity ) is highly suspicious for malignancy.9,21 another recent study demonstrated that gnas gene mutations were found in 66% of ipmns , and either gnas or k - ras mutations were present in 96% of ipmns.22 other biomarkers that have been analyzed include micro - ribonucleic acid ( mir ) . in one study , endoscopically acquired pancreatic cystic fluid was obtained from 38 patients who subsequently had surgical resection of the cystic lesion.23 levels of two specific mirs ( mir-21 and mir-221 ) were found in higher concentrations in the malignant versus benign cystic lesions . interpretation of the results can be difficult and variable depending upon the type of lesion , and the amount of fluid able to be aspirated at the time of the eus procedure . the information obtained from the cyst fluid is used in conjunction with the patient s clinical presentation , as well as specific cyst - related morphological features on imaging , in order to make an overall assessment in terms of the type of pcn present and its inherent risk of malignancy . with the development of new and emerging molecular markers , cyst fluid analysis is likely to become even more complex in its attempt to risk - stratify specific pcns . however , in the opinion of these authors , current data do not support the widespread use of molecular analysis in cyst fluid interpretation due to low overall specificity and sensitivity of the tests . for example , a recent study characterized the performance of molecular analysis ( dna ) in diagnosing mucinous lesions.24 dna analysis was performed on cyst fluid and compared with resection specimens . molecular analysis had a sensitivity of 50% and specificity of 80% in identifying mucinous lesions . diagnostic performance did increase when combined with cea and cytology ; however , the study clearly shows that currently available molecular analysis studies are insufficient when used alone . in routine clinical practice , lesions in which we may be searching for more information to guide a patient toward , or away from , surgical resection ( as opposed to continued surveillance ) . the various diagnostic modalities discussed above can be useful for narrowing down the diagnosis of the exact type of pcn ; however , definitive diagnosis is often times difficult without supporting histological evidence ( ie , by means of surgical resection ) . given this diagnostic challenge , a more practical approach to risk - stratification has been suggested and outlined in a recent paper by tanaka et al.9 within this publication , evidence - based guidelines were devised by expert physicians and surgeons of the international association of pancreatology ( iap ) . worrisome features and high - risk stigmata to determine management of these lesions based on their malignant potential . furthermore , they outline the stepwise use of multiple imaging modalities when further work - up is required in order to determine whether a pcn is appropriate for surgical resection . the evidence - based worrisome features based on multiple imaging modalities include cyst size 3 cm , thickening or hyperenhancement of the cyst walls , mpd size of 59 mm , mural nodules , abrupt change in the mpd caliber ( with distal pancreatic atrophy ) , and regional lymphadenopathy . features on ct , mri , or eus such as an obstructed common bile duct in a patient with a lesion of the pancreatic head , an enhanced solid component to the cyst , and mpd size 10 mm are highly suspicious for malignancy and are thus termed high - risk stigmata ( table 3).9 patients who demonstrate high - risk stigmata should be strongly considered for surgical resection unless clinically contraindicated due to high operative risk . furthermore , pcns that cause symptoms ( eg , abdominal pain , pancreatitis , and weight loss ) often necessitate strong consideration for surgical resection , as the presence of symptoms has been shown to confer a higher risk of malignant transformation.9 the following algorithm for risk - stratification and management of pcns has been proposed : any pcn with features showing high - risk stigmata high - risk stigmata are not present on noninvasive imaging studies , the next step is to assess for worrisome features . if worrisome features are suggested on ct or mri , an eus examination should be performed by an experienced endo scopist to assess for these features , looking specifically for the presence of a mural nodule , mpd abnormalities with gland atrophy , thickened or enhanced portions of the cyst wall , and the presence of undetected regional lymphadenopathy . in addition , eus - fna may be performed to obtain cyst fluid for cytological analysis which may be suspicious or positive for malignancy . based on these findings surgery may be considered . if none of these worrisome features are present , different intervals of surveillance should occur based on the cyst size . for cyst size 23 cm , patients should be evaluated with mri or eus as frequently as every 36 months , with consideration for resection in young , surgically fit individuals . for cyst size 12 cm , monitoring with ct or mri annually may be considered , with lengthening of the surveillance interval if no changes in cyst features are present . for small lesions < 1 cm in size , monitoring with ct or mri can be performed every 23 years.9 management of suspected ipmn lesions differs depending on the origination of the cystic lesion within the main duct or the branch ducts . according to a published series , main duct ipmns have a mean frequency of malignancy of 61.6% , and a mean frequency of invasive disease of 43.1%.4,9 in one series , the three factors most predictive of malignancy in md - ipmn and mixed - type ipmn lesions were the presence of symptoms , mural nodules , and mpd diameter 15 mm or greater.4,25 another large study of resected md - ipmns showed malignancy was associated with older age ( at least 6 years older than their benign counterparts ) , as well as the presence of jaundice or worsening diabetes at presentation.4,26 several patients , however , in both series had no obvious clinical or radiographic predictors of advanced disease , yet were found to have malignancy at the time of resection . given the high prevalence of malignancy in md - ipmn lesions , it is inferred that most md - ipmns go on to progress to malignancy . coupled with the low overall 5-year survival rates following surgical resection ( 31%54% ) , guidelines suggest removal of all md - ipmns in surgically fit patients with the aim of complete resection of the cyst with negative margins . long - term follow - up of patients with resected noninvasive md - ipmns has shown good long - term survival rates . resection of invasive ipmns results in a 5-year survival rate ranging from 36% to 60%.4,9 the management of branch duct ipmns is less clear and depends greatly on the clinical context . the malignant potential of a bd - ipmn is less than md - ipmn , with a mean frequency of malignancy of 25.5% , and a mean frequency of invasive cancer of 17.7%.9 sugiyama et al showed the two strongest predictors of malignancy in bd - ipmn lesions are the presence of a mural nodule and cyst size > 30 mm.4,25 furthermore , matsumoto et al demonstrated that bd - ipmns with size < 30 mm and the absence of mural nodules are highly unlikely to be malignant.27 these patients were followed for 33 months , and the majority remained asymptomatic without progression to advanced disease . in addition to size and the presence of mural nodules , a rapid rate of cyst growth is another high - risk factor.4,9 one study investigated bd - ipmns < 30 mm and without mural nodules.28 during follow - up , 17.4% of the patients underwent resection , and the malignant cysts had grown by a greater percentage ( 69.8% versus 19.4% ) , and at a greater rate ( 4.1 mm versus 1.0 mm per year ) , when compared with the nonmalignant cysts . overall , a cyst growth rate of more than 2 mm / year was associated with a higher risk of malignancy.28 in addition , high - grade cellular atypia on eus - fna results , as opposed to positive cytology , was also found to be a high - risk factor for malignancy in bd - ipmns.9 the results of these studies and many others are the basis for the algorithmic guidelines suggested by tanaka et al in regards to the management of bd - ipmn lesions.4,9 given the lower risk for malignancy when compared with md - ipmn lesions , conservative management with periodic surveillance is reasonable , particularly in older patients and those without worrisome features . mcns have an overall prevalence of invasive carcinoma of less than 15%.4,9 malignancy is usually absent in mcns < 34 cm in size . since these lesions commonly occur in the body and tail of the pancreas , surgical resection is more often less invasive , since a distal pancreatectomy may be performed as opposed to pancreaticoduodenectomy . in younger patients with cysts located in the distal pancreas , surgery the prognosis for patients undergoing resection of mcns prior to the development of invasive disease is excellent . once an mcn develops into a mucinous cystadenocarcinoma , resectability is difficult , which leads to poor prognosis.3,4,9 as described above , scas have an extremely low potential for malignant transformation . if the work - up of a pcn is strongly suggestive of an sca , and the patient has no symptoms due to the cyst , these lesions can be managed conservatively with observation . if the diagnosis is unclear , or if the sca causes symptoms ( usually seen with sca size > 4 cm29 ) , surgical resection should be considered.3,16 spns have a low but significant a total of 15% of the resected spns were malignant , without corresponding preoperative features predictive of malignancy.30 given the low - grade potential for malignancy in spn lesions , and the high cure rate if completely resected , the threshold for surgical resection in appropriate patients should be low ; especially since most of these patients are young women below the age of 45 years old.3,16 the type of resection depends on the location of the pcn and the extent of involvement . limited resections can be considered for mcns and bd - ipmns without findings suspicious for malignancy or invasion . however , limited resections are technically difficult and associated with complications such as leaks and positive margins . therefore , limited pancreatectomy should only be performed if negative margins can be definitively obtained.4 the standard treatment of any pcn with an invasive component is pancreaticoduodenectomy , distal pancreatectomy , or total pancreatectomy . in recent years , the mortality rate for pancreatic resection has fallen to less than 2% at high - volume centers . mcns are typically located in the tail of the pancreas and can be resected using a distal pancreatectomy ( with or without splenectomy ) . ipmns are frequently located in the head and may require pancreaticoduodenectomy , or total pancreatectomy if more extensive involvement of the ductal system is discovered intraoperatively.3 recent studies have examined nonsurgical methods for treating pcns . eus - guided mucosal ablation by ethanol injection into the cyst cavity is a novel technique that has been recently investigated . typical candidates for this investigative approach have been patients who are poor surgical candidates with worrisome - appearing cysts that lack communication with the mpd ( so as not to inject alcohol directly into the pancreatic ductal system ) . the initial pilot study performed by gan et al31 showed that ethanol ablation is safe and feasible , and a subset of patients ( 8 of 23 on follow - up ) underwent complete resolution of the cyst . a follow - up study by dewitt et al32 showed that eus - guided ethanol lavage resulted in a greater decrease in cyst size compared with saline lavage . follow - up using ct surveillance revealed no cyst recurrence for a median of 26 months , and the percentage of complete pancreatic cyst ablation was 33% . studies have also investigated eus - guided ethanol ablation followed by local injection of paclitaxel.33 overall , these findings are promising and may present an alternative therapy for patients unwilling or unfit for surgery . however , it should be noted that complications such as pain and pancreatitis are relatively common . more research at high - volume centers is needed before eus - guided ethanol ablation can be recommended to patients over surgical resection . several studies have investigated current trends in the evaluation and management of pcns among physicians . in one recent study , a comparison of practice habits and awareness of consensus guidelines was examined between general gastroenterologists and surgeons , and a specialist group of eus experts.34 awareness of the existence of published guidelines for the diagnosis and management of pcns was less common in the general group than the specialist group ( 64% versus 33% unaware , respectively ) . the american society for gastrointestinal endoscopy guidelines data for surveillance intervals of ipmns and other pcns are limited and depend largely on clinical judgment and the perceived risk for malignancy , comorbidities , and patient preference . surveillance of non - resected ipmns with eus or mri at appropriate intervals based on cyst size and other features as dictated by the iap guidelines has been discussed above.9 interval surveillance imaging after ipmn resection is strongly recommended , as multifocal disease is common and additional lesions may develop in the remnant pancreas . recurrence rates of new ipmn lesions following resection range from 0%20%.4,9,35 in patients with noninvasive disease that was completely resected , at least an annual examination of the remnant pancreas with mri or eus is encouraged . however , the risk of developing invasive disease in another ipmn lesion within the gland appears to be very low.35 surveillance for invasive ipmns after resection should mimic follow - up guidelines for pancreatic ductal adenocarcinoma . for mcns , given the nearly 100% cure rate following resection of noninvasive lesions , continued surveillance is unnecessary in most cases . malignant mcns should be followed frequently at 612 month intervals with either ct or mri.4,9 data for surveillance guidelines for the other pcn types is limited , and surveillance should be considered on an individual basis . the increasing discovery of pcns is largely due to the widespread use of new , cross - sectional imaging techniques . physicians and surgeons need to be aware of the different types of pancreatic cysts so that a determination may be made regarding the potential for malignant transformation . appropriate evaluation of a possible pcn includes a multidisciplinary approach among abdominal radiologists , gastroenterologists with a special expertise in eus , and pancreatic surgeons . updated published guidelines exist to help providers recognize higher risk lesions , and provide recommendations in terms of surveillance strategies and the need for possible pancreatic resection . much is still unknown about pcns , yet our knowledge on risk - stratification , optimal surveillance intervals , and post - surgical management is rapidly increasing .

pancreatic cystic lesions are commonly encountered today with the routine use of cross - sectional imaging modalities such as computed tomography ( ct ) and magnetic resonance imaging ( mri ) . the majority of patients discovered to have a pancreatic cyst are completely asymptomatic ; yet the presence of such a finding instills fear in the minds of both patient and physician , as the concern for malignant transformation to pancreatic cancer is great despite the relatively low overall likelihood of cyst progression . not all cysts in the pancreas represent pancreatic cystic neoplasms ( pcns ) , and not all pcns have significant malignant potential . mucinous pcns are the most concerning , as these lesions have the greatest potential for cancerous transformation to adenocarcinoma . within the group of mucinous pcns , intraductal papillary mucinous neoplasms ( ipmns ) involving the main pancreatic duct are the most worrisome , and surgical resection should be pursued if the patient has appropriate operative risks . ipmn lesions involving the branch ducts , and mucinous cystadenomas , have a lower likelihood for malignancy , and they may be closely followed for the development of any worrisome or high - risk features . surveillance of known pcns is performed with a combination of ct , mri and endoscopic ultrasound ( eus ) . eus - guided fine - needle aspiration ( eus - fna ) may be used to assess cyst fluid cytology , and also to detect cyst fluid amylase level , carcinoembryonic antigen level , and dna molecular analysis in certain cases . the presence or absence of specific cyst morphological features , as well as the cyst fluid analysis , is what enables the physician to guide the patient towards continued surveillance , versus the pursuit of surgical resection .

bromodomains are epigenetic reader modules of post - translational modifications , responsible for the recognition of -n - acetyllysine ( kac ) marks on histone proteins . in recent years , bromodomains have been implicated as both attractive and druggable targets for treating cancer , inflammation , and neurological disorders . inhibitors have been discovered against several bromodomains , including the bet ( bromodomain and extra - terminal ) family members ( brd2 , brd3 , brd4 , and brdt ) , baz2a / b , crebbp , ep300 , and smarca2/4 . however , very few reports have appeared on the discovery of inhibitors to other bromodomain targets that are implicated in cancer biology but likely more challenging to drug . one such bromodomain for which an inhibitor could be useful is that of the protein atad2 ( aaa domain - containing protein 2 ) , also known as atad2a or ancca ( aaa nuclear coregulator cancer - associated protein ) . the bromodomain of atad2 offers an exciting target for the design of novel cancer therapeutics for several reasons . overexpression of atad2 , a protein that possesses both aaa+ atpase ( atpases associated with various cellular activities ) and bromodomain functionalities , has been linked to poor prognosis in prostate , lung , and triple - negative breast cancers , as well as in hepatocellular carcinoma . atad2 serves as a coactivator for transcription factors such as e2f family members , myc , and estrogen and androgen receptors , whereby driving the expression of a subset of genes that promote cell proliferation and survival , including c - myc , cyclin d1 , e2f , and survivin . finally , downregulation of atad2 via sirna shows an increase in apoptotic activity , suggesting that a small - molecule inhibitor of atad2 could result in cancer cell death and tumor regression . while the cellular and genetic evidence suggests that atad2 inhibitors could be useful for cancer therapy , one computational study of the bromodomain family classified atad2 asdifficult to drug , as its kac binding site is quite dissimilar to other druggable bromdomains atad2 is composed of the typical left - handed four - helix bundle structure , with the za and bc loops determining the geometry of the kac binding pocket ( figure 1a ) . however , when compared to brd4 , only three of seven residues lining the kac binding pocket that interact with peptide are shared with atad2 ( figure 1b , supporting information ( si ) , table s1 , figure s1 ) . ( a ) x - ray structure of atad2 bound to histone 4-derived peptide ( h4kac5 ) , highlighting the conserved kac binding site hydrogen bond donor n1064 in purple spheres ( pdb i d 4quu ) . residues shown as sticks are those that interact with the kac residue of h4kac5 , with only three residues sharing identity to brd4 ( colored purple ) . polar contacts between the peptide and atad2 are shown as yellow dashes , and crystallographic water molecules are shown as blue spheres . to target challenging proteins , such as those involved in protein protein interactions , fragment - based drug discovery has been used to effectively identify hits that can be elaborated to high affinity molecules via structure - based design . while kac - reading bromodomains possess deeper and more - defined binding pockets than traditional protein protein interaction surfaces , fragment - based methods could be applied to those bromodomains that are deemed difficult to drug . while a number of techniques are possible for conducting a fragment - based screen , we rely on the use of protein - observed nmr spectroscopy because it offers several advantages over other biochemical or biophysical methods . because the binding observation by nmr is direct , weak binders up to several millimolar in affinity can be identified with very few false positives . in addition , the binding location of the hits can be ascertained if the resonance assignments of the protein are known . nmr can also be used to determine fragment binding affinities , even for very weak - binding hits , allowing for hits to be rank ordered without a secondary assay . herein we present the results of a fragment - based screen conducted by nmr against the bromodomain of atad2 . from this screen , hits were identified representing several chemotypes that have not been previously observed as bromodomain ligands . although relatively few hits were found , the calculated hit rate suggests that this bromodomain should be druggable . we also present the x - ray crystal structures of atad2 bound to the hits identified in the screen . these results serve as useful starting points toward the design of high affinity , selective chemical probes for atad2 in order to understand its role in cancer biology . because atad2 has been predicted to be challenging to drug , it may be difficult to identify hits from traditional high - throughput screening methods . indeed , it was recently reported that a screen of atad2 against a library of kac mimics revealed no potent hits . as an alternative , we chose a fragment - based approach in an effort to identify even weak binding hits that could provide a useful starting point for further optimization . to screen atad2 against an unbiased fragment library of approximately 13800 molecules , protein - observed h n sofast - hmqc nmr spectra were recorded on uniformly n - labeled atad2 in the presence of mixtures containing 12 fragments . a mixture was considered a hit if , relative to the absence of fragments , chemical shift changes were observed for the backbone resonances in the presence of fragments ( si , figure s2 ) . hit mixtures were then deconvolved as singletons to isolate the hit fragments , resulting in a total of 65 fragment hits ( figure 2 ) . the binding affinity range for the hits , as determined by sofast - hmqc titration experiments , is 350 m to > 2 mm , with 12 hits having a kd of less than 1 mm . taken together , very few weak - binding hits have been identified against atad2 , with a calculated hit rate of 0.1% . however , a previous analysis of fragment - based screens suggests that protein targets with a hit rate of at least 0.1% can result in the design of high affinity , drug - like molecules against the target . we therefore conclude that atad2 may be a druggable target , but based on the weak affinity of the hits identified , it is likely to still be difficult to drug . hits are clustered by chemotype and classified as being commonly observed ( cluster 1 ) or novel bromodomain ligands ( clusters 23 ) . although the assignments of atad2 s backbone resonances are not known , we observed that one distinct set of resonances shift in the spectra depending on the fragment hit bound to atad2 , and these residues likely correspond to those within the kac binding pocket ( si , figure s2 ) . the 65 hits identified have been clustered into three chemical clusters ( figure 2 ) . cluster 1 fragment hits ( 14 ) represent 5,6- and 6,6-fused ring chemotypes that have been previously identified as bromodomain inhibitors , including dihydroquinolinones , dihydroimidazolones , quinazolines , and benzoimidazolylanilines . cluster 2 hits ( 58 ) , on the contrary , are unique to atad2 and include tricyclic - containing small molecules such as diphenyltriazoles and diphenyloxadiazoles . a second unique cluster of atad2 hits , cluster 3 ( 912 ) , is composed of sulfur - containing fragments such as tetrahydrothiazoloazepinones and benzothiazoles . the listed binding affinities of 112 vary only 2-fold despite great differences in chemical structure ; however , relative to recently published atad2 ligands , the fragments identified herein are significantly greater in potency and thus may represent better starting points for medicinal chemistry optimization . on the basis of ligand efficiencies alone ( figure 2 ) , one may choose to optimize the more efficient members of cluster 1 . however , for selectivity purposes , medicinal chemistry optimizations of the unique fragments from clusters 23 may also be desirable . on the basis of high - resolution structures presented in the literature for bromodomains of the bet family and recently for atad2 , the binding orientations of cluster 1 molecules to atad2 could be modeled to aid in medicinal chemistry optimization of the fragment hits . however , because two clusters of hits identified from the screen have not been previously characterized as bromodomain inhibitors , it was important to understand how these molecules bind within the kac binding site . to obtain this information , we determined x - ray structures of atad2 when complexed with three different fragments ( 5-amino-1,3,6-trimethyl-1,3-dihydro-2h - benzo[d]imidazol-2-one ( 1 ) , 3-(5-phenyl-1h-1,2,4-triazol-3-yl)aniline ( 5 ) , and 2-amino-7,7-dimethyl-5,6,7,8-tetrahydro-4h - thiazolo[5,4-c]azepin-4-one ( 12 ) ) ( figure 3 , si , figure s3 ) , which served as representatives of each chemical cluster discussed above ( table 1 ) . all three complexes crystallized in the p6522 space group with one molecule in the asymmetric unit , allowing for direct comparisons between the complexes and also to the ligand - free atad2 structure ( pdb i d 3dai ) . as shown in figure 3 , all three fragments occupy the same general space within the kac binding pocket and make a critical hydrogen bond to the highly conserved n1064 residue ( figure 1a ) . when compared to ligand - free atad2 , minimal structural perturbations were observed upon binding of these three fragments , with the rmsd ranging from 0.117 to 0.176 , suggesting that structural changes to the pocket are not inducible . view of the kac binding site in the presence of fragment 1 ( a ) , 5 ( b ) , and 12 ( c ) ( pdb ids 4tyl , 4tz2 , and 4tz8 ) . dashed yellow lines depict polar contacts between the bound fragment and atad2 residues / crystallographic water molecules . fragment 1 represents a hit from cluster 1 and has been observed as part of elaborated small - molecule inhibitors for bet family bromodomains . on the basis of a map of intermolecular contacts ( pdb i d 4tyl ) , 1 is anchored within the kac binding site primarily due to the hydrogen bonding interactions between its carbonyl and both n1064 and a water molecule inside the pocket ( figure 3a ) . much of the six - membered ring is solvent exposed , with its amine substituent making hydrogen - bonding interactions with two crystallographic waters in the binding site . the remaining interactions are hydrophobic in nature ( v1008 , v1013 , v1018 , y1021 , y1063 , and i1074 ) . from cluster 2 , fragment 5 was chosen for crystallization with atad2 ( pdb i d 4tz2 ) . this tricyclic fragment takes advantage of the same contacts as 1 , being mainly hydrophobic in nature ( v1008 , v1013 , v1018 , y1021 , y1063 , and i1074 ) aside from the hydrogen bonding interactions with n1064 ( figure 3b ) . its kac - mimic triazole warhead does not form a hydrogen bond to any water molecules within the binding site , likely because this larger - sized fragment displaces the water molecule seen both in the complexes with 1 and 12 ( si , figure s4 ) . similar to 1 , the 5-phenyl portion of 5 is largely solvent exposed and likely contributing very little to binding affinity . however , unlike the binding of 1 , the 3-aminophenyl substituent binds deep within the kac binding pocket , displacing an additional three conserved water molecules ( si , figure s4a ) . the displacement of water may account for its higher affinity observed for binding to atad2 relative to fragments from clusters 1 and 3 . only one other example of water displacement is available in the pdb , and in this case , the inhibitor displaces just one water molecule from the kac binding pocket of brd2 ( pdb i d 2dvv ) . the 3-aminophenyl portion of 5 that binds deep inside the kac recognition pocket participates in hydrogen bonds to i1056 , m1029 , and a crystallographic water molecule . it should be noted that an elaborated analogue of 5 has been described as a dual kinase bromodomain inhibitor and has been crystallized with brd4 ( pdb i d 4o77 ) . however , the analogue and 5 do not share similar binding poses as the analogue does not bind deep within the kac pocket . a cluster 3 representative fragment ( 12 ) binds in a very similar fashion as to 1 , with the carbonyl of its seven - membered ring making hydrogen - bonding interactions to both n1064 and a water molecule ( pdb i d 4tz8 ) . the amine substituent and heteroatoms of the thiazole ring make hydrogen - bonding interactions to v1008 , k1011 , and two crystallographic waters in the binding site . this amine substituent takes the place of a water molecule deep inside the kac binding pocket , in agreement with one of the four waters also displaced by 5 ( si , figure s4b ) . this fragment also utilizes a number of hydrophobic interactions involving v1008 , v1013 , v1018 , y1021 , y1063 , and i1074 ( figure 3c ) . the dimethyl substituent on the seven - membered ring is largely solvent exposed and likely not contributing to binding affinity . following the submission of this manuscript , a group from the structural genomics consortium ( sgc ) published structures of atad2 bound to a histone - derived peptide ( h4kac5 ) , several solvent molecules , dna / rna bases , and their analogues . we have prepared structural overlays of our fragment - bound structures with these newly released structures to provide suggestions on how to improve the potency of the fragment hits identified from our nmr screen ( figure 4 , si , figure s5 ) . ( a ) binding pose of 1 ( green ) ( pdb ic 4tyl ) overlaid with 1-methylquinolin-2-one ( salmon ) ( pdb i d 4qst ) . ( b ) binding pose of 5 ( magenta ) ( pdb i d 4tz2 ) overlaid with thymidine ( yellow ) ( pdb i d 4qsv ) . ( c ) binding pose of 12 ( cyan ) ( pdb i d 4tz8 ) overlaid with thymidine ( yellow ) ( pdb i d 4qsv ) . residues depicted as sticks and water molecules summarize interactions between atad2 and the fragments 1 , 5 , and 12 . ( d ) overlay of binding poses for h4kac5 peptide ( orange ) ( pdb i d 4quu ) , thymidine ( yellow ) , 1 ( green ) , 5 ( magenta ) , and 12 ( cyan ) . on the basis of the chemical similarity of 1 to 1-methylquinolin-2-one ( pdb i d 4qst ) , the binding poses of these two molecules were overlaid for comparison of intermolecular interactions . as seen in figure 4a , the two small molecules overlay almost exactly in the kac binding site . both have a carbonyl group that makes a hydrogen bond to n1064 and a water molecule . binding affinity data was not provided for 1-methylquinolin-2-one ; however , we assume its kd is greater than 10 mm , as all of the molecules with reported kd values in the study were at best 10 mm . we rationalize the large difference in kd ( estimated 10 mm relative to 600 m for 1 ) is due to the additional two hydrogen bonds formed between 1 and crystallographic water molecules in the kac binding site . thus , an electron rich group that can participate in hydrogen bond formation is favored on the side of the pocket formed by the za loop . when the two binding poses of thymidine and 5 are overlaid , they do not share many common binding elements ( figure 4b ) , as expected . in the case of 5 , its kac - mimic warhead is the triazole ring , allowing the aniline nh2 group to plunge deeper into the kac binding site . in fact , 5 is positioned more than 3 further inside the pocket , and we reason that this unique binding pose drives the substantial difference in affinity for atad2 ( 350 m vs 10 mm ) . one interesting insight this provides is that introducing substituents around the aniline moiety may provide unwanted steric bulk and force the aniline to not bind as deep inside the pocket . although the aniline ring occupies a never before drugged portion of the kac binding site that could be optimized to increase interactions between the molecule and atad2 , we suggest only conservative changes to the ring be made to avoid a change in its binding pose . fragment 12 identified from our screen does not structurally resemble the solvent molecules or dna / rna bases that were crystallized with atad2 by the sgc . similar to 5 , we chose to overlay 12 with thymidine to highlight how the thiazole of 12 also binds deeper within the kac binding pocket ( figure 4c ) . the amino group of the thiazole ring binds nearly 3 deeper into the pocket than the methyl substituent of thymidine . in doing so , 12 displaces a water molecule present in both the structures with bound 1 and thymidine ( si , figure s4b ) . from an overlay of binding poses for the h4kac5 peptide , thymidine , 1 , 5 , and 12 ( figure 4d ) , it is apparent that 5 and 12 bind in a portion of the kac pocket that is unoccupied by the other molecules . these two fragments bind deep into the pocket , which may contribute to their higher affinity for atad2 . as changes are made to the portions of 5 and 12 that bind deep within the pocket , nmr hmqc spectra could be used to asses whether the binding mode changes significantly , as there appears to be a subset of resonances that report binding deeper inside the pocket ( si , figure s2 ) . from the structural overlays , it is also evident that the peptide and 5 do not fill the side of the kac pocket formed by the za loop . modifications to 5 and even 12 to better fill that portion of the kac pocket could also improve potency . we suggest the addition of a hydrogen - bond donor / acceptor group , such an amine substituent like seen in 1 , or a hydroxyl that , similar to thymidine , could bridge through hydrogen bonding to the za loop itself . bromodomains represent a highly druggable class of therapeutic targets that have received great interest in recent years . despite this interest in exploiting bromodomain inhibition for the treatment of several diseases , relatively few chemical probes or inhibitors for bromodomains outside of the bet family have been discovered . on the basis of cellular and genetic studies , inhibition of atad2 provides a useful strategy for the treatment of several types of cancer , including prostate , lung , and breast malignancies . we sought to identify small - molecule ligands for the bromodomain of atad2 from which chemical probes could be designed to validate atad2 as a cancer target . a total of 65 fragment hits for atad2 were identified by nmr spectroscopy , and about 1/4 of these fragments bind with affinities less than 1 mm . co - crystal structures of atad2 were determined for three fragment hits , revealing the contacts critical for each cluster of inhibitor . together with recently published data from the sgc , we propose several strategies to improve ligand binding affinities toward the design of a chemical probe . chemical probes will be useful to examine the biological impact of atad2 bromodomain inhibition and to determine whether this is a viable strategy for treating human cancers . all screened compounds were purchased from commercial vendors and the purity was determined to be 95% or higher . hplc data of all compounds were obtained using an agilant 1200 series sytem with a uv detector set to 220 nm . samples were injected onto a phenomenex 2.0 mm 50 mm , 2.6 m , c18 column at 45 c . a mobile phase of a being h2o + 0.1% trifluoroacetic acid and b being acetonitrile + 0.1% trifluoroacetic acid was used . a linear gradient from 5% to 95% b in 1.95 min was then followed by pumping 95% b for another 0.45 min having a flow rate of 1.0 ml / min . mass spectral data were obtained in positive ion mode using agilent 6140 single quadrupole mass spectrometer using an electrospray ionization ( esi ) source .

cellular and genetic evidence suggest that inhibition of atad2 could be a useful strategy to treat several types of cancer . to discover small - molecule inhibitors of the bromodomain of atad2 , we used a fragment - based approach . fragment hits were identified using nmr spectroscopy , and atad2 was crystallized with three of the hits identified in the fragment screen .

the two main types of skin cancer linked to ultraviolet ( uv ) radiation are nonmelanoma and malignant melanoma . the incidence and prevalence of both these skin cancers are increasing globally each year , with 2 - 3 million and 132,000 cases reported yearly , respectively . uv radiation is emitted from both the sun and artificial tanning devices , the latter of which has been referred to as sunlamps , sunbeds , and tanning salons , henceforth referred to as indoor tanning it [ 3 , 4 ] . such exposure is also associated with premature aging , cataracts , and immune suppression [ 3 , 4 ] . in climates such as australia and the united states of america ( usa ) , reports have shown skin cancer accounts for more than 80% and 50% , respectively , of all new cancers diagnosed each year [ 5 , 6 ] . surprisingly , within the united kingdom ( uk ) , where uv exposure in terms of both uv levels and hours of sunshine is significantly less , latest figures show skin cancer still accounts for more than 30% of new cancer diagnosed each year . such alarming figures are partly attributable to the growth and popularity of the it industry . despite the adverse health effects , it appears to be gaining considerable popularity and for many having a tan is considered highly desirable by many young females , for reasons such as enhancing appearance [ 812 ] . furthermore , specifically within the uk , where the weather is considerably mixed , using it to achieve the desired tanned look is both a more affordable and widely available alternative to young people than travelling to warmer climates . primary prevention interventions have predominantly focused on the health consequences of such behaviour , and whilst they have been successful at enhancing knowledge and attitudes related to uv exposure and protection , few have been successful in modifying uv exposure / protection behaviours [ 1317 ] , and even fewer have focused directly on it practices . there is now a growing body of literature on interventions targeting the appearance consequences ( e.g. , premature skin aging wrinkles and pigmented age spots ) associated with uv exposure [ 10 , 1820 ] . the first systematic review assessing the efficacy of such interventions revealed that the synthesis of the findings was promising . reviewed studies that had assessed changes in uv exposure / protection intentions and behaviours revealed significant findings in favour of appearance - focused interventions . these types of interventions may be a successful alternative ; however , the findings can not be easily generalised to other countries of differing climates , for example , the uk , without knowing if appearance is a primary motive of this behaviour . to aid our understanding of adherence and nonadherence to health - related behaviours , emphasis has been placed on theoretical models such as the theory of planned behaviour tpb . the basic principle of the tpb is that the immediate predictor of engagement in a volitional behaviour is intention and that an individual 's intention is influenced by three principal determinants , attitude , subjective norm ( sn ) , and perceived behavioural control ( pbc ) . few researchers have applied the tpb model to understand uv exposure and protection [ 2326 ] . their findings suggest that the model is a useful theoretical framework for understanding the motivations of such behaviour . however , even fewer studies have extended the model to include factors that have been at the forefront of research when referring to the motivations of tanning ( e.g. , appearance factors ) , none focus on a uk sample [ 27 , 28 ] . thus , whilst appearance - based interventions may be a breakthrough alternative to health consequence interventions , to date there is no comprehensive picture of the factors that influence the decisions to indoor tan within young people within the uk . to develop interventions that focus on appearance , further research is required to examine the factors that influence such decisions , in order to ensure that interventions are developed based on theoretical underpinnings of the behaviour . thus , the aim of the study was to examine young people 's it intentions and behaviour living within the uk using an extended tpb model that included variables on appearance reasons to tan , perceived susceptibility to damaging appearance , perceived susceptibility to health consequences , and tanning knowledge . prior to data collection , ethical approval from the institutional research ethics committee was obtained . all students at the uk university college within the west midlands were invited by email to take part in a prospective questionnaire survey on young adults ' views and behaviour towards it . of the 284 who volunteered ( approximately 10% of the entire student population ) , 210 young adults ( 44 male and 166 female mean age 21.37 3.30 years ) , indicated that they had indoor tanned within 12 months ( referred to as the tanner - t group ) ; a usual cut - off used within the tanning literature . whereas 71 young adults ( 23 males and 48 females , mean age 22.12 4.05 years ) reported not to have tanned within 12 months , thus referred to as the nontanners ( nt ) group . participants firstly completed a self - report questionnaire by email ( time 1 or t1 ) that measured the constructs of the tpb , the proposed additional variables , and demographic characteristics . two weeks later the sample were contacted by email and asked to report their it behaviour preceding the two weeks ( time 2 or t2 ) ; responses constituted a 71% response rate ; reminder emails were also sent out . as an incentive , participants had the opportunity to enter a prize draw to win gift vouchers . sample size was determined as having sufficient power ( 0.8 ) to detect for a moderate effect size with the alpha set at 0.05 . skin type ( e.g. , tan and burn tendencies ) was assessed according to method(s ) published elsewhere . for example , ( i ) i always sunburn , never tan , ( ii ) usually sunburn , tan with difficulty , ( iii ) sometimes mild sunburn , tan about average , ( iv ) rarely sunburn , tan with ease , ( v ) very rarely sunburn , natural brown skin , ( vi ) very rarely or never sunburn , natural black skin . the constructs of the tpb were developed in line with recommendations [ 31 , 32 ] , and several response items selected were similar to those used within the literature [ 25 , 33 ] . all construct items were then aggregated to form a composite variable ( e.g. , pbc , attitude , sn , knowledge ) . a reliability analysis was conducted for all multiitem constructs / variables measured at baseline ( t1 ) for the whole sample and the t and nt groups ( table 2 ) . in line with recommendations from a recent discussion paper highlighting the problems with cronbach 's alpha as a measure of internal consistency , both cronbach 's alpha and guttman 's lambda 2 were reported . intention was measured using one item ; participants were asked to state how many times they intend to use an it booth to obtain a tan in the next two weeks . attitude towards it was measured using eight bipolar adjective scales that tapped into both the instrumental and affective attitude concept ( i.e. , for me , using an it booth in the next two weeks to obtain a tan would be harmful - beneficial / wise - foolish / unhealthy - healthy / good - bad / unpleasant - pleasant / enjoyable - unenjoyable / unrelaxing - relaxing / fun - boring ) . sn was measured using two items that tapped into the injunctive and descriptive concept , using a scale ranging from strongly disagree to strongly agree ( i.e. , people in my social network , for example , family / friends , girlfriend / boyfriend , partner / spouse who are important to me would approve of me using it booths in the next two weeks to obtain a tan ) . pbc was measured using four items that tapped into both the controllability and self - efficacy concept ( i.e. , how confident are you that you could use an it booth in the next two weeks to obtain a tan if you wanted to ? not at all confident to very confident ) . appearance reasons to tan was measured using nine items from the appearance motives to tan and not tan scalesubscale appearance reasons to tan . in order to fit the aim and purpose of this research slight wording modifications were made ( e.g. , reference made to it ) and the scale ranged from strongly disagree to strongly agree . items consisted of statements , such as having a tan gives me more sex appeal , and perceived susceptibility to damaging appearance was measured using nine items from the appearance reasons to tan and not tan scalesubscale appearance reasons not to tan . again slight modifications were made to the wording and items were measured on a scale ranging from strongly disagree to strongly agree . items consisted of statements , such as i 'm concerned about getting blemished skin as a result of it , perceived susceptibility to health consequences ( e.g. , skin cancer ) was measured using three items on a scale ranging from strongly disagree to strongly agree ( i.e. , i am concerned if i use it booths frequently i will enhance my chances of skin cancer ) . again tanning knowledge consisted of 11 items based on the effects of it , uv safety , tanning fashion trends , and factual tanning statistics from websites on skin cancer [ 1 , 3 , 36 ] . items consisted of statements with a mixture of closed - ended and open - ended response options , such as it booths can not cause skin cancer , with response options of true / false / do not know or open - ended items , such as what is the minimum age you must be by law to use an it booth ? , with answers scored 1 for correct , 0 for incorrect or do not know . the interitem reliability of this scale was determined to be only moderate ; however low / moderate internal consistency does not necessarily mean that the scale items are not a composite measure of the variable of interest . tanning knowledge can be seen as multidimensional ( e.g. , as there are different dimensions to it knowledge of uv consequences , knowledge of protective behaviours ) , in that whilst some of the respondents may have good knowledge of how to protect themselves against uv rays , they may be less knowledgeable about the consequences of exposure to uv rays . it behaviour was measured using a single open - ended item , how many times did you use an it booth in the previous two weeks ? initial equivalence tests were conducted on the data obtained from those who had been tanners ( e.g. , t group ) and those who reported not to have used it booths in the previous 12 months , nontanners , ( e.g. , nt group ) , descriptive and simple statistical analysis procedures ( e.g. , independent t - tests , and chi - squares ) were performed on the demographic and the extended tpb questionnaire constructs / variables . welch 's t was reported instead of the more conventional independent t - value when the variances of the two groups differed by more than twice as the sample sizes were unequal . due to the violation of the chi - square , expected cell frequency assumption , the primary analyses were conducted using two hierarchical forced - entry regression analyses procedure ; this method is ideal when the predictors ( i.e. , ivs ) are selected based on previous research and when the researcher knows something about the predictability of some of the predictors ; thus the model allows the researcher to enter ivs into the model in a specific order . table 2 depicts t1 characteristics of the two groups ( e.g. , t versus nt ) and where possible , statistical analyses were carried out to assess for significant differences . none of the sample reported a personal diagnosis of skin cancer ; the t group revealed a greater percentage of family members being diagnosed with skin cancer than the nt group . skin type distribution revealed that a greater percentage of the t group reported skin type iii ( 43% , n = 90 ) , whereas 54% ( n = 38 ) of the nt group reported skin type iv or v. the skin type distribution for the t group was consistent with previous results for this population . as expected , significant differences for the tpb measures and the additional variables between the t and nt group emerged . significant differences were found for all but one variable , perceived susceptibility to damaging appearance . the t group revealed stronger positive perceptions towards it than the nt group on attitude , sn , pbc , appearance reasons to tan , the nt group revealed greater perceived susceptibility to health consequences than the t group ( mean of 5.1 and 4.2 resp . , p < 0.01 ) . to identify the prominent predictors of intention and behaviour to the use it booths , two hierarchical forced - entry regression analyses were used to analyse the t1 and t2 data . it intention was regressed on the tpb constructs in block one of the regression equation , and block two consisted of the additional variables ; appearance reasons to tan , perceived susceptibility to damaging appearance , perceived susceptibility to health consequences , and tanning knowledge . it behaviour was regressed on intention and pbc in block one , and block two consisted of the tpb and the additional variables . as depicted in table 4 , the tpb model ( pbc , attitude , sn ) made a significant contribution ( 8% , p < 0.001 ) towards explaining it intentions , with pbc and attitude making a significant unique contribution ( p < 0.01 and p < 0.05 resp . ) . entry of the additional variables in block two explained a further 9% of the variance in it intentions , with both pbc and the variable appearance reasons to tan making a significant contribution ( p < 0.05 and p < 0.001 resp . ) . the model accounted for 70% of the variance in behaviour ( p < 0.001 ) , with intention making a significant unique contribution ( p < 0.001 ) . entry of the additional variables accounted for a further 1% in block two , with the variables intention and appearance reasons to tan making a unique significant contribution ( p < 0.001 and p < 0.05 resp . ) . a reassuring finding was that the tanning group reported using it booths an average of 5 times in the last year , whereas student populations had been found to indoor tan up to 37 times per year . furthermore , over a two - week period the tanning group reported using an it booth an average of 1.1 times . other encouraging findings were that on average the tanning group had less favourable attitudes towards it . both family and friends were likely to disapprove of this behaviour and their perceived susceptibility to damaging appearance and health consequence was relatively high . knowledge and pbc was also high ; however the latter two outcomes should be interpreted with caution due to moderate / low inter - item reliability . consistent with the limited research on the tpb and it , the tpb model was successful in predicting behavioural intentions . however , the variance explained by the tpb model on it was relatively small compared to the previous research [ 25 , 27 ] . the tpb model ( pbc attitude and sn ) accounted for 8% of the variance in it intentions , with only pbc emerging as a significant unique predictor . this indicates that individual attitude and sn were not making a unique significant contribution to it intentions . when the model was extended to include the additional variables , a further 9% of the variance was explained with pbc and the variable appearance reasons to tan , both emerging as significant unique predictors , and the latter variable making a greater significant contribution . the magnitude of the latter variable was contrary to expectations , implying that the appearance reasons to tan outweighs two of the main constructs of the tpb , one 's attitudes towards the behaviour and friends and family perceptions towards it . the model predicted 70% of the variance in it behaviour , with intention making a significant unique contribution . overall , the findings suggest that the variable appearance reason to tan is the strongest predictor towards intention , and intention is the strongest predictor towards behaviour with appearance reason to tan a close second . the emphasis of appearance being the primary motivation to it is consistent with a number of authors who have been advocating the need for appearance - focused interventions to reduce uv exposure , as evidenced in a recent systematic review . however , the impact of this variable was not assessed on a uk sample ; thus it was not clear whether this variable was a primary predictor of this behaviour within the uk . thus considering the findings found from this study the authors now believe that appearance interventions are justified within a uk young adult population . however , further consideration will need to be given to how appearance perceptions can be altered , considering the importance of this to young adults ' daily lives . encouraging other alternative healthy approaches ( e.g. , diet , exercise , and clothing ) to address appearance enhancement has been put forth ; even sunless tanning lotion may be a more viable alternative . the authors feel that an in - depth exploration of the factors associated with appearance enhancement within young adults is also warranted and such findings would help to shape appearance - focused interventions . considering the baseline data of the two different groups ( e.g. , tanners the t group had stronger positive perceptions towards it than the nt group on attitude , sn , pbc , and appearance reasons to tan . although , the t group revealed greater tanning knowledge and of course greater intentions , the former is not out of the ordinary as research has shown that tanners are knowledgeable about this behaviour . the nt group revealed greater perceived susceptibility to health consequences than the t group . it may be interesting to also explore what motivates appearance enhancement of nt and the alternative methods they use to address appearance . a convenience sample was selected , which is associated with selection bias , with more of the volunteers being tanners or at least interested in it than the wider population . the sample was also young adults ( students ) from one university college ; this population was specifically chosen as young people are frequent it users and students have been reported to engage in a number of unhealthy practices . this may impact upon the generalisability of the findings to other higher education ( he ) institutions or nonstudent populations . therefore , as there is limited research using an extended tpb on it , and more specifically this is the first study to do so within the uk , a replication of the findings in other he institutions and nonstudent populations would provide insight into how representative the findings are . it is also important to note that a larger percentage of females participated in the study than males ; thus the findings may be more representative to females . behaviour was also measured by self - report , an objective measure such as change in skin colour using skin reflectance spectrophotometer equipment ; whilst expensive , it would enhance the validity of the findings . furthermore , it is important to note , while not unique to this paper , intention was only assessed using a single item . data collection was also conducted during a short time frame ( 2 weeks ) and only in one season . response rate for time 2 ( t2 ) was only 71% ( i.e. , < 80% ) notably , the findings highlighted the power of a tanned appearance for appearance enhancement in young adults within the uk and how at this current time in their lives this outweighs any appearance / health consequences . it is not unreasonable at this stage to conjecture that appearance - focused interventions may be a promising method to reduce uv exposure within a uk sample . as this is the first study to assess an extended model of the tpb ( which directly includes a measure of appearance reasons to tan ) on it in a uk young adult population , the findings are insightful , and the authors feel that interventions should now be developed to mirror such findings

the indoor tanning industry poses a long - term public health risk . despite the adverse health effects , indoor tanning seems to be gaining considerable popularity . the study examined indoor tanning intentions and behaviour within uk young adults using an extended theory of planned behaviour model , which included variables on appearance reasons to tan , perceived susceptibility to damaging appearance , perceived susceptibility to health consequences , and tanning knowledge . the model was successful in predicting indoor tanning intentions and behaviour ( explained 17% and 71% , resp . ) . an interesting outcome was the magnitude of the variable appearance reasons to tan . a current tanned appearance therefore seemed to outweigh any adverse future appearance or health consequences caused by indoor tanning . appearance - focused interventions to reduce such behaviour may now prove to be efficacious within a uk sample .

skin and soft tissue fungal infections were rare in survivors from 2004 indian ocean tsunami . only apophysomyces elegans , , fusarium solani and cladophialophora bantiana associated wound infections have been reported in tsunami survivors . cryptococcus gattii may be found worldwide , not only restricted to tropical and subtropical regions . c. gattii has been isolated from the environment e.g. decayed wood , soil around the trees . c. gattii can be divided into four molecular types vgi , vgii , vgiii and vgiv , . c. gattii associated infections usually manifest as pneumonia and meningitis . we report the first case of primary cutaneous cryptococcosis caused by cryptococcus gattii vgii in a tsunami survivor from thailand . a previously healthy 48-year - old thai woman was admitted to vichaiyut hospital in bangkok on december 27 , 2004 ( day 0 being the day of hospital admission ) for treatment of sustained injuries in the tsunami . one day earlier while walking on the beach in front of the hotel in lanta island , krabi province , thailand , she was hit and carried by the tsunami wave towards the hotel . although she was submerged under the sea water for some time , she did not pass out or aspirate the sea water . despite sustaining injury to her chin , right chest , back , right elbow , right index finger , left wrist and both lower legs , she was able to stand and walk to the high ground and later returned to her hotel . there were multiple skin abrasions of her back and anterior surface of both lower legs . rib cage x - ray revealed fracture of right 7th , 8th , 9th and 10th ribs . initially she was treated with broad spectrum antibiotics ciprofloxacin and clindamycin for 2 weeks . however , the wound of both lower legs gradually got worse . the skin of anterior surface of both lower legs appeared inflammed , red and tender . culture of pus from the left lower leg was sent on day 18 and on day 21 cryptococcus spp . ultrasound of the wound of left shin revealed thickening of skin and subcutaneous layer with irregular hypoechoic area about 0.93 cm . debridement of wounds of both lower legs was done under general anaesthesia on day 23 . moderate encapsulated yeast cells were found on the india ink stain of discharge of the left leg but not of the right leg . culture of pus from the left leg grew cryptococcus spp . which was identified as c. gattii serotypes b and c by using the canavanine - glycine - bromothymol blue agar and later it was determined to be c. gattii molecular type vgii by the ura5-rflp typing method ( fig . she was started on anti - fungal treatment on day 21 with iv amphotericin b 0.5 mg / kg / day and oral itraconazole 100 mg twice a day . on day 42 iv amphotericin b was discontinued and itraconazole was switched to oral fluconazole 200 mg daily . repeated india ink stain of pus on day 43 and day 56 still showed encapsulated yeast cells . on day 64 the wound finally closed and no relapse was seen during follow - up for the last nine years ( fig . the lungs and central nervous system are the most common organs affected by c. gattii acquired through the inhalation . primary skin and soft tissue cryptococcosis can occur after the traumatic inoculation . to our knowledge skin and soft tissue infection caused by c. gattii has not been reported in indian ocean tsunami survivors . our previously healthy patient most likely acquired infection during the 2004 tsunami as we found the small piece of woods inside her wound . culture of pus from the left leg grew c. gattii which belonged to vgii molecular type . cryptococcosis caused by c. gattii is uncommon and the most prevalent molecular type of c. gattii is vgii as in our case . vgii can be found in vancouver island , canada , northwestern united states , brazil , columbia and puerto rico . our patient was successfully treated by a combination of surgical drainage and antifungal treatment including three weeks of iv amphotericin b and three months of oral azoles . no relapse occurred on follow up for nine years . in summary , clinicians should be aware of possibility of c. gattii skin and soft tissue infection in tsunami survivors .

skin and soft tissue fungal infections with apophysomyces elegans , fusarium solani , cladophialophora bantiana have been reported in survivors from 2004 indian ocean tsunami . we report the first case of primary cutaneous cryptococcosis caused by cryptococcus gattii vgii in a tsunami survivor from thailand .

paragonimiasis is a parasitic infestation caused by the lung fluke , paragonimus westermani and related species . after ingested , juvenile worms of paragonimus migrate to the lung via tortuous tracks ( 1 ) . for this reason , although primary site of paragonimiasis is the lung , ectopic infestation can occur in unexpected sites such as the brain , subcutaneous tissue and muscles , omentum and mesentery , retroperitoneum , adrenal glands , ovary , epididymis and liver ( 1 - 13 ) . liver is known to be an organ in which ectopic paragonimiasis rarely occurs , and to our knowledge , only several cases of hepatic paragonimiasis have been reported in english and korean literatures ( 3 , 7 - 11 ) . in this case report , we described ct and mr findings of a case of hepatic paragonimiasis that showed characteristic imaging features . a 42-yr - old man visited our hospital with chronic cough , bloody sputum and weight loss of 5 kg for one year . chest ct scan showed a 2-cm , well - circumscribed nodule in the left upper lobe ( fig . liver ct scan at the arterial phase showed a cluster of small , rim - enhancing cysts at the subcapsular area of the segment vii and wedge - shaped enhancement of adjacent parenchyma ( fig . 2 ) . hepatic capsular enhancement and pleural thickening were also noted , adjacent to hepatic parenchymal lesion . at liver mri , the lesion appeared low signal intensity on t1-weighted images and bright signal intensity on t2-weighted images , indicating cystic nature ( fig . 3 ) . laboratory data including peripheral blood examination and blood chemistry revealed to be in normal range , and stool examination for various parasitic eggs was also negative . transthoracic needle biopsy was performed under ct guidance for histologic confirmation of the lung lesion . the biopsied specimens were obtained as yellowish and black pieces and were histologically diagnosed as chronic granulomatous inflammation with paragonimus eggs ( fig . he was treated with praziquantel with an oral dose of 75 mg / kg / day for 3 days , and gradually improved clinically . at follow - up the main pulmonary nodule in the left upper lobe showed a marked decrease in size , while most of smaller nodules resolved completely . human infestation by p. westermani occurs from ingestion of raw or incompletely cooked freshwater crabs or crayfish infected with metacercariae . the juvenile worms migrate into the abdominal muscles and lodge there for 5 - 7 days and come back into the abdominal cavity . about 2 - 8 weeks after infestation , they migrate through the diaphragm and the pleural cavity , and finally reach the lung where they become mature adult worms . during the peritoneal stage , the juvenile worms often cause damage to the liver capsule and parenchyma ( 1 ) . they fed metacercariae of p. westermani to dogs and found adult worms in the liver 3 to 49 days after ingestion . the number of adult worms found in the liver was correspondent to 5 percent of the ingested metacercariae . the cause of discrepancy between the incidence of hepatic paragonimiasis and frequency of hepatic damage in the experiment has not been explained . the diagnosis of paragonimiasis is usually made by detecting eggs in sputum and/or stool or by antibody test . the imaging studies may increase the confidence of the clinical diagnosis and demonstrate the extent of involvement . moreover , definitive diagnosis can be led only by imaging studies , if we are intimate to the characteristic findings of paragonimiasis , and thus allowing to avoid invasive procedure such as biopsy . in the review of previously reported ct findings , hepatic paragonimiasis was commonly seen as multiple low attenuating lesions which might be scattered or clustered ( 3 , 7 , 13 ) . in two case reports , it manifestated as a single , low attenuating lesion mimicking solid mass and mandated surgical resection ( 8 , 9 ) . on the other hand , hepatic paragonimiasis might be concomitantly found with intrahepatic cholelithiasis and incidentally diagnosed by hepatic resection ( 11 ) . in the present case , hepatic paragonimiasis manifestated as a cluster of rim - enhancing cysts , which was similar to the findings of cerebral ( 2 ) or pulmonary paragonimiasis ( 14 ) . in abdominopelvic cavity , similar ct appearances were reported in cases involving the liver , omentum ( 3 ) and retroperitoneum ( 5 ) . the ct and mr imaging features of clustered , rim - enhancing cysts are reflections of eosinophilic abscesses containing necrotic debris and paragonimus eggs histologically ( 2 ) . hepatic capsular enhancement and wedge - shaped perilesional enhancement at ct scan can be explained due to inflammatory changes , according to the previously reported microscopic examination in which acute inflammation of the hepatic capsule and adjacent parenchyma were described to be seen adjacent to eosinophilic abscess ( 12 ) . the peripheral location of the lesion reflects that the worms invade into the liver through the capsule and reside around the capsule rather than in the deep portion , as demonstrated by experimental studies in which metacercariae were present near to the surface in hepatic paragonimiasis ( 12 , 15 ) . when a cluster of small , rim - enhancing cysts at the subcapsular location in the liver is encountered at ct or mr imaging , the differential diagnoses include fascioliasis and pyogenic abscesses . in fascioliasis , eosinophilic microabscesses are arranged in tract - like fashion with one end of the tract subcapsular in location . and concomitant lung and pleura involvement is more common in hepatic paragonimiasis than in fascioliasis ( 16 ) . cystic lesions caused by paragonimus do not coalesce while clusters of microabscesses coalesce into a larger abscess cavity in pyogenic abscess . and pyogenic abscess has no preponderance of the subcapsular area . as shown in the present case , hepatic paragonimiasis has its own characteristic findings at ct or mri . thus , knowledge of these findings is helpful in differentiating various cystic lesions found in the liver .

hepatic paragonimiasis is a rare form of ectopic infestation caused by paragonimus . we experienced a case of hepatic paragonimiasis that showed characteristic imaging findings . ct and mr images showed a cluster of small cysts with rim enhancement in the subcapsular area of the liver . this finding seems to be characteristic for hepatic paragonimiasis , considering imaging findings in paragonimiasis involving other organs .

gastrointestinal stromal tumors ( gists ) are the most common sarcoma of the gastrointestinal tract . gists are malignancies that arise from intestinal cells of cajal , which are pace maker cells in the bowel wall , and the stomach is the most common location of gists [ 1 , 2 ] . surgical resection is the primary and only curative treatment of gists without dissemination or distant metastases . for relatively small gastric gists , laparoscopic resection has been reported to be a safe and effective treatment option associated with a significantly shorter hospital stay [ 3 , 4 ] . however , in cases with bulky or complicated gists , the indications for laparoscopic surgery remain controversial since laparoscopic procedures may be difficult in such cases , or even compromise the oncologic outcomes [ 5 , 6 , 7 ] . gists involving the stomach presenting as an intra - abdominal abscess are extremely rare , and only 7 cases have been reported in the english literature [ 8 , 9 , 10 , 11 , 12 , 13 , 14 ] . to the best of our knowledge , we herein report the first case treated by laparoscopic surgery for gastric gist presenting with an intra - abdominal abscess . a 70-year - old man presented with a 3-day history of acute upper abdominal pain . laboratory data showed a white blood cell count of 14,000/mm and c - reactive protein level of 11.8 mg / dl . a contrast - enhanced ct scan of the abdomen revealed a mass of mixed solid and fluid components , measuring 5.5 cm , adjacent to the lesser curvature of the stomach ( fig . he underwent gastrointestinal endoscopy , which revealed a submucosal tumor near the gastric angle without an ulcer or fistula . according to these findings , he was diagnosed as having an intra - abdominal abscess , and sulbactam / cefoperazone sodium 2 g / day was administered for 10 days . consequently , the fever decreased , his symptoms relieved and the laboratory data improved . a contrast - enhanced ct scan on the 7th day showed mass reduction ( 5.53.5 cm ) ( fig . an endoscopic ultrasound examination was performed on the 30th day , which showed a submucosal tumor with mixed hypoechoic changes ( fig . a fine - needle aspiration biopsy was performed , and the tumor was diagnosed as gist . 2a ) was performed to excise the tumor on the 70th day from the first presentation . laparoscopic findings showed that the tumor existed in the lesser curvature of the stomach and adhered to the omentum . wedge resection of the stomach was performed using a linear stapler , and the tumor was excised en bloc with the omentum and gastric wall ( fig . the excised tumor measured 3.0 2.5 cm in diameter and originated from the serosal layer of the stomach , without an obvious fistula between the gastric lumen and the tumor . the tumor was positive for c - kit ( cd 117 ) and cd 34 ( fig . 3b , c ) , with a ki-67 index of 8% and mitosis of < 5/50 hpf , and was accordingly diagnosed as low - risk gist . the postoperative course was uneventful , and the patient was discharged on postoperative day 12 . imatinib mesylate was administered as adjuvant treatment , and no recurrence has been noted for 2 years after operation . gists are understood to be specific kit- or pdfgra - driven mesenchymal gastrointestinal tract tumors , and a majority of these tumors ( more than 60% ) occur in the stomach [ 1 , 2 ] . the availability of a kit and pdgfra tyrosine kinase inhibitor , imatinib mesylate , has dramatically changed treatment strategies for metastatic or unresectable gists ; however , surgical resection remains the only curative treatment . cases of gists involving the stomach presenting as an intra - abdominal abscess are extremely rare , and only 7 cases have been reported in the english literature ( table 1 ) [ 8 , 9 , 10 , 11 , 12 , 13 , 14 ] . in some cases with gists , tumor degeneration or necrosis can result in bleeding into the gastrointestinal lumen or peritoneum space , and bleeding into the intra - abdominal space can cause an intra - abdominal abscess . five of 7 reported cases had a tumor with an ulcer or fistula in the lumen on macroscopic findings , and tumor necrosis was observed in 5 cases on histological examination . the presence of an ulcer , fistula , and necrosis may increase the risk of abscess formation ; however , the present case and the case reported by swain et al . we speculate that a minor hemorrhage occurred around the tumor , and subsequently bacterial infection via the gastrointestinal tract or blood stream might have resulted in abscess formation in these two cases . for relatively small gastric gists , laparoscopic resection has been reported to be associated with favorable short - term outcomes without compromising the oncologic results [ 3 , 4 ] . however , the indications for laparoscopic surgery should be carefully discussed for cases with bulky ( larger than 5 cm ) and high - risk tumors . therefore , most surgeons prefer conventional open surgery for large or complicated tumors to avoid tumor manipulation , which may cause dissemination or port site metastases [ 6 , 7 ] . in the present case , the tumor and abscess coexisted at the time of initial hospitalization ; however , the abscess was controlled by intravenous antibiotic therapy , and consequently the tumor was safely excised by laparoscopic surgery with sufficient surgical margins . to the best of our knowledge , this is the first report written in the english language of a case with gastric gist with an intra - abdominal abscess treated by laparoscopic surgery . we herein reported the case of stomach gist with an intra - abdominal abscess that was successfully excised by laparoscopic surgery .

gastrointestinal stromal tumors ( gists ) of the stomach presenting as an intra - abdominal abscess are extremely rare . we herein report a case that underwent successful laparoscopic resection of gastric gist presenting with an intra - abdominal abscess . a 70-year - old man presented with a 3-day history of acute upper abdominal pain with a fever . laboratory data showed an elevated white blood cell count and c - reactive protein level . a ct scan revealed a mass of mixed solid and fluid components , measuring 5.5 cm , in the adjacent lesser curvature of the stomach . he was diagnosed as having an intra - abdominal abscess , and antibiotic therapy was administered . consequently , his symptoms were relieved and a ct scan on the 7th day showed that the mass reduced and became homogenous . according to the results of a fine - needle aspiration biopsy , the tumor was suspected to be a gist . laparoscopic surgery with the 5-port approach was performed . the tumor existed in the lesser curvature of the stomach , and was excised en bloc with the omentum and gastric wall by wedge resection . the tumor measured 3.0 cm in diameter and originated from the serosal layer of the stomach . histological findings showed spindle cells with a stromal growth pattern that was positive for c - kit ( cd 117 ) and cd 34 . the postoperative course was uneventful , and no recurrence has been noted in the 2 years since the operation . this is the first report of a case with gastric gist with an intra - abdominal abscess that was successfully treated by laparoscopic surgery .

abdominal pain is one of the most common complaints during outpatient examinations . in particular , due to recent developments in diagnostic imaging , imaging tests are frequently performed in patients with complaints of abdominal pain . many cases in which abnormal findings are not observed are treated as cases of functional dyspepsia ( fd ) , irritable bowel syndrome ( ibs ) and other functional gastrointestinal disorders , or psychiatric disorders . we herein report a case of a middle - aged woman who was examined as an outpatient for complaints of repeated abdominal pain ; she was diagnosed with abdominal migraine as a result of a detailed medical interview and showed a dramatic improvement of her symptoms after the administration of medications . although there are very few reports on abdominal migraine occurring in adults , it is possible that many individuals remain undiagnosed . thus , we present this report because we believe that our experience of and findings on this condition are significant . a 52-year - old woman presented at our outpatient department with complaints of abdominal pain that persisted for approximately half a day . three months prior to presentation , she visited our hospital 's emergency department for a fever , diarrhea , and vomiting . at the time three weeks prior to presentation , she had experienced abdominal pain accompanied by nausea and vomiting that lasted for approximately half a day , but she had spontaneous remission . however , one week prior to presentation , she had experienced the same symptoms again and was seen by a local physician 5 days prior to presenting at our hospital . she was diagnosed with enteritis and prescribed medications : a proton pump inhibitor ( lansoprazole ) , an intestinal regulator ( bifidobacteria probiotic ) , and chinese herbal medicine ( daikenchuto ) , but she subsequently experienced repeated abdominal pain that lasted for approximately half a day and was thus examined at our department . the pain was located in the central portion of the trunk from the epigastric fossa to the lower abdomen , with no bias toward either the right or left side . the pain was dull , and the most frequently experienced concomitant symptoms were poor appetite , nausea , and vomiting . the pain was not associated with prodromal symptoms , scintillating scotoma , or sensitivity to light or sound , and the patient did not experience constipation , diarrhea , or weight loss . the pain was continuous and although it was severe enough to prevent her from performing housework , the symptoms disappeared during intervals of no pain . she had a history of migraine since 30 years of age and had undergone an ectopic pregnancy ( surgery ) at 32 years of age . she experienced menopause at 46 years of age and had no history of alcoholic beverage consumption or smoking . the physical findings were as follows : temperature , 36.6 ; blood pressure , 122/73 mmhg ; heart rate , 74/min ; and respiratory rate , 18/min . her abdomen was flat and pliable , and although she reported pressure pain in the area extending from the epigastric fossa to the lower abdomen , there was neither rebound tenderness nor muscular defense . blood tests showed slightly elevated amylase levels , but no other abnormalities ( table 1 ) . although several imaging modalities were performed ( abdominal ultrasound , abdominal contrast - enhanced ct , and upper and lower endoscopy ) , none showed abdominal findings that indicated any of the above disorders ( all imaging tests , except for lower endoscopy , were performed while the patient was experiencing pain ) . we suspected a functional disorder such as fd , and administered pharmaceuticals ; a stomachic ( oxethazaine ) , an intestinal regulator ( dimeticone ) , and an antiemetic ( domperidone ) . however , because there was no improvement in her symptoms and the pain recurred several times , the patient was examined three times as an outpatient during the week following her initial examination . during the third examination , she complained of a unilateral , throbbing headache in addition to her abdominal symptoms . her medical history suggested that the cause of the headache to be a migraine ; however , on reviewing her abdominal pain history , we discovered that it was marked by paroxysmal onset and went into spontaneous remission after approximately 12 hours of continuous pain . both the location of the abdominal pain and the concomitant symptoms met the diagnostic standards for the international classification of headache disorders , 2nd edition ( ichd - ii ) ( 1 ) and the rome iii criteria ( 2 ) for abdominal migraine ( fig . 1 ) . after administering calcium blockers ( lomerizine , 10 mg / day , as prophylactic treatment ) and analgesics ( loxoprofen as needed , 60 mg per use ) for a few days , the abdominal pain disappeared along with the headache symptoms . loxoprofen was tapered over the course of 2 weeks and she eventually used lomerizine alone ( fig . 2 ) . the symptoms initially appeared to recur when lomerizine was stopped , but after 6 months of continuous lomerizine therapy , her abdominal pain completely disappeared and lomerizine was therefore stopped . although she still experiences some occasional migraine headaches , they are being well controlled by occasional loxoprofen use , and there have been no episodes of abdominal pain . abdominal migraine falls under the subcategory of childhood periodic syndromes in the ichd - ii ( 1 ) and is classified as a childhood functional gastrointestinal disorder in the rome iii critera ( 2 ) . both of these have established diagnostic criteria for the disorder . both consider abdominal migraine to be a childhood disorder with the average age of onset at 8 years and a relatively high prevalence rate between 1% and 4% of children ( 3 ) . it has a relatively good prognosis because most patients with childhood onset of abdominal migraine go into spontaneous remission by the time they reach adulthood . however , although abdominal pain goes into remission , it shifts to a conventional migraine headache in many cases . dignan et al . observed patients with abdominal migraine for 10 years and reported that it shifted to migraine headache in 70% of them ( 4 ) . although this disease is referred to as abdominal migraine , the headache is absent or mild in most cases ( 5 ) . it is considered to be a migraine - related disorder for the following reasons : 1 ) a notable family history of migraine , 2 ) in many cases , the disorder shifts to migraine headache after reaching adulthood , 3 ) predominance in women , 4 ) a relatively clearly - defined beginning and end of symptoms , and 5 ) in many cases , migraine medication is effective . abdominal pain occurs in a poorly localized central abdominal area ( 6 ) and is often accompanied by concomitant symptoms such as nausea and vomiting , which are observed in cases of conventional migraine headaches . however , it is rarely associated with prodromal symptoms , scintillating scotoma , or sensitivity to light or sound . in the present case , the pain experienced by this patient met the diagnostic criteria for abdominal migraine listed in both the ichd - ii and the rome iii criteria . however , a careful workup through the differential diagnosis was required because the duration of the interval between attacks was atypical , there have been few reports on this disorder occurring in adults ( 7 - 13 ) , and it is a functional disorder . many patients who complain of epigastric symptoms during outpatient exams and who can not be diagnosed by imaging tests are treated for fd . the present case was also treated for fd , but showed no signs of improvement . using the rome iii criteria ( 14 ) , except for the fact that the abdominal pain lasted for a short time , the patient 's symptoms were consistent with epigastric pain syndrome ( eps ) . we believe that a diagnosis of abdominal migraine was accurate because 1 ) the abdominal pain was intense enough to significantly hinder the patient 's daily living activities , 2 ) the appearance and disappearance of symptoms was clearly delineated , and 3 ) proton pump inhibitors were completely ineffective , whereas migraine medication was effective . in addition , postprandial distress syndrome ( pds ) was ruled out because there was little relationship between food and the patient 's symptoms . by using the rome iii criteria , many patients who complain of abdominal pain are ultimately diagnosed with fd , but some of these patients have refractory disorders , and they may be suffering from abdominal migraine . ibs was also ruled out because it has little relationship to constipation and abdominal pain . in addition to fd and ibs , some diseases cause epigastric pain and are difficult to diagnose using imaging tests . eosinophilic gastroenteritis is distinguished by the fact that it causes paroxysmal abdominal pain ; however , in the present case , because there was no elevation in the eosinophil levels in the peripheral blood and random biopsies performed during upper and lower endoscopies did not indicate eosinophil infiltration , it did not satisfy the diagnostic criteria ( 15 ) for eosinophilic gastroenteritis and was thus ruled out . in addition , eosinophilic gastroenteritis was ruled out because there was no history of allergic disease and ct did not indicate ascites or thickening of the intestinal tract walls . chronic pancreatitis was ruled out because the patient had no history of alcohol consumption , i.e. , neither abdominal ultrasound nor ct indicated calcification of the pancreas , irregular margins , or pancreatic duct enlargement . moreover , chronic pancreatitis rarely presents with a paroxysmal onset of symptoms . because the serum amylase levels were slightly elevated but lipase levels were within normal range , we believed it was a case of nonspecific elevation . psychiatric disorders that may be accompanied by abdominal pain include some pain - related disorders such as depression and somatoform disorder . consultation of the diagnostic standards for these disorders ( dsm - iv ) ( 16 ) indicated that because the patient did not exhibit a depressed mood or a loss of interest or happiness , depression could be ruled out . because psychological factors are not related to pain onset , nausea , or continuous symptoms , pain disorder was also ruled out . as there is no evidence - based treatment for abdominal migraine , current treatments are based on experience and consist of medication therapy and the improvement of activities of daily living . improving the activities of daily living consists of receiving adequate sleep and avoiding stress , maintaining an adequate hydration level , and avoiding foods that contain vasoactive amines ( e.g. , cheese and wine ) . medication therapy includes ibuprofen , acetaminophen , and other analgesics , which can be highly effective if taken soon after the onset of pain . in addition , triptans ( 17 ) and ergotamines ( 18 ) have been reported to be effective , and roberts et al . reported that the effectiveness of triptans can be used in the differential diagnosis of abdominal migraine ( 7 ) . when accompanied by nausea and vomiting , antiemetics ( 8) are used . prophylactics include beta - blockers ( propranolol ) , antiallergic agents ( cyproheptadine ) , and pizotifen , which have been used experimentally ( 6,19 ) . research has indicated that the antiepileptic drugs valproic acid ( 20 ) and topiramate are also effective . when a calcium blocker ( lomerizine ) , the cost of which is covered by national health insurance in japan , was administered to the present patient , the patient experienced a marked improvement in her symptoms . thus , this drug can be expected to be used for the treatment of abdominal migraine . however , as none of these drugs are used for the treatment of other gastrointestinal disorders , to make an accurate diagnosis , we must keep abdominal migraine in mind and provide appropriate medications . a medline search of previous reports on abdominal migraine over the last 10 years using the key words abdominal migraine and adult revealed seven reports ( total of ten cases ) on adults ( 7 - 13 ) . a summary of the 11 cases of adult abdominal migraine ( ten cases plus the present case ) is shown in table 2 . five cases were aged in their twenties , two cases were in their thirties , one case was in his forties , and three cases , including the present case , were in their fifties . except for the present case , there was a period of 1 - 15 years between the onset of symptoms and the diagnosis , and in five of the ten cases , the onset of abdominal pain occurred when the patient had not yet reached adulthood . although there are very few studies on adult - onset abdominal migraine , long et al . reported that detailed interviews of 85 patients with abdominal pain ( median age , 37.6 years ; age range , 13 to 72 years ) whose pain could not be attributed to functional disorders revealed that 19 had a disease history implicating abdominal migraine and six presented the classical symptoms of abdominal migraine ( 21 ) . lundberg et al . reported that 12% of patients with migraine headache experience repeated intermittent abdominal pain , whereas only 1% of patients with muscle contraction headache experience abdominal pain ( 22 ) . one of the main reasons why there have been such few reports on adult - onset abdominal migraine is because the disease is not widely known , indicating that the number of reports on proactive suspicion of the disease may increase in the future . the present patient occasionally experienced headache in addition to abdominal pain , and five of the other ten cases also reported concomitant headache ( table 2 ) . the reason why abdominal migraine in adults is rarely accompanied by headache remains to be elucidated , which will be addressed through the study of additional cases in the future . because abdominal pain is severe in cases of abdominal migraine thus , when we meet a possible case of abdominal migraine , a careful interview process is required to determine the presence of concomitant headache . moskowitz 's trigeminovascular theory ( 23 ) is the strongest contender for an explanation of the onset mechanism underlying migraine headache . it proposes that pain transmission is activated via vasodilation of the carotid and cranial blood vessels due to the action of serotonin , and this then causes headache . internal serotonin is present in the brain and blood platelets , however , because > 90% of internal serotonin is present in the enterochromaffin cells and most serotonin receptors are also located in the enteric canal , it is thought that the pathological characteristics of abdominal migraine include the same abnormal serotonin dynamics as those observed in migraine headache ( 24 ) . in addition , from an anatomic point of view , it is believed that both central nervous system disorders and peripheral neuropathy ( hypersensitivity of visceral nerves ) are involved ( 24 ) . the present case had a history of migraine headache and was known to have experienced the sporadic onset of headaches , which were treated with single doses of nsaids . in general , most abdominal migraine experienced by patients during childhood shifted to migraine headache after the patient reached adulthood ( 1 ) , however , the present case presented a typical migraine headache that shifted to abdominal migraine during middle age . the present case had a history of infectious enteritis 3 months prior to the onset of abdominal migraine . although the mechanism of adult - onset abdominal migraine remains unclear , it was possible that infectious enteritis accelerated the hypersensitivity of the visceral nerves in the enteric canal , and as a result , caused adult - onset abdominal migraine . we would like to conduct future epidemiological research to determine whether the present case followed an extremely rare course or there are possibly many adult patients with abdominal migraine who remain undiagnosed . because the onset of migraine headache is considered most common between 20 and 40 years of age , it is important to question middle - aged or older patients undergoing examination for idiopathic abdominal pain about their history of migraine headache . furthermore , many patients do not consider it important to mention headaches during history - taking . if a physician asks open - ended questions about the patients ' medical history , patients may not mention migraine headaches . therefore , it is important to use close - ended questions such as have you ever been told that you have a migraine headache ? or are you prone to headaches ? we herein reported our experience of a middle - aged woman with abdominal migraine . although there are many reports on abdominal migraine in children , the possibility of this disease must also be considered in adults whose abdominal pain can not be attributed to any other disease , especially when the patient has a history of migraine headache .

a 52-year - old woman presented with recurrent , severe abdominal pain . laboratory tests and imaging were insignificant , and treatment for functional dyspepsia was ineffective . the poorly localized , dull , and severe abdominal pain , associated with anorexia , nausea , and vomiting , was consistent with abdominal migraine . the symptoms were relieved by loxoprofen and lomerizine , which are used in the treatment of migraine . we herein report a case of abdominal migraine in a middle - aged woman . abdominal migraine should be considered as a cause of abdominal pain as it might easily be relieved by appropriate treatment .

this study was approved by the institutional review boards of the national centre of excellence in molecular biology and the combined neuroscience institutional review board at the national institutes of health . written informed consent consistent with the tenets of the declaration of helsinki was obtained from participating individuals or their guardians before the study . families segregating arrd with three or more affected individuals were identified by visiting eye hospitals in pakistan , mostly in the punjab . blood samples were drawn from potentially informative family members , and genomic dna was extracted from leukocytes according to standard protocols . all participants underwent a detailed family , ophthalmic , and medical history , and selected individuals were evaluated by visual acuity , best - corrected visual acuity , slit - lamp biomicroscopy , ophthalmoscopy , fundus photography , and electroretinography ( erg ) . previously , as part of this project , 77 families had been mapped by linkage analysis to specific chromosomal locations ; the causative gene and mutations had been identified in 62 while for the remaining 15 only the locus had been identified . for the current homozygosity exclusion mapping study , 67 families were selected from the remaining unlinked families based on the availability of dna samples and consanguineous marriages of the parents of affected individuals . eight of these families have undergone whole genome linkage analysis without identified causative genes while the remaining 59 families were not screened . families with possible dominant or x - linked inheritance were excluded , and some families with only two affected offspring of consanguineous matings were included in the early parts of the study . one hundred eighty genes or loci associated with inherited retinal diseases were selected from retnet ( https://sph.uth.edu/retnet/ ) and screened by homozygosity exclusion mapping ( table 1 ) . homozygosity genotyping of 188 microsatellite markers was done in one affected individual of each family . loci homozygous in the first individual were genotyped in a second affected family member and , if also homozygous , in a third affected offspring of consanguineous parents . when when no single marker with a heterozygosity of 75% or greater was available , two markers were tested . we tested two markers within 1 to 2 cm of the candidate gene with heterozygosities over 50% , but approximately 50% of families would be expected to show discordant results for these markers . thus our assessment was that most discordant homozygosity would be the result of low information content rather than recombination between the two markers . families in which homozygosity was shared only by all affected siblings were further investigated by genotyping additional individuals for confirmation of cosegregation . a variant of the multiplexing short tandem repeat with tailed primers ( mstp ) approach described by oetting et . al . , using fluorescently labeled tagged primers homologous to extensions on initial primers in a two - pcr approach , was used to genotype these microsatellite markers . the pcr products were multiplex electrophoresed on an abi 3130 genetic analyzer ( applied biosystems , foster city , ca , usa ) , and fragment sizes were determined by genemapper version 4.0 ( applied biosystems ) . summary of mic rosatellite markers for homozygous mapping two - point linkage analyses were performed using the fastlink modification of the mlink program in the linkage program package . maximum logarithm of the odds ( lod ) scores were calculated using ilink , and linkmap was used for multipoint analysis . autosomal recessive rd was analyzed as a fully penetrant trait with an affected allele frequency of 0.00001 . haplotypes were generated using the cyrillic 2.1 program ( cyrillic software , wallingford , oxfordshire , uk ) and confirmed by inspection . based upon cosegregation of the risk haplotypes in a family , mutations in the exons and 100 bp of flanking intronic regions of the included known candidate gene associated with inherited retinal diseases were analyzed by sanger sequencing using abi prism 3130 automated sequencers ( applied biosystems ) and assembled and analyzed with seqman software ( dnastar lasergene 8 ; madison , wi , usa ) and mutation surveyor ( softgenetics , state college , pa , usa ) . a mutation was considered novel if it was not present in the human mutation database professional version on biobase ( https://portal.biobase-international.com/cgi-bin/portal/login.cgi ; in the public domain ) or the national center for biotechnology information dbsnp database ( http://www.ncbi.nlm.nih.gov/snp/ ; in the public domain ) , and sequence changes were considered pathogenic when they segregated with the disease in the family as well as their absence in 192 ethnically matched control chromosomes or at a frequency > 1% in the exac database ( http://exac.broadinstitute.org/ ; in the public domain ) ; and for missense changes were judged pathogenic in a computational test for mutations , including sorting intolerant from tolerant ( sift and provean , http://sift.jcvi.org/ ; in the public domain ) analysis , polymorphism phenotyping ( polyphen2 , http://genetics.bwh.harvard.edu/pph2/ ; in the public domain ) , and condel ( http://bg.upf.edu/fannsdb/ ; in the public domain ) . a sift score below the cutoff of 0.05 for a given substitution is classified as damaging while those with scores higher than this value are considered tolerated . in addition , we used condel ( consensus deleteriousness score of missense snvs ) , which computes a weighted average of the scores ( was ) of five tools : sift , polyphen2 , mapp ( multivariate analysis of protein polymorphism ) , logr pfam e - value , and mutation assessor . splicing changes were predicted using automated splice site analyses ( http://www.fruitfly.org/seq_tools/splice.html ; in the public domain ) . if the same variation was detected in more than one family , haplotypes of single nucleotide polymorphisms ( snps ) intragenic or within 1 mb of the mutated gene were genotyped . the frequency of the risk haplotype in the general population was calculated from 96 unrelated pakistani controls via the chm algorithm as implemented in the golden helix svs package ( golden helix , bozeman , mt , usa ) . this study was approved by the institutional review boards of the national centre of excellence in molecular biology and the combined neuroscience institutional review board at the national institutes of health . written informed consent consistent with the tenets of the declaration of helsinki was obtained from participating individuals or their guardians before the study . families segregating arrd with three or more affected individuals were identified by visiting eye hospitals in pakistan , mostly in the punjab . blood samples were drawn from potentially informative family members , and genomic dna was extracted from leukocytes according to standard protocols . all participants underwent a detailed family , ophthalmic , and medical history , and selected individuals were evaluated by visual acuity , best - corrected visual acuity , slit - lamp biomicroscopy , ophthalmoscopy , fundus photography , and electroretinography ( erg ) . previously , as part of this project , 77 families had been mapped by linkage analysis to specific chromosomal locations ; the causative gene and mutations had been identified in 62 while for the remaining 15 only the locus had been identified . for the current homozygosity exclusion mapping study , 67 families were selected from the remaining unlinked families based on the availability of dna samples and consanguineous marriages of the parents of affected individuals . eight of these families have undergone whole genome linkage analysis without identified causative genes while the remaining 59 families were not screened . families with possible dominant or x - linked inheritance were excluded , and some families with only two affected offspring of consanguineous matings were included in the early parts of the study . one hundred eighty genes or loci associated with inherited retinal diseases were selected from retnet ( https://sph.uth.edu/retnet/ ) and screened by homozygosity exclusion mapping ( table 1 ) . homozygosity genotyping of 188 microsatellite markers was done in one affected individual of each family . loci homozygous in the first individual were genotyped in a second affected family member and , if also homozygous , in a third affected offspring of consanguineous parents . when when no single marker with a heterozygosity of 75% or greater was available , two markers were tested . we tested two markers within 1 to 2 cm of the candidate gene with heterozygosities over 50% , but approximately 50% of families would be expected to show discordant results for these markers . thus our assessment was that most discordant homozygosity would be the result of low information content rather than recombination between the two markers . families in which homozygosity was shared only by all affected siblings were further investigated by genotyping additional individuals for confirmation of cosegregation . a variant of the multiplexing short tandem repeat with tailed primers ( mstp ) approach described by oetting et . using fluorescently labeled tagged primers homologous to extensions on initial primers in a two - pcr approach , was used to genotype these microsatellite markers . the pcr products were multiplex electrophoresed on an abi 3130 genetic analyzer ( applied biosystems , foster city , ca , usa ) , and fragment sizes were determined by genemapper version 4.0 ( applied biosystems ) . summary of mic rosatellite markers for homozygous mapping two - point linkage analyses were performed using the fastlink modification of the mlink program in the linkage program package . maximum logarithm of the odds ( lod ) scores were calculated using ilink , and linkmap was used for multipoint analysis . autosomal recessive rd was analyzed as a fully penetrant trait with an affected allele frequency of 0.00001 . haplotypes were generated using the cyrillic 2.1 program ( cyrillic software , wallingford , oxfordshire , uk ) and confirmed by inspection . based upon cosegregation of the risk haplotypes in a family , mutations in the exons and 100 bp of flanking intronic regions of the included known candidate gene associated with inherited retinal diseases were analyzed by sanger sequencing using abi prism 3130 automated sequencers ( applied biosystems ) and assembled and analyzed with seqman software ( dnastar lasergene 8 ; madison , wi , usa ) and mutation surveyor ( softgenetics , state college , pa , usa ) . a mutation was considered novel if it was not present in the human mutation database professional version on biobase ( https://portal.biobase-international.com/cgi-bin/portal/login.cgi ; in the public domain ) or the national center for biotechnology information dbsnp database ( http://www.ncbi.nlm.nih.gov/snp/ ; in the public domain ) , and sequence changes were considered pathogenic when they segregated with the disease in the family as well as their absence in 192 ethnically matched control chromosomes or at a frequency > 1% in the exac database ( http://exac.broadinstitute.org/ ; in the public domain ) ; and for missense changes were judged pathogenic in a computational test for mutations , including sorting intolerant from tolerant ( sift and provean , http://sift.jcvi.org/ ; in the public domain ) analysis , polymorphism phenotyping ( polyphen2 , http://genetics.bwh.harvard.edu/pph2/ ; in the public domain ) , and condel ( http://bg.upf.edu/fannsdb/ ; in the public domain ) . a sift score below the cutoff of 0.05 for a given substitution is classified as damaging while those with scores higher than this value are considered tolerated . in addition , we used condel ( consensus deleteriousness score of missense snvs ) , which computes a weighted average of the scores ( was ) of five tools : sift , polyphen2 , mapp ( multivariate analysis of protein polymorphism ) , logr pfam e - value , and mutation assessor . splicing changes were predicted using automated splice site analyses ( http://www.fruitfly.org/seq_tools/splice.html ; in the public domain ) . if the same variation was detected in more than one family , haplotypes of single nucleotide polymorphisms ( snps ) intragenic or within 1 mb of the mutated gene were genotyped . the frequency of the risk haplotype in the general population was calculated from 96 unrelated pakistani controls via the chm algorithm as implemented in the golden helix svs package ( golden helix , bozeman , mt , usa ) . a total of 67 probands and all affected siblings or ancestors who received a clinical diagnosis of arrp were included in this study . up to three affected individuals in each family were subjected to homozygosity screening in a stepwise manner . after screening the first affected patient with all 188 microsatellite markers , the average number of excluded markers for each family was 126 ( 126/188 = 67% efficiency ) , with exclusion rates of 85% after screening a second affected individual and 93.0% a third , so that the average number of markers to be analyzed was reduced from 188 to 13 in each family ( fig . 29 families were excluded from linkage to any known rp gene , making the exclusion rate 43.3% for families undergoing homozygosity screening . the remaining 38 families showed homozygosity and cosegregation in at least 1 of the 180 regions containing known arrd genes or loci . workflow of this study . after sequencing the included candidate genes for which the remaining 38 families showed homozygosity and cosegregation , in addition , variations considered benign by in silico prediction were detected in another four families ( table 3 ) . the two - point lod scores for markers in the homozygous chromosomal segments in these families are shown in table 4 . in seven families , no variations were identified by sequencing the known candidate genes within identified homozygous chromosomal regions , and their significance remains unclear . mutations detected in 67 arrd families subjected to homozygosity mapping variations of unknown significance detected in three genes of 67 arrd families two - point lod scores of arrd gene markers in 31 families overall , 24 causative mutations of 20 genes were identified in 27 families , including 12 missense , 6 nonsense , 4 indel - induced frameshift mutations , and 2 splice - site mutations ( the splicing site change in cngb1 was induced by an indel mutation ) . in all , 11 mutations were novel , and were not found in mutation databases or in 192 ethnically matched control chromosomes , while the remaining 13 mutations had been reported previously . each of the mutations was located within a homozygous region and cosegregated with the disease . for missense mutations , the substituted amino acid residues 27 ) , and in silico pathogenicity evaluation by polyphen2 , sift , and condel of the 12 missense mutations predicted these changes to be deleterious . some variations were detected in two families , including c.536a > g ( p.(k179r ) , family 61198 and 61199 ) and c.758t > g ( p.(m253r ) , family 61035 and 61126 ) in rdh5 , c.1466a > g ( p.(k489p ) , family 61301 and 61309 ) in tulp1 , and the probably nonpathogenic c.1946c > t ( p.(s649l ) , family 61237 and 61267 ) in prom1 . affected families who shared the same variations also shared a common haplotype of alleles at nearby intragenic snps , suggesting that the mutant allele was probably derived from a common ancestor ( supplementary table s2 ) . although genes or loci previously associated only with autosomal dominant inherited retinal disease were also screened in the study , no mutations were identified in these genes or loci . family 61220 , 61166 , 61219 , 61086 , 61042 and 61036 structure and haplotype of flanking markers of loci identified by homozygosity mapping . dna sequence tracings confirming the mutations are shown adjacent to the pedigrees , and cross - species conservation of amino acids showing missense mutations is shown on the right . family 61221 , 61192 , 61016 , 61015 and 61155 structure and haplotype of flanking markers of loci identified by homozygosity mapping . dna sequence tracings confirming the mutations are shown adjacent to the pedigrees , and cross - species conservation of amino acids showing missense mutations is shown on the right . family 61058 , 61076 , 61169 , 61150 , 61237 and 61267 structure and haplotype of flanking markers of loci identified by homozygosity mapping . dna sequence tracings confirming the mutations are shown adjacent to the pedigrees , and cross - species conservation of amino acids showing missense mutations is shown on the right . family 61217 , 61065 , 61198 , 61199,61035 and 61126 structure and haplotype of flanking markers of loci identified by homozygosity mapping . dna sequence tracings confirming the mutations are shown adjacent to the pedigrees , and cross - species conservation of amino acids showing missense mutations is shown on the right . family 61113 , 61262 , 61231 , 61312 and 61206 structure and haplotype of flanking markers of loci identified by homozygosity mapping . dna sequence tracings confirming the mutations are shown adjacent to the pedigrees , and cross - species conservation of amino acids showing missense mutations is shown on the right . family 61301 , 61309 and 61191 structure and haplotype of flanking markers of loci identified by homozygosity mapping . dna sequence tracings confirming the mutations are shown adjacent to the pedigrees , and cross - species conservation of amino acids showing missense mutations is shown on the right . information regarding the 24 sequence variations felt likely to be causative identified by homozygosity screening , and phenotypes of the 27 families in which they occurred , is provided in table 2 , and similar information for variations judged likely to be benign based on in silico predictions or presence in unaffected control individuals is provided in table 3 . the domain structure of the corresponding proteins and locations of the mutations with respect to these are shown in figures 8 and 9 , while the pedigree structure and corresponding haplotypes as well as sequence tracings and cross - species conservation for missense changes are shown in figures 2 through 7 , with the corresponding lod scores summarized in table 4 . domain structure and mutations of proteins in which mutations were identified by homozygosity mapping : cdhr1 , cep290 , cerkl , cnga3 , cngb1 , cngb3 , eys , grk1 , grm6 , lca5 . domain structure and mutations of proteins in which mutations were identified by homozygosity mapping : lrat , lrp5 , nr2e3 , prom1 , rbp3 , rdh5 , rdh12 , rp1 , rpe65 , rpgrip1 , tulp1 , ush2a . the c.847c > t , p.(r283 * ) mutation in cerkl seen in family 61219 is predicted to lead to truncation of the protein through a premature stop codon , and might be expected to lead to nonsense - mediated decay . however , this mutation has been shown to cause accumulation of truncated protein in the nucleus . the c.55c > t , p.(r19 * ) mutation in grk1 seen in family 61015 was predicted to lead to truncation of grk1 protein and result in nonsense - mediated decay , and seems likely to result in a variant form of oguchi disease , as described by zhang et al . , also predicted to result in nonsense - mediated decay ; however , detailed clinical data sufficient for this diagnosis could not be obtained , so it is listed in table 2 as having a stationary rd . family 61058 ( table 2 ) contains a c.652c > g , p.(r218 g ) mutation in lca5 segregating in a pseudo - dominant inheritance pattern , but the recessive nature of the mutation is confirmed by the sequencing results ( fig . 4 ) . the c.227g > a p.(r76q ) mutation in the dna - binding domain ( dbd ) of nr2e3 ( fig . the previously described missense variation c.1208g > a , p.(r403q ) in cngb3 seen in family 61221 ( fig . it was predicted to be probably damaging by polyphen2 but tolerated by sift and neutral by condel , so it seems most likely only to be a rare variation ( table 3 ) , although a modifying gene affecting the phenotype can not be excluded . the novel c.4268c > t , p.(p1423l ) variation in lrp5 was in an amino acid evolutionarily conserved from humans to zebrafish ( figs . 4 , 9 ) , had an allele frequency of 2.529 10 in the exac browser with no homozygotes listed in any population , and was not seen in 96 control individuals of pakistani ethnic extraction . however , in silico analysis with polyphen2 predicted that it would be benign , with sift showing that it was tolerated , and condel that it was neutral ( table 3 ) . hence , we treated this change as a rare variation even though the possibility that it might be responsible for disease in this family remains , especially since the phenotype in this family was progressive , consistent with a less severe mutation . a novel missense variation c.1946c > t , p.(s649l ) in prom1 was detected and cosegregated with the disease in families 61237 and 61267 ( table 3 ; fig . s649 is only weakly conserved from human to chicken , but the substitutions , from serine to threonine , are relatively conservative ( fig . thus , we presumed that this mutation is a rare variation , even though p.(s649l ) was not found in 192 control chromosomes from the pakistani population and had an overall frequency of 5.456 10 in the exac database , with all six variant alleles identified in the south asian population with no homozygotes . affected members of the two families share a common haplotype of alleles at 10 consecutive snps in and around prom1 suggesting that the variant allele is derived from a common ancestor ( supplementary table s2 ) . an overview of the clinical data from affected individuals in each family is shown in table 2 . the clinical symptoms , age of onset , and mode of inheritance in these families are generally consistent with the form of retinal degeneration described in the literature and on retnet . however , the clinical data available for this study are limited to ophthalmic history and examination , fundus photographs , and ergs , so that it is difficult to distinguish closely related forms of the retinal disease , for example , leber congenital amaurosis ( lca ) or cord from rp . thus , we use rd to represent those families in which the clinical findings are consistent with the previously described retinal disease , but were insufficient to distinguish unambiguously among the various diseases previously associated with that gene : rp , lca , congenital stationary night blindness ( csnb ) , or fundus albipunctatus , which together account for 8 of 27 ( 30% ) families . four of these had an early onset , but the differentiation between early - onset arrp and lca , difficult with the best documentation , could not be made reliably . one family was consistent with lca with typical fundoscopy , extinguished ergs , and a history of a congenital onset accompanied by nystagmus . four families that carried a homozygous rdh5 mutation received a diagnosis of fundus albipunctatus by fundus photographs . of the remaining two families , one showed the typical signs of csnb without progression , and the second was consistent with usher syndrome ( rp with deafness ) . although there was no indication of vestibular dysfunction in any affected individual in this family , suggesting usher type 2 and consistent with the causative gene , this was simply listed as usher syndrome because formal vestibular testing was not performed . a total of 67 probands and all affected siblings or ancestors who received a clinical diagnosis of arrp were included in this study . up to three affected individuals in each family were subjected to homozygosity screening in a stepwise manner . after screening the first affected patient with all 188 microsatellite markers , the average number of excluded markers for each family was 126 ( 126/188 = 67% efficiency ) , with exclusion rates of 85% after screening a second affected individual and 93.0% a third , so that the average number of markers to be analyzed was reduced from 188 to 13 in each family ( fig . 29 families were excluded from linkage to any known rp gene , making the exclusion rate 43.3% for families undergoing homozygosity screening . the remaining 38 families showed homozygosity and cosegregation in at least 1 of the 180 regions containing known arrd genes or loci . workflow of this study . after sequencing the included candidate genes for which the remaining 38 families showed homozygosity and cosegregation , in addition , variations considered benign by in silico prediction were detected in another four families ( table 3 ) . the two - point lod scores for markers in the homozygous chromosomal segments in these families are shown in table 4 . in seven families , no variations were identified by sequencing the known candidate genes within identified homozygous chromosomal regions , and their significance remains unclear . mutations detected in 67 arrd families subjected to homozygosity mapping variations of unknown significance detected in three genes of 67 arrd families two - point lod scores of arrd gene markers in 31 families overall , 24 causative mutations of 20 genes were identified in 27 families , including 12 missense , 6 nonsense , 4 indel - induced frameshift mutations , and 2 splice - site mutations ( the splicing site change in cngb1 was induced by an indel mutation ) . in all , 11 mutations were novel , and were not found in mutation databases or in 192 ethnically matched control chromosomes , while the remaining 13 mutations had been reported previously . each of the mutations was located within a homozygous region and cosegregated with the disease . for missense mutations , the substituted amino acid residues 27 ) , and in silico pathogenicity evaluation by polyphen2 , sift , and condel of the 12 missense mutations predicted these changes to be deleterious . some variations were detected in two families , including c.536a > g ( p.(k179r ) , family 61198 and 61199 ) and c.758t > g ( p.(m253r ) , family 61035 and 61126 ) in rdh5 , c.1466a > g ( p.(k489p ) , family 61301 and 61309 ) in tulp1 , and the probably nonpathogenic c.1946c > t ( p.(s649l ) , family 61237 and 61267 ) in prom1 . affected families who shared the same variations also shared a common haplotype of alleles at nearby intragenic snps , suggesting that the mutant allele was probably derived from a common ancestor ( supplementary table s2 ) . although genes or loci previously associated only with autosomal dominant inherited retinal disease were also screened in the study , no mutations were identified in these genes or loci . family 61220 , 61166 , 61219 , 61086 , 61042 and 61036 structure and haplotype of flanking markers of loci identified by homozygosity mapping . dna sequence tracings confirming the mutations are shown adjacent to the pedigrees , and cross - species conservation of amino acids showing missense mutations is shown on the right . family 61221 , 61192 , 61016 , 61015 and 61155 structure and haplotype of flanking markers of loci identified by homozygosity mapping . dna sequence tracings confirming the mutations are shown adjacent to the pedigrees , and cross - species conservation of amino acids showing missense mutations is shown on the right . family 61058 , 61076 , 61169 , 61150 , 61237 and 61267 structure and haplotype of flanking markers of loci identified by homozygosity mapping . dna sequence tracings confirming the mutations are shown adjacent to the pedigrees , and cross - species conservation of amino acids showing missense mutations is shown on the right . family 61217 , 61065 , 61198 , 61199,61035 and 61126 structure and haplotype of flanking markers of loci identified by homozygosity mapping . dna sequence tracings confirming the mutations are shown adjacent to the pedigrees , and cross - species conservation of amino acids showing missense mutations is shown on the right . family 61113 , 61262 , 61231 , 61312 and 61206 structure and haplotype of flanking markers of loci identified by homozygosity mapping . dna sequence tracings confirming the mutations are shown adjacent to the pedigrees , and cross - species conservation of amino acids showing missense mutations is shown on the right . family 61301 , 61309 and 61191 structure and haplotype of flanking markers of loci identified by homozygosity mapping . dna sequence tracings confirming the mutations are shown adjacent to the pedigrees , and cross - species conservation of amino acids showing missense mutations is shown on the right . information regarding the 24 sequence variations felt likely to be causative identified by homozygosity screening , and phenotypes of the 27 families in which they occurred , is provided in table 2 , and similar information for variations judged likely to be benign based on in silico predictions or presence in unaffected control individuals is provided in table 3 . the domain structure of the corresponding proteins and locations of the mutations with respect to these are shown in figures 8 and 9 , while the pedigree structure and corresponding haplotypes as well as sequence tracings and cross - species conservation for missense changes are shown in figures 2 through 7 , with the corresponding lod scores summarized in table 4 . domain structure and mutations of proteins in which mutations were identified by homozygosity mapping : cdhr1 , cep290 , cerkl , cnga3 , cngb1 , cngb3 , eys , grk1 , grm6 , lca5 . domain structure and mutations of proteins in which mutations were identified by homozygosity mapping : lrat , lrp5 , nr2e3 , prom1 , rbp3 , rdh5 , rdh12 , rp1 , rpe65 , rpgrip1 , tulp1 , ush2a . the c.847c > t , p.(r283 * ) mutation in cerkl seen in family 61219 is predicted to lead to truncation of the protein through a premature stop codon , and might be expected to lead to nonsense - mediated decay . however , this mutation has been shown to cause accumulation of truncated protein in the nucleus . the c.55c > t , p.(r19 * ) mutation in grk1 seen in family 61015 was predicted to lead to truncation of grk1 protein and result in nonsense - mediated decay , and seems likely to result in a variant form of oguchi disease , as described by zhang et al . , also predicted to result in nonsense - mediated decay ; however , detailed clinical data sufficient for this diagnosis could not be obtained , so it is listed in table 2 as having a stationary rd . family 61058 ( table 2 ) contains a c.652c > g , p.(r218 g ) mutation in lca5 segregating in a pseudo - dominant inheritance pattern , but the recessive nature of the mutation is confirmed by the sequencing results ( fig . 4 ) . the c.227g > a p.(r76q ) mutation in the dna - binding domain ( dbd ) of nr2e3 ( fig . the previously described missense variation c.1208g > a , p.(r403q ) in cngb3 seen in family 61221 ( fig . it was predicted to be probably damaging by polyphen2 but tolerated by sift and neutral by condel , so it seems most likely only to be a rare variation ( table 3 ) , although a modifying gene affecting the phenotype can not be excluded . the novel c.4268c > t , p.(p1423l ) variation in lrp5 was in an amino acid evolutionarily conserved from humans to zebrafish ( figs . 4 , 9 ) , had an allele frequency of 2.529 10 in the exac browser with no homozygotes listed in any population , and was not seen in 96 control individuals of pakistani ethnic extraction . however , in silico analysis with polyphen2 predicted that it would be benign , with sift showing that it was tolerated , and condel that it was neutral ( table 3 ) . hence , we treated this change as a rare variation even though the possibility that it might be responsible for disease in this family remains , especially since the phenotype in this family was progressive , consistent with a less severe mutation . a novel missense variation c.1946c > t , p.(s649l ) in prom1 was detected and cosegregated with the disease in families 61237 and 61267 ( table 3 ; fig . s649 is only weakly conserved from human to chicken , but the substitutions , from serine to threonine , are relatively conservative ( fig . 4 ) . this mutation does not occur in a transmembrane domain ( fig . 9 ) and is predicted to be benign by three in silico analyses . thus , we presumed that this mutation is a rare variation , even though p.(s649l ) was not found in 192 control chromosomes from the pakistani population and had an overall frequency of 5.456 10 in the exac database , with all six variant alleles identified in the south asian population with no homozygotes . affected members of the two families share a common haplotype of alleles at 10 consecutive snps in and around prom1 suggesting that the variant allele is derived from a common ancestor ( supplementary table s2 ) . an overview of the clinical data from affected individuals in each family is shown in table 2 . the clinical symptoms , age of onset , and mode of inheritance in these families are generally consistent with the form of retinal degeneration described in the literature and on retnet . however , the clinical data available for this study are limited to ophthalmic history and examination , fundus photographs , and ergs , so that it is difficult to distinguish closely related forms of the retinal disease , for example , leber congenital amaurosis ( lca ) or cord from rp . thus , we use rd to represent those families in which the clinical findings are consistent with the previously described retinal disease , but were insufficient to distinguish unambiguously among the various diseases previously associated with that gene : rp , lca , congenital stationary night blindness ( csnb ) , or fundus albipunctatus , which together account for 8 of 27 ( 30% ) families . four of these had an early onset , but the differentiation between early - onset arrp and lca , difficult with the best documentation , could not be made reliably . one family was consistent with lca with typical fundoscopy , extinguished ergs , and a history of a congenital onset accompanied by nystagmus . four families that carried a homozygous rdh5 mutation received a diagnosis of fundus albipunctatus by fundus photographs . of the remaining two families , one showed the typical signs of csnb without progression , and the second was consistent with usher syndrome ( rp with deafness ) . although there was no indication of vestibular dysfunction in any affected individual in this family , suggesting usher type 2 and consistent with the causative gene , this was simply listed as usher syndrome because formal vestibular testing was not performed . consanguineous matings have long been known to increase the risk of recessive diseases by increasing the fraction of the genome that is homozygous and identical by descent , and thus the number of potentially deleterious alleles descended from a common ancestor ; and homozygosity mapping provides an efficient means of localizing causative genes for recessive traits in these populations , particularly in populations with high consanguinity rates . pakistan , with consanguinity rates ranging from 17% to 38% , is an optimal country in which to implement this approach . one such was family 61015 , shown in table 2 as having a stationary form of rd . the phenotype in this family , as far as it could be ascertained , was consistent with mutations in grk1 causing oguchi disease and was similar to that in a previous pakistani family with a variant of oguchi disease due to deletion of exon 3 , although the degree of recovery from dark adaptation could not be ascertained in this family . thus , it is listed simply as a stationary rd , even though it is very likely to have oguchi type csnb . this gene primarily has been associated with enhanced s - cone syndrome and goldmann - favre syndrome . however , the phenotype in family 61150 is most consistent with progressive arrp , similar to that observed in a family of portuguese crypto - jews with a mutation in this gene . in addition , sharon et al . have described nr2e3 mutations in a series of patients with clumped pigmentary retinal degeneration , who also might be consistent with the phenotype observed in this family . taken together with our previous characterization of autosomal recessive retinal degenerations ( arrd ) in pakistan , we have studied a total of 144 consanguineous arrd families , in addition to 154 families with limited numbers of affected individuals or unclear inheritance patterns . of these 144 families , we have identified putative causative variations in 40 genes and 11 loci so far , and these genes and loci collectively account for disease in 104 of the 144 families ( 72.2% ) ( table 5 ; fig . 10 ) . the percentages of families who had variants in the 40 genes and 11 loci are shown in figure 10 , in decreasing order : rpe65 6.9% ( 10/144 ) , tulp1 6.9% ( 10/144 ) , rp1 4.9% ( 7/144 ) , pde6a and locus 4.9% ( 7/144 ) , ush2a and new locus 3.5% ( 5/144 ) , rdh5 2.8% ( 4/144 ) , 11p11.2-q13.2 locus 2.1% ( 3/144 ) , grm6 2.1% ( 3/144 ) , and 1p13.3 locus 2.1% ( 3/144 ) . it should be noted that this tabulation counts families sharing an intragenic haplotype as separate families . other genes or loci were identified in only one or two families in this cohort , respectively , accounting for less than 2% of the arrd population in pakistan . sequence variations identified in 144 unselected pakistani families contributions of specific genes and loci to pakistani families with arrp . the most frequently mutated genes in arrd differ remarkably among the populations of different ethnic origins . rpe65 and tulp1 were the genes most frequently mutated in pakistani patients with arrd , while the genes found to be most frequently mutated in other populations were rp1 in saudi arabians , rdh12 in spanish , and ush2a worldwide . the overall rate of variant detection was 61.8% ( 89/144 ) in this study , which is comparable to the worldwide variation detection rate of 60% . a recent report that summarized 103 published pakistani rd families found aipl1 and crb1 to be the most frequent causative genes , probably because lca families accounted for approximately 20% of the arrd families , while there were significantly fewer lca families in our patient cohort . in addition , the previous summary included families screened by sequencing previously identified candidate genes , which might tend to favor those genes identified early and/or widely publicized . in our study , sharing of variations by different families is likely to be the result of the variant allele being derived from a common ancestry , since all of the families that shared the same variation also shared common intragenic snp haplotypes for the associated gene . in addition , although we included the 79 genes / loci reportedly responsible only for autosomal dominant rd , no mutations were identified in genes previously associated only with adrd . taken together , we failed to uncover the pathologic variation in 27.8% of families in our arrd patient cohort . although 38 families showed homozygosity and cosegregation in at least 1 of the 180 regions containing known arrd genes or loci , no disease - causing mutations were identified in 11 of these , so that they are excellent candidates for identification of new arrd genes residing within linked regions in which no mutations were identified in the known candidate gene . the most promising approach to discover novel genes after such a systematic analysis of many consanguineous families is next - generation sequencing , although familial locus heterogeneity or compound heterozygous mutations might explain the phenotype of a small proportion of the families , as intrafamilial locus heterogeneity was detected in 15.3% of the families studied in a recent report of pakistani families with hearing impairment . in addition , compound heterozygous mutations were identified in 2.7% of genetically resolved arrd families in a review of all published retinal degeneration cases in pakistan . although homozygosity mapping has been proven effective , a major limitation is that this type of analysis will overlook familial locus heterogeneity or compound heterozygous mutations . in conclusion , our results provide a key bridge between bench and bedside and should make genetic diagnosis of arrd in patients more accessible and practical . this should greatly enhance the clinical genetic counseling , diagnosis , and early intervention of arrd in the pakistani population . these results also highlight the importance of analyzing the causative genes and their exons in different ethnic groups in a systematic and population - specific fashion .

purposethe pakistan punjab population has been a rich source for identifying genes causing or contributing to autosomal recessive retinal degenerations ( arrd ) . this study was carried out to delineate the genetic architecture of arrd in the pakistani population.methodsthe genetic origin of arrd in a total of 144 families selected only for having consanguineous marriages and multiple members affected with arrd was examined . of these , causative mutations had been identified in 62 families while only the locus had been identified for an additional 15 . the remaining 67 families were subjected to homozygosity exclusion mapping by screening of closely flanking microsatellite markers at 180 known candidate genes / loci followed by sequencing of the candidate gene for pathogenic changes.resultsof these 67 families subjected to homozygosity mapping , 38 showed homozygosity for at least one of the 180 regions , and sequencing of the corresponding genes showed homozygous cosegregating mutations in 27 families . overall , mutations were detected in approximately 61.8 % ( 89/144 ) of arrd families tested , with another 10.4% ( 15/144 ) being mapped to a locus but without a gene identified.conclusionsthese results suggest the involvement of unmapped novel genes in the remaining 27.8% ( 40/144 ) of families . in addition , this study demonstrates that homozygosity mapping remains a powerful tool for identifying the genetic defect underlying genetically heterogeneous arrd disorders in consanguineous marriages for both research and clinical applications .

about a century ago alois alzheimer and emil kraeplin among other neuropsychiatrists had ascribed arteriosclerotic dementia resulting from gradual strangulation of the blood supply to the brain as the main cause of dementia.1 otto binswanger should be recognized for introducing the notion of subclasses of vascular dementia ( vad ) . he described subcortical arteriosclerotic encephalopathy upon pathological verification of cerebral white matter disorder in a group of eight patients.1 currently we accept that cerebrovascular disease causes the second most common form of age - related dementia . vad can , however , result from all forms of cerebrovascular lesions that include post - stroke syndromes.2 the challenge of defining the pathological substrates of vad is complicated by the heterogeneous nature of cerebrovascular disease and co - existence of other pathologies including alzheimer type of lesions . blood vessel size , origin of vascular occlusion and genetic factors are critical factors in defining subtypes of vad ( table 1 ) . multi - infarct dementia is caused by large vessel disease whereas binswanger type of vad involving subcortical regions including the white matter results from small vessel changes . subcortical ischaemic vad appears the most significant subtype of vad.3 other factors that may define the subtype and degree of impairment include multiplicity , size , anatomical location , laterality and age of the lesion . for the purposes of prevention strategies and treatments it is imperative to recognise subtypes of vad yet not devise numerous categories to make the task unnecessarily complicated.4 there is now a general consensus that the clinical diagnosis of vad in demented patients with evidence of cerebrovascular lesions applies only when other causes of dementia are ruled out.5 the diagnosis of vad continues to be a challenge in the face of pathological outcomes ( fig . 1 ) . as with diagnosis of other causes of dementia criteria may be derived from a range of investigations including a detailed clinical history , timing of event , neuropsychometric tests , neuroimaging and neuropathological reports in accord with the dsm criteria . since the 1970s several clinical criteria for vad have been put forward.5 - 7 historically all the criteria have largely adopted the alzheimer type disease model despite the fact that vascular cognitive impairment ( vci ) or vad is a cluster of clinical syndromes related to complex interactions between cerebrovascular disease ( cvd ) , vascular risk factors , brain changes and host factors . the widely used criteria for vad include the dsm - iv,8 the icd-10,9 the addtc criteria10 and the ninds - airen criteria.4 the two cardinal elements implemented in the clinical criteria for vad are the nature of the cognitive syndrome11 and the type of vascular injury in the cause of the dementia.5,7,12 variation in definitions of these two elements has resulted in different prevalence estimates , vulnerable groups of subjects and consequently different types and distribution of brain lesions7,11,13 - 15 the ninds - airen criteria4 are the most widely used in randomised clinical trials . these criteria adequately ( 1 ) emphasise the heterogeneity of vad syndromes and pathologic subtypes including not only ischaemic stroke but also other causes of cvd such as cerebral hypoxicischaemic events , white matter lesions ( wmls ) and hemorrhagic strokes , ( 2 ) recognise the variability in clinical course , which may be static , remitting or progressive , ( 3 ) highlight the location of ischaemic lesions and the need to establish a causal relationship between vascular brain lesions and cognition , ( 4 ) recognise the need to establish a temporal relationship between stroke and dementia onset , ( 5 ) include specific findings early in the course that support a vascular rather than a degenerative cause , ( 6 ) emphasise the importance of brain imaging to support clinical findings , and ( 7 ) recognise the value of neuropsychological testing to document impairment in multiple cognitive domains . thus the ninds - airen criteria define vad as a syndrome with different aetiologies and clinical manifestations rather than a single entity . these criteria also incorporate three different levels of certainty of clinical diagnosis , namely probable , possible and definite.4 in a previous neuropathological series , the sensitivity of the ninds - airen criteria for probable and possible vad was determined to be 58% and the specificity 80%.16 the criteria successfully excluded ad in 91% of cases , and the proportion of mixed cases misclassified as probable vad was 29% . interestingly , compared to the addtc criteria,10 the ninds - airen criteria were more specific and better in excluding mixed cases ( 54% vs. 29% ) . despite these limitations , the frequencies of vad in autopsy series have been reported to be as low as 0.03% and as high as 58% with an overall mean rate of 17%17,18 that is consistent with estimates from clinical studies . in cases where currently accepted criteria have been applied the frequencies are even lower with a mean of 7% . whereas the incidence of vad cases in japan was reported to be 35%19 and 22%.20 taking these into account the worldwide frequency of vad in autopsy verified cases might be estimated to be between 10 and 15% . clinical and pathological evidence indicates combined neurodegenerative and vascular pathologies worsen presentation and outcome of dementia.21 - 24 a high proportion of individuals fulfilling the neuropathological diagnosis of ad have significant cerebrovascular lesions including silent infarcts upon imaging and extensive white matter disease.25 - 28 conversely , clinically diagnosed vad patients frequently show extensive ad - type neuropathological changes.28 - 30 for example , in one study31 87% of the patients clinically diagnosed by ninds - airen criteria were found to have ad either alone ( 58% ) or in combination with cerebrovascular disease ( 42% ) at post - mortem ( fig . hulette et al30 noted that " pure " vad is very uncommon because vad cases without co - existing neuropathological evidence of ad are rarely found . therefore current clinical diagnostic criteria serve to detect pathology but not " pure " pathology . early validation studies indicated that while mixed dementia could be distinguished from ad , it could not be separated from vad.32 recent studies suggest that 30 - 50% of mixed ad and vad cases are misclassified as vad16,33 and the neuroimaging component of the ninds - airen criteria does not distinguish between people with and without dementia in the context of cerebrovascular disease . the potential overlap of pathologies is therefore complex , with different types of cerebrovascular lesions including subcortical infarction and small vessel disease,22,34 and accompanying neurodegenerative changes including tau , amyloid and -synuclein pathology ( fig . while evidence - based pathological criteria for the diagnosis of mixed dementia remains to be perfected , the diagnosis35,36 should be made when a primary neurodegenerative disease known to cause dementia exists with one or more of the pathological lesions defining the vad subtypes ( table 1 ) . the diagnosis of mixed dementia ( ad and vad or less frequently dementia with lewy bodies and vad ) particularly among the oldest old ( > 85 years of age ) therefore also remains a challenge . atherothromboembolism may be responsible for up to 50% of all ischaemic strokes involving both cortical and subcortical infarctions . intracranial small vessel disease causes 25% of the infarcts.37 small vessel alternations involve arteriosclerosis , hyalionsis and are associated with lacunar infarcts and lacunes predominantly occurring in the subcortical structures . white matter disease or subcortical leukoencephalopathy with incomplete infarction and small vessel disease are common pathological changes in cerebrovascular disease . others features include borderzone ( watershed ) infarctions , laminar necrosis and amyloid angiopathy ( table 2 ) . complicated angiopathies such as fibromuscular dysplasia , arterial dissections , granulomatous angiitis , collagen vascular disease and giant - cell arteritis are rarer causes of cerebrovascular disease37 and vad . previous studies have recorded ischaemic , oedematous and haemorrhagic lesions affecting the brain circulation or perfusion to be associated with vad ( table 3 ) . in four studies where vad was diagnosed 75% of the cases revealed cortical and subcortical infarcts suggesting other vascular pathologies involving incomplete infarction or borderzone infarcts could be important factors . among other lesions 25% of the cases had cystic infarcts whereas 50% of the cases had lacunar infarcts or microinfarcts . lacunar infarcts , however , appear to be a common category of infarcts , and currently recognised as the most frequent cause of stroke ( dissections , granulomatous angiitis , collagen vascular disease and giant - cell arteritis are rarer causes of cerebrovascular disease).37 severe amyloid angiopathy was present in 10% of the cases . most interestingly , in one study 55% of the cases revealed hippocampal atrophy.38 ischaemic vascular disease appears to correlate with widespread small ischaemic lesions distributed throughout the cns.38 the main subtypes of vad included in current classifications are cortical vad or mid ( also referred as post - stroke vad ) and subcortical vascular dementia ( svd ) or small vessel disease related dementia.4,39 - 42 strategic infarct dementia , hypoperfusive dementia resulting from global cerebrovascular insufficiency are less common types.4,39,41,43 further , subtypes include hemorrhagic dementia and hereditary vascular dementias . cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy or cadasil is probably the most common form of familial svd . not surprisingly , the clinical presentation of cortical and subcortical forms of vad show remarkable differences . large vessel occlusions resulting in large cortical infarcts produce cognitive and other deficits that depend on the location of the infarcts , while svd may have a relatively characteristic neuropsychological profile that includes early impairment of attention and executive function , with slowing of motor performance and information processing.2,3,44 clinically , svd is characterised by pure motor hemiparesis , bulbar signs and dysarthria , gait disorder , variable depressive illness , emotional lability , and deficits in executive functioning.45 - 49 the early clinical phase of svd includes episodes of mild upper motor neuron signs ( drift , reflex asymmetry , incordination ) , gait disorder ( apractic - atactic or small - stepped ) , imbalance and falls , urinary frequency and incontinence , dysarthria , dysphagia as well as extrapyramidal signs such as hypokinesia and rigidity.44,47,48,50 however , these focal neurological signs are often subtle.51,52 upon imaging patients with svd have multiple lacunes and extensive wmls and often reveal clinical history of " prolonged tia " or " multiple tias " which mostly are small strokes without residual symptoms and only mild focal findings ( e.g. drift , reflex asymmetry , gait disturbance ) . this supports the importance of neuroimaging requirements in the criteria.44 the early cognitive syndrome of svd is characterized by a dysexecutive syndrome with slowed information processing , usually mild memory deficit and behavioural symptoms.3 the dysexecutive syndrome includes impairment in goal formulation , initiation , planning , organising , sequencing , executing , set - shifting and set - maintenance , as well as in abstraction.40,47,53 the memory deficit in svd is usually milder than in ad , and is specified by impaired recall , relative intact recognition , less severe forgetting and better benefit from cues.53 episodic memory may be relatively spared , compared with ad . there is more learning impairment that can be partially corrected by providing salient cues to encourage learning and promote recognition.54 therefore , memory deficits in vad appear to be caused by problems in retrieval of information;55 - 57 in turn , memory retrieval deficits are due to aberrant frontal and subcortical mechanisms . in contrast , in ad involvement of the hippocampus by neurofibrillary tangles ( braak stage iii ) prevents storage of new information causing amnesic mild cognitive impairment . the svd syndrome may be readily confused with ad in view of the neuronal loss and coexisting vascular factors . the onset can be variable as suggested previously.45 sixty percent of the patients had a slow , less abrupt onset , and only 30% an acute onset of cognitive symptoms . the course was gradual without ( 40% ) and with ( 40% ) acute deficits , and fluctuating in only 20%.45 behavioural and psychological symptoms in svd include depression , personality change , emotional lability , loss of volition ( apathy),42 incontinence , as well as inertia , emotional bluntness and psychomotor retardation.4,40,47 these may include greater tendencies of aggression and agitation.2 such symptomatology is attributed to damage to the prefrontal subcortical circuits.44,58 svd incorporates two entities " the lacunar state " and " binswanger 's disease."3 svd is accredited to small vessel disease and is characterised by lacunar infarcts , focal and diffuse ischaemic wmls , and incomplete ischaemic injury.47 - 50,59 the infarcts and wmls are expected consequences of small vessel disease . small vessels including intracerebral end - arteries and arterioles undergo progressive age - related changes,59,60 which may result in lacunar or microinfarcts . these range from wall thickening by hyalinosis , reduction or increment of the intima to severe arteriosclerosis and fibroid necrosis ( fig . uncomplicated hyalinosis is characterised by almost complete degeneration of vascular smooth muscle cells ( becomes acellular ) with concentric accumulation of extracellular matrix components like the collagens and fibroblasts.60 these changes are most common in the small vasculature of the white matter ( fig . small vessel changes likely promote occlusion or progressive stenosis with consequent acute or chronic ischaemia of the tissue behind it.60 alternatively , arteriolsclerotic changes located in small vessels in the deep white matter and basal ganglia ( lenticulostriate ) may loose their elasticity to dilate and constrict in response to variations of systemic blood pressure or loss of auto - regulation . the deep cerebral structures would be rendered most vulnerable because the vessels are end arteries almost devoid of anastomoses . small vessel pathology could lead also oedema and damage of the blood - brain barrier ( bbb ) with chronic leakage of fluid and macromolecules in the white matter.61 lacunes as complete or cavitating infarcts as defined above , measuring up to 15 mm in diameter , are seen radiologically and upon gross examination at autopsy ( table 2 ; fig . these lesions are largely confined to the cerebral white matter and subcortical structures including the thalamus , basic ganglia and brainstem . most lacunes , remnants of small infarcts , are detected in the cystic or chronic stage with no viable central tissue but could have perifocal regions with incomplete infarction , particularly in the white matter . a few lacunes may represent healed or re - absorbed as minute or petechial haemorrhages . microlacunes have also been described which essentially should be thought of as large cystic microinfarcts . to distinguish perivascular cavities it has been suggested that lacunes be classed into three subtypes : lacunar infarcts , lacunar haemorrhages and dilated perivascular spaces.63 lacunar infarcts usually result from progressive small vessel disease manifested as hypertensive angiopathy that may involve stenosis caused by hyalinosis . small vessel disease in a perforating artery for example , may also reveal regions of incomplete infarction , attenuation or rarefaction usually recognised by pallor upon microscopic examination . lacunes are associated with small perivascular cavities up to 2 mm in diameter often found in the basal ganglia and the white matter.59,61 perivascular cavities or empty spaces resulting from distortion or elongation of small arteries collectively referred to as tat lacunaire in the grey matter and tat crible in the white matter may be numerous in older subjects.62 microinfarcts have been variably described but widely thought to be small ishaemic lesions visible only upon light microscopy ( fig . these lesions of up to 5 mm diameter may or may not involve a small vessel at its centre but are foci with pallor , neuronal loss , axonal damage ( white matter ) and gliosis . microinfarcts have also been described as attenuated lesions of indistinct nature occurring in both cortical or subcortical regions . microinfarcts and lacunar infarcts appear central to the most common cause of vad and predict poor outcome in the elderly.37,38,63 interestingly , in the autopsied older japanese - american men the importance of microvascular lesions as a likely explanation for dementia was nearly equal to that of alzheimer lesions.64 microinfarction in the subcortical structures has been emphasised as substrate of cognitive impairment , and correlated with increased alzheimer type of pathology but cortical microinfarcts also appear to contribute to significantly to the progression of cognitive deficits in brain aging.65 furthermore microinfarcts even in borderzone ( watershed ) regions may aggravate the degenerative process as indicated by worsening impairment in ad.66 thus multiple microinfarction appears strongly correlated with dementia indicated by several studies.17 white matter lesions ( or subcortical leucoencephalopathy ) incorporating myelin loss are considered a consequence of vascular disease . the frequency of white matter changes is increased in patients with cerebrovascular disease and those at risk for vascular disease including arterial hypertension , cardiovascular disease and diabetes mellitus.2 lesions in the deep white matter have been correlated with dementia including vad.2,28 conflicting data with respect to periventricular lesions may depend on definition of the boundaries between the periventricular and deep white matter if the coursing of the fibres are used as markers . lacunar infracts are produced when the ischaemic damage is focal and of sufficient severity to result in a small area of necrosis whereas diffuse white matter change is considered a form of rarefaction or incomplete infarction where there may be selective damage to some cellular components . while the u - fibres are usually spared white matter disease comprises several patterns of alterations including pallor or swelling of myelin , loss of oligodendrocytes , axons and myelin fibres , cavitations with or without presence of macrophages and areas of reactive astrogliosis,61 where the astrocytic cytoplasm and cell processes may be visible with standard stains . lesions in the white matter also include spongiosis i.e. vacuolisation of the white matter structures and widening of the perivascular spaces . cerebral amyloid angiopathy ( caa ) also falls in to the category of small vessel diseases and contributes to the svd syndrome . current evidence shows age rather than gender , history of hypertension or other vascular disease to be the strongest risk factor for sporadic occurrence of caa.67,68 several familial forms of caa presenting with ischaemic and haemorrhagic infarcts or oligaemia are perhaps the most studied among the hereditary cerebrovascular disorders.69,70 the clinical features comprise focal neurological signs including spasticity , ataxia , facial paralysis , occasional seizures and cognitive impairment often leading to dementia.71 - 73 consideration of the hereditary cerebral haemorrhage with amyloidosis of the dutch type ( hchwa - d ) disease provides certain clues to a link between caa and stroke . it is thought that the first stroke - like episode triggers multiple cerebral bleeds which may be preceded by diffuse white matter changes that in turn lead to rapid decline of cognitive functions.71,74 the degree of caa is strongly correlated with the presence or absence of dementia while this was not true for diffuse plaques or neurofibrillary tangles . this suggests caa alone causes dementia in hchwa - d,73 a notion likely to be the case in other hereditary amyloid angiopathies.71 however , cognitive impairment is also a consistent feature in sporadic caa cases in the absence of other pathologies as verified by mri and neuropathological assessment.69 ischaemic wmls associated with lipohyalinosis and narrowing of the lumen of the small perforating arteries and arterioles , which nourish the deep white matter , often occur in ad and are common in vad . upon mri these correspond best with deep with matter hyperintensities , which may increases the likelihood of correct diagnosis.69 in a few studies biopsy proven caa was associated with extensive diffuse hyperintensities presenting as multifocal non - haemorrhagic leukoariosis.67,71 pathological studies also supported the robust relationship between caa and wmls irrespective of the presence of alzheimer type of pathology.75,76 several early reports suggest the existence of several familial stroke disorders unrelated to atherosclerotic disease . most of these disorders may be classed as svd involving small vessels of the subcortical structures.77 cadasil is perhaps the most common form of these hereditary cvds leading to cognitive decline and dementia.78 - 81 it is difficult to estimate the world - wide prevalence of cadasil but in western countries cadasil cases occur ~5 in 100,000 . there are estimated to be at least 500 cadasil families worldwide suggesting that this disorder is much more common than familial ad.77 cadasil begins with migraine as the first symptom in up to 40% of the patients.38,78,79 the age of onset is usually ascribed by the age at which the first ever stroke occurs rather than the migraine attack . cadasil may be manifest well before the first stroke on the basis of characteristic white matter hyperintensities upon mri.82,83 migraine usually with aura may begin even before the age of 10 years , but more commonly during the third decade.38,79 more severe manifestations including tias , recurrent strokes and depressive illness follow quickly . motor deficits , an ataxic hemiparesis , hemianopsia and dysarthria may accompany these principal events . neocortical strokes are rare and they usually do not cover a wide territory.83 large artery infarcts such as those of the middle or posterior cerebral artery are uncommon.77 dichgans and colleagues80 have suggested that the men are at greater risk for early immobilisation and death . however , smoking and high homocysteine ( > 15 mol / l ) can increase risk for more strokes and migraine.81 there is not a clear consensus for genotype - phenotype correlations80 and the apolipoprotein e 4 allele does not influence disease progression81 ( rk et al , unpublished observations ) . pathological features include severe arteriopathy with the presence of granular osmiophilic material in the arterial walls of both the brain and systemic organs.84 loss of brain vascular smooth muscle cells leads to wall thickening and fibrosis in small and medium - sized penetrating arteries.77,84 this would reduce both cerebral blood flow and blood volume in affected white matter with effects on the haemodynamic reserve by decreasing the vasodilatory response . affected vessels presumably progress to obliteration or thrombose as evident by the appearance of lacunar infarcts , mainly in the basal ganglia and fronto - temporal white matter.84 these pathologies initiate cognitive deficits , which progress to dementia of the subcortical vascular type . cadasil is caused by single missense mutations or exon deletions in the notch 3 gene.85 the gene encodes a type 1 transmembrane protein ( notch 3 ) , which is essential during development and regulating cellular differentiation . in adults notch 3 appears to be expressed only in vascular smooth muscle cells and may promote cell survival by inhibiting apoptosis but the exact function remains to be elucidated.77 notch 3 mutations consistently result in either a gain or loss of one ( or more odd number of ) cysteine residue(s ) in one of the 34 epidermal growth factor - like repeats in the amino - terminal region of the molecule.85 it is not entirely clear which step in the notch signalling pathway leads to the charactersitic vascular pathology of cadasil . the clinical diagnosis of cadasil is relied upon positive family history and hyperintense lesions upon t2 mri particularly in the temporal pole , whereas confirmed by notch 3 gene screening or the presence of gom in skin or nerve - muscle biopsies.77 the two main subtypes of the vad syndrome include large cortical infarction or mid and svd or small vessel disease related dementia . svd is clinically characterised by pure motor hemiparesis , bulbar signs and dysarthria , gait disorder , variable depressive illness , emotional lability , and deficits in executive functioning . the pathological features of svd involve small vessel degeneration , lacunes , multiple microinfarcts in the subcortical structures and white matter lesions extending into the deep white matter . cadasil is an example of familial svd that presents with recurrent subcortical strokes and slowly progressing course leading to cognitive impairment and dementia . the svd syndrome may be readily confused with ad in view of the neuronal loss and coexisting vascular factors . however , the symptomatology in svd is attributed to damage to the frontal- subcortical neuronal circuits .

atherothromboembolism and intracranial small vessel disease are considered to be the main causes of cerebrovascular injury , which may lead to cognitive impairment and vascular dementia ( vad ) . vad appears to be the second most common type of dementia with prevalence estimates of 10 - 15% . cortical or multi - infarct dementia and subcortical vascular dementia ( svd ) are suggested to be the two main forms of vad . the main clinical features of svd comprise decreased motor performance , early impairment of attention and executive function with slowing of information processing . svd results from lacunar infarcts or multiple microinfarcts in the basal ganglia , thalamus , brainstem and white matter and are associated with more than 50% of the vad cases . white matter changes including regions of incomplete infarction are usually widespread in vad but their contribution to impairment of subcortical regions is unclear . while most of vad occurs sporadically only a small proportion of cases bear clear familial traits . cadasil is likely the most common form of hereditary vad , which arises from subcortical arteriopathy . svd needs unambiguous definition to impact on preventative and treatment strategies , and critical for selective recruitment to clinical trials .

the total synthesis of two key analogues of vancomycin containing single - atom exchanges in the binding pocket ( residue 4 amidine and thioamide ) are disclosed as well as their peripherally modified ( 4-chlorobiphenyl)methyl ( cbp ) derivatives . their assessment indicates that combined pocket amidine and cbp peripherally modified analogues exhibit a remarkable spectrum of antimicrobial activity ( vssa , mrsa , vana and vanb vre ) and impressive potencies ( mic = 0.060.005 g / ml ) against both vancomycin - sensitive and -resistant bacteria and likely benefit from two independent and synergistic mechanisms of action . like vancomycin , such analogues are likely to display especially durable antibiotic activity not prone to rapidly acquired clinical resistance .

numerous wild edible plant species have been used by different communities in ethiopia , mainly as supplement to conventional foods [ 18 ] . however , the biodiversity is threatened through replacement of forests with agricultural expansion and deforestation without cultivation and domestication of potential species . diversification of production and consumption habits to include a broader range of plant species , particularly those currently identified as underutilized , could significantly contribute to improve health and nutrition , livelihoods , and ecological sustainability . edible wild and traditional vegetables have played an important role in supplementing staple foods by supplying trace elements , vitamins , and minerals . as wild food plants grow in natural conditions , they are easily accessed and freely harvested for their human food and nutrition values . they are relevant in household food security and nutrition in some rural areas , particularly during seasonal food shortage periods , and provide good nutritional supplies , notably micronutrients . although less prevalent in higher - income populations , these deficiencies do occur in such groups , especially among premature infants , children , and the elderly . several sources revealed that edible wild plant and traditional vegetable species increase the nutritional quality by providing minerals , fiber , vitamins , and essential fatty acids and enhance taste and color in rural diets . underutilized green leafy vegetables are a good source of many nutrients like iron , calcium , ascorbic acid , and -carotene that could help in overcoming micronutrient malnutrition and easily accessed by the community at a low cost . because micronutrient deficiencies ( such as vitamin a ) are associated with low intake of foods such as vegetables , as opposed to starchy ( energy rich ) staples which provide the majority of energy intake in typical african diets , increment in energy production and consumption will likely do little to ameliorate the problem of micronutrient deficiency unless identification , proper evaluation , and domestication of nutritionally potential lesser known vegetables are integrated into the diets of the population . moreover , the roles of edible wild plants and lesser known crops in human nutrition are potentially valuable to maintain a balance between population growth and agricultural productivity , particularly in the tropical and subtropical areas of the world . hence , continuous search for new source of nutrient especially from plant foods is a basis for selecting promising species for further studies on green leafy vegetables to meet the nutritional requirements . evaluation of the nutrient and antinutrient compositions of wild edible plants helps to identify foods rich in minerals and acquiring knowledge on the methods of appropriate preparation to enhance bioavailability of nutrients . as the consumption of plant products low in bioavailable minerals is high in rural communities of ethiopia , the presence of antinutritional factors that limits the optimal utilization of wild and traditional vegetables and the extent to which the household food preparation methods could reduce them need investigations . therefore , the objective of this work was to assess mineral contents and their bioavailability as a function of local household preparation methods of some wild and traditional vegetables of the gumuz ethnic community located in benishangul gumuz state western ethiopia . the study plants were collected from agalo meti district in kemash zone of benishangul gumuz regional state located at 530 km west of the capital addis ababa . selection of species for our investigation was based on ethnobotanical study findings in the study area . dioscorea abyssinica ( tuber ) , oxytenanthera abyssinica munro ( young shoot ) , abelmoschus esculentus ( l. ) moench ( immature pod ) , and portulaca quadrifida ( aerial part ) were selected based on their wider utilization by the community in the study area . the first two species are wild and the last two exist both under cultivation and wild stand . the selected vegetables were recommended for domestication according to an association in benishangul gumuz called tikuret le gumuz limat mahiber for the reason that they are widely consumed by the community and because they are most suited for domestication . each species was collected at the time when edible parts are acceptable for consumption by the local community . the specimens were identified by a botanist from bioversity international and ethiopian public health institute ( ephi ) using standard procedures and deposited in the national herbarium of addis ababa university ( aau ) . edible parts of the selected vegetables were harvested manually from their natural habitat in different areas of agalo meti woreda . five - kilogram edible parts of the respective species were collected from 20 different plants of the same species to ensure the representativeness . the sample extract solution was transferred to polyethylene bottle and stored until use for determinations of minerals . blank was prepared without sample by taking the same amount of reagents under the same condition . the minerals , namely , calcium , iron , zinc , and cu , were analyzed using shimadzu atomic absorption spectrophotometer ( aa-6800/aa wizard software ) . sodium and potassium contents were determined using flame photometer ( jenway , pf 7 , essex , uk ) according to the method described by aoac 2005 , 966.16 and 965.30 , respectively . phosphorus was determined using uv - visible spectrophotometer ( cecil instruments , cambridge england , deuterium f 500 ma , power t3 . absorbance of standard , blank , and samples was read at 660 nm using uv - visible spectrophotometer . absorbance versus concentration calibration curve was constructed and the equation obtained was used to calculate the unknown phosphorus concentration in the samples:(1)phosphorus in mg/100 gm = asabdilution factorextracted volume100slopeweight of sample1000,where as is the absorbance of sample , ab is the absorbance of blank , and slope is calculated from the calibration curve . briefly , 0.5 gm of dried sample was extracted with 10 ml of 0.2 n hcl for 1 hour at an ambient temperature and centrifuged ( 3000 rpm for 30 m ) . two ml of wade reagent ( 0.03% fecl36h2o and 0.3% sulfosalicylic acid ) was added to 3 ml of the supernatant sample solution . the amount of phytic acid was calculated using phytic acid standard curve prepared in the same condition and the result was expressed as phytic acid mg/100 g dry weight . the oxalate content of the samples was determined using titration method [ 16 , 17 ] . two g of finely ground samples was placed in a 250 ml conical flask containing 190 ml of distilled water . ten ml 6 m hcl solution was added to each of the samples and the suspension was digested at 100c for 1 h. the samples were then cooled and made up to 250 ml mark of the flask . the samples were filtered and duplicate portion of 125 ml of the filtrate was measured into beaker and four drops of methyl red indicator was added , followed by the addition of concentrated nh4oh solution ( drop wise ) until the solution was changed from pink to yellow color . each portion was then heated to 90c , cooled , and filtered to remove the precipitate containing ferrous ion . each of the filtrates was again heated to 90c and 10 ml of 5% cacl2 solution was added to each of the samples with stirring consistently . after cooling , the samples were left overnight . the supernatants were decanted and the precipitates were completely dissolved in 10 ml 20% h2so4 . the total filtrates resulting from digestion of 2 g of each of the samples were made up to 200 ml . aliquots of 125 ml of the filtrates were heated until near boiling and then titrated against 0.02 m standardized kmno4 solutions to a pink color which persisted for 30 sec . the analysis was carried out in duplicate , and the results were expressed in dry basis . the suggested critical values used to predict the bioavailability were calcium : phytate < 6 , phytate : iron > 1 , phytate : zinc > 15 , and phytate : calcium / zinc > 0.5 [ 19 , 20 ] . descriptive statistics such as means and standard deviation were calculated using spss version 16 software . one - way analysis of variance ( anova ) was used to see the effect of household processing methods on mineral contents and the bioavailability of minerals in selected vegetables . multiple comparison tests using least significant difference technique ( lsd , p < 0.05 ) were applied to compare the means of each parameter between different household preparation practices using spss version 16 software . paired comparison t - test was used to determine if there was a significant mean difference between raw and processed vegetables for each parameter . from the results presented in table 1 , it is noticeable that the concentration of different macroelements ( ca , p , na , and k ) and trace elements ( fe , zn , and cu ) of the wild and traditional vegetables was high and the vegetables studied could be regarded as appreciably important sources of these essential elements . potassium is abundant in all vegetables analyzed followed by calcium , phosphorus , and sodium from the macroelements . from the trace elements , iron level was relatively higher followed by zinc and copper . as presented in table 2 , the iron and zinc contents of raw a. esculentus ( okra ) were 2.3 mg/100 g and 0.68 mg/100 g , respectively . iron content in immature pods of raw a. esculentus obtained in the present study was higher than findings of for different a. esculents varieties reported within the range of 0.870.97 mg/100 g fw but the zinc content ( 0.68 0.04 mg/100 g fw ) obtained in this study was slightly lower compared to the value reported ( 1.291.37 mg/100 g fw ) in their study . the variations in the contents of the minerals in a. esculentus may be due to the varietal difference or genetic factor , environmental factor , and their interactions . significant loss of iron and zinc was observed in the cooked a. esculentus but not statistically significant between raw and sun dried young pods of a. esculentus . sun drying is the gradual loss of water through evaporation and can not support leaching . iron level in raw ( whole ) dioscorea abyssinica was 27 mg/100 g fw . however , cooking and peeling the bark of the tuber significantly reduced the iron content to 1.79 mg/100 g fw . the iron content of d. abyssinica obtained in this study in the raw sample was higher when compared with the value reported for dioscorea pentaphylla ( 8 mg/100 g fm ) . zinc concentration of d. abyssinica was also significantly reduced during cooking and debarking from 0.5 mg/100 g to 0.3 mg/100 g fw . the statistically significant ( p < 0.05 ) reduction of iron and zinc concentration upon cooking and peeling of the tuber may be because of the presence of minerals in the outer nonedible part of the tuber which was removed by peeling after boiling the tuber . the same reason may apply to the minerals which showed enormous reduction in the boiled and peeled d. abyssinica . reduction in zinc content was also reported in peeled and boiled tubers of dioscorea cayenensis . the potassium level in raw tubers of d. abyssinica was 341.16 mg/100 g. statistically significant losses were observed when the tubers were cooked compared to the raw samples . this loss may be attributed to the leaching out of minerals including potassium into the cooking water . the high potassium content in this tuber could help to maintain normal blood pressure and can be labeled as heart protective vegetable . the minerals content of raw and cooked juvenile shoots of oxytenanthera abyssinica is presented in table 1 . the iron and zinc contents of juvenile shoots of o. abyssinica were 0.6 mg/100 g and 0.9 mg/100 g , respectively . all minerals tested were decreased in the cooked samples with the reduction being statistically significant in zn , cu , p , na , and k but fe and ca were not significantly ( p < 0.05 ) lost in the cooked samples . the variation in the degree of loss might be related to the chemical forms of minerals in the food matrix . the respective potassium contents determined in raw and cooked samples of o. abyssinica were 456.2 mg/100 g and 273.2 mg/100 g on fresh weight basis . the potassium content of raw o. abyssinica obtained in the current study is well agreed with the value reported for other bamboo species such as bambusa tulda ( 408 mg/100 g , fw ) and dendrocalamus hamiltonii ( 416 mg/100 g , fw ) . raw shoots of o. abyssinica had the highest phosphorus content compared to other study vegetables . sodium intakes more than 1 g per day tend to aggravate a genetically determined susceptibility to hypertension , and intakes above 7 g / day may induce hypertension even in individuals who have no specific genetic susceptibility . in this context , all the vegetables contained safe sodium levels . the recommended dietary allowance ( rda ) for iron is 10 mg / day for adults . the raw immature pods of a. esculentus could provide 116.5% of the rda requirement for iron if 500 g fresh vegetable is consumed per day , considering it would be fully bioavailable . the cooked and sun dried a. esculentus could provide 34% and 129% rda requirement for iron from 500 g fw meal . cooked d. abyssinica and bamboo shoots can also provide 89.5% and 30% rda requirement of iron from fresh 500 g meal , respectively , and only 125 g fresh leaves and steams of portulaca quadrifida can provide 100% rda requirement for iron . this rda calculation did not consider the inhibitory effect of different antinutritional factors that affect the bioavailability of iron . five hundred grams of meal ( fresh basis ) from cooked a. esculentus could provide 23% of the zinc rda requirement for lactating mothers . similarly , cooked d. abyssinica and juvenile shoots of o. abyssinica in the cooked form could provide 12.1 and 22.5% rda requirement for zinc , respectively . portulaca quadrifida could provide 35% of rda for zinc considering the requirement for lactating woman . calcium content ranged from 22.9 mg/100 g fw in cooked bamboo shoot to 140.4 mg/100 g fw in sun dried a. esculentus . raw immature pods of a. esculentus contained 131.7 mg/100 g fresh weight basis . the calcium level obtained in this study for a. esculentus grown in ethiopia was higher than the values reported for different a. esculentus varieties in nigeria . raw aerial part of p. quadrifida contained higher ( 118 mg/100 g fw ) calcium content next to sun dried and raw a. esculentus . the calcium content in raw and cooked o. abyssinica was 24.5 mg/100 g and 22.9 mg/100 g fw , respectively . the calcium content reported in india for the different bamboo species was within the range of 21.17180.69 mg/100 g ( 29 ) ; the present value obtained for o. abyssinica was similar to bambusa balcooa ( 24.01 mg/100 g ) . the calcium content shows no significant ( p > 0.05 ) variation between raw and boiled d. abyssinica and o. abyssinica ( table 1 ) . however , significant reduction was observed when a. esculentus was cooked but sun drying did not affect the calcium content . consumption of 500 g cooked a. esculentus , cooked d. abyssinica , and o. abyssinica and raw p. quadrifida can contribute 52.12 , 20.37 , 11.5 , and 59.0% rda requirement for calcium , respectively . copper was the element found in a trace amount in all wild and traditional vegetables analyzed which ranged from 0.07 to 0.46 mg/100 g fw . this is important due to the fact that copper is required by body at a trace level for many metabolic activities ( 25 ) . phosphorus content in this study ranged from 23.7 mg/100 g to 57.3 mg/100 g fw . raw a. esculentus , d. prehensilis , and o. abyssinica contained 39.1 , 40.7 , and 57.3 mg/100 g phosphorus , respectively . cooking resulted in significant losses ( 7.341.76% ) of phosphorus with variable degree of proportion among the vegetables ( table 1 ) . consumption of 500 g cooked a. esculentus , d. abyssinica , and o. abyssinica and raw p. quadrifida could contribute by meeting 25.9 , 16.9 , 27.9 , and 28.4% rda requirement for phosphorus , respectively . antinutritional components such as oxalates , tannins , polyphenols , and phytic acid ( myoinositol hexaphosphate ) present in plant foods are known to have adverse effects on human nutrition by inhibiting iron and zinc absorption . the molar ratios along with the suggested critical values for predicting the bioavailability calcium , iron , and zinc are presented in table 2 . the calculated phytate / zinc molar ratios for raw and processed weps were within the range of 2.0314.22 , which were in the range of the suggested critical level ( < 15 regarded as favorable for zinc absorption ) . ratios 15 are associated with low zinc bioavailability . according to who cut - offs phytic acid to zinc mole ratio 15 , 515 , and < 5 is equal to zinc bioavailability as low ( 1015% ) , moderate ( 3035% ) , and high ( 5055% ) , respectively . in this context , a. esculentus and p. quadrifida had moderate zinc bioavailability , whereas the molar ratios suggested that high zinc bioavailability could be achieved from d. abyssinica and juvenile shoots of o. abyssinica . however , the critical phytate : zinc molar ratio may also depend on dietary calcium levels because of the kinetic synergism between calcium and zinc ions resulting in ca : zn : phy complex which is less soluble than phytate complexes formed by either of the ions alone , suggesting that ca : phy / zn molar ratio is better predictor of zinc bioavailability than phy : zn molar ratio alone . calcium phytate / zinc molar ratios of cooked and sun dried a. esculents and p. quadrifida were above the critical level ( 0.5 mol / kg ) as indicated in table 2 , suggesting that calcium interference was more likely to affect zinc bioavailability . the ca phy / zn molar ratio reported in the earlier study for different a. esculentus varieties in nigeria was within the range of 0.2930.436 . higher calcium content in a. esculentus that grows in ethiopia compared to nigeria could be attributed to genetic differences , environmental variability , and interaction of the genetic factor with the environment . considering both phy / zn and ca phy / zn molar ratios , zinc could adequately be absorbed in the body from d. abyssinica and shoots of o. abyssinica . as indicated in table 2 , phytate / iron molar ratios were > 1 ( indicative of poor iron bioavailability ) for all raw and processed study plants except d. abyssinica . this might be due to the reported higher phytate content and insufficient phytic acid degradation ( 4.527.5% ) by boiling alone . the calcium / phytate molar ratios in all the raw and processed study plants were > 6 , which is regarded as favorable for calcium absorption , predicting that a good calcium bioavailability could be achieved from all the selected vegetables . in addition to phytic acid , oxalic acid in insoluble form is responsible for interference of divalent metals absorption particularly calcium by forming insoluble salts . the oxalate / calcium molar ratios of all the selected raw and processed vegetables were below the critical level of 2.5 known to significantly impair calcium bioavailability suggesting that they are good calcium bioresources for the local populace . immature pods of abelmoschus esculentus , aerial parts of portulaca quadrifida , and juvenile shoots of oxytenanthera abyssinica consumed by the gumuz community indicated that they are appreciably important sources of essential minerals . except d. abyssinica , reducing duration of cooking time and using other processing methods such as fermentation ( d. abyssinica and o. abyssinica ) might alleviate the deterioration of the nutrients . the predicted mineral bioavailability shows adequacy in terms of calcium and zinc ( moderately bioavailable ) but not in iron . hence , there is a need for some enhancers to increase iron absorption in all species . the study results further revealed that a. esculentus and p. quadrifida are rich sources of bioavailable calcium . with the exception of d. abyssinica , the vegetables are rich in potassium and can contribute in maintaining normal blood pressure and its heart protective role . to be able to justify the overall nutritional value of the wild and semiwild edible vegetables , proper assessment of the type and concentration of their antinutrients is necessary . the rate in reduction of antinutritional factors depended upon the type of processing ( cooking and sun drying ) and vegetable . however , the reduction of phytic acid , oxalate , and tannins by traditional cooking methods alone was not adequate to the level that could improve iron and zinc bioavailability . the vegetables have higher moisture content and are mostly used after cooking as side dishes by the community . the combination effects of these factors might reduce the actual impact of antinutritional factors in impairing bioavailability of nutrients and/or causing ill health . however , appropriate processing methods that are known to reduce the antinutritional factors ( such as fermentation ) could be encouraged in the community .

edible parts of some wild and traditional vegetables used by the gumuz community , namely , portulaca quadrifida , dioscorea abyssinica , abelmoschus esculentus , and oxytenanthera abyssinica , were evaluated for their minerals composition and bioavailability . mineral elements , namely , ca , fe , zn , and cu , were analyzed using shimadzu atomic absorption spectrophotometer . effects of household processing practices on the levels of mineral elements were evaluated and the bioavailability was predicted using antinutrient - mineral molar ratios . fe , zn , ca , cu , p , na , and k level in raw edible portions ranged in ( 0.64 0.0227.0 6.24 ) , ( 0.46 0.020.85 0.02 ) , ( 24.49 1.2131.7 8.3 ) , ( 0.11 0.010.46 0.04 ) , ( 39.13 0.3457.27 0.94 ) , ( 7.34 0.4220.42 1.31 ) , and ( 184.4 1.31816.3 11.731 ) mg/100 g fw , respectively . although statistically significant losses in minerals as a result of household preparation practices were observed , the amount of nutrients retained could be valuable especially in communities that have limited alternative sources of these micronutrients . the predicted minerals ' bioavailability shows adequacy in terms of calcium and zinc but not iron .

acute mountain sickness ( ams ) is a disorder which is seen in ascent to altitudes higher than 3000 meters . it is a clinical syndrome with headache and one or more other symptoms including gastrointestinal symptoms ( poor appetite , nausea , vomiting ) , fatigue and/or weakness , dizziness / lightheadedness , and sleep disturbances [ 1 , 2 ] . its diagnosis is based on environmental symptom questionnaires , hackett or lake louise ams score [ 3 , 4 ] . several studies in different countries have been done so far , and incidence rates have been reported from 25% to 69% [ 2 , 511 ] . some effective factors have been considered in several studies , such as change of altitude related to residence altitude [ 3 , 5 , 11 ] , speed of mountain climbing [ 1 , 2 , 11 ] , very low and very high range of ages [ 5 , 12 ] , positive history of mountain sickness [ 8 , 11 ] , beginning mountain climbing at night , sleep in altitudes more than 3 hours in altitude , and water and fluid intake . however , the relation of some factors like age and gender to the incidence of the disease is in doubt . mount damavand has four main paths for mountaineers ' ascent . in our previous study , the incidence rate of disease was seen in more than 60% of mountaineers who ascend from the south . iranian expert mountaineers believe that ascent from southern path is accompanied by higher disease occurrence , but there is no documented data for this belief . on the other hand , some of the trekkers plan the beginning of mountain climbing at night . in the literature review , we did not find any investigation on beginning of ascending time ( except our previous study ) and effect of path on incidence of ams in other altitudes . so we designed this study for evaluating the incidence of ams in different paths of mount damavand and the effect of beginning time of ascent on ams incidence in iranian trekkers . this study was a descriptive cohort study which was performed on trekkers of mount damavand during 8 weekends in summer 2007 . mount damavand , iran 's highest mountain is located in the alborz mountain range and it has special characteristic such as dormant volcano and single peak . all trekkers who climb over 4200 m were included in this study . the first questionnaire was related to personal information and ascent characteristics ( including sex , age , and home altitude , medical history , past history of ams , beginning time of mountain climbing , number of acclimatization programs before this trekking , and duration of sleeping at over 4200 m ) , and the second questionnaire was lake louise score questionnaire . ams was defined as a lake louise score more than 5 ( table 1 ) . sleeping more than 3 hours was considered as a risk factor for ams . interviewers were trained physicians when trekkers came back to the camps of 4200 m. according to our previous study , sample size was calculated to be 280 trekkers in all four paths ( 70 trekkers for each path ) . in this period , all men and women who ascended mount damavand from northern , western , eastern , and southern paths and passed 4200 m altitude were included in the study . exclusion criteria included mountaineers who had used drugs for ams prevention ( acetazolamide , dexamethasone , or aspirin ) or individuals who could not ascend to the top because of climbing - related injuries . convenient sampling was done with the presence of research team at the altitude of about 4200 meters in different paths . according to our previous experience , the participants were divided into three groups by their age , under 15 y , 1650 y , and over 50 y. trekkers were divided into 3 groups based on the number of ascents they had made to altitudes above 4000 m in the last 6 months : experienced ( more than 3 ascents ) , semiexperienced ( 1 - 2 ascents ) , and inexperienced ( less than 1 ascents ) . this study was approved by research committee of sports medicine research center and ethics committee of tehran university of medical sciences . qualitative data were summarized as frequencies , whereas continuous data were expressed as mean and standard deviation . categorical variables were analyzed with the chi - square and fisher exact test and numerical variables were analyzed with the independent t - test . multiple logistic regression analysis was used to explore the independent predicting variables for ams . for inferential purpose , 95% confidence interval was presented for the estimated effects ( e.g. , odds ratio ) . from 360 completed questionnaires , 9 persons were excluded because of drug utilization and none of the studied persons was injured and finally 351 persons were studied ( including 300 male and 51 female mountaineers ) . mean age of participants was 35.08 years , and its range was from 12 to 69 years . ams occurred in 188 trekkers , so the overall incidence rate of ams was 53.6% . the incidence rate under 15 y , 1650 y , and over 50 y was 33.8% , 48.5% , and 44.2% , respectively , but these differences were also not statistically significant ( p = 0.7 ) . forty - nine percent of male participants and 41.2% of female participants were affected , and this difference was also not statistically significant ( p = 0.2 ) . the highest incidence rate of ams was in southern path . table 2 shows the number of mountaineers and the incidence of ams in each path . the difference between the incidences among different paths was highly significant ( p < 0.001 ) . although ams incidence was significantly higher in southern path in comparison to others , there was no difference between the incidences of ams in other paths ( table 3 ) . seventeen to thirty - one percent of trekkers in north , west , and east paths were inexperienced , while this percent in south path was about 47% . there is no significant difference between experience of trekkers in north , west , and east paths , but this variable was significantly different in south path in comparison to others ( p = 0.01 ) . the minimum incidence of the disease as it is shown by table 4 was in individuals who started climbing between 6 and 12 am ( p = 0.02 ) , but the time of mountain top arrival had no influence on the disease incidence ( p = 0.4 ) . this difference tended to be statistically significant ( p = 0.06 ) . in any case the mean acclimatization programs in nonpatients were greater or equal to 2.17 ( sd = 1.46 ) , while in patients were less or equal to 1.76 ( sd = 1.25 ) , and statistically significant ( p = 0.025 , ci = 0.050.76 ) . cigarette smoking and positive family history of ams had no statistically significant influence on the incidence of the disease ( p = 0.5 for each one ) . initial model includes beginning time of mountain climbing , sleeping at 4200 m altitude , home altitude , ams history , and ams history in damavand . multiple logistic regression analysis reveals that the southern path and ams history on mount damavand increases the incidence of ams in damavand ( p = 0.019 and p < 0.001 , resp . ) . the risk of ams occurrence for trekkers who ascended from southern path was 1.9 ( ci = 1.13.3 ) ( table 5 ) . the main goal of this study was assessment of acute mountain sickness incidence in different paths of damavand . according to our review , the novel finding of this study is that the incidence of ams had association with ascending route . as a whole , the incidence rate of ams in this study and in all four paths was the highest among studies on ams . in previous studies , the incidence of ams is reported as 68% in the study of mountaineers who climbed over 4200 m in himalaya and 69% in professional astronomy staff on mauna kea . in other studies at about 4800 m or less which were mainly on alps and rocky mountains climbers , the incidence rate was lower than 40% [ 2 , 5 , 10 , 14 , 15 ] . as a whole , high incidence rate of ams in our study can be due to specific characteristics of mount damavand or genetic characteristics of iranian trekkers . the incidence of ams was 10.4 to 34.5% in a study on mount tochal ( another mountain in the alborz range at 3450 m ) [ 15 , 16 ] . in our previous study which was also reported on the southern path of damavand , incidence rate was 60.8% . our findings in this study showed that the incidence rate of ams was unequal in difference routes of mountain . our study showed that ams in southern path climbers was significantly higher than any other path , but there is not significant difference among any other paths . by multiple logistic regression analysis a hypothesis is that the individual characteristics of damavand in southern path ( such as higher substantial amount of malodorous sulfur compounds and high - steep path in comparison with other paths ) have caused this . furthermore , a study on sulfur concentration in each path of mount damavand needs to be done . on the other hand , the southern route has less distance and it is the easiest route , so trekkers can ascent faster and less - experienced trekkers select this rout for ascending ( as it is seen in section 3 ) . faster mountain climbing and higher - inexperienced trekkers in south route can be other factors that influence higher rate of ams in this route . often trekkers believe the best time for trekking on the damavand is at the early morning ( before 4 am ) , and this time is accompanied by lower ams rate . the lowest incidence of ams was in trekkers who start climbing between 6 am and 12 md . if it was found in the damavand , it may be due to environmental factors ( such as the higher concentration of sulfur during night period ) , and if it was found in other mountains , it may be due to personal character ( such as fatigue at the night ) . . meaningful influence of positive past history of ams on frequent ams has been reported in other studies [ 5 , 10 , 11 ] . influence of physiologic and genetic characteristics of individual suffering from ams are guessed as important factors [ 19 , 20 ] . however , in this study there was an association between ams and positive history of ams in the univariate analysis , but not in multiple logistic regression analysis . on the other hand , ams history in damavand was a positive factor for ams occurrence in both the univariate and multiple logistic regression analyses . this finding confirms the similar finding in our previous study , and the iranian expert trekkers think that there is a specific character in mount damavand which is such as high sulfur gas concentration in the air . similar to our previous study and other studies , there was no relation between sex , age , and smoking and incidence rate of ams [ 1 , 11 , 21 ] . however , a higher incidence of ams has been reported in children and a lower rate in the elderly [ 1 , 5 , 22 , 23 ] . the finding of this study in relation to the role of residence altitude in the incidence rate of the disease was the same as other studies [ 5 , 11 ] . speed of mountain climbing was an effective factor in the occurrence of ams in other studies [ 1 , 2 , 22 , 24 ] , but in our study there was no meaningful difference between mean climbing time in patients and healthy groups . similarly , in our study we found that acclimatization programs cause more decrease in ams . in this study like the previous one , there is a significant inverted relationship between sleeping in altitudes and the occurrence of ams . fatigue due to short time of sleeping may be the reason for higher ams , while insomnia is a symptom of ams . however , the average time of sleep in affected and nonaffected individuals had no statistically significant difference . it seems that other factors such as experience of climbers or fatigue affect this finding . in our study , we did not find any association between sleep in altitude and incidence of ams in multiple logistic regression analysis . none of the factors such as sleeping at 4200 m altitude , home altitude , and ams history was significant predictors of ams ( southern path as a significant predictor of ams doubles its risk in comparison to other routes ) . our study was performed on mount damavand with the characteristics such as dormant volcano , single peak , and sulfur odors especially on south route . on the other hand , we did not find other study in this mount except our previous study . moreover , no previous studies have evaluated the influence of the time of beginning mountain climbing on occurrence of ams . the incidence of ams above 4200 m , this rate might be higher if climbers / trekkers who started climbing but turned around below 4200 m were included . although several known factors cause ams , there are also some unknown factors which do the same . besides other factors , the path and the beginning time of climbing can affect the incidence of ams . the risk of ams occurrence was doubled for trekkers who ascended from southern path . in our study ams history in general , ams history on damavand in particular , the beginning time of climbing , sleeping at 4200 m altitude , and home altitude had significant effect on ams incidence .

background . this study was designed to evaluate the incidence of acute mountain sickness ( ams ) occurring on different climbing routes on mount damavand and the effect of beginning time of ascent in iranian trekkers . methods . this study was a descriptive cohort investigation , performed in summer 2007 . all trekkers who ascended mount damavand from northern , western , eastern , and southern paths and passed 4200 m altitude were included in the study . two questionnaires were completed for each trekker ( personal information and lake louise score questionnaire ) . multiple logistic regression analysis was used to explore the independent predicting variables for ams . results . overall incidence rate of ams was 53.6% . this rate was the highest in south route ( 61.5% ) ( p < 0.001 ) . there was no difference in the incidence of ams on other paths . ams history , ams history on damavand , the beginning time of climbing , sleeping at 4200 m altitude , and home altitude had significant effect on ams incidence , but by multiple logistic regression analysis south route and ams history on mount damavand had positive effect on incidence of ams ( p = 0.019 and p < 0.001 ) . conclusion . the path and the beginning time of ascent can affect incidence of ams . the risk of occurrence of ams was 1.9 times as large for trekkers who ascended from southern route .

limb salvage surgery and revision arthroplasty are more demanding procedures than primary total arthroplasty . components of these procedures are a stable fixation of the prostheses to the host bone , joint line restoration , and a stable range of motion consistent with the patient 's daily activities . therefore , implant selection should be based on the amount of bone loss , the status of ligaments , and the soft - tissue stabilizing structures [ 15 ] . in cases of global insufficiency or complete loss of all joint ligaments the increasing constraint from a posterior stabilized prosthesis to a nonlinked constrained , hinged or rotating hinge device is required [ 1 , 2 , 611 ] . in cases of gross segmental bone loss , modular prostheses or allograft - prosthesis composites modular knee prostheses with a rotating hinge articulation are used for reconstruction following tumor resections around the knee , complex primary knee arthroplasty , and revision total knee arthroplasties . such devices provide a stable reconstruction of the knee when the intrinsic soft - tissue stability had been lost as a result of surgical intervention [ 1315 ] . the hinge axis allows flexion - extension motion , and the vertically oriented post - in - channel axis provides internal and external rotations . furthermore , the post - in - channel design allows distraction during flexion and extension , which is restricted by the restraint of the remaining soft tissues . if distraction of a rotating hinge prosthesis occurs , the angular laxity depends on the design of the central rotational stem ( length and taper ) and the design of the tibial rotational cylinder . dislocation of rotating hinge prostheses due to implant 's breakage or fatigue failures is occasionally observed in patients following total knee arthroplasty for tumoral indications and revisions total knee arthroplasty [ 2 , 1622 ] . based on an earlier published biomechanical study , we tested six further implants of american and european manufacturers . the aim of the biomechanical study was to establish the association between design of the central rotational stem and implant 's stability . therefore , the angular laxity of the peg with increasing amounts of distraction as well as the maximum amount of distraction before the stem 's dislocation was determined . the hypothesis of the study was that implant designs with cylindrical shaped pegs are more stable than implant designs with conical shaped pegs concerning stability and maximum amount of distraction before dislocation . we performed a biomechanical analysis using a custom made biomechanical apparatus on a test bench . therefore , the lengths and tapers of the peg of six different rotating hinge knee implants ( limb preservation system lps / m.b.t . ( depuy , warsaw , in ) ; s - rom noiles ( depuy ) , global modular resection system gmrs ( stryker , mahwah , nj ) , rt - plus ( plus orthopedics , mdling , austria ) , and nexgen ( zimmer , kiel , germany ) ) were determined with a standard calliper rule ( figure 1 , table 1 ) . the zimmer nexgen was tested twice , one time with the thinnest and one time with the thickest polyethylene inlay available because the length of the peg varies with the thickness of the polyethylene inlay . additionally , we intended to test the link endo model ( waldemar link gmbh and co.kg , hamburg , germany ) and the genux ( implantcast gmbh , buxtehude , germany ) , but both implants were not suitable for the biomechanical analysis because of their mechanical antidislocation feature which prevents distraction at all . the degree of tilting of the central rotational stem within the vertical post - in channel was measured in a theoretical setting by extending the distraction , as well as the maximum amount of distraction before the stem 's dislocation . a custom made biomechanical apparatus was used to imitate distraction , but physiological load was not applied . nevertheless , the central rotational stem / polyethylene inlay was fixed to the proximal ( femoral ) base plate of the biomechanical apparatus ( figure 2 ) , which was mobile to imitate the distraction ( figure 2 ) . the hinged bearing inserts were fixed with a retainer to the proximal base plate with a mobile screw in a horizontal gliding slot to simulate the angulation . the tibial component was edged into the distal ( tibial ) part of the biomechanical apparatus with the rotational surface horizontally aligned . the vertical alignment of the components in the neutral position was verified with the centerline of a goniometer . laterally directed , not standardized pressure , which was produced by each observer , was used to generate the angulation of the peg within the tibial rotational cylinder . displacement of the central rotational stem to each side was measured with a standard goniometer . distraction was increased by 5 mm increments using 1 mm metal platelets , which were stacked , up to the increment that allowed dislocation ( figures 3(a)3(c ) ) . the point of dislocation was defined as the point at which any laterally directed force would cause the central rotational stem to jump off the tibial component ( figure 3(c ) ) . after femorotibial disengagement , the measurements were repeated with 1 mm increments from the last 5 mm increment before the increment that allowed determining dislocation . for the second approach , the fixation screw at the horizontal gliding slot was removed and the vertical post - in channel was filled with the metal platelets under the tip of the peg to simulate distraction as performed in the study of ward et al . . the purpose of this procedure was to evaluate if the horizontal gliding slot would influence the results of method 1 . the remaining steps of measurement were repeated the same as in the first method . in the third method , the measurements were visualized with sketches of the prosthesis components which were overlaid for each increment of distraction . three observers ( andreas leithner , senior orthopedic surgeon , mathias glehr , resident , and joerg friesenbichler , resident ) with different physiological strength performed three cycles of measurements with each implant and method . the degrees of angular laxity were plotted at each level of the component 's distraction . the slope of the curves reflects the extent to which the angular laxity increases , with increasing distraction . the end point of each curve demonstrates the last measureable angle before the implant 's dislocation ( figure 4 ) . multiple one - way analyses of variance ( anova ) were performed at 10 mm , 15 mm , and 25 mm of distraction for methods 1 and 2 to determine if there were differences between the tested implants concerning stability against lateral directed forces ( angular laxity ) . furthermore , an intraclass - correlation coefficient ( icc ) was calculated at 15 mm and 25 mm of distraction to determine the observer 's reliability . we calculated observed power according to the magnitude of distraction - angular displacement at 10 mm , 15 mm , and 25 mm of distraction in both methods according to hoenig and heisey . for statistical analysis the pasw statistics 17.0 program ( spss inc . , chicago , il , usa ) was used , and a p value < 0.05 was considered to be statistically significant . using the thinnest polyethylene inlays available , the gmrs , nexgen , and rt - plus implant designs , with truly cylindrical central rotational stems , were superior to the lps / m.b.t . ( part cylindrical and part conical ) and the s - rom noiles ( conical ) implant designs , with regard to stability and maximum amount of distraction before dislocation ( 38 versus 36 versus 30 versus 27 versus 26 mm , resp . ) ( table 2 ) . the gmrs , nexgen , and rt - plus implant designs with long central rotational stems ( 47 , 46 , and 38 mm ) required 38 , 36 , and 30 mm of distraction do dislocate . in comparison , the lps / m.b.t . and s - rom noiles ( both have a stem length of 46 mm ) dislocated at 27 mm and 26 mm , respectively , of distraction ( table 2 ) . the nexgen rotating hinge device with a 26 mm polyethylene inlay and a stem length of 60 mm dislocated at 42 mm of distraction , but the implant was quite unstable from 40 mm of distraction until dislocation . despite a shorter central rotational stem , the rt - plus device dislocated later than the lps / m.b.t . and the s - rom noiles implants ( 30 versus 27 versus 26 mm ) . the gmrs , rt - plus , and nexgen ( with a 26 mm pe - inlay ) implants were the only designs with tilting angles less than 10 degrees at any given amount of distraction until dislocation . this circumstance could be verified with methods 1 and 2 but not with method 3 . at 25 mm of distraction , the lps / m.b.t . and s - rom noiles devices had an angular laxity of 11.2 and 18.4 degrees in case of method 1 and 11.3 and 17 degrees in case of method 2 , while at the same increment the other tested implants showed tilting angles ranging from 2.1 to 3.2 degrees for method 1 and 2.3 to 3.3 degrees for method 2 ( table 3 ) . the moments , forces , and the elasticity on the devices , respectively , during the measurements with methods 1 and 2 could not be reproduced using the graphic delineations ( method 3 ) . therefore , it was not possible to determine the correct angular laxity with this method . the distraction - angular displacement curve of the s - rom noiles design showed an inferior increment ( high laxity ) when compared to the other implant designs , which showed steep rising slopes ( lower laxity ) ( figure 4 ) . these findings confirmed that shorter and more tapered pegs have a greater angular laxity at any given amount of distraction . the multiple one - way anova analyses revealed statistical significant differences between the tested implants concerning stability ( angular laxity ) against lateral directed forces at 10 mm , 15 mm , and 25 mm of distraction for both methods of measurement ( method 1 : p < 0.001 at all tested increments ; method 2 : p = 0.031 at 10 mm , p < 0.001 at 15 mm , and 20 mm of distraction ) , and the magnitude of difference between angular laxity at 10 mm , 15 mm , and 20 mm in both methods was large enough that post hoc power analysis could show over 80% power . furthermore , the intraclass - correlation coefficient ( icc ) revealed excellent results for both methods of measurement . at 15 mm of distraction , the icc at 25 mm of distraction resulted 0.994 ( p < 0.001 ) for method 1 and 0.993 ( p < 0.001 ) for method 2 . determining the interobserver correlation at 15 mm and 25 mm of distraction also showed high precision although not using a standardized pulley system to generate angular laxity . using a custom made biomechanical apparatus , the current study demonstrated that rotating hinge designs with long , cylindrical , and central rotational stems ( gmrs , nexgen , and rt - plus ) were superior to implant designs with shorter and/or more tapered rotational stems in the theoretical setting . the lps / m.b.t . and s - rom noiles implants require at least 26 mm and 27 mm , respectively , of distraction to dislocate . in contrast , the gmrs , the nexgen ( with a 12 mm polyethylene inlay ) , and the rt - plus devices with truly cylindrical , nontapered central rotational stems required 38 mm , 36 mm , and 30 mm of distraction to dislocate . the nexgen rotating hinge knee with a 26 mm polyethylene inlay dislocated at 42 mm of distraction . the implants with cylindrical , nontapered central rotational stems also had the lowest tilting angles at any given amount of distraction until dislocation , while the s - rom noiles implant showed the highest angular laxity throughout the biomechanical analysis ( table 3 ) . one main limitation of the study was the manual pressure , which was used to generate the lateral tilting of the central rotational stem within the tibial rotational cylinder and was not standardized by using a loaded pulley system . therefore , each observer influenced the measurements with his physical strength , similar to the study of ward et al . furthermore , in clinical setting the forces transmitted to the prostheses are influenced by many variables such as patient 's age , height , weight , length of the lower extremities , muscle strength , cementation technique of the implant , and the patient 's daily habits which were not minded in the current study . nevertheless , determining the interitem correlation matrix revealed high interobserver agreement and could therefore diminish this bias . this study showed that devices with longer , nontapered pegs had provided the best results which could be proven in the current biomechanical study using another different rotating hinge designs . the authors concluded that short and great tapered pegs have greater instability and higher risk of dislocation at any given amount of distraction . on the contrary , the endo - model ( waldemar link gmbh and co.kg ) has the shortest peg , and nevertheless , this implant seems to be a safe design because of the mechanical antidislocation feature [ 15 , 18 ] . investigations showed that instability , relative to soft - tissue compromise and ligamentous insufficiency , is most apparent to the patients in flexion when they lift the lower limb out of a seated position or leave the leg dangling [ 2 , 18 , 20 , 24 ] . et al . tested the rotational stability of a rotating hinge device and demonstrated the importance of the soft tissues and the newly formed periprosthetic scar , to protect the prosthesis from excessive rotational stresses , especially when using devices without a rotational stop . ligamentous imbalance , component malalignment , or loosening and polyethylene wear are the most common reasons for this complication . depending on the severity of instability , several repair techniques and treatment options have been described . further opportunities to enhance the stability are advancement of the collateral ligaments , muscle , and/or tendon transfers . additionally , collateral ligament insufficiency can be treated with soft - tissue reconstruction and/or with implants that provide inherent stability in the coronal plane . dislocation of rotating hinge knee prostheses is a rare complication . in the majority of cases , except for those of ward et al . , petrou et al . , and joshi and navarro - quilis , dislocation occurred due to breakage of any prosthesis ' component or fatigue of the tibial antidislocation device . all authors supposed excessive flexion gap instability and posterior dislocating forces causing the mechanical failure of the prostheses [ 2 , 1620 ] . . emphasized the importance of the ligamentous balance , especially the flexion gap , and suggested constrained prosthesis with an antidislocation device in cases of ligamentous insufficiency [ 18 , 19 ] . [ 15 , 20 ] recommended rotating hinge devices with a long ( > 5 cm ) , cylindrical peg or an effective mechanical antidislocation feature in case of severe articular compromise . the results of the biomechanical analysis showed that the design of the peg plays a major role in the stability of a rotating hinge device . we conclude that rotating hinge prostheses with shorter and markedly tapered pegs have the highest angular laxity at any given amount of distraction , and they may become unstable under conditions of mild joint distraction , theoretically . prosthetic designs with longer , cylindrical pegs might be useful in patients with severe articular compromise because the intrinsic design of such devices allows less tilting under mild joint distraction . nevertheless , none of the implants allowed dislocation until at least 25 mm of distraction .

purpose . rotating hinge knee prostheses should provide a stable situation following reconstruction . we performed a biomechanical analysis to establish the association between design of the central rotational stem ( peg ) and implant 's stability , in a theoretical setting . methods . six different rotating hinge designs were tested , and three observers performed two different measurements with a custom made biomechanical apparatus and laterally directed pressure . the aim was to assign the degree of tilting of the peg within the vertical post - in channel by extending the distraction as well as the maximum amount of distraction before the peg 's dislocation . an intraclass - correlation coefficient ( icc ) was calculated to determine the observer 's reliability . results . implant designs with cylindrical pegs of different lengths were superior to implant designs with conical or other shaped pegs concerning stability and maximum amount of distraction before dislocation , showing steep rising distraction - angular displacement curves . the icc at 15 mm and 25 mm of distraction revealed high interobserver reliability ( p < 0.001 ) . conclusion . the biomechanical analysis showed that rotating hinge prostheses with long and cylindrical pegs have the highest stability at any given amount of distraction . designs with shorter and markedly tapered pegs may become unstable under conditions of mild joint distraction which has to be proven in future in vivo investigations .

the causative agent of melioidosis , burkholderia pseudomallei , is endemic in the northern part of australia and southeast asia including malaysia . acute melioidosis may present as localized or septicaemic infections and can be fatal if left untreated . it was the common cause of community - acquired pneumonia in northeastern thailand and was attributed as the cause of fatal community - acquired bacteremic pneumonia in northern australia [ 1 , 2 ] . latent infection may remain asymptomatic for years only to be reactivated from a latent focus when the host is immunocompromised . therefore , it is important to treat melioidosis with prolonged course of antibiotics so as to avoid disease relapses which are commonly associated with short courses of antibiotics . some patients may default treatment or take improper dosage of antibiotics because of the long duration of treatment , and this may contribute to the relapse or the development of resistance . burkholderia pseudomallei is intrinsically resistant to a wide range of antibiotics which include some -lactam antibiotics , aminoglycosides , and macrolides . the antibiotics that are currently being used for the therapy of melioidosis are ceftazidime , imipenem , meropenem , amoxicillin / clavulanate , cefoperazone / sulbactam , trimethoprim - sulfamethoxazole ( tmp - smx ) , doxycycline , and chloramphenicol . the antibiotic regime for the treatment of melioidosis varies from one country to another . in malaysia , ceftazidime alone , or in combination with tmp - smx or cefoperazone / sulbactam alone or in combination with tmp - smx or imipenem , is the recommended antibiotic of choice for the intensive phase of treatment followed by oral tmp - smx plus doxycycline or amoxicillin / clavulanate in the maintenance phase . the development of resistance of burkholderia pseudomallei to some of these antibiotics has been reported in neighbouring countries such as singapore and thailand [ 4 , 5 ] . reports on the antibiotic susceptibility of burkholderia pseudomallei in malaysia have been limited to a few selected antibiotics and a smaller number of tested strains and from restricted demographic areas [ 6 , 7 ] . therefore , the aim of the study was to determine the in vitro antibiotic susceptibility patterns of clinical isolates of burkholderia pseudomallei to a panel of antibiotics used for the treatment of melioidosis and also to the potential alternative antibiotics such as tigecycline , ampicillin / sulbactam , and piperacillin / tazobactam . a total of 170 burkholderia pseudomallei nonrepeat clinical isolates were collected from the year 2001 until the year 2009 , from the microbiology laboratories of 29 government hospitals situated in 11 out of 14 states in malaysia ( table 1 ) . these isolates were sent from these hospitals for the confirmation of identification at the bacteriology unit , institute for medical research . species identification was carried out by gram - staining , motility , api 20ne ( biomrieux ) , and polymerase chain reaction technique using specific 16rrna primers as described by brook et al . 1997 . the strains were stored at 80c in 20% glycerol and were revived by subculturing onto blood agar plates before being further used . minimum inhibitory concentrations ( mic ) of the antibiotics were determined by e - test ( biomrieux ) following the manufacturer 's instructions . eleven antibiotics were tested , namely , ceftazidime , imipenem , meropenem , amoxicillin / clavulanic acid , tmp - smx , ciprofloxacin , doxycycline , chloramphenicol , piperacillin / tazobactam , ampicillin / sulbactam , and tigecycline . a 0.5 mcfarland suspension was made for each bacterial isolates and then inoculated on mueller - hinton ( mh ) ( bd ) agar . the e - test strips of each antibiotic were placed on the mh agar and incubated overnight at 37c . the mic ( g / ml ) interpretation for susceptible ( s ) , intermediate ( i ) , and resistant ( r ) for amoxicillin - clavulanic acid ( s 8/4 , i 16/8 , r 32/16 ) , ceftazidime ( s 8 , i 16 , r 32 ) , imipenem ( s 4 , i 8 , r 16 ) , doxycycline ( s 4 , i 8 , r 16 ) , and tmp - smx ( s 2/38 , - , r 4/76 ) was carried out following the clsi approved guideline m45-a2 . for ciprofloxacin ( s 1 , i 2 , r 4 ) , chloramphenicol ( s 8 , i 16 , r 32 ) , piperacillin / tazobactam ( s 16/4 , i 32/464/4 , r 128/4 ) , and ampicillin / sulbactam ( s 8/4 , i 16/8 , r 32/16 ) , the mic ( g / ml ) for enterobacteriaceae was referred . for tigecycline , the us fda approved breakpoints for enterobacteriaceae were used ( s 2 , i 4 , r 8) . for meropenem , the interpretative criteria outlined by the e - test manufacturer for aerobes were followed ( s 4 , i 8 , r 16 ) . any values which were in between the sensitive and resistant breakpoints were interpreted as intermediates . sensitivity to ceftazidime , imipenem , amoxicillin / clavulanic acid , ampicillin / sulbactam , and doxycycline was noted in 169 ( 99.4)% of the isolates . one isolate was shown to have heterogenous population with ceftazidime susceptibility of 8 g / ml and ceftazidime resistance of 256 g / ml . the isolate that was intermediately resistant to imipenem ( mic 6 g / ml ) was also resistant to amoxicillin / clavulanic acid and ampicillin / sulbactam . for chloramphenicol and tmp - smx , 164 ( 96.5% ) and 153 ( 90% ) of the strains were susceptible , respectively . only 72 ( 42.3% ) isolates were susceptible to ciprofloxacin while 94 ( 55.3% ) isolates showed intermediate resistance with mic of 1.53.0 g / ml . susceptibility to tigecycline was observed in 110 isolates ( 64.7% ) while intermediate resistance was noted in 59 ( 34.7% ) of the isolates . one isolate was resistant to tigecycline at the mic of 8.0 g / ml . the minimum concentration of antibiotics that inhibited 50% ( mic50 ) and 90% ( mic90 ) of the isolates and the range of mics of the tested antibiotics were shown in table 2 . the mic90 of most of the antibiotics were within the range of 0.38 to 4.0 g / ml . chloramphenicol was shown to have higher mic50 and mic90 values than the other antibiotics ( mic50 , 6.0 g / ml ; mic90 8.0 g / ml ) . imipenem ( mic50 , 0.38 g / ml ; mic90 , 0.38 g / ml ) was more active towards these strains than meropenem ( mic50 , 0.75 g / ml and mic90 , 1.0 g / ml ) . the resistance pattern observed among these strains was mainly monoresistance where 7 strains showed resistance to a single antibiotic , either to ceftazidime ( 1 strain ) , ciprofloxacin ( 1 strain ) , or trimethoprim / sulfamethoxazole ( 5 strains ) . two strains showed resistance to 2 different types of antibiotics ( 1 isolate to ciprofloxacin & trimethoprim / sulfamethoxazole ; 1 strain to amoxicillin / clavulanic acid and ampicillin / sulbactam ) . one strain showed multiple resistances to 4 antibiotics namely ciprofloxacin , chloramphenicol , trimethoprim / sulfamethoxazole , and tigecycline and intermediate resistance to doxycycline . this study showed that the local strains were highly susceptible to meropenem and piperacillin / tazobactam . for ceftazidime , imipenem , amoxicillin / clavulanic acid , and doxycycline , only 0.6% of the isolate was resistant to these antibiotics . this was a blood isolate from a patient who had no past record of melioidosis and had passed away a day after admission before the culture result was obtained . primary resistance to ceftazidime is rare and studies had documented that the development of resistance emerge mainly during treatment [ 11 , 12 ] . the susceptibility to trimethoprim / sulfamethoxazole is slightly lower compared to 97.5% in the another local study on 80 burkholderia pseudomallei . only 4.0% of our isolates was resistant to trimethoprim / sulfamethoxazole compared to 13% resistance rate in thailand . an open label study in thailand had shown that the combination of chloramphenicol , tmp - smx , and doxycycline was associated with higher adverse effect compared to tmp - smx and doxycycline only . the antibiotic susceptibility rate of ciprofloxacin was low at 42.3% , and 90% of the isolates were inhibited at the intermediate mic value of 2 g / ml , which implied that ciprofloxacin was less effective towards these strains . fluoroquinolones have been shown to be less effective clinically [ 15 , 16 ] . a study on the activity of tigecycline against burkholderia pseudomallei in thailand showed low mic50 and mic90 ( 0.5 g / ml and 2.0 g / ml ) . in contrast , the isolates tested in this study were less susceptible to tigecycline , where higher mic50 and mic90 values ( 0.75 g / ml and 4.0 g / ml ) were observed . the susceptibility of tigecycline was only 64.7% , and 34.7% of the strains were inhibited at the intermediate range of mic of 3.06.0 g / ml . this is in concordance with another study in australia , where 85.5% of the isolates were inhibited at an intermediate mic of 4.0 g / ml with lower susceptibility rate of 14.5% . the isolate that was intermediately resistant to imipenem with mic 6 g / ml , also had co - resistance to amoxicillin / clavulanic acid and ampicillin / sulbactam . this isolate was susceptible to meropenem at mic 1.5 g / ml . the mic50 and mic90 of meropenem were noted to be higher than those in imipenem ; however , meropenem is still effective against all the strains tested . in malaysia , meropenem or imipenem has been used for severe infection or in the event of treatment failure with ceftazidime . all the multiply resistant strains were still susceptible to ceftazidime but some of these strains were resistant to the antibiotics used in the maintenance therapy . , the burkholderia pseudomallei isolates from malaysia were highly susceptible to the antibiotics used in the treatment of melioidosis namely ceftazidime , trimethoprim / sulfamethoxazole , imipenem , meropenem , amoxicillin - clavulanate , and doxycycline . the presence of isolates that were resistant to the antibiotics used for maintenance therapy is of concern because this could affect the treatment outcome and may lead to the relapse of infection .

acute melioidosis may present as localised or septicaemic infections and can be fatal if left untreated . burkholderia pseudomallei resistant to antibiotics used for the treatment of melioidosis had been reported . the aim of this study was to determine the in vitro antibiotic susceptibility patterns of burkholderia pseudomallei isolated in malaysia to a panel of antibiotics used for the treatment of melioidosis and also to potential alternative antibiotics such as tigecycline , ampicillin / sulbactam , and piperacillin / tazobactam . a total of 170 burkholderia pseudomallei isolates were subjected to minimum inhibitory concentration determination using e - test method to eleven antibiotics . all isolates were sensitive to meropenem and piperacillin / tazobactam . for ceftazidime , imipenem , amoxicillin / clavulanic acid , and doxycycline resistance was observed in 1 isolate ( 0.6% ) for each of the antibiotics . trimethoprim / sulfamethoxazole resistance was observed in 17 ( 10% ) isolates . for other antibiotics , ampicillin / sulbactam , chloramphenicol , tigecycline , and ciprofloxacin resistance were observed in 1 ( 0.6% ) , 6 ( 3.5% ) , 60 ( 35.3% ) and 98 ( 57.7% ) isolates respectively . one isolate b170/06 exhibited resistance to 4 antibiotics , namely , ciprofloxacin , chloramphenicol , trimethoprim / sulfamethoxazole , and tigecycline . in conclusion , the malaysian isolates were highly susceptible to the current antibiotics used in the treatment of melioidosis in malaysia . multiple resistances to the antibiotics used in the maintenance therapy are the cause for a concern .

both embryonic and adult stem cells are sources of undifferentiated cells , with the ability to repair and regenerate tissue . some of the stem cell types that can be used to improve infarcted heart are as follows : embryonic , cardiac , cardiomyocyte progenitor cells , hematopoietic cells , bone marrow mesenchymal stem cells , skeletal myoblast , fetal and umbilical cord blood cells.[913 ] several routes can be used to deliver such cells to the human myocardium or to the coronary circulation . these routes include intravenous infusion , direct injection into the ventricular wall , or transepicardial / transendocardial infusions . however , questions relating to dose , timing , and route of administration need to be addressed . escs hold promise for myocardial cellular therapy , but the clinical use of these cells raises ethical issues and political controversies . the ethical problems specific to esc research , however , can be avoided by reprogramming somatic cells to produce induced pluripotent stem cells . takahashi and yamanaka demonstrated the induction of pluripotent stem cells from mouse embryonic or adult fibroblasts by introducing four factors ( oct3/4 , sox2 , c - myc , and klf4 ) . the induced pluripotent stem cells showed the morphology and growth properties of esc and expressed esc marker genes . more recently , zhang et al . have shown that human - induced pluripotent stem cells can differentiate into functional cardiomyocytes and these cells may be suitable for therapeutic application . the results showed that human escs have the ability to differentiate into myocytes with the structural and functional properties of cardiomyocytes . the preclinical studies have shown that esc transplant results in improvement in cardiac structure and function.[1820 ] caspi et al . evaluated the ability of human escs and their cardiomyocyte derivatives to engraft and improve myocardial performance in the rat chronic infarction model . the results of this study showed the formation of stable cardiomyocyte grafts , attenuation of the remodeling process , and functional benefit , highlighting their potential for myocardial cell therapy strategies . currently , adult stem cells are the only stem cell types that can be used for transplant to treat diseases like mi . these unspecialized cells are found in within a tissue or organ and have the ability to develop into specialized cells . the purpose of these cells is to primarily maintain and repair the tissue in which they are found . the adult human heart contains small populations of indigenous committed cardiac stem cells or multipotent cardiac progenitor cells . the markers used to identify cardiac stem cells and their biologic functions in human are ssea-1 , oct-3/4 , isl-1 , c - kit ( cd117 , scfr ) , sca-1 ( ly 6 ) , mdr-1 ( abcb1 , pgp ) , abcg2 ( mxr1 , bcrp ) , cd133 ( prominin ) , cd90 ( thy-1 ) , cd105 ( endoglin ) , cd34 , cd31(pecam-1 ) , and cd45 ( lca ) . to date , at least five apparently different cardiac stem and/or progenitor cell types have been described . in addition , the adult bone marrow stem cells consist of hematopoietic stem cells , endothelial progenitor cells , and mesenchymal stem cells . genetically engineered rat mesenchymal stem cells using ex vivo retroviral transduction and these stem cells repaired infarcted myocardium . recent study showed that granulocyte colony - stimulating factor promoted bone marrow cells to migrate into the infarcted heart and differentiate into cardiomyocytes ; however , proportional contribution of cells from bone marrow was small when compared with non - bone marrow in the infarction model . zhang et al . identified a single non - hematopoietic mesenchymal stem cell subpopulation isolated from human bone marrow , clonally purified , and compared the effects with unpurified mesenchymal stem cell , mononuclear cells , or peripheral blood mononuclear cells on myocardial repair after induction of mi in rats . they observed that transplantation with single clonally purified mesenchymal stem cells was more beneficial to the cardiac repair when compared with other stem cells . recent in vivo studies indicate that engrafted bone marrow cells survive and grow within the spared myocardium after infarction by forming junctional complexes with resident myocytes . these cells generate de novo myocardium composed of integrated cardiomyocytes and coronary vessels . previously , we tried to find out if bone marrow - derived stem cells from patients can differentiate into cardiomyocyte precursors when cocultured with atrial tissue condition medium , and we observed that these cells resembled smooth muscles probably cardiomyocytes . more recently , a randomized , double - blind , placebo - controlled , dose - escalation study ( reperfused mi patients [ n = 53 ] ) showed that intravenous allogeneic human mesenchymal stem cells are safe in patients after mi . studies have also shown that adipose tissue - derived cells have the ability to give rise to functional cardiomyocyte - like cells . adipose tissue contains a population of adult multipotent mesenchymal stem cells and endothelial progenitor cells with extensive proliferative capacity . these cells have the potential to differentiate into several lineages including cardiomyocytes , suggesting their potential as cell source for repairing the damaged tissues . more recently , bai et al . showed that both freshly isolated human adipose tissue - derived cells and cultured human adipose tissue - derived stem cells engrafted into infarcted myocardium survived and improved myocardial function . the results suggest that both cells are promising alternative source for myocardial repair post - mi . to explore the clinical application of human stem cells , kim et al . transplanted human unrestricted somatic stem cells into porcine hearts post - mi . four weeks after transplantation , the engrafted cells were detected in the infarct region , resulting in improved regional and global function of the porcine heart , suggesting positive potential for their implantation for cellular cardiomyoplasty . these cells have a potential for cell - based therapy to treat acute mi by contributing to the repair processes post - mi , thus providing a useful therapeutic option . wu et al . investigated the therapeutic potential of human umbilical cord - derived stem cells in a rat mi model . more recently , schlechta et al . have shown that umbilical cord blood - derived hematopoietic precursor cells can be reproducibly expanded ex vivo and retain their potential to improve cardiac function post - mi . escs hold promise for myocardial cellular therapy , but the clinical use of these cells raises ethical issues and political controversies . the ethical problems specific to esc research , however , can be avoided by reprogramming somatic cells to produce induced pluripotent stem cells . takahashi and yamanaka demonstrated the induction of pluripotent stem cells from mouse embryonic or adult fibroblasts by introducing four factors ( oct3/4 , sox2 , c - myc , and klf4 ) . the induced pluripotent stem cells showed the morphology and growth properties of esc and expressed esc marker genes . more recently , zhang et al . have shown that human - induced pluripotent stem cells can differentiate into functional cardiomyocytes and these cells may be suitable for therapeutic application . the results showed that human escs have the ability to differentiate into myocytes with the structural and functional properties of cardiomyocytes . the preclinical studies have shown that esc transplant results in improvement in cardiac structure and function.[1820 ] caspi et al . evaluated the ability of human escs and their cardiomyocyte derivatives to engraft and improve myocardial performance in the rat chronic infarction model . the results of this study showed the formation of stable cardiomyocyte grafts , attenuation of the remodeling process , and functional benefit , highlighting their potential for myocardial cell therapy strategies . currently , adult stem cells are the only stem cell types that can be used for transplant to treat diseases like mi . these unspecialized cells are found in within a tissue or organ and have the ability to develop into specialized cells . the purpose of these cells is to primarily maintain and repair the tissue in which they are found . the adult human heart contains small populations of indigenous committed cardiac stem cells or multipotent cardiac progenitor cells . the markers used to identify cardiac stem cells and their biologic functions in human are ssea-1 , oct-3/4 , isl-1 , c - kit ( cd117 , scfr ) , sca-1 ( ly 6 ) , mdr-1 ( abcb1 , pgp ) , abcg2 ( mxr1 , bcrp ) , cd133 ( prominin ) , cd90 ( thy-1 ) , cd105 ( endoglin ) , cd34 , cd31(pecam-1 ) , and cd45 ( lca ) . to date , at least five apparently different cardiac stem and/or progenitor cell types have been described . in addition , the adult bone marrow stem cells consist of hematopoietic stem cells , endothelial progenitor cells , and mesenchymal stem cells . genetically engineered rat mesenchymal stem cells using ex vivo retroviral transduction and these stem cells repaired infarcted myocardium . recent study showed that granulocyte colony - stimulating factor promoted bone marrow cells to migrate into the infarcted heart and differentiate into cardiomyocytes ; however , proportional contribution of cells from bone marrow was small when compared with non - bone marrow in the infarction model . zhang et al . identified a single non - hematopoietic mesenchymal stem cell subpopulation isolated from human bone marrow , clonally purified , and compared the effects with unpurified mesenchymal stem cell , mononuclear cells , or peripheral blood mononuclear cells on myocardial repair after induction of mi in rats . they observed that transplantation with single clonally purified mesenchymal stem cells was more beneficial to the cardiac repair when compared with other stem cells . recent in vivo studies indicate that engrafted bone marrow cells survive and grow within the spared myocardium after infarction by forming junctional complexes with resident myocytes . previously , we tried to find out if bone marrow - derived stem cells from patients can differentiate into cardiomyocyte precursors when cocultured with atrial tissue condition medium , and we observed that these cells resembled smooth muscles probably cardiomyocytes . more recently , a randomized , double - blind , placebo - controlled , dose - escalation study ( reperfused mi patients [ n = 53 ] ) showed that intravenous allogeneic human mesenchymal stem cells are safe in patients after mi . studies have also shown that adipose tissue - derived cells have the ability to give rise to functional cardiomyocyte - like cells . adipose tissue contains a population of adult multipotent mesenchymal stem cells and endothelial progenitor cells with extensive proliferative capacity . these cells have the potential to differentiate into several lineages including cardiomyocytes , suggesting their potential as cell source for repairing the damaged tissues . more recently , bai et al . showed that both freshly isolated human adipose tissue - derived cells and cultured human adipose tissue - derived stem cells engrafted into infarcted myocardium survived and improved myocardial function . the results suggest that both cells are promising alternative source for myocardial repair post - mi . to explore the clinical application of human stem cells , kim et al . transplanted human unrestricted somatic stem cells into porcine hearts post - mi . four weeks after transplantation , the engrafted cells were detected in the infarct region , resulting in improved regional and global function of the porcine heart , suggesting positive potential for their implantation for cellular cardiomyoplasty . these cells have a potential for cell - based therapy to treat acute mi by contributing to the repair processes post - mi , thus providing a useful therapeutic option . wu et al . investigated the therapeutic potential of human umbilical cord - derived stem cells in a rat mi model . they showed that transplanted cells provide benefit in cardiac function recovery after acute mi . more recently , schlechta et al . have shown that umbilical cord blood - derived hematopoietic precursor cells can be reproducibly expanded ex vivo and retain their potential to improve cardiac function post - mi . the best cell type as a source for transplantation into the ischemic heart remains a controversy because of lack of well - defined cell markers . studies need to address the source and the best types of stem cells for clinical applications [ table 1 ] . gene therapy is an interesting option , but the use of viral vectors for gene delivery is a concern due to safety issues . both in vitro and in vivo studies performed in animal models have demonstrated that the transplanted cells undergo differentiation in an attempt to repair damaged myocardium . previous studies in the animal models have suggested that esc transplantation post - mi is beneficial . in rats , escs differentiated into cardiomyocytes and improved cardiac function . singla et al . have concluded that transplanted mouse escs have the ability to regenerate infarcted myocardium and improve cardiac structure and function . have examined the fate and functional impact of bone marrow cells and esc - derived cardiomyocytes after transplantation into the infarcted mouse heart . the results have shown that esc - based therapy is a promising approach for the treatment of impaired myocardial function ; this therapy also provides better results when compared with the bone marrow - derived cells . have genetically engineered rat mesenchymal stem cells that have the ability to repair infarcted myocardium to nearly normal cardiac performance . a new therapeutic strategy for cardiac tissue regeneration cultured adipose tissue - derived mesenchymal stem cells and transplanted the monolayered cells onto the scarred myocardium that improved cardiac function in rats . the results from the clinical studies have demonstrated a good safety profile with improved cardiac function , and favorable effects in patients with mi.[4449 ] the randomized controlled clinical trial ( 60 patients ; 6-month follow - up ) has shown that intracoronary transfer of autologous bone - marrow cells promoted improvement of left - ventricular systolic function in mi patients . the trial ( 59 patients with acute mi ) has demonstrated that intracoronary infusion of progenitor cells ( circulating progenitor or bone marrow - derived progenitor cells ) is safe and feasible in patients after acute mi successfully revascularized by stent implantation . in a prospective , nonrandomized , open - label study with five heart transplant candidates with severe ischemic heart failure , the autologous bone - marrow mononuclear cell injections were performed safely and resulted in improved exercise capacity ( 6-month follow - up evaluation ) . in the cell - randomized clinical trial ( 27 patients ) , granulocyte colony - stimulating factor therapy with intracoronary infusion of peripheral blood stem cells have shown improved cardiac function , and promoted angiogenesis in mi patients . i / ii study have shown that intracoronary administration of enriched bone marrow cd133 + cells in patients with acute mi ( 19/35 patients ) although feasible is associated with increased incidence of coronary events . this process involves implantation of bone marrow stem cells into the infarcted area of heart in an effort to restore the viability of the tissue . stamm et al . have injected autologous ac133 bone marrow cells into the infarct area of patients with mi and restored tissue viability . a clinical trial in patients with old mi and ischemic coronary artery disease ( n = 12 ) has shown that treatment with skeletal myoblast in conjunction with coronary artery bypass is safe and feasible . a randomized controlled trial ( 69 patients with acute mi ) has shown that transplantation of bone marrow mesenchymal stem cells might improve cardiac function and is safe and feasible with no deaths or malignant arrhythmias . in another clinical trial , have shown that intracoronary transplantation of mesenchymal stem cells and endothelial progenitors is feasible , safe , and may also contribute to regional regeneration of myocardial tissue early or late following mi . in a phase i clinical study have shown that cd133 -enriched stem cell transplantation improved cardiac function in all patients . li et al . have shown that autologous peripheral blood stem cell transplantation by intracoronary infusion in patients with acute a phase i / iia double - blind , randomized , placebo - controlled , dose - escalating trial of intramyocardial injection of autologous cd34 cells in patients with intractable angina ( n = 24 ) provided evidence for feasibility , safety , and bioactivity . furthermore , a larger phase iib study is underway to evaluate this therapy . in the doppler substudy of the randomized , double - blind , placebo - controlled reinfusion of enriched progenitor cells and infarct remodeling in acute myocardial infarction trial , microvascular function of the infarct - related artery was restored after intracoronary transplantation of bone marrow progenitor cells in patients with reperfused acute mi . in an open - labeled prospective clinical trial , choi et al . have shown that intracoronary transplantation of autologous peripheral blood stem cells ( mobilized by granulocyte colony - stimulating factor ) was safe in patients who underwent percutaneous coronary intervention and improved myocardial function , but could not significantly improve left ventricular function compared with standard reperfusion treatment . balogh et al . transplanted autologous bone marrow - derived cd34 stem cells in patients with mi , and these cells i clinical study has shown cd133 -enriched stem cell transplantation into ischemic myocardium without coronary artery bypass grafting that was feasible , safe , and improved cardiac function in all patients . more recently , in a prospective , randomized study ( 60 patients ) , gyngysi et al . have compared the safety and feasibility of early and late delivery of bone marrow mononuclear stem cells after mi with combined delivery approach . they have shown that this approach induces a moderate but significant improvement in myocardial infarct size and left ventricular function . have evaluated evidence from randomized control trials on the effectiveness of adult bone marrow - derived stem cells to treat acute mi ( 13 rcts ; 811 participants ) . the results of this systematic review have suggested that there is little evidence to assess the clinical effects of this treatment . have investigated the efficacy of autologous bone marrow - derived mesenchymal stem cells in improving the heart function in patients ( n = 8) with old mi . they have shown that this transplantation is safe , feasible , and that the cells improved the cardiac function without serious adverse effects . have shown that the autologous mononuclear bone marrow cell transplantation ( 60 patients ) provided sustained improvement in global left ventricular systolic function in patients with acute mi . more recently , in a phase i clinical trial , krause et al . have shown that ( 12-month follow - up ) left ventricular electromechanical mapping and percutaneous intramyocardial cell injection in patients with acute mi was a safe procedure . the paradigm that the adult heart is a postmitotic organ was established in the 1970s . later , this concept was changed because of the growing body of evidence challenging this traditional view . in adults , myocardium can regenerate from the cardiac stem cells that enter the cell cycle , contributing to changes in the myocardial mass . a low level of cardiomyocyte renewal may occur throughout life , which may be in response to cardiac stem cell activity or migration of stem cells from distant tissues . such repair following mi is grossly inadequate to compensate for the severe loss of functional cardiomyocytes . existing therapies lower the early mortality rates , prevent additional damage to the heart muscle , and reduce the risk of further heart attacks . however , there is need for a treatment to improve the clinical conditions by replacing the damaged heart cells . thus , the cardiac tissue regeneration with the application of stem cells may be an effective therapeutic option . the regeneration of cardiomyocytes ( heart muscle cells ) to improve postinfarcted heart with the application of stem cells has been broadly studied . recently , interest has been concentrated on myocardial regeneration with the application of stem cells . studies have shown that both embryonic and adult stem cells have the ability to repair and regenerate tissue , and in turn , these cell types can be used in the cardiomyocyte regeneration . however , there are major hurdles for the therapeutic use of adult stem cells which include harvesting and expansion of cells in vitro , advanced methods to obtain desired cell types , optimization of route of administration of stem cells , and specific induction of heart tissue regeneration . escs hold promise for myocardial cellular therapy , but the clinical uses of these cells raise ethical issues and political controversies . the ethical issues specific to esc research can be avoided by reprogramming somatic cells to produce induced pluripotent stem cells . in vitro human escs give rise to cardiomyocytes ; however , the regenerative capacity of undifferentiated human escs after in vivo engraftment needs to be established . the major hurdle for the clinical application of escs is the formation of teratoma by undifferentiated cells , as they may not be directed to form new myocardium after transplantation . although escs have potential to repair the damaged tissue , the use of escs carries a threat for neoplastic transformation due to inherent risk for unguided differentiation . the malignant tumorigenic potential of escs needs to be defined , and the risk of teratoma formation has to be eliminated before esc - based therapy is considered . guided cardiopoiesis uses the definitive engagement of stem cells to generate cardiac tissue avoiding teratomas . behfar et al . demonstrated that use of cardiopoietic cells eliminates reliance on host heart signaling for differentiation . these cells delivered into infarcted hearts generated cardiomyocytes integrating with host myocardium for tumor - free repair . other challenges include the need for preparations of high cardiac purity , improved delivery methods , and overcome immune rejection and graft cell death . research has been focused on understanding how stem cells target injured tissue . a review by smart and riley focuses on existing insights into the trafficking of stem cells in the context of cardiac regenerative therapy . in addition , the transplanted stem cells into injured tissue express paracrine signaling factors ( cytokines , chemokines , and growth factors ) involved in the process of stem cell - driven repair . future studies need to address the mechanistic basis for stem cell - mediated paracrine enhancement . in conclusion , stem cell transplantation appears to be a safe and effective option for treating the postinfarcted heart . to date , there are several preclinical studies that have demonstrated the potential of stem cell - based therapy in the treatment of mi . these clinical studies have demonstrated a good safety profile , improved cardiac function , and favorable effects in patients with mi . the results obtained from animal studies are promising , and the data obtained from the human clinical trials are even more encouraging .

permanent loss of cardiomyocytes and scar tissue formation after myocardial infarction ( mi ) results in an irreversible damage to the cardiac function . cardiac repair ( replacement , restoration , and regeneration ) is , therefore , essential to restore function of the heart following mi . existing therapies lower early mortality rates , prevent additional damage to the heart muscle , and reduce the risk of further heart attacks . however , there is need for treatment to improve the infarcted area by replacing the damaged cells after mi . thus , the cardiac tissue regeneration with the application of stem cells may be an effective therapeutic option . recently , interest is more inclined toward myocardial regeneration with the application of stem cells . however , the potential benefits and the ability to improve cardiac function with the stem cell - based therapy need to be further addressed . in this review , we focus on the clinical applications of stem cells in the cardiac repair .

a 50-year - old female patient was referred to our department of oral medicine and radiodiagnosis with a complaint of a painless swelling over the right mandibular region since 3 months . intraoral examination revealed a swelling in relation to the right mandibular posterior region extending from the region of 45 to 48 posteriorly and left mandibular posterior region extending from 35 to 38 [ figures 1 and 2 ] . on palpation , a panoramic radiograph was taken to view the apparently isolated lesion on the mandibular right posterior region . the radiolucency observed in the region of right mandibular posteriors was multilocular with sclerotic borders extending posteriorly up to the anterior border of the ramus with the superior wall scalloping between the roots of 44 and 47 region . in addition to the suspected multilocular radiolucency , orthopantomographs revealed bountiful lesions ; multiple radiolucencies were noted in the anterior maxilla and lower left body of the mandible [ figure 3 ] . computed tomography ( ct ) scan revealed the bilateral involvement of the posterior aspect of mandible [ figure 4 ] and anterior aspect of maxilla [ figure 5 ] showing bicortical expansion in relation to right and left posterior mandible . a clinical diagnosis of the traumatic bone cyst with differential diagnosis of central giant cell granuloma and odontogenic keratocyst was rendered . intraoral examination revealing swelling in the right mandibular posterior region intraoral examination revealing swelling in the left mandibular posterior region orthopantomographs showing multiple radiolucencies computed tomography image showing mandibular lesion computed tomography image showing maxillary lesion patient was offered a choice of surgical exploration and biopsy . curettage of the cavities was done , and the obtained connective tissue material was sent for histopathological examination , which revealed densely inflamed connective tissue stroma with extravasated red blood cells , necrotic changes , and chronic inflammatory cells . surgical exploration of the lesion in right mandible surgical exploration of the lesion in left mandible surgical exploration of the lesion in maxilla patient made an unremarkable recovery after surgery . follow - up radiographs after a period of 6 months showed 90% of bone regeneration [ figure 9 ] . comparing our data with the literature review , an interesting finding of sbcs in an older age group that were not associated with cement - osseous dysplasia is rare , such variation in the clinical presentation of sbcs may be relevant to the better understanding of this underexplored lesion . these lesions showed radiographic characteristics that suggested differential diagnoses of ameloblastoma , odontogenic keratocyst tumor or a central giant cell lesion in view of their location . due to these radiographic characteristics , ct imaging is important for an accurate diagnosis , as this may avoid unnecessarily aggressive surgical approaches , like en bloc resections of the mandible . found that most recurrences were seen within 5 months of the surgical treatment , and this group recommended that follow - up should be continued until healing was confirmed radiographically within 3 years . in our sample , evidence of new bone formation after surgery was noticed within 3 months of the surgery and complete healing was noticed radiographically . the most common radiographic finding in multifocal sbcs was scalloping , consistent with what is traditionally described in single lesions . bony expansion and multilocular radiolucent patterns were observed . in general , sbcs have a tendency to grow along the long axis of the bone , causing minimal expansion ; however , expansion of the involved bone can occur and is more common with larger lesions . in general , the mandibular body is by far the most common location for sbcs , with the maxilla representing an uncommon site . one explanation for this lower prevalence in the maxilla could be related to the possibility that maxillary lesions are obscured by the overall radiolucent appearance imparted by the maxillary sinus . another possible explanation could be due to differences in the quality and amount of bone marrow and vascularity in the maxilla compared to the mandible . in this case , we noted that multiple sbcs in the mandibular body , as well as maxilla . curettage of the bone walls is the most widely accepted approach to the management of sbcs of the jaws , the goal being to promote the formation of a blood clot and subsequent bone repair . in addition , application of gelfoam , grafting of allogenic bone with platelet - rich plasma and intralesional injections of a mixture of blood , porous hydroxyapatite , and bone fragments have been reported to produce good results . in general , all maxillofacial lesions present with a standard set pattern of clinical or radiographic presentations . however , this case defies the regular standards set for a traumatic bone cyst . proper recording of case history , knowledge of symptomatology , and correct radiographic interpretation will be helpful for proper diagnosis and management .

simple bone cyst is an oft - described entity , which goes by many sobriquets and has been presented in the literature as early as 1926 . it is a lesion of unclear etiology with many proposed hypothesis for its pathogenesis and nonspecific histopathological presentation . we present a case of a solitary bone cyst , which clinically presented as a solitary lesion , but radiological examination revealed multiple lesions peppering the maxilla and the mandible , thereby highlighting the importance of atypical presentation and surprise lesions that may show up on radiological examination .

persons with epilepsy , even those with well - controlled seizures , may face emotional distress , low self - esteem , reduced social interactions , decreased job opportunities , and problems with daily activities . a growing body of evidence implicates the socioeconomic status as a risk factor for epilepsy in adults . the improvement in the quality of life of patients with epilepsy is now widely accepted as a highly important therapeutic goal . quality of life has been defined by the world health organization as a reference to a person 's well - being and the individual 's perception of their position in life . however , studies on adults with epilepsy in china and abroad seldom focus on the effect of occupational status on the patients quality of life . this study attempted to explore the effects of occupational status on the quality of life . it aimed to provide an objective basis for the possible relationship between occupational status and the quality of life of patients with epilepsy . a total of 11 professional epilepsy centers or neurological clinics were randomly selected at beijing . the inclusion criteria were as follows : ( 1 ) diagnosis of epilepsy based on the 2014 international league against epilepsy for more than 1 year , ( 2 ) age 1865 years , ( 3 ) educational attainment higher than primary school , and ( 4 ) signed informed consent . meanwhile , the exclusion criteria were as follows : ( 1 ) progression of central nervous system ( cns ) disease or acute stage and other severe chronic cns diseases ; ( 2 ) disturbance of consciousness , cognitive impairment , and language dysfunction ; ( 3 ) seizure occurrence of more than once within 72 h of screening ; ( 5 ) history of schizophrenia ; ( 6 ) history of cranial surgery ; and ( 7 ) history of drug abuse . the term occupation is defined by webster 's new international dictionary as follows : fixed occupation : fixed occupation refers to work with stable compensation , complete insurance provisions , and several government benefitsfree occupation : a person with a free occupation works independently of any organization . some individuals engage in free occupations as their long - term careerspeasantry : this occupation refers to the long - term involvement in agricultural production ( i.e. , occupations of nomads , fishermen , and farmers)student ( 18 years old ) : students receive university education and are not completely integrated into the workforceworker : manual and mental workers are individuals employed in enterprises , institutions , and organizations , who receive wages as their main source of income . fixed occupation : fixed occupation refers to work with stable compensation , complete insurance provisions , and several government benefits free occupation : a person with a free occupation works independently of any organization . some individuals engage in free occupations as their long - term careers peasantry : this occupation refers to the long - term involvement in agricultural production ( i.e. , occupations of nomads , fishermen , and farmers ) student ( 18 years old ) : students receive university education and are not completely integrated into the workforce worker : manual and mental workers are individuals employed in enterprises , institutions , and organizations , who receive wages as their main source of income . on the basis of the inclusion and exclusion criteria , qualified investigators surveyed 819 adults with epilepsy diagnosed in the neurology departments of 11 public hospitals in beijing . the quality of life in epilepsy-31 ( qolie-31 ) is a health - related quality of life survey for adults ( > 18 years ) with epilepsy . derived from the qolie-89 , this scale contains domains that include seven subscales : overall quality of life , seizure worry , emotional well - being , cognitive function , energy / fatigue , medication effects , and social function . the responses can yield seven individual scores ( per subtest ) and a total ( composite ) score . the raw scores are then rescaled from 0 to 100 , with higher values indicating better qolie-31 scores . the survey was conducted by trained researchers on adult patients with epilepsy ; the questionnaire contained questions on patient - related information and the qolie-31 scale ( chinese version ) . data were inputted using epidata 3.0 ( the epidata association , odense denmark , 2003 ) and analyzed using spss 17.0 ( ibm , chicago , il , usa ) . the two sample means were compared using a t - test ( or rank - sum test ) . three sets of measurement data were compared using analysis of variance ( anova , or rank - sum test ) , and multiple comparisons were conducted using bonferroni correction . a value of p 0.05 was considered statistically significant . according to a previous study conducted by de boer , a simple sampling method was used in this study . assuming a similar rate for the employed patients with epilepsy , a minimum of 460 patients should be recruited to achieve a two - tailed 95% significance level and 15% loss rate of a questionnaire . data were inputted using epidata 3.0 ( the epidata association , odense denmark , 2003 ) and analyzed using spss 17.0 ( ibm , chicago , il , usa ) . the two sample means were compared using a t - test ( or rank - sum test ) . three sets of measurement data were compared using analysis of variance ( anova , or rank - sum test ) , and multiple comparisons were conducted using bonferroni correction . a value of p 0.05 was considered statistically significant . according to a previous study conducted by de boer , a simple sampling method was used in this study . assuming a similar rate for the employed patients with epilepsy , a minimum of 460 patients should be recruited to achieve a two - tailed 95% significance level and 15% loss rate of a questionnaire . on the basis of the inclusion and exclusion criteria , 819 adult patients with epilepsy were enrolled in the present study . among these subjects , 513 were males ( 62.14% ) , whereas 306 were females ( 37.86% ) . a total of 546 individuals ( 77.28% ) held educational attainments of high school diploma and higher . meanwhile , 353 subjects ( 43.10% ) received an average household income of 5000 per month over the past year . a total of 726 subjects ( 88.64% ) received specialist medical treatment , and 603 patients ( 73.62% ) experienced seizures over the past year . the average age at which the patients suffered their first seizure episode was 19.49 13.04 years ( 163 years ) . the number of employed subjects with high school and/or higher education ( 79.85% ) were greater than the number of unemployed subjects ( 20.15% ) . furthermore , the majority of patients with income 5000 per month were employed ( 85.92% ) , and the difference between the employed and unemployed patients was statistically significant ( p < 0.05 ) . no significant differences in gender , age , and marital status were observed between the employed and unemployed subjects ( p > 0.05 ) . the age at a first seizure episode was higher in the patients with occupation ( 20.49 13.16 years ) than in those without occupation ( 16.98 12.41 years ) . the duration of disease was also significantly shorter in the employed ( 12.96 12.49 years ) than in the unemployed patients ( 15.99 12.06 years ) ( p < 0.01 ) . in contrast , no significant difference in the occupational status was observed between the patients who experienced seizure attacks over the year and those who did not , as well as those with varying degrees of compliance ( p > 0.05 ) [ table 1 ] . comparison of demographic and disease data between the employed and unemployed adult patients with epilepsy ( n = 819 ) qolie-31 : quality of life in epilepsy-31 ; sd : standard deviation . the scores of the employed adult patients with epilepsy on overall quality of life , energy / fatigue , emotional well - being , seizure worry , cognition , social function , medication effects , and qolie-31 were higher than the scores of the unemployed patients [ table 2 ] . comparison of the quality of life between employed and unemployed adult patients with epilepsy values are expressed as the mean standard deviation . * with statistically significant difference between employed and unemployed patients ; p<0.01 . qolie-31 : quality of life in epilepsy-31 . on comparing the differences in scores between employed and unemployed patients for each factor on the quality of life , the highest discrepancy was noted in social function , followed by cognition and energy / fatigue . the lowest difference was observed in the overall quality of life [ table 3 ] . the adult patients with epilepsy were classified into two groups on the basis of the occupational type as follows : fixed occupation ( 466 patients ) and freelance occupation ( 70 patients ) . no significant disparity in the overall quality of life and related factors was found between the patients in fixed and freelance occupations ( p > 0.05 ) [ table 4 ] . the adult patients with epilepsy in occupations as student and worker attained significantly higher qolie-31 scores ( 68.04 14.01 and 63.95 14.52 , respectively ) than those as a peasant ( 56.26 14.04 ) ( p < 0.05 ) [ table 5 ] . table 6 shows that the patients with occupations as student and peasant significantly differed in qolie-31 score , cognition , emotional well - being , overall quality of life , social function , and presence of energy / fatigue . in contrast , the patients with the three occupations showed no significant difference in seizure worry and medication effects [ table 6 ] . differences in quality of life and related factors between employed and unemployed adult patients with epilepsy ci : confidence interval ; qolie-31 : quality of life in epilepsy-31 . comparison of the overall quality of life and related factors between adult patients with epilepsy in fixed and freelance occupations values are expressed as the mean standard deviation . qolie-31 : quality of life in epilepsy-31 . comparison of the overall quality of life and related factors between patients with epilepsy in different occupations values are expressed as the mean standard deviation . qolie-31 : quality of life in epilepsy-31 . diversity factor analysis on the variation in the qolie-31 score of the patients with epilepsy in different special occupations qolie-31 : quality of life in epilepsy-31 ; ci : confidence interval . occupational status might reflect income , social status , and social prestige ; hence , it is a highly important factor related to socioeconomic status . the group found that lack of education , single marital status , divorce or deceased spouse , increasing age , and low per capita income can reduce the quality of life in patients with epilepsy . people with epilepsy can suffer from difficulties in social interactions , which , though not always readily apparent , may significantly impact their social function and ability to form quality social relationships and networks . socioeconomic status and occupation the present results showed no significant difference in gender , age , marital status , seizure occurrence for the past year , and degrees of medication adherence ( p > 0.05 ) among patients in different occupations . in contrast , patients in different occupations significantly differed in level of education , income , type of diagnosis , treatment , age at first onset , and disease course ( p < 0.05 ) . different kinds of occupations receive different incomes and consequently , afford different treatments , resulting in the disparity in the quality of life . employed adult patients with epilepsy scored higher in qolie-31 , overall quality of life , energy / fatigue , emotional well - being , seizure worry , cognition , social function , and medication effects than the unemployed patients . low socioeconomic status is associated with risk factors such as infection and poor nutrition , thereby worsening the quality of life . however , the effect of employment varies for each factor and the overall quality of life . through analysis , the difference between the employed and unemployed patients was found to be higher in social function , cognition , and energy / fatigue than in the other factors . social functioning , however , is a key aspect in the quality of life and it is frequently referenced that social connections and networks in both professional work and personal relationships are the single most important factor and predictor of well - being , happiness , and life satisfaction . some studies showed that higher educational levels are related to better scores on the social function subscale . patients with epilepsy might feel unsafe , uncomfortable , anxious , and disappointed , which can cause problems for them in relation to their peers and reduce their social relations , which is a problem for many persons with epilepsy . employment can provide satisfaction and communication opportunities , which can significantly improve social function , individual cognition , and energy . by participating in special employment programs , people with epilepsy can be guided toward the labor market , thus improving the quality of their lives . hence , adult patients with epilepsy should be actively encouraged to seek job opportunities and not dwell on anxiety , fear , and inferiority . as previously discussed , no difference in the quality of life was noted in the patients with fixed and freelance occupations . freelance occupations are relatively new compared with traditional jobs , but most freelance occupations resemble fixed occupations in many aspects such as salary , social status , and educational background . therefore , adult patients with epilepsy might still attain positive benefits from employment , regardless of whether fixed or freelance work is involved . three different kinds of occupations were assessed in terms of their effect on the quality of life of adult patients with epilepsy . the three different groups belong to different social classes and present varying economic statuses and education levels . according to the analysis , economic status and educational level were the two largest independent risk factors that affected the qolie-31 score of the adult patients with epilepsy . in a previous study , economic status was found to obviously influence the qolie-89 total and subscale scores ; higher economic statuses were noted to correlate with higher qolie-89 total scores . in the present study , the students and farmers differed in the qolie-31 scores , emotional well - being , overall quality of life , social function , and maximum energy . in contrast , the two groups did not significantly differ in seizure worry and drug compliance . this finding might be attributed to the lack of involvement of other occupations , which might have affected the results . in this study , the quality of life of adult patients with epilepsy was probably found to be associated with occupation . unemployment and long - term sick leave might lead to loss of social connections and isolation , causing depression and anxiety . psychic and emotional distress may decrease the probability of returning to work , causing a vicious circle . the overall quality of life of employed patients is higher than that of unemployed patients . meanwhile , the influence of different kinds of occupations varies among different factors related to the quality of life . however , the present study was a cross - sectional study with no follow - up for the quality of life of chinese adult patients with epilepsy . adult patients with epilepsy should actively seek a job to concurrently achieve a significant enhancement in their quality of life .

background : epilepsy is one of the most common serious neurological disorders . the present study aimed to investigate the influence of occupational status on the quality of life of chinese adult patients with epilepsy.methods:this study surveyed 819 subjects clinically diagnosed with epilepsy for more than 1 year in 11 hospitals in beijing ; 586 were employed ( 71.55% ) . all subjects completed the case report form with inquiries on demographic data , social factors , and illness . the patients quality of life was assessed using the quality of life in patients with epilepsy-31 items ( qolie-31 ) questionnaire.results:the qolie-31 score in the employed group was significantly higher than that in the unemployed group . furthermore , the scores in all the sections ( overall quality of life , energy / fatigue , emotional well - being , seizure worry , cognition , social function , and medication effects ) of the employed group were higher than those of the unemployed group . both the employed and unemployed groups achieved the highest difference in social function . the qolie-31 score of students was higher than those of farmers and workers . both the students and workers scored higher in the quality of life compared with the adult peasants living with epilepsy . the students and farmers showed significant differences in qolie-31 score , cognition , emotional well - being , overall quality of life , energy / fatigue , and social function . in contrast , no significant difference was noted in seizure worry and medication effects across the three different kinds of occupation.conclusion:occupational status might affect the quality of life of chinese adult patients with epilepsy , and social function is the most important contributing factor .

this changeable virus is responsible for several pandemic events such as 1947 , 1976 , 1977 , and 2009 pandemics . the novel h1n1 virus was detected in 2009 pandemic that was reported in mexico first and then spreading around the world . formal statistics reports explained that 3,672 known cases of influenza were confirmed from june to december 30 , 2009 . most of patients presented mild symptoms and their disease was self - limited but some of them presented with severe complications including deaths . during the epidemic distribution of infection diseases such as influenza , health care workers are responsible for delivering good quality management and treatment . their knowledge and correct behavior can play an important role in disease spreading among individuals , and also protecting them from illness . thus , medical and dental students especially residents education about preventive strategies , effective treatment and follow - up is critical , as well as their actions and behaviors in these fields . medical and dental residents and fellowships usually donot have any longtime experiences and therefore they may have a greater risk comparison to other health care workers . proper knowledge , attitude , and practices are crucial to prevent and control the disease , particularly among residents who has a greater risk of infection . the current study reports the knowledge , attitudes and practices ( kap ) toward the pandemic h1n1 among the medical and dental residents and fellowships of shiraz university of medical sciences . also , we associated some demographic factors with residents knowledge , attitudes and practices , to determine the probable predictors . this study was a cross - sectional survey conducted from september through october 2009 , within shiraz school medicine in iran . shiraz university of medical sciences is ranked as one of the best medical universities in iran . it is the home for 13 hospitals , which makes it a regional health care service and the reference medical center in south half of iran . the self - administered questionnaire was offered to 125 residents and fellowships regarding influenza sign and symptoms , preventive and treatment efforts , as well as precautions activities . annually,153 medical residents and 21 fellowships are accepted to studying in shirazuniversity of medicalsciences as post graduate students in different majors and faculties . every year , in shiraz university of medical sciences , education development center ( edc ) invite all of the new medical and dental residents and fellowships to teach them several key points about their educational role toward medical students . all of the participants in the edc programs were included in our study applying convenient sampling . after coordinating with edc master , the researchers went to the meetings and waited to gather the completed questionnaires . the researches described participants about the importance of their participation and request them to fill the questionnaires completely and carefully . the questionnaire had two components : a guideline to filling the questionnaire for participants and their demographic factors ; and knowledge , attitudes , and practices questions about pandemic swine influenza disease . some expert professors in influenza field and some expert professors in kap study were consult to improve the validity of the questionnaire . a pilot study was performed on 50 participants in order to establish the reliability of the questionnaire using cronbach 's alpha test that was 0.83 for knowledge item , 0.74 for attitudes item and 0.66 for practices item . demographic factors including age , gender , marital status , educational faculty ( medicine , dental ) , and educational grade ( resident , fellowship ) were asked . one close question on the necessity of educational programs from participants point of view was designed ( yes / no ) . they were allowed to choose answers correct , incorrect , and do not know . the score value of 1 was allocated to answers that were agreed to who and cdc guidelines . all other responses were give 0 score value . in 7 questions among 35 knowledge questions , twelve questions were assessed participants attitudes . according to the subject importance from participant 's perspective the responses were made on a 5 point likert scale ( very high importance , high importance , moderate importance , low importance , doesnot have importance ) . therefore , participants score for each question ranged 1 - 5 , and the attitudes score for each participant ranged in between 12 - 60 . the participants score for each question ranged in between 1 - 5 and the total score of practice questions ranged 12 - 60 . finally , one question was asked about the participants knowledge resource that they could choose more than one selection among conferences and pamphlets , specialized internet resources , unspecialized internet resources , scholarly journals , public journals , books , media , and other resources . independent sample t - test was used to determine the significant differences in the means of knowledge , attitudes , and practices according to demographic factors . to compute the correlation between knowledge and attitudes , knowledge and practices , attitudes and practice , as well as age with knowledge , attitudes , and practices we analyzed data using spearman correlation coefficient statistic method . finally to determine the significant predictor factors of the level of knowledge , attitudes , and practices we used multivariate linear regression and multiple multivariate analyses . this study was a cross - sectional survey conducted from september through october 2009 , within shiraz school medicine in iran . shiraz university of medical sciences is ranked as one of the best medical universities in iran . it is the home for 13 hospitals , which makes it a regional health care service and the reference medical center in south half of iran . the self - administered questionnaire was offered to 125 residents and fellowships regarding influenza sign and symptoms , preventive and treatment efforts , as well as precautions activities . annually,153 medical residents and 21 fellowships are accepted to studying in shirazuniversity of medicalsciences as post graduate students in different majors and faculties . every year , in shiraz university of medical sciences , education development center ( edc ) invite all of the new medical and dental residents and fellowships to teach them several key points about their educational role toward medical students . all of the participants in the edc programs were included in our study applying convenient sampling . after coordinating with edc master , the researchers went to the meetings and waited to gather the completed questionnaires . the researches described participants about the importance of their participation and request them to fill the questionnaires completely and carefully . the questionnaire had two components : a guideline to filling the questionnaire for participants and their demographic factors ; and knowledge , attitudes , and practices questions about pandemic swine influenza disease . some expert professors in influenza field and some expert professors in kap study were consult to improve the validity of the questionnaire . a pilot study was performed on 50 participants in order to establish the reliability of the questionnaire using cronbach 's alpha test that was 0.83 for knowledge item , 0.74 for attitudes item and 0.66 for practices item . demographic factors including age , gender , marital status , educational faculty ( medicine , dental ) , and educational grade ( resident , fellowship ) were asked . one close question on the necessity of educational programs from participants point of view was designed ( yes / no ) . they were allowed to choose answers correct , incorrect , and do not know . the score value of 1 was allocated to answers that were agreed to who and cdc guidelines . all other responses were give 0 score value . in 7 questions among 35 knowledge questions , twelve questions were assessed participants attitudes . according to the subject importance from participant 's perspective the responses were made on a 5 point likert scale ( very high importance , high importance , moderate importance , low importance , doesnot have importance ) . two reverse phrases were asked to prevent from halo effects . therefore , participants score for each question ranged 1 - 5 , and the attitudes score for each participant ranged in between 12 - 60 . the participants score for each question ranged in between 1 - 5 and the total score of practice questions ranged 12 - 60 . finally , one question was asked about the participants knowledge resource that they could choose more than one selection among conferences and pamphlets , specialized internet resources , unspecialized internet resources , scholarly journals , public journals , books , media , and other resources . independent sample t - test was used to determine the significant differences in the means of knowledge , attitudes , and practices according to demographic factors . to compute the correlation between knowledge and attitudes , knowledge and practices , attitudes and practice , as well as age with knowledge , attitudes , and practices we analyzed data using spearman correlation coefficient statistic method . finally to determine the significant predictor factors of the level of knowledge , attitudes , and practices we used multivariate linear regression and multiple multivariate analyses . the response rate of this survey was very high ; about 98%.the mean age of the overall responders was 30.62(5.17 ) years . approximately , 40.8% of participants were female and54% of participants were married . among responders , 79.7%were physician , 20.3% were dentist also 81.3% of all population was medical residents . ninety five percent of them believed that educational course about influenza is necessary and they should pass it to improve their information . the mean score of knowledge , attitude and practices did not differ significantly among males and females . forty five percentof participants used conferences and pamphlets for improving their knowledge about influenza infection , 7.1% used specialized internet resources , and 12.4% unspecialized internet resources . approximately,16.8% , 31.9% , 27.4% used scholarly journals , public journals , and books , respectively . student t - test analysis didnot show any statistical significant differences among several demographic categories ; except knowledge scores that were greater in married than single participants , physicians than dentists and fellowships than residents[table 1 ] . calculating the correlation showed the presence of significant linear positive correlation between knowledge and practice items ( correlation coefficient : 0.45 , p value < 0.001 ) and attitude with practice ( correlation coefficient : 0.50 , p value < 0.001 ) . analysis the correlation between age and participant 's knowledge , attitude and practices showed significant positive linear correlation between age and knowledge and behavior scores ( knowledge correlation coefficient : 0.49 , p value < 0.001 , practice correlation coefficient : 0.352 , p value 0.002 ) . tables 2 , 3 and 4 show the frequency of participants responses to knowledge , attitude and practice questions , respectively . table 5 shows multivariate linear regression and multiple multivariate analyses of the kap scores in relation to several independent variables . the educational major , age , and sex were significant predictors for responders knowledge score in multivariate linear regression model . age was the only significant predictor for both attitude and practice scores and aging was associated with getting better scores in these items . after applying the multiple multivariate analysis , age and sex was the main predictors of knowledge , attitude and practices . mean of knowledge , attitude and practice scores towards pandemic influenza a ( h1n1 ) in different groups the frequency of responses of participants to knowledge questions regarding pandemic influenza a the frequency of responses of participants to attitude questions regarding pandemic influenza a the frequency of responses of participants to practice questions regarding pandemic influenza a multivariate linear regression and multiple multivariate analyses of knowledge , attitudes and practice towards pandemic influenza a the health care workers immediate and appropriate responses have a crucial role to control h1n1 influenza pandemic . in iran , medical residents and fellowships are important health care providers because they visit patients immediately after hospitalization and often manage patients in major referral hospitals . dentists have an important role by their contacts with several patients and their essential roles in disease transmission trends . medical staffs and students one study in thailand also showed the high level of scores in these items regarding influenza h1n1 among medical students . data analysis showed that knowledge was influenced by educational major and degree that physicians had better awareness than dentists , in addition fellowships also aware more . this pattern is expected because medical students and fellowships spend more courses about infectious diseases than dental students and medical residents , respectively . the attitude and knowledge scores were not different among several groups , so the high knowledge is not sufficient alone for improving attitudes and practices . using motivational educating models can be helpful to convert individual 's knowledge to correct attitudes and behaviors , subsequently . analyzing the correlation between age and knowledge , attitude and practice showed that younger students had low experiences , little awareness , less study knowledge about the current medical subjects . there was a significant correlation between knowledge and attitude and also attitude and practice in this study . another study among health care workers showed that knowledge correlated significantly with attitude and practices . thus , we should emphasize on medical and also dental students and motivate male students to improve their knowledge about pandemic flu diseases . younger students and physicians are important target groups for advanced educational programs about infection diseases . most of participants in this study reported media as their references for their information about influenza disease . this study has some limitations , such as the convenient sampling method and self - reported questionnaires that may cause several biases including recall and social desirability bias . it would be helpful to policy makers if further researches for providing the effective educational and motivational methods would perform . the residents and fellowships knowledge , attitude and behaviors about the influenza disease are important due to the residents and fellowships role model to general population and also their essential role in distributing the disease to the community . governments should provide advanced and motivational education methods to educate medical and dental residents and fellowships for possible future pandemics

background : influenza disease is one of the oldest medical problems that can cause severe illness and high mortality rates , worldwide . in flu pandemics , medical and dental students knowledge , attitudes , and practices ( kap ) is critical to save patients life . the aim of this study was to determine the score of kap toward the pandemic h1n1 and their predictor factors among the medical and dental residents and fellowships of shiraz university of medical sciences , iran.methods:in 2009 , 125 participants were recruited in a convenient sampling cross - sectional survey . self - reported questionnaire were used and results were analyzed applying appropriate statistical tests.results:the mean score of participants knowledge , attitude and practice were 22.6 , 21.1 and 26.5 respectively . participants practice had significant linear positive correlation with knowledge and attitude . also , their age was significantly and directly correlated to knowledge and practice . the educational major , age , and sex were significant predictors of responder 's knowledge score and age was the only significant predictor of both attitude and practice scores.conclusions:high knowledge is not sufficient lonely for improve attitude and practices . it seems that traditional educational models are not efficient and governments should emphasize to advanced and motivational education methods including health belief model and motivational interview at postgraduate levels . perhaps younger students , dentists and males have less motivation to change their attitude and behavior , so we can focuses our interventions in these groups .

lymphocytic thrombophilic arteritis ( lta ) is a newly described medium vessel arteritis of the skin . it overlaps considerably with polyarteritis nodosa ( pan ) , but lacks systemic features . to the best of our knowledge , a 53-year - old south indian lady presented with a 5-year history of reddish patches over both lower limbs and arms . since past 10 days , they were mildly itchy and painful . the lesions first started on the lower limb as small red patches , which slowly increased in size and healed with residual hyperpigmentation . she applied topical creams for the past 3 - 4 months , without any benefit . there were multiple erythematous patches and hyperpigmented macules associated with livedo racemosa on both legs and arms [ figure 1a and b ] . on palpation , focal areas of mild induration were noted , but no nodules were palpable . her laboratory investigations , which included testing for anti - nuclear antibodies , anti - dsdna , anca , and anti - phospholipid antibodies were negative . the medium - sized arteries in the deep dermis showed thickening of their walls with a hyalinised fibrin ring involving the entire circumference of the vessel [ figure 2a and b ] . nuclear dust was noted in the vessel walls and within the lumina [ figure 2c ] . ( a ) involvement of medium sized arteries in deep dermis ( h and e , 100 ) . note the perivascular and intraluminal lymphocytic infiltrate and nuclear dust ( h and e , 200 ) the patient was treated with dapsone , 100 mg / day for 3 months , and doxycycline , 100 mg / day for 1 month . about 80% of the lesions cleared in 4 months . cutaneous pan ( cpan ) is characterised by necrotising arteritis presenting as tender nodules or livedo reticularis . systemic manifestions like myalgia , neuropathy , hypertension , and weight loss are noted occasionally , but viscera are spared . in 2003 , the term macular arteritis was first used to describe a form of medium vessel vasculitis in african american patients presenting as hyperpigmented macules , without the classic features such as palpable purpura or erythematous and tender nodules . all these patients had longstanding ( 4 months to 15 years ) hyperpigmented lesions , some of them with a reticular pattern and no palpable nodules . histologically , they were all characterised by fibrinoid necrosis of medium - sized arteries with lymphocytic infiltrates , leading to endarteritis obliterans . all these patients had an indolent clinical course , with no demonstrable improvement despite treatment . in 2008 , lee and kossard used the term lta to describe a similar vasculitis in 5 young women with a characteristic intraluminal hyalinised fibrin ring and lymphocytic infiltrates with a scarcity of neutrophils . clinically , these patients had progressive pigmentation , livedo racemosa , and palpable mild induration . they felt that lta is a better suited term than macular arteritis , since the lesions are not just macular and also to underline the associated thrombophilic status . the lesions are asymptomatic and have a predilection for lower extremities , which is usually the first to be involved , and they slowly progress to involve the upper limbs , although to a lesser extent . the most important differential diagnoses to be considered are anti - phospholipid syndrome ( aps ) and cutaneous pan ( cpan ) . the characteristic clinicopathological features of lta , cpan , and aps are summarized in table 1 . differential diagnosis of lta ( adapted from lee et al . and gupta et al . ) some patients of lta show increased levels of anti - phospholipid antibodies , but did not satisfy the clinical or laboratory criteria ; the significance of this finding is unknown . it characteristically presents as painful subcutaneous nodules , associated with mild constitutional symptoms . while many authors believe that cpan is a distinct entity , several others view it just as an evolutionary stage in classic pan . the first , or acute stage , shows neutrophilic infiltration of vessels at the dermis . there is no fibrinoid necrosis or disruption of the internal elastic lamina ( iel ) . this stage shares a lot of microscopic similarities with lta and has led to the idea that lta is just a form of cpan . while there is certainly a histologic overlap , the iel is stated to be intact in macular arteritis , in contrast to cpan at a similar stage . our case showed a preserved iel . in the third and fourth ( healing ) stages , cpan shows obliteration of the lumen , fibroblastic reaction , and proliferation of small vessels in the perivascular area . despite lesions being present for 5 years in our patient , there were no signs of healing and the biopsy , with its fibrin ring , seems to represent an active lesion . in order to see the characteristic microscopic features of cpan , one needs to biopsy the nodular / active areas and , often , multiple step sections are necessary . in contrast , the biopsy site was randomly chosen in our patient and the changes were easily identified in the initial section . the presence of concentric layers of elastic fibres among the smooth muscle bundles characterizes a vein and elastic stains should be employed in all cases with vascular pathology of the medium calibre vessels . , we did not find any granulomatous inflammation and the clinical presentation was incompatible with that of tuberculosis ; this might be just a chance concurrence . in all the cases described so far , the course appears indolent without any systemic signs . lta ( or macular arteritis ) is an idiopathic , indolent form of cutaneous medium vessel arteritis without a systemic component and a characteristic hyaline fibrin ring encircling the lumen . while there are microscopic differences from cpan , the small numbers of cases reported so far do not amount to conclusive testimony . it is important to recognize these features to accumulate evidence to support its candidature as a distinct vasculitis or consider it as a morphologic pattern in the spectrum of pan . despite the striking histopathological features the bottom - line is that patients with such a hyalinised fibrinring need to be screened for occult , potentially serious systemic causes of vasculitis .

a 55-year - old woman presented with a 5-year history of livedo racemosa on her limbs . histology showed vasculitis of medium - sized arteries with a circumferential , hyalinised , intraluminal fibrin ring . her laboratory investigations did not indicate any underlying systemic disease . the findings were consistent with lymphocytic thrombophilic arteritis ( lta ) , alias macular arteritis , which is a recently described entity . the importance of lta lies in the fact that it is a close clinical and microscopic mimic of polyarteritis nodosa ( pan ) . lta is believed to be a distinct entity by some and as a form of pan by others . we have discussed this case in our report .

it is an important element to inspire thoughts out - of - the - box , or , more practically , to find new ways of organizing and correcting problems otherwise not considered . at a time when there is a debate of reforming the portuguese health - care system , the canadian system , though different , may provide answers to some of the questions that are being asked . this article is based on a report on a 1-month observership in family medicine that took place in march 2013 in edmonton , alberta , canada . every canadian citizen is assigned a personal health number ( phn ) which guarantees free access to health care at any health institution in canada . on the first approach , this may prove to be difficult regarding organizing healthcare resources ; however , patients usually choose one doctor as their family physician , thus guaranteeing the continuity of care . most physicians accept only patients without a family doctor and will not accept patients with physicians already . it rests on the physician to decide whether or not he is willing to accept new patients , giving him freedom of choice regarding the number of patients , he is willing to see and follow ( usually between 1500 and 2000 patients ) . this decision might not only be settled at individual level but also at organizational level as is it possible for a clinic to refuse new patients usually if their panel is full . furthermore , physicians are also allowed to refuse to treat a patient further in case the patient - physician relationship and interaction are compromised . besides regular patients , there are also the walk - ins , which are patients who require medical examination due to an acute situation . here , again , the physician may choose not to see walk - in patients or the clinic is allowed to state that it does not accept any walk - ins . for this matter , there are specified walk - in clinics that center their care on acute conditions and usually have easier accessibility due to longer opening hours . many clinics have specified walk - in times for their patients on top of booked appointments . as for the articulation of primary care and secondary care , it is quite well established in canada . usually , primary care physicians reference patients to a specialist by letter , which is registered in the computer system . the waiting time to get an appointment varies from 1 to 6 months ; however , in case it 's urgent , the primary care physician may also phone the specialist and book an appointment for the very same day . the whole reference process happens at individual level , that is , the primary care physician references a patient to a specialist , individually . therefore , it is also possible for the patients to choose the specialist they wish to go to . in case it is required , the primary care physician may also phone the specialist and discuss a patient 's case with him or her . in case it 's urgent , the specialist on call at the hospital can always be contacted by phone for advice . all physicians usually work at the hospital or at a clinic ( a building where physicians may rent an office to practice in ) . for the latter , it is required that physicians cover the rent and any expenses they may have with personnel , such as nurses and secretaries . medical services are always covered by the provincial government , so the patient is not required to pay for the visits . in case of an acute onset of symptoms , patients may go to their family physician , to the walk - in clinics , or directly to the hospital emergency room . at the hospital , patients are triaged by a nurse and if wait times are long and the patient is triaged low , they may choose to book an appointment with his family physician instead . describing the activity of the family physician , their approach is family - centered , rather than at individual level , as the patients physicians accept are members of a family . risk groups ( children , pregnant women , diabetic patients , etc . ) are seen in a special type of appointment throughout the day . other types of special appointments include the general physical examination , which consists in a yearly evaluation , from head to toe , of the patient 's overall health . during this visit , it is normal practice to review the patient 's personal history and habits to keep track of any change that may have occurred . in case , the patient suffers from a common chronic condition ( e.g. : hypertension , diabetes , chronic obstructive pulmonary disease , asthma , etc . ) it includes the problem list , lifestyle issues , the current medication , the involvement of other health care professionals , the advised scheduling of medical services throughout the year , end of life discussion , and the goals set by the patient and the physician . it is reviewed on a yearly basis and is expected to help the patient get a better grasp of his condition and manage medical visits accordingly . patients are advised to book their appointments according to their condition and their motive , always having the possibility of booking an appointment on the same day in case of an acute condition . home visits are optional , but most family doctors do not perform them . if the clinic has the necessary conditions , family physicians are also able to perform deliveries , small surgeries ( e.g. circumcisions , vasectomies , cyst removals , ingrown toenail incisions ) , biopsies ( includes endometrial biopsies ) , casts , use of liquid nitrogen , insert intrauterine devices , aspirations , and joint injections . with special training , family physicians are allowed other procedures , such as performing c - sections or anesthesia . family physicians also have the option to become involved in hospital care and stay responsible for a ward of 2025 patients . every major hospital has family medicine wards that admit patients with different types of pathologies such as hip fracture , delirium , sepsis , congestive heart failure , etc . , the family doctor is responsible for these patients , having to check on them on a daily basis and if required , ask for the collaboration from another specialist . , family physicians can do emergency work as well without extra training . in the larger centers , family physicians require 1 extra year of residency training in emergency room medicine and are almost considered equivalent to a specialist emergency room physician . regarding medication , it is sold in units and physicians may , in one prescription , prescribe chronic medication that lasts for a year . as patients are advised to pick up their medication at the same pharmacy , physicians may call up the pharmacy at any point and discuss the patient 's medication with the pharmacist . what 's more , pharmacists may also renovate prescriptions ( except for narcotics ) , which , consequently , make many patients not to go and see the doctor . nurses at the primary care clinics assist the doctors and have other duties , such as giving injections , ear wash , wart treatment , filling cognitive assessment forms , take away casts , review chronic disease management plans , collect the history in newborns , and obtain the ankle brachial index . the canadian health - care service is fundamentally public ; however , clinics are owned by a physician . physicians get their income according to a fee - for - service model , that is , the physician bills the government after seeing a patient . afterward , the physician has to pay about 30% of their income to the physician who owns the clinic , to cover staff salaries , rent , etc . , income taxes cover 38% of a physician 's income , and physicians have to pay their insurance fee . physicians are considered self - employed ; therefore , professionals do not have a right to pension or to sickness leave . there are also some salaried positions in the public system ( alternate relationship plan ) , but they are mainly reserved for academic physicians to promote research and teaching . as for working hours , this individual modeling could prove an obstacle for the management of the clinic ; however , there is a record of each doctor 's activity plan together with the number of patients he or she is currently seeing and the patients the physician should be seeing . in some provinces , there are initiatives to promote primary health creating structures such as the primary care network ( pcn ) . a pcn may include one clinic with several physicians and support staff or many physicians in various clinics across a health region . each network works toward developing programs and providing services to best meet the needs of local patients . in a pcn , patients have access to multiple services such as geriatrics long - term , women 's health clinic , shared care maternity , pediatric asthma , cast clinic , and after - hours clinic . overall , pcns aim to provide better services and to engage the community in activities to promote the role of physicians within primary care . regarding continued medical education , it is required that every doctor submits 250 mainpro ( maintenance of proficiency ) credits every 5 years - one credit is equal to 1 h of learning . these credits are authorized by the college of family physicians of canada , and they can be gained in any scientific activity participated besides practice , i.e. research , teaching , courses , reading journals , certified podcasts , group sessions , congress attendance , master 's degree , etc . every portuguese citizen has a health care number which allows them to have access to health care , after paying a token duty . the latter does not apply in certain situations , such as patients that demonstrate financial hardship ( it is mandatory to declare one 's income to the state ) as well as their dependents , pregnant women and women in labor , children until the age of twelve , patients with an inability level of 60% or higher , transplant patients , military and former military personnel who have been permanently incapacitated due to their military service , and certain unemployed people . blood , cell , tissue or organ donors , as well as firemen , are exempted of co - payment in case of primary care . demonstrating financial hardship proves a barrier to vulnerable groups such as homeless people as they have no means of proving they have a right to be exempted from the usual co - payment . furthermore , excepting emergency room visits , portuguese citizens may only use health - care resources that are available in their living area . when a family physician starts working at a health - care center , he / she is given a list of about 18002000 patients , which is made out of members of various families . the physician is expected to follow these patients throughout life , occasionally accepting a new family member by request from the family . thus , there is a very little variation in the patients the physician usually sees . however , this assures the continuity of care , aids the physician to get more familiar with the patients , and to establish a closer patient doctor relationship . in case doctor relationship is compromised , the physician may ask another physician , of the same health care unit , to see and follow a patient . because of the list system , there is a considerable amount of citizens that do not have access to primary care , let alone a family physician . as the portuguese health - care system is a mix between a public and a private system , citizens with higher incomes however , patients have to pay out - of - pocket for the full amount of that visit as they are not covered by the state . some public health care subsystems have an agreement with some doctors and partially cover for the visits , but only specific groups of the population ( such as police , firemen , military , and state workers ) have access to them . for those with fewer resources , access to health care equals , for the most part , paying a visit to the emergency room at the nearest hospital . outside the hospital , there are some centers that offer a public health care service ( sap ) , but they only cover matters of an acute onset . there has been an effort to cover the primary care health requirements of citizens that are not included in a family physician 's list . this is being accomplished by asking some family physicians to follow vulnerable risk groups , such as children and pregnant women , in addition to their usual clinical activity . although not enough , this measure slightly helps to alleviate the difficult access to family physicians . on the other hand , matters are quite different for citizens that have a family physician . in a health care practice , the physician 's schedule is organized by the type of visit and it includes allocated times for normal visits , urgencies , addressing risk groups ( children , pregnant women , family planning , diabetes , and hypertension ) , and home visits . a visit usually lasts for 1015 min and the patient may book a visit according to his current need . there are also appointments that do not require the presence of the patient and thus have a lower token duty than the one to pay for a regular visit . they include the renovation of a prescription , prescribing exams , a telephone contact , or filling out any other type of document that does not require the patient to be in the room . the longest a medicine prescription lasts is 6 months , and patients need to go to their physician to get a refill . when prescribing , physicians may only prescribe a whole package of pills , which causes patients to store many packages at home . it is not customary for pharmacies to keep track of a patient 's medication ; therefore , there is no dialog with the physician on the patient 's medication . when it comes to risk groups , all visits start with an evaluation by the nurse , followed by the visit to the physician . nurses play an important role in primary care as , besides giving injections and doing wound treatments , they are responsible for the teaching and giving advice to risk groups . they also perform home visits and in case they consider it to be necessary , may request the physician to reassess a patient . in some health - care centers , nurses may become a family nurse and work in partnership with one family physician . this means that a family nurse only follows the patients that are included in the physician 's list . family physicians do not usually perform any surgical or invasive activities unless they underwent a specific training in that area . this is the case for very few physicians hence when required , most physicians reference patients to the local hospital through a computer program . this application is the main means of referral between primary and secondary care as patients need to be referred through it , to have an appointment at a public hospital . when making a referral , the family physician chooses the required specialty instead of an individual doctor , and he may only refer to the local hospital . a patient is only transferred to a different hospital in case the local one considers it does not have the necessary means to provide the best care to the patient . apart from this application , the communication between primary and secondary care is scarce and difficult to establish . reports from the hospital specialists have to be requested by special notice and most of the time ; patients have to carry the notices themselves . work is being done to establish a national platform on the internet for this purpose , but it is not yet fully functional . a family physician 's schedule is made out of a weekly 40 h labor journey . family physicians only work on weekdays and do not have to make night shifts . like any other physician , family physicians are state workers and are paid a fixed salary at the end of each month , having the right for pension or sickness leave . the percentage of the salary that has to be paid to the state varies with the income of every physician . every canadian citizen is assigned a personal health number ( phn ) which guarantees free access to health care at any health institution in canada . on the first approach , this may prove to be difficult regarding organizing healthcare resources ; however , patients usually choose one doctor as their family physician , thus guaranteeing the continuity of care . most physicians accept only patients without a family doctor and will not accept patients with physicians already . it rests on the physician to decide whether or not he is willing to accept new patients , giving him freedom of choice regarding the number of patients , he is willing to see and follow ( usually between 1500 and 2000 patients ) . this decision might not only be settled at individual level but also at organizational level as is it possible for a clinic to refuse new patients usually if their panel is full . furthermore , physicians are also allowed to refuse to treat a patient further in case the patient - physician relationship and interaction are compromised . besides regular patients , there are also the walk - ins , which are patients who require medical examination due to an acute situation . here , again , the physician may choose not to see walk - in patients or the clinic is allowed to state that it does not accept any walk - ins . for this matter , there are specified walk - in clinics that center their care on acute conditions and usually have easier accessibility due to longer opening hours . many clinics have specified walk - in times for their patients on top of booked appointments . as for the articulation of primary care and secondary care , it is quite well established in canada . usually , primary care physicians reference patients to a specialist by letter , which is registered in the computer system . the waiting time to get an appointment varies from 1 to 6 months ; however , in case it 's urgent , the primary care physician may also phone the specialist and book an appointment for the very same day . the whole reference process happens at individual level , that is , the primary care physician references a patient to a specialist , individually . therefore , it is also possible for the patients to choose the specialist they wish to go to . in case it is required , the primary care physician may also phone the specialist and discuss a patient 's case with him or her . in case it 's urgent , the specialist on call at the hospital can always be contacted by phone for advice . all physicians usually work at the hospital or at a clinic ( a building where physicians may rent an office to practice in ) . for the latter , it is required that physicians cover the rent and any expenses they may have with personnel , such as nurses and secretaries . medical services are always covered by the provincial government , so the patient is not required to pay for the visits . in case of an acute onset of symptoms , patients may go to their family physician , to the walk - in clinics , or directly to the hospital emergency room . at the hospital , patients are triaged by a nurse and if wait times are long and the patient is triaged low , they may choose to book an appointment with his family physician instead . describing the activity of the family physician , visits their approach is family - centered , rather than at individual level , as the patients physicians accept are members of a family . risk groups ( children , pregnant women , diabetic patients , etc . ) are seen in a special type of appointment throughout the day . other types of special appointments include the general physical examination , which consists in a yearly evaluation , from head to toe , of the patient 's overall health . during this visit , it is normal practice to review the patient 's personal history and habits to keep track of any change that may have occurred . in case , the patient suffers from a common chronic condition ( e.g. : hypertension , diabetes , chronic obstructive pulmonary disease , asthma , etc . ) it includes the problem list , lifestyle issues , the current medication , the involvement of other health care professionals , the advised scheduling of medical services throughout the year , end of life discussion , and the goals set by the patient and the physician . it is reviewed on a yearly basis and is expected to help the patient get a better grasp of his condition and manage medical visits accordingly . patients are advised to book their appointments according to their condition and their motive , always having the possibility of booking an appointment on the same day in case of an acute condition . home visits are optional , but most family doctors do not perform them . if the clinic has the necessary conditions , family physicians are also able to perform deliveries , small surgeries ( e.g. circumcisions , vasectomies , cyst removals , ingrown toenail incisions ) , biopsies ( includes endometrial biopsies ) , casts , use of liquid nitrogen , insert intrauterine devices , aspirations , and joint injections . with special training , family physicians family physicians also have the option to become involved in hospital care and stay responsible for a ward of 2025 patients . every major hospital has family medicine wards that admit patients with different types of pathologies such as hip fracture , delirium , sepsis , congestive heart failure , etc . , the family doctor is responsible for these patients , having to check on them on a daily basis and if required , ask for the collaboration from another specialist . , family physicians can do emergency work as well without extra training . in the larger centers , family physicians require 1 extra year of residency training in emergency room medicine and are almost considered equivalent to a specialist emergency room physician . regarding medication , it is sold in units and physicians may , in one prescription , prescribe chronic medication that lasts for a year . as patients are advised to pick up their medication at the same pharmacy , physicians may call up the pharmacy at any point and discuss the patient 's medication with the pharmacist . what 's more , pharmacists may also renovate prescriptions ( except for narcotics ) , which , consequently , make many patients not to go and see the doctor . nurses at the primary care clinics assist the doctors and have other duties , such as giving injections , ear wash , wart treatment , filling cognitive assessment forms , take away casts , review chronic disease management plans , collect the history in newborns , and obtain the ankle brachial index . the canadian health - care service is fundamentally public ; however , clinics are owned by a physician . physicians get their income according to a fee - for - service model , that is , the physician bills the government after seeing a patient . afterward , the physician has to pay about 30% of their income to the physician who owns the clinic , to cover staff salaries , rent , etc . , income taxes cover 38% of a physician 's income , and physicians have to pay their insurance fee . physicians are considered self - employed ; therefore , professionals do not have a right to pension or to sickness leave . there are also some salaried positions in the public system ( alternate relationship plan ) , but they are mainly reserved for academic physicians to promote research and teaching . as for working hours , family physicians are free to plan their weekly schedule . this individual modeling could prove an obstacle for the management of the clinic ; however , there is a record of each doctor 's activity plan together with the number of patients he or she is currently seeing and the patients the physician should be seeing . in some provinces , there are initiatives to promote primary health creating structures such as the primary care network ( pcn ) . a pcn may include one clinic with several physicians and support staff or many physicians in various clinics across a health region . each network works toward developing programs and providing services to best meet the needs of local patients . in a pcn , patients have access to multiple services such as geriatrics long - term , women 's health clinic , shared care maternity , pediatric asthma , cast clinic , and after - hours clinic . overall , pcns aim to provide better services and to engage the community in activities to promote the role of physicians within primary care . regarding continued medical education , it is required that every doctor submits 250 mainpro ( maintenance of proficiency ) credits every 5 years - one credit is equal to 1 h of learning . these credits are authorized by the college of family physicians of canada , and they can be gained in any scientific activity participated besides practice , i.e. research , teaching , courses , reading journals , certified podcasts , group sessions , congress attendance , master 's degree , etc . every portuguese citizen has a health care number which allows them to have access to health care , after paying a token duty . the latter does not apply in certain situations , such as patients that demonstrate financial hardship ( it is mandatory to declare one 's income to the state ) as well as their dependents , pregnant women and women in labor , children until the age of twelve , patients with an inability level of 60% or higher , transplant patients , military and former military personnel who have been permanently incapacitated due to their military service , and certain unemployed people . blood , cell , tissue or organ donors , as well as firemen , are exempted of co - payment in case of primary care . demonstrating financial hardship proves a barrier to vulnerable groups such as homeless people as they have no means of proving they have a right to be exempted from the usual co - payment . furthermore , excepting emergency room visits , portuguese citizens may only use health - care resources that are available in their living area . when a family physician starts working at a health - care center , he / she is given a list of about 18002000 patients , which is made out of members of various families . the physician is expected to follow these patients throughout life , occasionally accepting a new family member by request from the family . thus , there is a very little variation in the patients the physician usually sees . however , this assures the continuity of care , aids the physician to get more familiar with the patients , and to establish a closer patient doctor relationship . in case doctor relationship is compromised , the physician may ask another physician , of the same health care unit , to see and follow a patient . because of the list system , there is a considerable amount of citizens that do not have access to primary care , let alone a family physician . as the portuguese health - care system is a mix between a public and a private system , citizens with higher incomes opt to see specialists at their offices directly . however , patients have to pay out - of - pocket for the full amount of that visit as they are not covered by the state . some public health care subsystems have an agreement with some doctors and partially cover for the visits , but only specific groups of the population ( such as police , firemen , military , and state workers ) have access to them . for those with fewer resources , access to health care equals , for the most part , paying a visit to the emergency room at the nearest hospital . outside the hospital , there are some centers that offer a public health care service ( sap ) , but they only cover matters of an acute onset . there has been an effort to cover the primary care health requirements of citizens that are not included in a family physician 's list . this is being accomplished by asking some family physicians to follow vulnerable risk groups , such as children and pregnant women , in addition to their usual clinical activity . although not enough , this measure slightly helps to alleviate the difficult access to family physicians . on the other hand , matters are quite different for citizens that have a family physician . in a health care practice , the physician 's schedule is organized by the type of visit and it includes allocated times for normal visits , urgencies , addressing risk groups ( children , pregnant women , family planning , diabetes , and hypertension ) , and home visits . a visit usually lasts for 1015 min and the patient may book a visit according to his current need . there are also appointments that do not require the presence of the patient and thus have a lower token duty than the one to pay for a regular visit . they include the renovation of a prescription , prescribing exams , a telephone contact , or filling out any other type of document that does not require the patient to be in the room . the longest a medicine prescription lasts is 6 months , and patients need to go to their physician to get a refill . when prescribing , physicians may only prescribe a whole package of pills , which causes patients to store many packages at home . it is not customary for pharmacies to keep track of a patient 's medication ; therefore , there is no dialog with the physician on the patient 's medication . when it comes to risk groups , all visits start with an evaluation by the nurse , followed by the visit to the physician . nurses play an important role in primary care as , besides giving injections and doing wound treatments , they are responsible for the teaching and giving advice to risk groups . they also perform home visits and in case they consider it to be necessary , may request the physician to reassess a patient . in some health - care centers , nurses may become a family nurse and work in partnership with one family physician . this means that a family nurse only follows the patients that are included in the physician 's list . family physicians do not usually perform any surgical or invasive activities unless they underwent a specific training in that area . this is the case for very few physicians hence when required , most physicians reference patients to the local hospital through a computer program . this application is the main means of referral between primary and secondary care as patients need to be referred through it , to have an appointment at a public hospital . when making a referral , the family physician chooses the required specialty instead of an individual doctor , and he may only refer to the local hospital . a patient is only transferred to a different hospital in case the local one considers it does not have the necessary means to provide the best care to the patient . apart from this application , the communication between primary and secondary care is scarce and difficult to establish . reports from the hospital specialists have to be requested by special notice and most of the time ; patients have to carry the notices themselves . work is being done to establish a national platform on the internet for this purpose , but it is not yet fully functional . a family physician 's schedule is made out of a weekly 40 h labor journey . family physicians only work on weekdays and do not have to make night shifts . like any other physician , family physicians are state workers and are paid a fixed salary at the end of each month , having the right for pension or sickness leave . the percentage of the salary that has to be paid to the state varies with the income of every physician . regarding the health - care system , accessing health care seems easier in canada than in portugal . thanks to the phn , citizens have free access to and choice of any health care institution or health professional in canada , even across provinces . on the other hand , accessibility is limited in portugal due to the geographic restriction and the co - payment that is requested to have access to any type of health care . when it comes to primary health care , all of these are aggravated by the static list of primary care physicians and the shortage of family doctors . this picture is very concerning as citizens with fewer resources , and thus , more vulnerable , are the ones who have the most difficulty to be granted access to health care . comparing the work of family physicians , the patients , a canadian doctor sees and follows may vary more than in portugal , which could pose a problem for continuity of care . in practice , however , patients tend to stay loyal to one doctor . as for their abilities , when they undergo a special training , they are allowed more complex interventions that could otherwise only be performed by a hospital specialist . besides that , they also have to be on call 24/7 and prepared to follow a patient that is hospitalized , especially if they work in a rural area . however , most family physicians in canada do not do any home visits , which is common practice in portugal . nurses play a more active role in portugal than in canada as they complement the work of family physician . on the other hand , the collaboration with other health professionals ( especially pharmacies ) is more established in canada than in portugal . overall , one may consider the canadian primary health - care system to be very good , and the way it is organized may inspire other countries that are experiencing difficulties in primary healthcare delivery , such as is happening in portugal . observing and experiencing other health - care services provides new insight on organization , planning , and structure of one owns health - care service . it is a highly valuable experience that enriches participants and actively contributes for the development and improvement of primary care . hereby , the author declares that she presents a conflict of interest as she has been involved in both the establishment and management of the family medicine 360 exchange program over the past years . hereby , the author declares that she presents a conflict of interest as she has been involved in both the establishment and management of the family medicine 360 exchange program over the past years .

background : the work of a family physician is quite different in each country , and if we consider different continents , differences are even more remarkable . social and cultural contexts justify a particular organization , not only of the health - care system but also its providers as well.objectives:by analyzing different health - care systems , new ideas may come about which may trigger positive changes in a health - care service to diminish healthcare disparities.methods:description and comparison of the primary healthcare service in canada and portugal.results:although both health - care systems are mainly public , organizational differences can be found that condition primary healthcare access.conclusion:exchanges in other health - care systems contribute for an active knowledge exchange that prompts participants to analyze options on how to improve healthcare access to citizens . this ultimately , leads to the development of primary care , the pillar of a well - functioning health - care system .

direct drug delivery to the neural axis is an effective treatment for primary disorders of the central nervous system ( cns ) , as well as for a growing number of systemic conditions , when other means have failed or resulted in intolerable side effects.1,2 neuromodulation in chronic pain patients traditionally has involved opiate - based drugs ; however , the treatment of certain pain states may be improved with a mixture of opiates and local anesthetics or infused local anesthetics alone . this review discusses the role of intrathecal ( it ) infusion of local anesthetic , specifically bupivacaine , in patients suffering from chronic , intractable head and neck pain . as a group , chronic pain patients may challenge even the most adept physician . due to the high number of chronic pain generators and the disparate emotional and physical responses that are manifest in patients , therapeutic options have evolved to allow for individualized treatments.3,4 to add further to the complexities , significant alterations in neural circuitry lead to changing medication requirements ; this , coupled with ever increasing tolerance and systemic side effects , makes the management of chronic pain quite dynamic . when conventional means of effective pain management fail to treat patients effectively or side effects progress to bar further use , an alternative mode of analgesic delivery should be sought . brain barrier , bypassing systemic metabolism that may yield adverse drug interactions , use of fractionated doses of medication , the ability to achieve constant level of active drug , and immediate access to receptors . august bier first described cocainization of the spinal cord in 1898 and phenol was used in the management of malignant pain in the 1960s.5 cns opioid receptors were found in the early 1970s , and it morphine injections , first in animals6 and then in humans,7 each displayed safety and effective analgesia . while it opioids at first were used primarily to treat patients with cancer - related pain , they are now used in the treatment of a wide range of malignant and nonmalignant causes of primarily nociceptive pain . current us food and drug administration ( fda)-approved drugs for it use in chronic pain patients are morphine sulfate and ziconotide . epidural bupivacaine hydrochloride , a local anesthetic of the amide group , has proved to be an effective epidural analgesic in obstetrical patients as well in those suffering from malignant and nonmalignant spine pain.8 the success of the fda - approved it analgesics has fostered a search for other primary it medications and for other drugs that could augment the effects of morphine via co - administration . combinations of opioids and local anesthetic are now used extensively in both malignant and a growing proportion of nononcogenic pain patients.911 toxicology studies have demonstrated the safety of chronic it bupivacaine infusions in numerous models including the dog , rabbit , and cat.1214 furthermore , a 1989 study by burm displayed only mild neurologic toxicity with it bupivacaine concentrations 100 times higher than those necessary for clinical effect.15 animal studies with local anesthetics and opioids have demonstrated a synergistic potentiation of antinociceptive effects.16 the available safety data with chronic infusion in humans has proven favorable . in addition , postmortem spinal tissue histology in terminal cancer patients did not show neurotoxicity after the administration of chronic it morphine sulfate in combination with bupivacaine.17,18 however , there have been no randomized control studies looking at the effectiveness or safety of it bupivacaine administration . as progress has been made in the use of it opioids with or without local anesthetic for those suffering from low back pain , the search for effective treatment of patients with refractory head and neck pain has proved much more difficult . studies have described the use of both anesthetic and neurosurgical means , including electrical stimulation of both brain and spine,19 c2 - 5 posterior root rhizotomies , c2 cordotomy , stereotactic medial thalamotomy,20 intraventricular morphine,21 and the intracisternal administration of destructive agents22 and analgesics.23 results have been limited secondary to ineffective analgesia and/or unacceptable side effects . high cervical it ( hcit)/intracisternal bupivacaine , however , has a limited but expanding number of single case series reports supporting its effectiveness in treating patients with refractory head and neck pain . in 1994 , crul et al reported on two patients with end - stage oral cancer refractory to conventional pain management.24 each patient had a catheter placed from a lateral c1 - 2 approach into the cisterna magna followed by hcit morphine infusion . in both patients , the it morphine failed to significantly assuage their pain and bupivacaine was added to the solution . the addition allowed the first patient the ability to sleep and significantly reduced the paroxysmal pain as well as the baseline discomfort . the patient was able to be released from the hospital and died 12 days later from her cancer . the second patient also had significantly better pain control after the addition of hcit bupivacaine . in this case , transient increased disequilibrium was eliminated with titration of the bupivacaine dose from 9.6 to 6.6 mg/24 hr . after 6 months , the patient reported near complete pain relief ( visual analog scale [ vas ] , 12 ) as well as undisturbed sleep . baker et al reported on six patients who received hcit bupivacaine with diamorphine clonidine and/or baclofen in a palliative setting for a mean of 67 ( 1387 ) days via an external reservoir system.25 all patients had severe neuropathic pain secondary to tumor invasion , two of which included a trigeminal distribution . in all cases , pain was reduced significantly , allowing for reduction in systematic opioids and/or adjuvant analgesic doses . no major complications were reported ( one patient reported arm weakness / numbness but preferred that to the pain ) , and all side effects were mitigated by changes in medication dosages ( hypotension was reduced by decreasing clonidine infusion ) . the mean dose of bupivacaine in the combination solution was 46 mg/24 hr ( 2075 ) . in 1996 , appelgren et al reported on 13 patients afflicted with head and/or neck pain ( four from nonmalignant causes , nine malignant ) all treated with continuous hcit bupivacaine infusion.26 the same group recently reported an update of their experience spanning from 19902005.27 the 40 neuropathic or mixed pain patients had a median age of 67 ( 2784 ) , 15 were noncancer patients , and the median duration of pain was 1.8 years ( 1 month18 years ) . inclusion criteria included the following : ( 1 ) pain dominating patient s life ; ( 2 ) traditional analgesia had failed ; and ( 3 ) unacceptable side effects . depression , senility , and severe physical conditions were not considered contraindications ; moribund patients and those with overt psychoses were excluded . catheters were placed from an interlaminar approach ( 35 : c7-t4 and 4 : t4 - 9 ) , save one direct occipital catheter . all but three catheter tips were placed into the cisterna magna or at the c1/c2 theca . all catheters , once in final position , were subcutaneously tunneled to the subclavicular space and a 1 ml bolus ( bupivacaine 5 mg / ml ) was given and then connected to a filter and external reservoir . once connected , a bupivacaine infusion ( 5 mg / ml ) was commenced at 0.10.2 mg / hr , using a patient - controlled bolus dose of 0.10.2 mg with a 15-minute lockout , titrated to patient needs . the study reported on 2570 hcit treatment days , 1567 of which occurred at the patient s home . most patients ( 27/40 ) experienced complete pain relief , and partial pain relief was attained in four ( median change in vas was from 10 to 2 ) . improvement in sleep quality was identified in half of the patients , and a majority reported improvement in their ability to ambulate . the mean daily doses ranged from 10 mg to 59 mg amongst the disparate groups ; however , individual differences were quite small . opioid reduction varied significantly , but those individuals who responded to hcit saw significant reductions in systematic analgesics . the most common complication in the series was facial , cervical , or extremity paresthesias . these and other mild side effects could be effectively treated adjusting the infusion rate . two exit - site infections were treated with removal of the catheter , and the chief reason for removal of the system was death . the key to success in any procedure is appropriate patient selection ; this is perhaps the most critical aspect of neuromodulation . this is often done best with a multidisciplinary team that considers the needs of each individual . the patient s immediate support group ( family and other loved ones ) should be involved , not only to assess the appropriateness of the intervention , but to also educate them as to what to expect after the procedure is completed . once there is agreement to move forward , the techniques involved with high cervical catheter placement have been previously described.24,28 it is of paramount importance to localize the catheter tip at the level that corresponds to the patient s pain.29 bupivacaine s local anesthetic effects are quite localized , and if the catheter tip is not to the correct level , the result could be diminished pain relief . beyond the side effects heretofore discussed , there are others reported in the literature related specifically to it bupivacaine administration , including reports of aseptic meningitis.3032 if dosing errors do occur and superfluous drug is introduced intrathecally , cerebrospinal fluid lavage with normal saline or lactated ringer s solution has been successful in rapid elimination of the local anesthetic.33 nearly all other complications can be mitigated or eliminated with titration of it bupivacaine dosing . though not often used , it bupivacaine solutions appear to offer properly selected head and neck pain patients a means of increased pain relief when other options have failed . as more authors report their results , we hope to attain a better understanding of the indications , techniques , and complications related to this treatment modality .

direct central nervous system ( cns ) analgesic delivery is a useful option when more traditional means of dealing with chronic pain fail . solutions containing local anesthetic have been effective in certain disease states , particularly in patients suffering from intractable head and neck pain . this review discusses historical aspects of cns drug delivery and the role of intrathecal bupivacaine - containing solutions in refractory head and neck pain patients .

tryptoline ( tetrahydro--carboline ) and 1,2,3,4-tetrahydroisoquinoline ( thiq ) are substructures of numerous bioactive natural products that contain additional fused rings ( scheme 1 ) . the desire to build such highly substituted polycyclic compounds with complete stereocontrol has inspired the development of numerous synthetic methods . here , we report a redox - neutral annulation strategy that enables the asymmetric synthesis of relevant core structures from simple thiq or tryptoline and readily available , highly enantioenriched 4-nitrobutyraldehydes . as part of our ongoing efforts to develop practical methods for the c h functionalization of amines , we recently reported the first examples of what may be termed a redox - annulation of amines ( scheme 2 ) . conceptually , an amine such as thiq engages an aldehyde with a pendent carbon nucleophile to form product 2 via reductive n - alkylation / oxidative c c bond formation in an overall redox - neutral process . condensation of the two components initially forms an azomethine ylide intermediate 1 that , following proton transfer and ring closure , provides product 2 . for instance , thiq and indole aldehyde 3 form annulation product 4 in 64% yield . while this is a clear demonstration of the utility of this approach for the facile preparation of polycyclic ring systems , the method requires relatively high reaction temperatures . more serious limitations are the need for using nonenolizable aldehydes and electron - rich aromatic nucleophiles ( e.g. , indole , -naphthol ) . in order to be applicable to the synthesis of structures related to those shown in scheme 1 , the redox - annulation would have to proceed with enolizable aldehydes capable of installing a fully saturated ring bearing variable substituents . these considerations , coupled with our goal to perform amine redox - annulations in asymmetric fashion , led to the identification of 4-nitrobutyraldehydes 5 as ideal reaction partners . these precursors are easily prepared in a single step and nearly enantiopure form from aldehydes and nitroalkenes by means of well - established organocatalytic methods . the corresponding annulation products 6 are equipped with substituents in relevant positions and , due to the presence of a nitro group , offer numerous opportunities for further product manipulation . we began our investigation into the feasibility of the proposed annulation process by employing 5a and thiq as model substrates ( table 1 ) . the original annulation conditions developed for product 4 failed to provide any desired product ( entry 1 ) . addition of acetic acid ( 1 equiv ) , an additive previously shown to be an excellent promoter for amine -oxygenation and -sulfenylation , did not provide any improvements ( entry 2 ) . gratifyingly , the use of 5 equiv of acetic acid under otherwise identical conditions allowed for the isolation of the desired product as a 1.8:1 mixture of diastereomers 6a and 7a in 45% overall yield . the formation of 7a is readily rationalized by the intermediacy of enamines , causing epimerization of the stereogenic center in -position of the aldehyde prior to the annulation step . excess acetic acid apparently reduces the concentration of enamine intermediates , facilitating the redox - isomerization and minimizing undesired side reactions . indeed , further increase of the amount of acetic acid to 10 equiv led to an increase in yield to 65% ( entry 4 ) . with 2-ethylhexanoic acid ( 2-eha ) , remarkably , with benzoic acid , previously shown to be an excellent catalyst for amine -functionalizations , no desired product was formed ; only epimerization of the starting material was observed ( entry 6 ) . addition of molecular sieves had a beneficial effect , and the amount of thiq could be lowered to 2 equiv ( entry 7 ) . a reduction in temperature from 150 to 120 c , while reducing the reaction time from 15 to 2 min , led to a further increase in yield to 71% ( entry 8) . a reaction performed under simple reflux conditions was completed in only 1 h and gave rise to nearly identical results ( entry 9 ) . even at a temperature of only 60 c , the reaction progressed in an efficient manner ( entry 10 ) . for the sake of convenience and to keep reaction times brief , subsequent experiments were performed under reflux in toluene . reactions were performed with 5a ( 0.2 mmol ) and thiq ( 4 equiv ) . partially epimerized starting material was recovered ( dr = 1:1 ) . performed with 5a ( 0.6 mmol ) and 2 equiv of thiq in the presence of 4 ms . the scope of the asymmetric redox - annulation of thiq was explored with a range of 4-nitrobutyraldehydes ( scheme 3 ) . ,-disubstituted 4-nitrobutyraldehydes 5 with various substitution patterns readily underwent the title reaction , providing products 6/7 in moderate to good yields and with diastereoselectivities of up to 5:1 . notably , in all cases , separation of the two diastereomeric products was readily accomplished by standard column chromatography . 4-nitrobutyraldehydes without -substituents gave rise to products 6 with good to excellent levels of diastereoselectivity . importantly , the reaction of mono- and disubstituted 4-nitrobutyraldehydes was also applicable to substituted thiqs and tryptoline . reactions were performed on a 0.6 mmol scale . for products 6a/7a6j/7j , yields correspond to combined , isolated yields of both diastereomers . for products 6k t , yields correspond to isolated yields of the major diastereomer . during the development of the title reaction , we occasionally observed small amounts of the regioisomeric annulation products 8 and 9 ( scheme 4 ) . following extensive experimentation , conditions were identified that provided 8 and 9 as the major products with regioselectivities of up to nearly 9:1 . diastereoselectivities were similar to those observed in redox - annulations that involve the benzylic position , with -monosubstituted 4-nitrobutyraldehydes providing products 8 in highly diastereoselective fashion . key to accomplishing redox - annulations at the less reactive -c h bond is performing the reaction at higher temperature ( reflux in xylenes ) and to maintain a low concentration of 5 . redox - annulations of thiq were also performed with parent 4-nitrobutyraldehyde 10eqs 1 and 2 . diastereomeric products 11 and 12 were obtained in a 2:1 ratio and 55% overall yield ( eq 1 ) . notably , product 13 was obtained as a single diastereo- and regioisomer in 61% yield ( eq 2).1 products derived from the redox - annulation could be readily modified . for instance , reduction of 8k with zn / hcl provided amine 15 in 83% yield ( eq 3 ) . removal of the nitro group in 6k via hydrogenolysis provided heterocycle 16 in 75% yield ( eq 4 . importantly , the enantiomeric purity of the material was not affected by this transformation . finally , compound 11 was alkylated with methyl vinyl ketone in the presence of dbu to yield product 17 as a single diastereomer in 69% yield ( eq 5).3 the asymmetric redox - annulation was applied to a short synthesis of the natural product ( )-protoemetinol ( scheme 5 ) . condensation of 6,7-dimethoxy - thiq and 18 resulted in the formation of 19 and two of its diastereomers in 61% overall yield . the major diastereomer was converted to ( )-protoemetinol in a single operation that served to remove both the nitro and benzyl groups . to shed light on the mechanism of the redox - annulations discussed above and to rationalize the high regioselectivities of eqs 1 and 2 , we carefully analyzed the reaction between thiq and 4-nitrobutyraldehyde ( 10 ) by dft calculations [ m06 - 2x - d3/def2-qzvp / iefpcm//m06-l - d3/6 - 31+g(d , p)/iefpcm ] . although the uncatalyzed reaction of thiq and 10 results in complex mixtures , knowledge of this pathway is important to understand any potential background reaction ( scheme 6 ) . consequently , the uncatalyzed pathway was evaluated first . in the first step of this transformation , thiq and 10 form the hemiaminal 20 in an almost thermoneutral reaction . water can be eliminated from 20 in two different orientations yielding the azomethine ylides 21a and 21b in highly endergonic transformations ( g = + 18.9 and + 30.1 kcal mol ) . due to the direct conjugation of the azomethine ylide with the benzene ring , ylide 21a we were unable to locate any transition states for this elimination , and all attempts starting from different transition state guesses resulted in a barrierless addition of water to the corresponding azomethine ylides . therefore , it was concluded that this reaction occurs without a significant barrier which can be rationalized with the high reactivities of the formed azomethine ylides . in principle , the hemiaminal 20 could also form the corresponding enamine ( not shown in scheme 6 ) through another elimination of water . this transformation is thermoneutral ( g = 0.1 kcal mol ) and can account for the epimerization of -substituted aldehydes ( table1 ) . next , an intramolecular proton transfer takes place between the nitroalkane moiety and the ylide . in both transition states ts01a and ts01b , the proton transfers occur through five - membered transition states and are very high in energy ( g = + 42.9 and + 47.1 kcal mol ) . in an alternative pathway , the zwitterions 22 could also be formed via a zwitterionic intermediate with an exocyclic double bond ( not shown in scheme 6 ) . as this isomer is formed in a highly endergonic step ( g = + 25 kcal mol ) and requires a highly unfavorable 1,3-proton shift , this pathway seems less likely . the zwitterions 22a and 22b subsequently undergo intramolecular cyclization reactions to either form the diastereomeric products 11 and 12 or the regioisomers 13 and 14 . these reactions occur without a significant barrier , which can be explained by the high reactivities of nitronate anions and iminium cations . our calculations predict a small thermodynamic preference ( g = 0.9 kcal mol ) for the cis - product 11 over the trans - product 12 , which is in good agreement with the experimental 2:1 ratio . a much stronger preference ( g = 2.0 kcal mol ) for the cis - isomer was calculated for 13 compared to 14 in line with the exclusive isolation of 13 . as the intramolecular proton transfers yielding the zwitterions 22 are very high in energy , we were wondering whether acetic acid could act as a proton shuttle to bypass ts1a and ts1b as seen in related oxygenation and sulfenylation reactions . however , starting from many different transition - state guesses , we were unable to locate any transition states for such mechanisms . instead , these structures relaxed to hydrogen - bonded adducts between acetic acid and different polar groups of the azomethine ylides furthermore , acetic acid could also facilitate the formation of iminium ions as intermediates in these transformations , but based on our calculations , iminium ions are very high in energy ( g > 47 kcal mol ; see the supporting information for more details ) and were thus ruled out . instead , we considered a concerted elimination of acetic acid from acetylated hemiaminals ( n , o - acetals ) as an alternative mechanism ( e.g. , 23 , 24 , and 27 ) , as previously suggested by yu and co - workers in the formation of pyrroles from pyrroline and aldehydes . according to the calculated free energy profile in scheme 6 , the combination of thiq , 10 , and acoh results in the formation of the n , o - acetal 23 in a slightly endergonic reaction . subsequently , acetic acid is eliminated in a concerted yet highly asynchronous transition state ( ts02 , see the supporting information ) yielding the azomethine ylide 21a . next , acetic acid adds across the other terminus of the ylide ( ts03 ) , resulting in the formation of the more stable isomeric n , o - acetal 24 . this intermediate can now react via two pathways to form either the product 11 ( or 12 ) or the regioisomer 13 . for the formation of 11 , the next step involves an intramolecular deprotonation of an acidic proton in -position of the nitro group . this reaction proceeds through a six - membered transition state ( ts04 ) and yields the ammonium nitronate 25 . this endergonic step is also reflected in a very late transition state with a short n h bond ( 1.14 ) . next , acetic acid is eliminated through ts05 ( g = 30.5 kcal mol ) , which is also the rate - determining step for the formation of 11 . this reaction proceeds in a concerted yet highly asynchronous fashion in which the cleavage of the c o bond lacks behind the proton transfer . after cyclization of the generated zwitterion 22a , the annulation product 11 is formed in an overall exergonic reaction . the alternate regioisomer 13 could also be obtained from n , o - acetal intermediate 23 . this pathway is identical to the one discussed for the formation of 11 for the transformation of 23 to 24 . however , from there it continues with an almost isoenergetic isomerization of the n , o - acetal 24 to the n , o - acetal 27 . this two - step process consists of elimination and subsequent addition of acetic acid through transition states ts06 and ts07 . in principle , 27 could also be obtained in a sequence of elimination and addition of acoh ( 23 21b 27 , not shown in scheme 6 ) , and we have calculated the transition states ( g = 30.8 and 29.9 kcal mol , respectively , supporting information ) for these transformations as well . as they are significantly higher in energy than the barriers for the pathway involving 26 ( scheme 6 ) , we have to conclude that the azomethine ylide 21b does not lie on the reaction coordinate for the formation of either 11 or 13 . next , the ammonium nitronate 28 is formed through ts08 before the rate - determining elimination of acetic acid takes place ( ts09 , g = + 30.8 kcal mol ) . alternatively , zwitterions 22 could also be obtained directly from the azomethine ylide 26 through an intramolecular proton transfer . however , these reactions would occur with significantly higher barriers ( g = + 40.4 kcal mol for 26 22a and g = + 37.6 kcal mol for 26 22b ) and thus appear to play no role in this transformation . the calculated barriers might be slightly overestimated as in the experimental studies both acetic acid and thiq are used in excess . the results of eqs 1 and 2 can also be rationalized using the computational data in scheme 6 . although the energetic difference between both rate - determining steps ts05 and ts09 is rather small , the formation of 11 and 12 is kinetically preferred . the conditions of eq 1 favor the formation of the kinetically preferred products 11 and 12 , while the thermodynamically more stable product 13 is obtained exclusively at higher temperatures and longer reaction times . we have reported the first examples of asymmetric redox - annulations of tetrahydroisoquinolines and tryptoline with readily available , highly enantioenriched 4-nitrobutyraldehydes . reactions proceed under operationally convenient conditions and do not require the use of expensive catalysts . this strategy enabled the asymmetric preparation of polycyclic amines that contain the core structure of various bioactive compounds in just two steps from commercial materials . simply by changing the reaction conditions , regioselective redox - annulations can be achieved at the nonbenzylic position of thiq , enabling the rapid exploration of new chemical space . for the computational investigations , the conformational space for each structure was explored using the opls-2005 force field and a modified monte carlo search algorithm implemented in macromodel 10.6 . an energy cutoff of 20 kcal mol was employed for the conformational analysis , and structures with heavy - atom root - mean - square deviations ( rmsd ) less than 2 after the initial force field optimizations were considered to be the same conformer . the remaining structures were subsequently optimized with the dispersion - corrected m06-l functional with grimme s dispersion - correction d3 and the double- basis set 6 - 31+g(d , p ) . solvation by toluene was taken into account by using the integral equation formalism polarizable continuum model ( iefpcm ) for all calculations . following the intrinsic reaction coordinates ( irc ) confirmed that all transition states connected the corresponding reactants and products on the potential energy surface . thermal corrections were obtained from unscaled harmonic vibrational frequencies at the same level of theory for a standard state of 1 mol l and 298.15 k. entropic contributions to the reported free energies were derived from partition functions evaluated with the quasiharmonic approximation by truhlar and co - workers . electronic energies were subsequently obtained from single - point calculations of the m06-l - d3 geometries employing the meta - hybrid m06 - 2x functional , grimme s dispersion - correction d3 ( zero - damping ) , the large quadruple- basis set def2-qzvp , and iefpcm for toluene , a level expected to give accurate energies . an ultrafine grid was used throughout this study for numerical integration of the density . starting materials , reagents , and solvents were purchased from commercial sources and used as received unless stated otherwise . propionaldehyde , isovaleraldehyde , hydrocinnamaldehyde , and trans - cinnamaldehyde were purified by distillation prior to use . powdered molecular sieves ( 4 ) were activated before use by heating in a furnace to 300 c for 2 h and were stored in a desiccator . analytical thin - layer chromatography was performed on em reagent 0.25 mm silica gel 60 f254 plates . visualization was accomplished with uv light , potassium permanganate , and dragendorff munier stains followed by heating . purification of reaction products was carried out by flash column chromatography using sorbent technologies standard grade silica gel ( 60 , 230400 mesh ) . chemical shifts in proton nuclear magnetic resonance spectra ( h nmr ) are reported in ppm using the solvent as an internal standard ( cdcl3 at 7.26 ppm , cd2cl2 at 5.30 ppm ) . data are reported as app = apparent , s = singlet , d = doublet , t = triplet , q = quartet , m = multiplet , comp = complex , br = broad ; coupling constant(s ) in hertz . chemical shifts in proton - decoupled carbon nuclear magnetic resonance spectra ( c nmr ) are reported in ppm using the solvent as an internal standard ( cdcl3 at 77.0 ppm , cd2cl2 at 54.0 ppm ) . hrms spectrometry data were recorded on a spectrometer operating on esi - fticr ( mecn as solvent ) . optical rotations were measured using a 1 ml cell with a 1 dm path length at 589 nm and at 25 c . ( s)-diphenylprolinol trimethylsilyl ether was prepared according to a literature procedure . to a stirred solution of nitroalkene ( 3 mmol , 1 equiv ) , ( s)-diphenylprolinol trimethylsilyl ether ( 0.15 mmol , 5 mol % ) , and p - nitrophenol ( 0.15 mmol , 5 mol % ) in toluene ( 3 ml ) was added aldehyde ( 4.5 mmol , 1.5 equiv ) at room temperature . after completion , the reaction was quenched by the addition of 1 m hcl ( 10 ml ) and the resulting mixture extracted with etoac ( 2 10 ml ) . solvent was removed under reduced pressure and the residue purified by silica gel chromatography . to a stirred solution of trans - cinnamaldehyde ( 3 mmol , 1 equiv ) , ( s)-diphenylprolinol trimethylsilyl ether ( 0.3 mmol , 10 mol % ) , and benzoic acid ( 0.6 mmol , 20 mol % ) in meoh ( 6 ml ) was added nitromethane ( 9 mmol , 3 equiv ) at room temperature . after completion , the reaction was quenched by the addition of saturated nahco3 ( 10 ml ) and the resulting mixture extracted with etoac ( 2 10 ml ) . solvent was removed under reduced pressure and the residue purified by silica gel chromatography . to a stirred solution of nitroalkene ( 3 mmol , 1 equiv ) and ( s)-diphenylprolinol trimethylsilyl ether ( 0.3 mmol , 10 mol % ) in dioxane ( 0.6 ml ) the reaction mixture was stirred at room temperature , and the reaction progress was monitored by tlc . after completion , the reaction was quenched by the addition of 1 m hcl ( 10 ml ) and the resulting mixture extracted with etoac ( 2 10 ml ) . solvent was removed under reduced pressure and the residue purified by silica gel chromatography . the title compound was synthesized following general procedure a. the product was obtained as a yellow oil in 78% yield ( mixture of two diastereomers ) . compound 5a was previously reported , and its published characterization data matched our own in all respects . the enantiomeric excess was determined by hplc with daicel chiralcel od - h : n - hexane / i - proh = 90/10 , flow rate = 1 ml / min , uv = 210 nm , tr = 22.4 min ( minor ) and tr = 32.9 min ( major ) , 96% ee . the title compound was synthesized following general procedure a. the product was obtained as a yellow oil in 78% yield ( mixture of two diastereomers ) . compound 5b was previously reported , and its published characterization data matched our own in all respects . the title compound was synthesized following general procedure a. the product was obtained as a yellow solid in 70% yield ( mixture of two diastereomers ) . compound 5c was previously reported , and its published characterization data matched our own in all respects . the title compound was synthesized following general procedure a. the product was obtained as a yellow oil in 70% yield ( mixture of two diastereomers ) . compound 5d was previously reported , and its published characterization data matched our own in all respects . the title compound was synthesized following general procedure a. the product was obtained as a yellow oil in 68% yield ( mixture of two diastereomers ) . compound 5e was previously reported , and its published characterization data matched our own in all respects . the title compound was synthesized following general procedure a. the product was obtained as a yellow oil in 57% yield ( mixture of two diastereomers ) . compound 5f was previously reported , and its published characterization data matched our own in all respects . the title compound was synthesized following general procedure a. the product was obtained as a yellow oil in 66% yield ( mixture of two diastereomers ) . compound 5 g was previously reported , and its published characterization data matched our own in all respects . the title compound was synthesized following general procedure a. the product was obtained as a white solid in 57% yield ( mixture of two diastereomers ) . compound 5h was previously reported , and its published characterization data matched our own in all respects . the title compound was synthesized following general procedure b. the product was obtained as a yellow oil in 74% yield . compound 5k was previously reported , and its published characterization data matched our own in all respects . the product was reduced to the corresponding alcohol using nabh4 for hplc analysis : daicel chiralcel od - h , n - hexane / i - proh = 90/10 , flow rate = 1 ml / min , uv = 230 nm , tr = 17.2 min ( minor ) and tr = 21.9 min ( major ) , 95% ee . the title compound was synthesized following general procedure c. the product was obtained as a yellow oil in 44% yield . compound 5l was previously reported , and its published characterization data matched our own in all respects . the title compound was synthesized following general procedure c. the product was obtained as a yellow oil in 34% yield . compound 5 m was previously reported , and its published characterization data matched our own in all respects . the title compound was synthesized following general procedure c. the product was obtained as a yellow solid in 72% yield . compound 5n was previously reported , and its published characterization data matched our own in all respects . the title compound was synthesized following general procedure c. the product was obtained as a yellow oil in 56% yield . compound 5o was previously reported , and its published characterization data matched our own in all respects . the title compound was synthesized following general procedure c. the product was obtained as a yellow solid in 69% yield . compound 5p was previously reported , and its published characterization data matched our own in all respects . the title compound was synthesized following general procedure c. the product was obtained as a yellow oil in 42% yield . compound 5q was previously reported , and its published characterization data matched our own in all respects . to a mixture of aldehyde ( 0.6 mmol , 1 equiv ) , 4 ms ( 0.2 g ) and acoh ( 6 mmol , 10 equiv ) in toluene ( 6 ml ) was added the amine ( 1.2 mmol , 2 equiv ) at room temperature . the mixture was heated under reflux for 1 h. subsequently , the reaction mixture was allowed to cool to room temperature , diluted with etoac ( 20 ml ) , and washed with saturated aqueous nahco3 ( 3 10 ml ) . the combined aqueous layers were extracted with etoac ( 2 10 ml ) and the combined organic layers dried over anhydrous na2so4 . solvent was removed under reduced pressure and the residue purified by silica gel chromatography . a solution of amine ( 1.2 mmol , 2 equiv ) and 2-eha ( 6 mmol , 10 equiv ) in xylenes ( 6 ml ) was heated under reflux . the aldehyde ( 0.6 mmol , 0.12 m solution in xylenes ) was delivered through the top of the reflux condenser over 15 h via syringe pump . the reaction was then kept under reflux for a further 0.51 h at which time the aldehyde was consumed as judged by tlc analysis . subsequently , the reaction mixture was allowed to cool to room temperature , diluted with etoac ( 20 ml ) , and washed with saturated aqueous nahco3 ( 3 20 ml ) . the combined aqueous layers were extracted with etoac ( 2 10 ml ) and the combined organic layers dried over anhydrous na2so4 . solvent was removed under reduced pressure and the residue purified by silica gel chromatography . a 10 ml microwave reaction tube was charged with a 10 8 mm sic passive heating element , aldehyde ( 0.6 mmol , 1 equiv ) , toluene ( 6 ml ) , amine ( 1.2 mmol , 2 equiv ) , 3 ms ( 0.2 g ) , and acoh ( 6 mmol , 10 equiv ) . the reaction tube was sealed with a teflon - lined snap cap and heated in a microwave reactor at 120 c ( 015 psi ) for 3 min . after being cooled with compressed air flow , the reaction mixture was diluted with etoac ( 20 ml ) and washed with saturated aqueous nahco3 ( 3 20 ml ) . the combined aqueous layers were extracted with etoac ( 2 10 ml ) and the combined organic layers dried over anhydrous na2so4 . solvent was removed under reduced pressure and the residue purified by silica gel chromatography . following general procedure a , products 6a and 7a were obtained in a 2:1 ratio ( 71% combined yield ) . yellowish oil ; rf = 0.32 in 20% etoac / hex ; [ ]d 31.9 ( c 0.5 , chcl3 ) ; ir ( kbr ) 3063 , 3028 , 2959 , 2927 , 2828 , 1539 , 1492 , 1452 , 1311 , 1264 , 1153 , 1094 , 750 , 703 cm ; h nmr ( 500 mhz , cdcl3 ) 7.337.28 ( comp , 2h ) , 7.277.25 ( m , 1h ) , 7.197.15 ( comp , 4h ) , 7.07 ( app t , j = 7.5 hz , 1h ) , 6.87 ( app d , j = 7.8 hz , 1h ) , 4.97 ( dd , j = 11.2 , 9.8 hz , 1h ) , 4.39 ( d , j = 9.8 hz , 1h ) , 3.34 ( ddd , j = 11.4 , 9.8 , 4.9 hz , 1h ) , 3.19 ( dd , j = 13.7 , 4.1 hz , 1h ) , 3.11 ( app t , j = 11.4 hz , 1h ) , 3.05 ( dd , j = 9.6 , 6.7 hz , 1h ) , 3.002.98 ( m , 1h ) , 2.962.92 ( comp , 2h ) , 2.302.23 ( m , 1h ) , 0.73 ( d , j = 6.5 hz , 3h ) ; c nmr ( 125 mhz , cdcl3 ) 138.2(2 ) , 134.2(1 ) , 132.9 , 129.5 , 129.1 , 128.1 , 128.0 , 127.0 , 126.3 , 91.7 , 77.6 , 77.3 , 77.1 , 63.0 , 62.1 , 56.0 , 46.5 , 31.2 , 29.9 , 16.7 ; hrms ( esi ) m / z calcd for c20h23n2o2 [ m + h ] 323.1754 , found 323.1765 ; hplc daicel chiralcel od - h , n - hexane / i - proh = 90/10 , flow rate = 1 ml / min ; uv = 230 nm , tr = 7.1 min ( major ) and tr = 8.0 min ( minor ) , 95% ee . yellowish solid ; mp 180182 c ; rf = 0.46 in 20% etoac / hex ; [ ]d 130.8 ( c 0.5 , chcl3 ) ; ir ( kbr ) 3061 , 3031 , 2957 , 2957 , 2927 , 2893 , 2811 , 2767 , 1546 , 1489 , 1452 , 1351 , 1299 , 1146 , 1111 , 738 , 696 cm ; h nmr ( 500 mhz , cdcl3 ) 7.327.29 ( comp , 2h ) , 7.277.20 ( comp , 2h ) , 7.207.14 ( comp , 3h ) , 7.11 ( app t , j = 7.6 hz , 1h ) , 6.90 ( app d , j = 7.9 hz , 1h ) , 5.09 ( dd , j = 11.7 , 9.0 hz , 1h ) , 4.20 ( d , j = 9.0 hz , 1h ) , 3.82 ( dd , j = 11.7 , 5.0 hz , 1h ) , 3.25 ( dd , j = 12.2 , 3.5 hz , 1h ) , 3.12 ( ddd , j = 14.8 , 9.4 , 5.0 hz , 1h ) , 3.05 ( app dt , j = 10.9 , 4.4 hz , 1h ) , 2.98 ( dd , j = 12.2 , 1.8 hz , 1h ) , 2.86 ( ddd , j = 11.1 , 9.4 , 3.5 hz , 1h ) , 2.76 ( app dt , j = 15.4 , 3.5 hz , 1h ) , 2.182.09 ( m , 1h ) , 0.95 ( d , j = 7.2 hz , 3h ) ; c nmr ( 125 mhz , cdcl3 ) 138.5 , 136.0 , 133.0 , 129.5 , 128.7 , 128.2 , 127.6 , 127.5 , 126.5 , 126.3 , 88.3 , 66.0 , 62.0 , 52.7 , 50.8 , 35.8 , 31.2 , 15.2 ; m / z ( esi - ms ) 323.2 [ m + h ] ; hplc daicel chiralcel od - h , n - hexane / i - proh = 90/10 , flow rate = 1 ml / min ; uv = 230 nm , tr = 6.3 min ( minor ) and tr = 6.9 min ( major ) , 95% ee . following general procedure a , products 6b and 7b were obtained in a 2:1 ratio ( 68% combined yield ) . yellowish oil ; rf = 0.32 in 20% etoac / hex ; [ ]d 32.6 ( c 0.5 , chcl3 ) ; ir ( kbr ) 3066 , 3033 , 2959 , 2920 , 2843 , 1608 , 1543 , 1509 , 1457 , 1363 , 1311 , 1225 , 1161 , 1096 , 733 cm ; h nmr ( 500 mhz , cdcl3 ) 7.237.18 ( m , 1h ) , 7.167.13 ( comp , 3h ) , 7.07 ( app t , j = 7.5 hz , 1h ) , 7.016.98 ( comp , 2h ) , 6.85 ( app d , j = 7.8 hz , 1h ) , 4.90 ( dd , j = 11.2 , 9.8 hz , 1h ) , 4.37 ( d , j = 9.8 hz , 1h ) , 3.32 ( ddd , j = 11.2 , 9.4 , 4.9 hz , 1h ) , 3.18 ( dd , j = 13.7 , 4.1 hz , 1h ) , 3.10 ( app t , j = 11.2 hz , 1h ) , 3.03 ( dd , j = 9.8 , 6.7 hz , 1h ) , 2.992.90 ( comp , 3h ) , 2.252.17 ( m , 1h ) , 0.72 ( d , j = 6.5 hz , 3h ) ; c nmr ( 125 mhz , cdcl3 ) 162.4 ( d , j = 246.2 hz ) , 134.2 , 134.0 ( d , j = 3.3 hz ) , 132.8 , 129.6 , 128.2 , 126.9 , 126.4(0 ) , 126.3(6 ) , 116.1 ( d , j = 21.5 hz ) , 91.8 , 63.0 , 62.0 , 55.3 , 46.5 , 31.3 , 29.9 , 16.6 ; m / z ( esi - ms ) 341.1 [ m + h ] . yellowish solid ; mp 100103 c ; ( rf = 0.47 in 20% etoac / hex ) ; [ ]d 126.5 ( c 0.5 , chcl3 ) ; ir ( kbr ) 3063 , 2969 , 2935 , 2910 , 2814 , 2769 , 1608 , 1546 , 1506 , 1427 , 1348 , 1299 , 1222 , 1161 , 1109 , 842 , 812 , 753 , 696 cm ; h nmr ( 500 mhz , cdcl3 ) 7.21 ( app t , j = 7.3 hz , 1h ) , 7.187.09 ( comp , 4h ) , 7.016.98 ( comp , 2h ) , 6.88 ( app d , j = 7.9 hz , 1h ) , 5.02 ( dd , j = 11.7 , 9.0 hz , 1h ) , 4.17 ( d , j = 9.0 hz , 1h ) , 3.80 ( dd , j = 11.7 , 4.9 hz , 1h ) , 3.22 ( dd , j = 12.2 , 3.2 hz , 1h ) , 3.12 ( ddd , j = 14.6 , 9.3 , 4.6 hz , 1h ) , 3.02 ( app dt , j = 10.9 , 4.6 hz , 1h ) , 2.96 ( dd , j = 12.2 , 1.7 hz , 1h ) , 2.84 ( app td , j = 10.9 , 3.2 hz , 1h ) , 2.74 ( app dt , j = 15.5 , 3.2 hz , 1h ) , 2.152.05 ( m , 1h ) , 0.94 ( d , j = 7.1 hz , 3h ) ; c nmr ( 125 mhz , cdcl3 ) 162.2 ( d , j = 245.9 hz ) , 136.1 , 134.2(9 ) , 134.2(6 ) , 133.0 , 129.7 ( d , j = 8.0 hz ) , 129.5 , 127.6 , 126.5 , 126.2 , 115.7 ( d , j = 21.4 hz ) , 88.6 , 66.0 , 61.9 , 52.1 , 50.8 , 35.8 , 31.2 , 15.0 ; m / z ( esi - ms ) 341.1 [ m + h ] . following general procedure a , products 6c and 7c were obtained in a 2:1 ratio ( 71% combined yield ) . yellowish solid ; mp 130132 c ; rf = 0.37 in 20% etoac / hex ; [ ]d 38.6 ( c 0.5 , chcl3 ) ; ir ( kbr ) 3058 , 3024 , 2954 , 2920 , 2831 , 1731 , 1546 , 1484 , 1452 , 1371 , 1304 , 1245 , 1143 , 1099 , 1074 , 1012 , 958 , 889 , 817 , 738 , 674 cm ; h nmr ( 500 mhz , cdcl3 ) 7.457.40 ( comp , 2h ) , 7.20 ( app t , j = 7.4 hz , 1h ) , 7.177.13 ( m , 1h ) , 7.097.05 ( comp , 3h ) , 6.85 ( app d , j = 7.9 hz , 1h ) , 4.90 ( app t , j = 9.7 hz , 1h ) , 4.36 ( d , j = 9.7 hz , 1h ) , 3.31 ( ddd , j = 11.6 , 10.9 , 4.5 hz , 1h ) , 3.18 ( dd , j = 13.7 , 3.9 hz , 1h ) , 3.09 ( app t , j = 11.6 hz , 1h ) , 3.053.01 ( m , 1h ) , 2.992.89 ( comp , 3h ) , 2.252.16 ( m , 1h ) , 0.72 ( d , j = 6.4 hz , 3h ) ; c nmr ( 125 mhz , cdcl3 ) 137.3 , 134.2 , 132.7 , 132.3 , 129.6 , 128.2 , 126.9 , 126.4 , 121.9 , 91.5 , 63.0 , 62.0 , 55.5 , 46.5 , 31.2 , 29.9 , 16.6 ; m / z ( esi - ms ) ( br ) 401.1 [ m + h ] , ( br ) 403.1 [ m + h ] . yellowish solid ; mp 185190 c ; rf = 0.48 in 20% etoac / hex ; [ ]d 134.9 ( c 0.5 , chcl3 ) ; ir ( kbr ) 3061 , 3026 , 2964 , 2927 , 2895 , 2809 , 2764 , 1541 , 1492 , 1447 , 1425 , 1351 , 1291 , 1148 , 1109 , 1072 , 1037 , 1007 , 837 , 807 , 731 cm ; h nmr ( 500 mhz , cdcl3 ) 7.457.41(comp , 2h ) , 7.21 ( app t , j = 7.4 hz , 1h ) , 7.177.14 ( m , 1h ) , 7.11 ( app t , j = 7.6 hz , 1h ) , 7.077.03 ( comp , 2h ) , 6.87 ( app d , j = 7.9 hz , 1h ) , 5.02 ( dd , j = 11.6 , 9.0 hz , 1h ) , 4.18 ( d , j = 9.0 hz , 1h ) , 3.78 ( dd , j = 11.6 , 4.9 hz , 1h ) , 3.22 ( dd , j = 12.2 , 3.2 hz , 1h ) , 3.11 ( ddd , j = 14.8 , 9.3 , 4.7 hz , 1h ) , 3.073.00 ( m , 1h ) , 2.982.94 ( m , 1h ) , 2.892.81 ( m , 1h ) , 2.782.70 ( m , 1h ) , 2.142.06 ( m , 1h ) , 0.94 ( d , j = 7.1 hz , 3h ) ; c nmr ( 125 mhz , cdcl3 ) 137.6 , 136.1 , 132.9(0 ) , 131.9(2 ) , 129.9 , 129.6 , 127.7 , 126.5 , 126.2 , 121.5 , 88.3 , 66.0 , 61.9 , 52.3 , 50.8 , 35.7 , 31.2 , 15.0 ; m / z ( esi - ms ) ( br ) 401.1 [ m + h ] , ( br ) 403.1 [ m + h ] . following general procedure a , products 6d and 7d were obtained in a 2:1 ratio ( 69% combined yield ) . yellowish oil ; rf = 0.35 in 20% etoac / hex ; [ ]d 34.8 ( c 0.5 , chcl3 ) ; ir ( kbr ) 3056 , 3021 , 2972 , 2925 , 2905 , 2878 , 2809 , 2767 , 1548 , 1509 , 1492 , 1445 , 1371 , 1346 , 1296 , 1267 , 1254 , 1151 , 1114 , 1037 , 951 , 832 , 802 , 733 cm ; h nmr ( 500 mhz , cdcl3 ) 7.227.18 ( m , 1h ) , 7.177.14 ( m , 1h ) , 7.137.09 ( comp , 2h ) , 7.097.04 ( comp , 3h ) , 6.87 ( app d , j = 7.9 hz , 1h ) , 4.94 ( dd , j = 11.2 , 9.8 hz , 1h ) , 4.38 ( d , j = 9.8 hz , 1h ) , 3.34 ( ddd , j = 11.2 , 9.6 , 4.9 hz , 1h ) , 3.18 ( dd , j = 13.7 , 4.1 hz , 1h ) , 3.113.01 ( comp , 2h ) , 3.002.91 ( comp , 3h ) , 2.31 ( s , 3h ) , 2.282.21 ( m , 1h ) , 0.73 ( d , j = 6.5 hz , 3h ) ; c nmr ( 125 mhz , cdcl3 ) 137.5 , 135.2 , 134.2 , 133.0 , 129.8 , 129.5 , 128.1 , 127.0 , 126.3 , 105.0 , 91.8 , 63.0 , 62.1 , 55.7 , 46.4 , 31.2 , 29.9 , 21.3 , 16.7 ; m / z ( esi - ms ) 337.2 [ m + h ] . yellowish oil ; rf = 0.52 in 20% etoac / hex ; [ ]d 157.6 ( c 0.5 , chcl3 ) ; ir ( kbr ) 3056 , 3024 , 2962 , 2920 , 2895 , 2809 , 2767 , 1546 , 1509 , 1494 , 1450 , 1368 , 1348 , 1294 , 1264 , 1148 , 1114 , 1039 , 948 , 837 , 800 , 726 cm ; h nmr ( 500 mhz , cdcl3 ) 7.237.19 ( m , 1h ) , 7.177.14 ( m , 1h ) , 7.127.10 ( comp , 3h ) , 7.097.05 ( comp , 2h ) , 6.90 ( app d , j = 7.9 hz , 1h ) , 5.07 ( dd , j = 11.7 , 9.0 hz , 1h ) , 4.18 ( d , j = 9.0 hz , 1h ) , 3.78 ( dd , j = 11.7 , 4.9 hz , 1h ) , 3.23 ( dd , j = 12.2 , 3.3 hz , 1h ) , 3.12 ( ddd , j = 14.8 , 9.3 , 4.9 hz , 1h ) , 3.04 ( app dt , j = 11.1 , 4.4 hz , 1h ) , 2.97 ( dd , j = 12.2 , 1.8 hz , 1h ) , 2.85 ( ddd , j = 11.1 , 9.3 , 3.5 hz , 1h ) , 2.75 ( app dt , j = 15.5 , 3.5 hz , 1h ) , 2.31 ( s , 3h ) , 2.132.10 ( m , 1h ) , 0.96 ( d , j = 7.1 hz , 3h ) ; c nmr ( 125 mhz , cdcl3 ) 137.1 , 136.1 , 135.5 , 133.2 , 129.5 , 129.4 , 128.1 , 127.6 , 126.5 , 126.3 , 88.5 , 66.0 , 62.0 , 52.4 , 50.8 , 35.9 , 31.3 , 21.3 , 15.2 ; m / z ( esi - ms ) 337.2 [ m + h ] . following general procedure a , products 6e and 7e were obtained in a 1.5:1 ratio ( 65% combined yield ) . yellowish solid ; mp 110113 c ; rf = 0.24 in 20% etoac / hex ; [ ]d 24.5 ( c 0.5 , chcl3 ) ; ir ( kbr ) 2954 , 2932 , 2902 , 2833 , 2804 , 1615 , 1546 , 1541 , 1462 , 1380 , 1249 , 1175 , 1116 , 1035 , 837 , 740 cm ; h nmr ( 500 mhz , cdcl3 ) 7.217.17 ( m , 1h ) , 7.167.14 ( m , 1h ) , 7.097.05 ( comp , 3h ) , 6.866.82 ( comp , 3h ) , 4.90 ( dd , j = 11.0 , 9.8 hz , 1h ) , 4.37 ( d , j = 9.8 hz , 1h ) , 3.77 ( s , 3h ) , 3.33 ( ddd , j = 11.3 , 9.8 , 4.9 hz , 1h ) , 3.18 ( dd , j = 13.7 , 4.1 hz , 1h ) , 3.093.01 ( comp , 2h ) , 2.992.90 ( comp , 3h ) , 2.262.17 ( m , 1h ) , 0.73 ( d , j = 6.5 hz , 3h ) ; c nmr ( 125 mhz , cdcl3 ) 159.2 , 134.1 , 132.9 , 130.2 , 129.5 , 128.1 , 126.9 , 126.3 , 114.5 , 91.9 , 62.9 , 62.1 , 55.4 , 55.2 , 46.5 , 31.3 , 29.9 , 16.7 ; m / z ( esi - ms ) 353.2 [ m + h ] . yellowish solid ; mp 120122 c ; rf = 0.41 in 20% etoac / hex ; [ ]d 178.1 ( c 0.5 , chcl3 ) ; ir ( kbr ) 2954 , 2927 , 2833 , 1613 , 1543 , 1506 , 1452 , 1361 , 1341 , 1247 , 1180 , 1027 , 820 , 745 cm ; h nmr ( 500 mhz , cdcl3 ) 7.227.18 ( m , 1h ) , 7.167.15 ( m , 1h ) , 7.127.09 ( comp , 3h ) , 6.89 ( app d , j = 7.9 hz , 1h ) , 6.856.81 ( comp , 2h ) , 5.03 ( dd , j = 11.5 , 9.2 hz , 1h ) , 4.19 ( d , j = 9.2 hz , 1h ) , 3.78 ( s , 3h ) , 3.76 ( dd , j = 8.0 , 4.9 hz , 1h ) , 3.243.22 ( m , 1h ) , 3.11 ( ddd , j = 14.6 , 9.1 , 4.7 hz , 1h ) , 3.083.02 ( m , 1h ) , 2.982.95 ( m , 1h ) , 2.872.84 ( m , 1h ) , 2.782.75 ( m , 1h ) , 2.152.04 ( m , 1h ) , 0.95 ( d , j = 7.1 hz , 3h ) ; c nmr ( 125 mhz , cdcl3 ) 158.9 , 136.0 , 133.2 , 130.5 , 129.5 , 129.2 , 127.6 , 126.5 , 126.3 , 114.1 , 88.6 , 65.9 , 61.9 , 55.4 , 52.0 , 50.8 , 35.9 , 31.1 , 15.2 ; m / z ( esi - ms ) 353.1 [ m + h ] . following general procedure a , products 6f and 7f were obtained in a 1:1 ratio ( 48% combined yield ) . yellowish oil ; rf = 0.28 in 10% etoac / hex ; [ ]d 37.4 ( c 0.5 , chcl3 ) ; ir ( kbr ) 3061 , 3024 , 2952 , 2920 , 2870 , 1598 , 1541 , 1497 , 1452 , 1371 , 1314 , 1272 , 1143 , 1096 , 941 , 750 , 698 cm ; h nmr ( 500 mhz , cdcl3 ) 7.307.26 ( comp , 2h ) , 7.247.17 ( comp , 2h ) , 7.187.12 ( comp , 3h ) , 7.10 ( app td , j = 7.6 , 1.5 hz , 1h ) , 6.89 ( app d , j = 7.8 hz , 1h ) , 4.82 ( dd , j = 11.2 , 9.9 hz , 1h ) , 4.30 ( d , j = 9.9 hz , 1h ) , 3.17 ( ddd , j = 11.4 , 9.9 , 4.6 hz , 1h ) , 3.092.99 ( comp , 2h ) , 2.92 ( app dt , j = 16.3 , 3.9 hz , 1h ) , 2.882.81 ( comp , 2h ) , 2.642.50 ( comp , 2h ) , 2.212.15 ( m , 1h ) , 2.061.96 ( m , 1h ) , 1.941.85 ( m , 1h ) , 1.621.55 ( m , 1h ) , 1.01 ( d , j = 6.4 hz , 3h ) ; c nmr ( 125 mhz , cdcl3 ) 141.7 , 134.2 , 133.2 , 129.6 , 128.7 , 128.5 , 128.1 , 126.9 , 126.3 , 89.5 , 62.8 , 62.0 , 47.4 , 46.2 , 30.9 , 30.8 , 29.8 , 27.5 , 16.3 ; m / z ( esi - ms ) 351.2 [ m + h ] . yellowish oil ; rf = 0.42 in 10% etoac / hex ; [ ]d 91.8 ( c 0.5 , chcl3 ) ; ir ( kbr ) 3063 , 3031 , 2930 , 2891 , 2809 , 2767 , 1605 , 1536 , 1498 , 1455 , 1356 , 1299 , 1245 , 1146 , 1104 , 736 , 694 cm ; h nmr ( 500 mhz , cdcl3 ) 7.297.32 ( comp , 2h ) , 7.237.20 ( m , 1h ) , 7.197.17 ( comp , 3h ) , 7.137.09 ( comp , 2h ) , 6.91 ( app d , j = 7.9 hz , 1h ) , 4.41 ( dd , j = 11.0 , 9.2 hz , 1h ) , 4.05 ( d , j = 9.2 hz , 1h ) , 3.093.03 ( m , 1h ) , 3.00 ( dd , j = 12.2 , 3.4 hz , 1h ) , 2.982.94 ( m , 1h ) , 2.90 ( dd , j = 12.2 , 2.3 hz , 1h ) , 2.812.74 ( comp , 2h ) , 2.742.67 ( m , 1h ) , 2.542.48 ( m , 1h ) , 2.482.40 ( m , 1h ) , 2.212.15 ( m , 1h ) , 1.711.62 ( m , 1h ) , 1.601.53 ( m , 1h ) , 1.12 ( d , j = 7.1 hz , 3h ) ; c nmr ( 125 mhz , cdcl3 ) 141.4 , 136.1 , 133.6 , 129.5 , 128.7 , 128.5 , 127.5 , 126.5 , 126.3 , 125.9 , 91.5 , 77.5 , 77.3 , 77.0 , 65.2 , 61.8 , 50.9 , 44.7 , 32.3 , 31.1 , 30.4 , 29.9 , 13.8 ; m / z ( esi - ms ) 351.2 [ m + h ] . following general procedure a , products 6 g and 7 g were obtained in a 1.8:1 ratio ( 70% combined yield ) . yellowish solid ; mp 6466 c ; rf = 0.27 in 10% etoac / hex ; [ ]d 61.4 ( c 0.5 , chcl3 ) ; ir ( kbr ) 3063 , 3026 , 2917 , 2851 , 2754 , 1600 , 1543 , 1492 , 1452 , 1368 , 1311 , 1257 , 1143 , 1099 , 738 , 696 cm ; h nmr ( 500 mhz , cdcl3 ) 7.387.35 ( comp , 2h ) , 7.317.23 ( comp , 5h ) , 7.207.17 ( comp , 2h ) , 7.147.11 ( m , 1h ) , 7.07 ( app t , j = 7.5 hz , 1h ) , 7.016.99 ( comp , 2h ) , 6.86 ( app d , j = 7.8 hz , 1h ) , 4.94 ( app t , j = 10.4 hz , 1h ) , 4.36 ( d , j = 9.7 hz , 1h ) , 3.29 ( app t , j = 11.4 hz , 1h ) , 3.213.16 ( m , 1h ) , 3.09 ( dd , j = 13.7 , 4.0 hz , 1h ) , 2.982.87 ( comp , 3h ) , 2.75 ( app dt , j = 10.6 , 5.0 hz , 1h ) , 2.60 ( dd , j = 13.7 , 2.8 hz , 1h ) , 2.502.38 ( m , 1h ) , 2.16 ( dd , j = 13.7 , 10.5 hz , 1h ) ; c nmr ( 125 mhz , cdcl3 ) 139.4 , 138.0 , 134.4 , 132.8 , 129.5 , 129.3 , 129.0 , 128.7 , 128.3 , 128.0 , 126.8 , 126.5 , 126.4 , 92.5 , 63.1 , 59.4 , 54.7 , 46.7 , 38.3 , 37.8 , 29.9 ; m / z ( esi - ms ) 399.2 [ m + h ] . yellowish oil ; rf = 0.48 in 10% etoac / hex ; [ ]d 156.6 ( c 0.5 , chcl3 ) ; ir ( kbr ) 3061 , 3024 , 2952 , 2927 , 2902 , 2804 , 2754 , 1600 , 1546 , 1492 , 1452 , 1356 , 1299 , 1252 , 1143 , 760 , 701 cm ; h nmr ( 500 mhz , cdcl3 ) 7.377.34 ( comp , 2h ) , 7.317.21 ( comp , 6h ) , 7.217.17 ( comp , 2h ) , 7.177.11 ( m , 1h ) , 6.976.95 ( comp , 2h ) , 6.91 ( d , j = 7.9 hz , 1h ) , 5.16 ( dd , j = 11.7 , 8.9 hz , 1h ) , 4.21 ( d , j = 8.9 hz , 1h ) , 3.97 ( dd , j = 11.7 , 4.9 hz , 1h ) , 3.26 ( ddd , j = 14.7 , 10.7 , 4.9 hz , 1h ) , 2.87 ( dd , j = 7.9 , 3.0 hz , 1h ) , 2.84 ( dd , j = 6.9 , 2.5 hz , 1h ) , 2.832.79 ( comp , 2h ) , 2.782.70 ( comp , 2h ) , 2.46 ( dd , j = 13.0 , 3.5 hz , 1h ) , 2.232.17 ( m , 1h ) ; c nmr ( 125 mhz , cdcl3 ) 140.9 , 138.1 , 136.3 , 133.2 , 129.6 , 129.2 , 128.9 , 128.6 , 128.3 , 127.7 , 127.6 , 126.6 , 126.3 , 126.1 , 89.2 , 66.5 , 57.1 , 52.9 , 50.8 , 43.3 , 33.2 , 31.6 ; m / z ( esi - ms ) 399.2 [ m + h ] . following general procedure a , products 6h and 7h were obtained in a 5:1 ratio ( 37% combined yield ) . yellowish solid ; mp 180184 c ; rf = 0.45 in 20% etoac / hex ; [ ]d 23.0 ( c 0.5 , chcl3 ) ; ir ( kbr ) 3063 , 3026 , 2952 , 2922 , 2868 , 2823 , 1546 , 1497 , 1450 , 1368 , 1306 , 1141 , 1099 , 1005 , 750 , 726 , 696 cm ; h nmr ( 500 mhz , cdcl3 ) 7.327.29 ( comp , 2h ) , 7.257.23 ( m , 1h ) , 7.217.14 ( comp , 4h ) , 7.07 ( app t , j = 7.5 hz , 1h ) , 6.85 ( app d , j = 7.9 hz , 1h ) , 4.92 ( app t , j = 9.8 hz , 1h ) , 4.35 ( d , j = 9.8 hz , 1h ) , 3.42 ( app t , j = 11.6 hz , 1h ) , 3.383.29 ( m , 1h ) , 3.19 ( dd , j = 13.4 , 3.7 hz , 1h ) , 3.132.96 ( comp , 3h ) , 2.942.87 ( m , 1h ) , 2.242.15 ( m , 1h ) , 1.501.44 ( m , 1h ) , 0.90 ( d , j = 7.0 hz , 3h ) , 0.83 ( d , j = 7.0 hz , 3h ) ; c nmr ( 125 mhz , cdcl3 ) 138.0 , 134.2 , 132.9 , 129.5 , 129.2 , 128.1 , 128.0 , 126.9 , 126.4 , 93.0 , 62.9 , 54.1 , 52.2 , 46.7 , 40.3 , 29.9 , 26.6 , 21.5 , 15.6 ; m / z ( esi - ms ) 351.2 following general procedure a , products 6i and 7i were obtained in a 1.5:1 ratio ( 66% combined yield ) . yellowish solid ; mp 145148 c ; rf = 0.35 in 30% etoac / hex ; [ ]d 11.9 ( c 0.5 , chcl3 ) ; ir ( kbr ) 3063 , 3026 , 3001 , 2957 , 2922 , 2902 , 2828 , 1610 , 1539 , 1504 , 1452 , 1378 , 1309 , 1252 , 1146 , 1035 , 859 , 817 , 760 , 743 , 701 cm ; h nmr ( 500 mhz , cdcl3 ) 7.317.28 ( comp , 2h ) , 7.277.21 ( m , 1h ) , 7.177.16 ( comp , 2h ) , 6.78 ( app d , j = 8.6 hz , 1h ) , 6.686.65 ( m , 1h ) , 6.62 ( dd , j = 8.6 , 2.5 hz , 1h ) , 4.92 ( dd , j = 11.2 , 9.8 hz , 1h ) , 4.32 ( d , j = 9.7 hz , 1h ) , 3.75 ( s , 3h ) , 3.353.26 ( m , 1h ) , 3.17 ( dd , j = 13.7 , 4.1 hz , 1h ) , 3.08 ( app t , j = 11.4 hz , 1h ) , 3.052.98 ( m , 1h ) , 2.972.87 ( comp , 3h ) , 2.302.18 ( m , 1h ) , 0.71 ( d , j = 6.5 hz , 3h ) ; c nmr ( 125 mhz , cdcl3 ) 159.2 , 138.2 , 135.7 , 129.1 , 128.0 , 128.0 , 125.3 , 114.4 , 112.2 , 91.9 , 62.6 , 62.1 , 55.9 , 55.4 , 46.4 , 31.2 , 30.2 , 16.7 ; m / z ( esi - ms ) 353.1 [ m + h ] . yellowish solid ; mp 130133 ; rf = 0.45 in 30% etoac / hex ; [ ]d 102.0 ( c 0.5 , chcl3 ) ; ir ( kbr ) 3061 , 3028 , 2959 , 2932 , 2907 , 2863 , 2806 , 2767 , 1613 , 1546 , 1494 , 1455 , 1356 , 1269 , 1151 , 1116 , 1039 , 827 , 743 , 698 cm ; h nmr ( 500 mhz , cdcl3 ) 7.317.28 ( comp , 2h ) , 7.277.21 ( m , 1h ) , 7.187.15 ( comp , 2h ) , 6.81 ( app d , j = 8.2 hz , 1h ) , 6.686.66 ( comp , 2h ) , 5.05 ( dd , j = 11.6 , 9.0 hz , 1h ) , 4.13 ( d , j = 9.0 hz , 1h ) , 3.80 ( dd , j = 10.2 , 5.0 hz . 1h ) , 3.78 ( s , 3h ) , 3.22 ( dd , j = 12.2 , 3.0 hz , 1h ) , 3.09 ( ddd , j = 14.8 , 9.2 , 4.5 hz , 1h ) , 3.052.99 ( m , 1h ) , 2.96 ( app d , j = 12.2 hz , 1h ) , 2.83 ( ddd , j = 12.2 , 10.2 , 3.3 hz , 1h ) , 2.742.66 ( m , 1h ) , 2.162.07 ( m , 1h ) 0.94 ( d , j = 7.1 hz , 3h ) ; c nmr ( 125 mhz , cdcl3 ) 158.8 , 138.6 , 137.6 , 128.7 , 128.2 , 127.5 , 127.4 , 125.4 , 114.4 , 112.2 , 88.5 , 65.8 , 62.0 , 55.4 , 52.6 , 50.7 , 35.9 , 31.5 , 15.1 ; m / z ( esi - ms ) 353.1 [ m + h ] . following general procedure a , products 6j and 7j were obtained in a 2:1 ratio ( 42% combined yield ) . yellowish oil ; rf = 0.36 in 20% etoac / hex ; [ ]d 17.5 ( c 0.5 , chcl3 ) ; ir ( kbr ) 3407 , 3058 , 3026 , 2952 , 2905 , 2838 , 2806 , 1724 , 1548 , 1492 , 1450 , 1378 , 1361 , 1267 , 1165 , 740 , 698 cm ; h nmr ( 500 mhz , cdcl3 ) 7.59 ( br s , 1h ) , 7.50 ( app d , j = 7.5 hz , 1h ) , 7.357.32 ( comp , 3h ) , 7.317.27 ( m , 1h ) , 7.197.14 ( comp , 3h ) , 7.137.07 ( m , 1h ) , 4.914.87 ( m , 1h ) , 4.26 ( d , j = 9.4 hz , 1h ) , 3.233.18 ( m , 2h ) , 3.052.93 ( comp , 2h ) , 2.902.81 ( comp , 2h ) , 2.692.61 ( m , 1h ) , 2.352.21 ( m , 1h ) , 0.77 ( d , j = 6.5 hz , 3h ) ; c nmr ( 125 mhz , cdcl3 ) 137.5 , 136.8 , 129.2 , 128.3 , 126.7 , 122.7 , 120.0 , 118.5 , 111.6 , 111.2 , 93.2 , 62.7 , 61.8 , 56.5 , 51.3 , 34.9 , 22.1 , 16.9 ; hrms ( esi ) m / z calcd for c22h24n3o2 [ m + h ] 362.1863 , found 362.1875 . yellowish solid ; mp 160163 c ; rf = 0.40 in 20% etoac / hex ; [ ]d 25.7 ( c 0.5 , chcl3 ) ; ir ( kbr ) 3409 , 3061 , 3031 , 2964 , 2898 , 2838 , 2774 , 1697 , 1632 , 1541 , 1452 , 1343 , 1269 , 1170 , 1114 , 1072 , 1007 , 968 , 736 , 696 cm ; h nmr ( 500 mhz , cdcl3 ) 7.61 ( br s , 1h ) , 7.51 ( app d , j = 7.8 hz , 1h ) , 7.357.32 ( comp , 2h ) , 7.28 ( app d , j = 7.3 hz , 1h ) , 7.247.21 ( comp , 2h ) , 7.207.15 ( m , 1h ) , 7.137.09 ( m , 1h ) , 5.28 ( dd , j = 11.8 , 9.4 hz , 1h ) , 4.184.12 ( m , 1h ) , 3.72 ( dd , j = 11.8 , 4.5 hz , 1h ) , 3.153.07 ( comp , 2h ) , 3.062.97 ( comp , 2h ) , 2.882.74 ( comp , 2h ) , 2.222.14 ( m , 1h ) , 1.01 ( d , j = 7.1 hz , 3h ) ; c nmr ( 125 mhz , cdcl3 ) 136.9 , 128.8 , 128.4 , 127.8 , 126.7 , 122.7 , 120.0 , 118.5 , 111.6 , 87.7 , 63.8 , 62.1 , 52.9 , 52.7 , 35.9 , 22.1 , 14.5 ; m / z ( esi - ms ) 362.2 [ m + h ] . yellowish solid ; mp 150152 c ; rf = 0.42 in 30% etoac / hex ; [ ]d 56.3 ( c 0.5 , chcl3 ) ; ir ( kbr ) 3059 , 3028 , 2931 , 2862 , 2862 , 2834 , 1714 , 1630 , 1551 , 1489 , 1447 , 1364 , 1036 , 1143 , 1108 , 1081 , 752 , 700 cm ; h nmr ( 500 mhz , cdcl3 ) 7.337.30 ( comp , 2h ) , 7.297.19 ( comp , 4h ) , 7.187.14 ( m , 1h ) , 7.08 ( app t , j = 7.3 hz , 1h ) , 6.87 ( app d , j = 7.8 hz , 1h ) , 4.97 ( dd , j = 11.1 , 9.8 hz , 1h ) , 4.37 ( d , j = 9.8 hz , 1h ) , 3.56 ( app td , j = 12.7 , 4.4 hz , 1h ) , 3.383.22 ( comp , 3h ) , 3.07 ( ddd , j = 15.8 , 9.3 , 6.3 hz , 1h ) , 2.98 ( app dt , j = 16.4 , 4.1 hz , 1h ) , 2.90 ( ddd , j = 9.9 , 6.1 , 3.6 hz , 1h ) , 2.172.09 ( m , 1h ) , 1.831.77 ( m , 1h ) ; c nmr ( 125 mhz , cdcl3 ) 139.9 , 134.2 , 133.0 , 129.5 , 129.2 , 128.1 , 128.0 , 127.5 , 127.0 , 126.3 , 91.0 , 63.1 , 54.5 , 49.0 , 45.9 , 29.8 , 28.2 ; m / z ( esi - ms ) 309.1 [ m + h ] ; hplc daicel chiralcel od - h , n - hexane / i - proh = 90/10 , flow rate = 1 ml / min , uv = 230 nm , tr = 11.1 min ( major ) and tr = 13.9 min ( minor ) , 94% ee . yellowish solid ; mp 150152 c ; rf = 0.31 in 30% etoac / hex ; [ ]d 50.1 ( c 0.5 , chcl3 ) ; ir ( kbr ) 3021 , 2941 , 2914 , 2845 , 2824 , 1644 , 1541 , 1485 , 1369 , 1309 , 1143 , 1102 , 1067 , 1105 , 811 , 766 , 755 cm ; h nmr ( 500 mhz , cdcl3 ) 7.317.27 ( comp , 2h ) , 7.247.19 ( m , 1h ) , 7.177.15 ( comp , 3h ) , 7.08 ( app t , j = 7.2 hz , 1h ) , 6.85 ( app d , j = 7.8 hz , 1h ) 4.91 ( app t , j = 9.6 hz , 1h ) , 4.37 ( d , j = 9.6 hz , 1h ) , 3.54 ( app td , j = 12.8 , 4.4 hz , 1h ) , 3.373.22 ( comp , 3h ) , 3.113.03 ( m , 1h ) , 3.012.97 ( m , 1h ) , 2.91 ( ddd , j = 10.1 , 5.9 , 3.9 hz , 1h ) , 2.152.05 ( m , 1h ) , 1.831.76 ( m , 1h ) ; c nmr ( 125 mhz , cdcl3 ) 138.2 , 134.1 , 133.9 , 132.5 , 129.6 , 129.4 , 128.8 , 128.3 , 126.9 , 126.4 , 90.9 , 63.0 , 54.4 , 48.3 , 46.1 , 29.7 , 28.1 ; m / z ( esi - ms ) ( cl ) 343.2 [ m + h ] , ( cl ) 345.2 yellowish solid ; mp 150153 c ; rf = 0.31 in 30% etoac / hex ; [ ]d 48.9 ( c 0.5 , chcl3 ) ; ir ( kbr ) 3021 , 2941 , 2914 , 2855 , 2824 , 1644 , 1541 , 1485 , 1451 , 1364 , 1309 , 1143 , 1102 , 1067 , 1105 , 811 , 766 , 749 cm ; h nmr ( 500 mhz , cdcl3 ) 7.467.40 ( comp , 2h ) , 7.247.18 ( m , 1h ) , 7.16 ( d , j = 7.1 hz , 1h ) , 7.127.05 ( comp , 3h ) , 6.84 ( app d , j = 7.8 hz , 1h ) , 4.89 ( dd , j = 11.2 , 9.6 hz , 1h ) , 4.34 ( d , j = 9.6 hz , 1h ) , 3.53 ( app td , j = 12.8 , 4.4 hz , 1h ) , 3.343.18 ( comp , 3h ) , 3.04 ( ddd , j = 15.5 , 9.1 , 6.2 hz , 1h ) , 2.97 ( app dt , j = 16.3 , 4.2 hz , 1h ) , 2.922.85 ( m , 1h ) , 2.112.02 ( m , 1h ) , 1.791.75 ( m , 1h ) ; c nmr ( 125 mhz , cdcl3 ) 138.9 , 134.2 , 132.8 , 132.3 , 129.6 , 129.2 , 128.2 , 126.9 , 126.3 , 121.9 , 90.8 , 63.1 , 54.4 , 48.5 , 46.0 , 29.8 , 28.2 ; m / z ( esi - ms ) ( br ) 387.1 [ m + h ] , ( br ) 389.1 [ m + h ] . yellowish solid ; mp 154156 c ; rf = 0.48 in 30% etoac / hex ; [ ]d 29.6 ( c 0.5 , chcl3 ) ; ir ( kbr ) 3021 , 2948 , 2917 , 2855 , 2824 , 1648 , 1548 , 1510 , 1364 , 1306 , 1140 , 1102 , 1067 , 1036 , 942 , 804 , 752 cm ; h nmr ( 500 mhz , cdcl3 ) 7.237.20 ( m , 1h ) , 7.197.16 ( m , 1h ) , 7.147.11 ( comp , 4h ) , 7.08 ( app t , j = 7.5 hz , 1h ) , 6.88 ( app d , j = 7.9 hz , 1h ) , 4.994.89 ( m , 1h ) , 4.37 ( d , j = 9.6 hz , 1h ) , 3.583.48 ( m , 1h ) , 3.363.30 ( m , 1h ) , 3.28 ( dd , j = 13.2 , 2.1 hz , 1h ) , 3.263.21 ( m , 1h ) , 3.103.04 ( m , 1h ) , 3.022.95 ( m , 1h ) , 2.912.87 ( m , 1h ) , 2.32 ( s , 3h ) , 2.172.06 ( m , 1h ) , 1.831.73 ( m , 1h ) ; c nmr ( 125 mhz , cdcl3 ) 137.6 , 137.0 , 134.2 , 133.1 , 129.9 , 129.5 , 128.1 , 127.3 , 127.0 , 126.3 , 91.2 , 63.1 , 54.5 , 48.6 , 45.9 , 29.9 , 28.3 , 21.3 ; m / z ( esi - ms ) 323.1 yellowish solid ; mp 120124 c ; rf = 0.26 in 30% etoac / hex ; [ ]d 31.3 ( c 0.5 , chcl3 ) ; ir ( kbr ) 3063 , 3031 , 2999 , 2927 , 2860 , 2833 , 1610 , 1546 , 1514 , 1452 , 1366 , 1306 , 1249 , 1175 , 1143 , 1101 , 1035 , 827 , 731 cm ; h nmr ( 500 mhz , cdcl3 ) 7.227.17 ( m , 1h ) , 7.177.10 ( comp , 3h ) , 7.06 ( app t , j = 7.5 hz , 1h ) , 6.896.80 ( comp , 3h ) , 4.88 ( dd , j = 11.2 , 9.7 hz , 1h ) , 4.34 ( d , j = 9.7 hz , 1h ) , 3.77 ( s , 3h ) , 3.543.45 ( m , 1h ) , 3.343.18 ( comp , 3h ) , 3.05 ( ddd , j = 15.7 , 9.3 , 6.3 hz , 1h ) , 2.96 ( app dt , j = 16.4 , 4.1 hz , 1h ) , 2.922.85 ( m , 1h ) , 2.09 ( ddd , j = 15.0 , 12.8 , 4.7 hz , 1h ) , 1.801.74 ( m , 1h ) ; c nmr ( 125 mhz , cdcl3 ) 159.2 , 134.2 , 133.0 , 132.0 , 129.5 , 128.5 , 128.1 , 127.0 , 126.3 , 114.5 , 91.4 , 63.1 , 55.4 , 54.5 , 48.2 , 45.9 , 29.8 , 28.3 ; m / z ( esi - ms ) 339.2 [ m + h ] . following general procedure a , product 6p was obtained in 60% yield . yellowish solid ; mp > 190 c ; rf = 0.45 in 30% etoac / hex ; [ ]d 12.5 ( c 0.5 , chcl3 ) ; ir ( kbr ) 3055 , 3017 , 2927 , 2855 , 2810 , 1600 , 1537 , 1496 , 1440 , 1357 , 1309 , 1140 , 1102 , 1063 , 939 , 856 , 745 cm ; h nmr ( 500 mhz , cd2cl2 ) 7.867.85 ( comp , 3h ) , 7.71 ( s , 1h ) , 7.567.45 ( comp , 2h ) , 7.40 ( app d , j = 8.4 hz , 1h ) , 7.267.22 ( comp , 2h ) , 7.09 ( app t , j = 6.2 hz , 1h ) , 6.86 ( app d , j = 7.8 hz , 1h ) , 5.11 ( app t , j = 9.6 hz , 1h ) , 4.43 ( d , j = 9.6 hz , 1h ) , 3.75 ( app td , j = 12.4 , 4.3 hz , 1h ) , 3.41 ( app td , j = 11.2 , 4.6 hz , 1h ) , 3.383.33 ( m , 1h ) , 3.29 ( dd , j = 13.0 , 3.2 hz , 1h ) , 3.152.99 ( comp , 2h ) , 2.992.88 ( m , 1h ) , 2.292.21 ( m , 1h ) , 1.871.85 ( m , 1h ) ; c nmr ( 125 mhz , cd2cl2 ) 137.6 , 134.8 , 133.7 , 133.3 , 133.0 , 129.6 , 128.9 , 128.0 , 127.9 , 127.8 , 126.9 , 126.5(8 ) , 126.5(5 ) , 126.2 , 126.0 , 125.1 , 91.0 , 63.1 , 54.3 , 49.1 , 45.7 , 29.8 , 28.0 ; m / z ( esi - ms ) 359.2 [ m + h ] . following general procedure a , product 6q was obtained in 50% yield . yellowish solid ; mp 128130 c ; rf = 0.35 in 30% etoac / hex ; [ ]d 35.2 ( c 0.5 , chcl3 ) ; ir ( kbr ) 3118 , 3062 , 3021 , 2952 , 2921 , 2858 , 1548 , 1496 , 1454 , 1361 , 1309 , 1146 , 1102 , 1081 , 1012 , 932 , 742 cm ; h nmr ( 500 mhz , cdcl3 ) 7.357.30 ( m , 1h ) , 7.227.17 ( m , 1h ) , 7.167.12 ( m , 1h ) , 7.07 ( app t , j = 7.5 hz , 1h ) , 6.84 ( app d , j = 7.8 hz , 1h ) , 6.26 ( dd , j = 3.3 , 1.9 hz , 1h ) , 6.11 ( app d , j = 3.3 hz , 1h ) , 4.92 ( app t , j = 9.8 hz , 1h ) , 4.32 ( d , j = 9.8 hz , 1h ) , 3.74 ( app td , j = 12.6 , 4.4 hz , 1h ) , 3.363.17 ( comp , 3h ) , 3.05 ( ddd , j = 16.3 , 9.9 , 6.5 hz , 1h ) , 2.93 ( app dt , j = 16.4 , 3.6 hz , 1h ) , 2.86 ( ddd , j = 11.3 , 6.4 , 2.9 hz , 1h ) , 2.222.14 ( m , 1h ) , 1.901.83 ( m , 1h ) ; c nmr ( 125 mhz , cdcl3 ) 152.9 , 142.5 , 134.1 , 132.7 , 129.5 , 128.2 , 127.0 , 126.2 , 110.5 , 106.7 , 88.9 , 62.7 , 53.8 , 45.4 , 41.8 , 29.7 , 24.8 ; m / z ( esi - ms ) 299.2 [ m + h ] . following general procedure a , product 6r was obtained in 44% yield . yellowish solid ; mp 150152 c ; rf = 0.29 in 30% etoac / hex ; [ ]d 35.1 ( c 0.5 , chcl3 ) ; ir ( kbr ) 3062 , 3024 , 2997 , 2927 , 2858 , 2834 , 1610 , 1541 , 1506 , 1454 , 1364 , 1306 , 1271 , 1250 , 1143 , 1032 , 763 , 693 cm ; h nmr ( 500 mhz , cdcl3 ) 7.327.29 ( comp , 2h ) , 7.25 ( d , j = 7.1 hz , 1h ) , 7.247.20 ( comp , 2h ) , 6.79 ( d , j = 8.6 hz , 1h ) , 6.68 ( d , j = 2.3 hz , 1h ) , 6.63 ( dd , j = 8.6 , 2.6 hz , 1h ) , 4.92 ( dd , j = 11.1 , 9.7 hz , 1h ) , 4.31 ( d , j = 9.7 hz , 1h ) , 3.76 ( s , 3h ) , 3.54 ( app td , j = 12.6 , 4.3 hz , 1h ) , 3.333.25 ( comp , 2h ) , 3.23 ( ddd , j = 13.7 , 4.5 , 1.8 hz , 1h ) , 3.03 ( ddd , j = 15.7 , 9.2 , 6.3 hz , 1h ) , 2.94 ( app dt , j = 15.7 , 4.0 hz , 1h ) , 2.892.85 ( m , 1h ) , 2.152.06 ( m , 1h ) , 1.841.74 ( m , 1h ) ; c nmr ( 125 mhz , cdcl3 ) 159.2 , 140.0 , 135.7 , 129.2 , 128.1 , 127.9 , 127.5 , 125.4 , 114.4 , 112.1 , 91.3 , 62.8 , 55.4 , 54.5 , 48.9 , 45.9 , 30.2 , 28.3 ; m / z ( esi - ms ) 339.1 [ m + h ] . following general procedure yellowish solid ; mp 148151 c ; rf = 0.42 in 50% etoac / hex ; [ ]d 18.9 ( c 0.5 , chcl3 ) ; ir ( kbr ) 3059 , 3028 , 2990 , 2945 , 2921 , 2889 , 2883 , 2855 , 2834 , 1610 , 1544 , 1520 , 1447 , 1354 , 1261 , 1226 , 1143 , 1112 , 1019 , 873 , 759 , 704 cm ; h nmr ( 500 mhz , cdcl3 ) 7.307.28 ( comp , 2h ) , 7.257.22 ( m , 1h ) , 7.227.18 ( comp , 2h ) , 6.61 ( s , 1h ) , 6.37 ( s , 1h ) , 4.89 ( dd , j = 11.2 , 9.6 hz , 1h ) , 4.27 ( d , j = 9.6 hz , 1h ) , 3.83 ( s , 3h ) , 3.72 ( s , 3h ) , 3.51 ( app td , j = 12.7 , 4.3 hz , 1h ) , 3.303.18 ( comp , 3h ) , 2.992.91 ( m , 1h ) , 2.892.83 ( comp , 2h ) , 2.10 ( ddd , j = 15.1 , 12.7 , 4.8 hz , 1h ) , 1.78 ( app ddt , j = 13.7 , 4.3 , 2.4 hz , 1h ) ; c nmr ( 125 mhz , cdcl3 ) 148.7 , 147.3 , 139.8 , 129.1 , 128.0 , 127.4 , 126.6 , 125.1 , 111.8 , 109.8 , 91.7 , 63.0 , 56.0(1 ) , 55.9(9 ) , 54.5 , 49.0 , 46.2 , 29.5 , 28.2 ; m / z ( esi - ms ) 369.1 [ m + h ] . following general procedure yellowish solid ; mp 144146 c ; ( rf = 0.31 in 30% etoac / hex ) ; [ ]d 38.5 ( c 0.5 , chcl3 ) ; ir ( kbr ) 3409 , 3056 , 3031 , 2947 , 2917 , 2846 , 2806 , 2757 , 1729 , 1598 , 1546 , 1492 , 1455 , 1356 , 1311 , 1264 , 1168 , 738 , 701 cm ; h nmr ( 500 mhz , cdcl3 ) 7.65 ( br s , 1h ) , 7.557.50 ( m , 1h ) , 7.397.33 ( comp , 2h ) , 7.337.29 ( m , 1h ) , 7.277.21 ( comp , 3h ) , 7.17 ( app td , j = 7.6 , 1.3 hz , 1h ) , 7.147.10 ( m , 1h ) , 4.88 ( dd , j = 11.2 , 9.5 hz , 1h ) , 4.25 ( d , j = 9.5 hz , 1h ) , 3.443.33 ( m , 1h ) , 3.253.18 ( comp , 2h ) , 3.052.93 ( comp , 2h ) , 2.922.78 ( comp , 2h ) , 2.212.13 ( m , 1h ) , 1.99 ( app ddt , j = 13.6 , 4.7 , 2.5 hz , 1h ) ; c nmr ( 125 mhz , cdcl3 ) 139.1 , 136.8 , 130.2 , 129.3 , 128.3 , 127.5 , 126.7 , 122.7 , 120.0 , 118.5 , 111.6 , 111.2 , 92.8 , 61.9 , 55.1 , 51.3 , 49.7 , 31.5 , 22.1 ; m / z ( esi - ms ) 348.2 [ m + h ] . following general procedure b , products 8a and 9a were obtained in a 1.2:1 ratio ( 62% combined yield ) . yellowish oil ; rf = 0.37 in 20% etoac / hex ; [ ]d + 24.8 ( c 0.5 , chcl3 ) ; ir ( kbr ) 3066 , 3031 , 2977 , 2895 , 2816 , 2776 , 2757 , 2678 , 1546 , 1494 , 1452 , 1375 , 1289 , 1151 , 1119 , 1072 , 1039 , 751 , 696 cm ; h nmr ( 400 mhz , cdcl3 ) 7.367.30 ( comp , 2h ) , 7.297.27 ( m , 1h ) , 7.217.12 ( comp , 4h ) , 7.087.03 ( comp , 2h ) , 4.66 ( dd , j = 11.1 , 8.2 hz , 1h ) , 4.03 ( d , j = 15.2 hz , 1h ) , 3.56 ( d , j = 15.2 hz , 1h ) , 3.19 ( dd , j = 18.5 , 10.4 hz , 1h ) , 3.012.92 ( comp , 2h ) , 2.932.85 ( m , 1h ) , 2.842.77 ( m , 1h ) , 2.262.13 ( comp , 2h ) , 0.78 ( d , j = 6.1 hz , 3h ) ; c nmr ( 75 mhz , cdcl3 ) 137.6 , 132.9 , 131.3 , 129.0 , 128.2 , 127.9 , 126.8 , 126.3 , 125.8 , 96.2 , 62.8 , 60.6 , 57.4 , 54.0 , 34.3 , 32.8 , 16.9 ; hrms ( esi ) m / z calcd for c20h23n2o2 [ m + h ] 323.1754 , found 323.1765 . yellowish solid ; mp 182185 c ; rf = 0.48 in 20% etoac / hex ; [ ]d 31.6 ( c 0.5 , chcl3 ) ; ir ( kbr ) 3068 , 3028 , 2975 , 2895 , 2813 , 2778 , 2763 , 1546 , 1494 , 1455 , 1375 , 1364 , 1289 , 1156 , 1115 , 1032 , 750 , 698 cm ; h nmr ( 400 mhz , cdcl3 ) 7.357.32 ( comp , 2h ) , 7.287.23 ( comp , 3h ) , 7.207.13 ( comp , 2h ) , 7.127.02 ( comp , 2h ) , 5.09 ( dd , j = 12.3 , 9.1 hz , 1h ) , 3.96 ( d , j = 15.1 hz , 1h ) , ( dd , j = 12.3 , 4.5 hz , 1h ) , 3.53 ( d , j = 15.1 hz , 1h ) , 3.163.02 ( comp , 2h ) , 2.922.82 ( comp , 2h ) , 2.70 ( dd , j = 11.5 , 3.2 hz , 1h ) , 2.312.14 ( m , 1h ) , 1.00 ( d , j = 7.2 hz , 3h ) ; c nmr ( 75 mhz , cdcl3 ) 138.1 , 133.2 , 131.5 , 128.6 , 128.2 , 127.7 , 127.5 , 126.7 , 126.2 , 125.8 , 89.7 , 62.0 , 61.7 , 57.8 , 49.8 , 35.0 , 33.1 , 13.3 ; m / z ( esi - ms ) 323.2 [ m + h ] . following general procedure b , products 8c and 9c were obtained in a 1.2:1 ratio ( 49% combined yield ) . yellow solid ; mp 180182 c ; rf = 0.31 in 20% etoac / hex ; [ ]d + 36.7 ( c 0.5 , chcl3 ) ; ir ( kbr ) 3068 , 3026 , 2962 , 2930 , 2883 , 1971 , 1929 , 1897 , 1714 , 1650 , 1546 , 1489 , 1452 , 1375 , 1262 , 1153 , 1007 , 815 , 758 , 738 cm ; h nmr ( 400 mhz , cdcl3 ) 7.497.43 ( comp , 2h ) , 7.207.12 ( comp , 2h ) , 7.097.04 ( comp , 4h ) , 4.59 ( dd , j = 11.4 , 8.4 hz , 1h ) , 4.02 ( d , j = 15.1 hz , 1h ) , 3.55 ( d , j = 15.1 hz , 1h ) , 3.213.13 ( m , 1h ) , 3.042.73 ( comp , 4h ) , 2.242.06 ( comp , 2h ) , 0.78 ( d , j = 6.1 hz , 3h ) ; c nmr ( 75 mhz , cdcl3 ) 136.6 , 132.7 , 132.1 , 131.1 , 128.2 , 126.7 , 126.3 , 125.8 , 121.8 , 95.9 , 62.6 , 60.5 , 57.2 , 53.4 , 34.2 , 32.7 , 16.7 ; m / z ( esi - ms ) ( br ) 401.1 [ m + h ] , ( br ) 403.1 [ m + h ] . yellow solid ; mp > 200 c ; rf = 0.53 in 20% etoac / hex ; [ ]d 32.2 ( c 0.5 , chcl3 ) ; ir ( kbr ) 3066 , 3025 , 2964 , 2928 , 2896 , 2811 , 2762 , 1892 , 1730 , 1652 , 1546 , 1489 , 1456 , 1377 , 1363 , 1306 , 1287 , 1257 , 1153 , 1108 , 1073 , 1009 , 825 , 800 , 749 cm ; h nmr ( 400 mhz , cdcl3 ) 7.517.44 ( comp , 2h ) , 7.207.14 ( comp , 2h ) , 7.147.03 ( comp , 4h ) , 5.02 ( dd , j = 12.3 , 8.9 hz , 1h ) , 3.96 ( d , j = 15.1 hz , 1h ) , 3.61 ( dd , j = 12.3 , 4.5 hz , 1h ) , 3.52 ( d , j = 15.1 hz , 1h ) , 3.123.01 ( comp , 2h ) , 2.942.80 ( comp , 2h ) , 2.68 ( dd , j = 11.6 , 3.2 hz , 1h ) , 2.242.13 ( m , 1h ) , 0.98 ( d , j = 7.2 hz , 3h ) ; c nmr ( 75 mhz , cdcl3 ) 137.1 , 133.1 , 131.8 , 131.4 , 129.4 , 128.2 , 126.7 , 126.3 , 125.8 , 121.5 , 89.5 , 61.8 , 61.6 , 57.7 , 49.3 , 34.8 , 33.1 , 13.2 ; m / z ( esi - ms ) ( br ) 401.2 [ m + h ] , ( br ) 403.2 [ m + h ] . following general procedure b , products 8e and 9e were obtained in a 1.3:1 ratio ( 54% combined yield ) . yellow solid ; mp 115117 c ; rf = 0.28 in 20% etoac / hex ; [ ]d + 25.1 ( c 0.5 , chcl3 ) ; ir ( kbr ) 3024 , 2965 , 2928 , 2894 , 2821 , 2762 , 2680 , 1959 , 1902 , 1796 , 1593 , 1545 , 1495 , 1373 , 1362 , 1255 , 1093 , 1011 , 933 , 818 , 745 cm ; h nmr ( 500 mhz , cdcl3 ) 7.187.13 ( comp , 2h ) , 7.127.07 ( comp , 2h ) , 7.097.02 ( comp , 2h ) , 6.886.84 ( comp , 2h ) , 4.59 ( dd , j = 11.4 , 8.7 hz , 1h ) , 4.01 ( d , j = 15.2 hz , 1h ) , 3.78 ( s , 3h ) , 3.54 ( d , j = 15.2 hz , 1h ) , 3.233.13 ( m , 1h ) , 2.992.88 ( comp , 2h ) , 2.962.80 ( comp , 2h ) , 2.222.10 ( comp , 2h ) , 0.78 ( d , j = 6.2 hz , 3h ) ; c nmr ( 125 mhz , cdcl3 ) 159.3 , 133.1 , 131.5 , 129.7 , 128.5 , 127.0 , 126.5 , 126.1 , 114.5 , 96.7 , 63.1 , 60.8 , 57.6 , 55.4 , 53.5 , 34.6 , 33.1 , 17.1 ; m / z ( esi - ms ) 353.2 [ m + h ] . yellow solid ; mp > 200 c ; ( rf = 0.45 in 20% etoac / hex ) ; [ ]d 31.6 ( c 0.5 , chcl3 ) ; ir ( kbr ) 3026 , 3013 , 2992 , 2969 , 2955 , 2899 , 2835 , 2780 , 2761 , 1966 , 1922 , 1884 , 1613 , 1548 , 1512 , 1456 , 1442 , 1399 , 1380 , 1355 , 1310 , 1300 , 1254 , 1178 , 1028 , 829 , 748 cm ; h nmr ( 500 mhz , cdcl3 ) 7.197.13 ( comp , 4h ) , 7.117.06 ( m , 1h ) , 7.067.02 ( m , 1h ) , 6.896.85 ( comp 2h ) , 5.03 ( dd , j = 12.3 , 9.2 hz , 1h ) , 3.95 ( d , j = 15.1 hz , 1h ) , 3.79 ( s , 3h ) , 3.58 ( dd , j = 12.3 , 4.5 hz , 1h ) , 3.52 ( d , j = 15.1 hz , 1h ) , 3.123.00 ( comp , 2h ) , 2.932.79 ( comp , 2h ) , 2.68 ( dd , j = 11.5 , 3.2 hz , 1h ) , 2.212.13 ( m , 1h ) , 1.00 ( d , j = 7.2 hz , 3h ) ; c nmr ( 125 mhz , cdcl3 ) 159.1 , 133.4 , 131.7 , 130.3 , 129.0 , 128.4 , 126.9 , 126.4 , 126.0 , 114.3 , 90.2 , 62.2 , 61.9 , 58.0 , 55.5 , 49.3 , 35.3 , 33.4 , 13.6 ; m / z ( esi - ms ) 353.2 [ m + h ] . following general procedure b , product 8k was obtained in 48% yield . yellow solid ; mp 160163 c ; rf = 0.47 in 30% etoac / hex ; [ ]d + 27.2 ( c 0.5 , chcl3 ) ; ir ( kbr ) 3061 , 3026 , 2932 , 2865 , 2799 , 2749 , 1721 , 1650 , 1541 , 1437 , 1366 , 1240 , 1141 , 1141 , 738 cm ; h nmr ( 400 mhz , cdcl3 ) 7.377.30 ( comp , 2h ) , 7.307.27 ( m , 1h ) , 7.257.20 ( comp , 2h ) , 7.197.13 ( comp , 2h ) , 7.097.02 ( comp , 2h ) , 4.62 ( dd , j = 11.4 , 8.6 hz , 1h ) , 4.03 ( d , j = 15.1 hz , 1h ) , 3.58 ( d , j = 15.1 hz , 1h ) , 3.30 ( app td , j = 11.8 , 4.8 hz , 1h ) , 3.23 ( app dt , j = 11.8 , 3.0 hz , 1h ) , 3.022.91 ( comp 2h ) , 2.892.78 ( m , 1h ) , 2.49 ( app td , j = 11.8 , 3.6 hz , 1h ) , 2.161.95 ( comp , 2h ) ; c nmr ( 75 mhz , cdcl3 ) 139.2 , 132.9 , 131.3 , 129.0 , 128.2 , 128.0 , 127.2 , 126.8 , 126.3 , 125.8 , 95.7 , 60.6 , 57.5 , 55.1 , 47.2 , 32.9 , 30.9 ; m / z ( esi - ms ) 309.1 [ m + h ] . yellow solid ; mp 165167 c ; rf = 0.48 in 30% etoac / hex ; [ ]d + 18.2 ( c 0.5 , chcl3 ) ; ir ( kbr ) 3042 , 2965 , 2928 , 2894 , 2821 , 2762 , 2680 , 1959 , 1902 , 1796 , 1593 , 1543 , 1495 , 1373 , 1362 , 1351 , 1255 , 1093 , 1085 , 1011 , 933 , 818 , 745 cm ; h nmr ( 500 mhz , cdcl3 ) 7.347.29 ( comp , 2h ) , 7.187.13 ( comp , 4h ) , 7.107.04 ( comp , 2h ) , 4.56 ( dd , j = 11.3 , 8.6 hz , 1h ) , 4.03 ( d , j = 15.2 hz , 1h ) , 3.58 ( d , j = 15.2 hz , 1h ) , 3.29 ( app td , j = 11.4 , 5.5 hz , 1h ) , 3.253.18 ( m , 1h ) , 3.022.91 ( comp , 2h ) , 2.872.79 ( comp , 2h ) , 2.47 ( app td , j = 11.6 , 4.4 hz , 1h ) , 2.101.99 ( comp , 2h ) ; c nmr ( 125 mhz , cdcl3 ) 137.6 , 133.7 , 132.8 , 131.1 , 129.2 , 128.5 , 128.1 , 126.7 , 126.2 , 125.7 , 95.4 , 60.4 , 57.3 , 54.9 , 46.5 , 32.8 , 30.7 ; m / z ( esi - ms ) ( cl ) 343.2 [ m + h ] , ( cl ) 345.2 [ m + h ] . yellow solid ; mp 175178 c ; rf = 0.51 in 30% etoac / hex ; [ ]d + 19.4 ( c 0.5 , chcl3 ) ; ir ( kbr ) 3021 , 2947 , 2922 , 2894 , 2820 , 2759 , 2687 , 2356 , 1924 , 1897 , 1548 , 1515 , 1496 , 1456 , 1376 , 1351 , 1256 , 1152 , 811 , 748 cm ; h nmr ( 400 mhz , cdcl3 ) 7.217.12 ( comp , 6h ) , 7.117.05 ( comp , 2h ) , 4.61 ( dd , j = 11.4 , 8.7 hz , 1h ) , 4.04 ( d , j = 15.2 hz , 1h ) , 3.59 ( d , j = 15.2 hz , 1h ) , 3.29 ( dd , j = 11.8 , 4.7 hz , 1h ) , 3.23 ( app dt , j = 11.8 , 3.2 hz , 1h ) , 3.042.93 ( comp , 2h ) , 2.86 ( dd , j = 13.7 , 7.0 hz , 1h ) , 2.48 ( app td , j = 11.8 , 3.5 hz , 1h ) , 2.34 ( s , 3h ) , 2.151.99 ( comp , 2h ) ; c nmr ( 75 mhz , cdcl3 ) 137.6 , 136.2 , 132.9 , 131.3 , 129.7 , 128.2 , 127.0 , 126.8 , 126.3 , 125.8 , 95.8 , 60.6 , 57.5 , 55.1 , 46.8 , 32.9 , 30.9 , 21.1 ; m / z ( esi - ms ) 323.1[m + h ] . following general procedure c , products 11 and 12 were obtained in a 2:1 ratio ( 55% combined yield ) . yellowish oil ; rf = 0.40 in 50% etoac / hex ; ir ( kbr ) 3056 , 3033 , 2932 , 2851 , 2804 , 2754 , 1529 , 1489 , 1452 , 1368 , 1306 , 1143 , 740 cm ; h nmr ( 500 mhz , cdcl3 ) 7.12 ( app t , j = 7.4 hz , 1h ) , 7.05 ( app d , j = 7.5 hz , 1h ) , 7.00 ( app t , j = 7.5 hz , 1h ) , 6.78 ( app d , j = 7.5 hz , 1h ) , 4.744.62 ( m , 1h ) , 4.20 ( d , j = 9.3 hz , 1h ) , 3.153.07 ( m , 1h ) , 3.042.93 ( comp , 2h ) , 2.902.85 ( comp , 2h ) , 2.75 ( app dt , j = 11.1 , 5.3 hz , 1h ) , 2.302.23 ( comp , 2h ) 1.821.72 ( m , 1h ) , 1.631.55 ( m , 1h ) ; c nmr ( 125 mhz , cdcl3 ) 134.4 , 133.3 , 129.6 , 127.9 , 126.6 , 126.3 , 86.0 , 62.6 , 53.8 , 46.6 , 31.1 , 29.3 , 20.0 ; m / z ( esi - ms ) 233.2 yellowish oil ; rf = 0.50 in 50% etoac / hex ; ir ( kbr ) 3061 , 3026 , 2932 , 2865 , 2799 , 2749 , 1721 , 1650 , 1541 , 1437 , 1366 , 1240 , 1141 , 1141 , 738 cm ; h nmr ( 500 mhz , cdcl3 ) 7.227.13 ( comp , 3h ) , 7.127.09 ( m , 1h ) , 5.18 ( app dt , j = 4.5 , 2.3 hz , 1h ) , 3.68 ( s , 1h ) , 3.16 ( ddd , j = 16.9 , 12.2 , 5.3 hz , 1h ) , 3.123.07 ( m , 1h ) , 3.00 ( ddd , j = 11.1 , 5.3 , 1.7 hz , 1h ) , 2.692.60 ( comp , 2h ) , 2.592.50 ( m , 1h ) , 2.41 ( app td , j = 12.0 , 3.0 hz , 1h ) , 2.262.15 ( m , 1h ) , 1.94 ( ddd , j = 18.5 , 9.3 , 4.9 hz , 1h ) , 1.721.61 ( m , 1h ) ; c nmr ( 125 mhz , cdcl3 ) 135.9 , 133.8 , 129.4 , 126.8 , 126.2 , 124.4 , 82.7 , 65.6 , 56.5 , 52.7 , 29.5 , 28.9 , 21.1 ; m / z ( esi - ms ) 233.2 [ m + h ] . following general procedure b ( 5 equiv of 2-eha was used ) , product 13 was obtained in 61% yield . yellowish solid ; mp 7577 c ; ( rf = 0.43 in 50% etoac / hex ) ; ir ( kbr ) 3066 , 3028 , 2950 , 2820 , 2754 , 2687 , 2361 , 2329 , 1964 , 1919 , 1842 , 1805 , 1546 , 1496 , 1463 , 1455 , 1357 , 1347 , 1277 , 1237 , 1218 , 1117 , 1105 , 744 cm ; h nmr ( 400 mhz , cdcl3 ) 7.177.10 ( comp , 2h ) , 7.086.99 ( comp , 2h ) , 4.454.40 ( m , 1h ) , 3.95 ( d , j = 15.2 hz , 1h ) , 3.53 ( d , j = 15.2 hz , 1h ) , 3.123.05 ( m , 1h ) , 2.912.80 ( comp , 3h ) , 2.402.32 ( m , 1h ) , 2.27 ( app td , j = 12.1 , 2.8 hz , 1h ) , 2.091.99 ( m , 1h ) , 1.951.87 ( m , 1h ) , 1.78 ( ddd , j = 16.3 , 8.4 , 3.5 hz , 1h ) ; c nmr ( 75 mhz , cdcl3 ) 133.0 , 131.5 , 128.2 , 126.6 , 126.2 , 125.8 , 89.8 , 59.9 , 57.5 , 54.7 , 32.7 , 30.1 , 22.8 ; m / z ( esi - ms ) 233.2 [ m + h ] . compound 8k was reduced following an adapted literature procedure . to a stirred solution of 8k ( 0.09 mmol , 0.053 g ) in i - proh ( 2 ml ) was added 1 m hcl ( 1.8 mmol , 1.8 ml ) followed by zn dust ( 5.45 mmol , 0.356 g ) . the reaction mixture was stirred at room temperature for 2 h. the reaction was then quenched by addition of saturated nahco3 ( aq ) . the resulting mixture was stirred vigorously for 20 min and then filtered through a plug of celite and washed with etoac . the filtrate was extracted with etoac , and the combined organic layers were dried over na2so4 , filtered , and evaporated under reduced pressure . the residue was purified by flash chromatography to give the title compound as a white solid in 83% yield . mp 122125 c ; rf = 0.25 in 5% meoh / etoac ; [ ]d + 66.8 ( c 0.5 , chcl3 ) ; ir ( kbr ) ; 3409 , 3061 , 3024 , 2919 , 2801 , 2755 , 1949 , 1599 , 1588 , 1553 , 1493 , 1453 , 1362 , 1346 , 1120 , 1096 , 1011 , 863 , 748 cm ; h nmr ( 500 mhz , cdcl3 ) 7.387.32 ( comp , 2h ) , 7.307.24 ( comp , 3h ) , 7.177.10 ( comp , 3h ) , 7.077.02 ( m , 1h ) , 3.95 ( d , j = 15.2 hz , 1h ) , 3.50 ( d , j = 15.2 hz , 1h ) , 3.31 ( dd , j = 16.5 , 4.4 hz , 1h ) , 3.19 ( app dt , j = 11.5 , 3.3 hz , 1h ) , 2.872.77 ( comp , 2h ) , 2.492.41 ( m , 1h ) , 2.38 ( app td , j = 12.2 , 2.8 hz , 1h ) , 2.23 ( app dq , j = 10.5 hz , 4.3 hz , 1h ) , 2.05 ( app qd , j = 12.8 , 3.7 hz , 1h ) , 1.931.86 ( m , 1h ) , 1.24 ( br s , 2h ) ; c nmr ( 125 mhz , cdcl3 ) 143.2 , 133.6 , 133.3 , 128.8 , 128.3 , 127.8 , 126.9 , 126.4 , 125.8 , 125.7 , 64.8 , 59.6 , 58.1 , 55.8 , 51.3 , 34.0 , 32.2 ; m / z ( esi - ms ) 279.2 [ m + h ] . to a stirred solution of 6k ( 31 mg , 0.1 mmol , 94% ee ) in etoh ( 2 ml ) was added pd(oh)2/c ( 20 wt % , 75 mg ) . the reaction mixture was purged twice with hydrogen gas and then placed under 1 bar of hydrogen gas at 60 c for 14 h. after being cooled to room temperature , the mixture was filtered through a celite pad and washed with ethyl acetate . the solvent was removed under reduced pressure , and the crude material purified by flash chromatography to give the desired product as an off - white solid in 75% yield . mp 124127 c ; rf = 0.41 in 40% etoac / hex ; [ ]d 27.2 ( c 0.5 , chcl3 ) ; ir ( kbr ) 3058 , 3032 , 2942 , 2915 , 2801 , 2742 , 1598 , 1494 , 1450 , 1348 , 1296 , 1151 , 1131 , 1104 , 1064 , 1039 , 963 , 758 , 733 , 696 cm ; h nmr ( 500 mhz , cdcl3 ) 7.367.27 ( comp , 4h ) , 7.257.18 ( comp , 2h ) , 7.167.09 ( comp , 3h ) , 3.32 ( app d , j = 11.0 hz , 1h ) , 3.23 ( ddd , j = 17.0 , 12.1 , 6.1 hz , 1h ) , 3.15 ( app dt , j = 11.4 , 3.1 hz , 1h ) , 3.06 ( dd , j = 11.4 , 6.0 hz , 1h ) , 2.872.72 ( comp , 2h ) , 2.60 ( app td , j = 11.6 , 3.9 hz , 1h ) , 2.542.49 ( comp , 2h ) , 2.031.87 ( comp , 2h ) , 1.70 ( app q , j = 12.4 hz , 1h ) ; c nmr ( 125 mhz , cdcl3 ) 146.4 , 138.1 , 134.7 , 129.2 , 128.7 , 127.2 , 126.5 , 126.3 , 126.0 , 125.0 , 63.5 , 57.1 , 52.6 , 43.7 , 39.2 , 33.3 , 29.9 ; m / z ( esi - ms ) 264.3 [ m + h ] ; hplc daicel chiralcel od - h , n - hexane / i - proh / diethylamine = 94.95/5/0.05 , flow rate = 1 ml / min , uv = 254 nm , tr = 6.2 min ( major ) and tr = 22.1 min ( minor ) , 94% ee . alkylation of 11 was achieved following an adapted literature procedure . to a stirred solution of 11 ( 0.1 mmol , 0.023 g ) and methyl vinyl ketone ( 0.13 mmol , 0.01 ml ) in mecn ( 1 ml ) was added dbu ( 0.1 mmol , 0.015 ml ) at room temperature . solvent was removed under reduced pressure , and the crude material purified by flash chromatography to give the title compound as a colorless oil in 69% yield . the relative stereochemistry was determined by gcosy and noesy nmr : rf = 0.32 in 20% etoac / hex ; ir ( kbr ) 3073 , 3021 , 2962 , 2900 , 2814 , 2759 , 1719 , 1645 , 1521 , 1450 , 1358 , 1299 , 1252 , 1141 , 1106 , 1052 , 745 cm ; h nmr ( 400 mhz , cdcl3 ) 7.197.11 ( m , 1h ) , 7.087.03 ( comp , 2h ) , 6.69 ( app d , j = 7.9 hz , 1h ) , 4.27 ( s , 1h ) , 3.183.06 ( m , 1h ) , 3.032.99 ( m , 1h ) , 2.90 ( app dd , j = 10.8 , 3.0 hz , 1h ) , 2.792.63 ( comp , 2h ) , 2.592.45 ( comp , 4h ) , 2.362.21 ( m , 1h ) , 2.152.02 ( comp , 4h ) , 1.951.87 ( m , 1h ) , 1.801.61 ( comp , 2h ) ; c nmr ( 75 mhz , cdcl3 ) 207.6 , 137.4 , 132.1 , 129.2 , 127.0 , 126.2 , 125.6 , 94.5 , 69.7 , 55.7 , 51.1 , 37.8 , 34.7 , 30.9 , 30.0 , 22.5 , 22.4 ; m / z ( esi - ms ) 303.2 [ m + h ] . to a stirred solution of ( e)-(((4-nitrobut-3-en-1-yl)oxy)methyl)benzene ( 0.858 g , 4.14 mmol , 1 equiv ) , ( s)-diphenylprolinol trimethylsilyl ether ( 0.135 g , 0.414 mmol , 10 mol % ) , and p - nitrophenol ( 0.028 g , 0.207 mmol , 5 mol % ) in toluene ( 4.14 ml ) was added n - butyraldehyde ( 1.12 ml , 12.42 mmol , 3 equiv ) at room temperature . the reaction mixture was stirred for 14 h and quenched by the addition of 1 m hcl ( 10 ml ) , and the resulting mixture was extracted with etoac ( 2 10 ml ) . solvent was removed under reduced pressure and the residue purified by silica gel chromatography to give the title compound as a colorless oil in 73% yield ( dr = 6.5:1 ) . colorless oil ; rf = 0.35 in 10% etoac / hex ; [ ]d + 3.76 ( c 0.25 , chcl3 ) ; ir ( kbr ) 3032 , 2967 , 2876 , 2735 , 1722 , 1555 , 1496 , 1454 , 1382 , 1204 , 1100 , 1027 , 739 , 698 cm ; h nmr ( 500 mhz , cdcl3 ) 9.67 ( d , j = 1.2 hz , 1h ) , 7.387.28 ( m , 5h ) , 4.62 ( dd , j = 12.8 , 6.6 hz , 1h ) , 4.464.45 ( comp , 2h ) , 4.42 ( dd , j = 12.8 , 6.5 hz , 1h ) , 3.51 ( t , j = 5.8 hz , 2h ) , 2.902.84 ( m , 1h ) , 2.492.40 ( m , 1h ) , 1.861.62 ( comp , 3h ) , 1.541.46 ( m , 1h ) , 1.00 ( t , j = 7.4 hz , 3h ) ; c nmr ( 125 mhz , cdcl3 ) 202.9 , 137.8 , 128.4 , 127.7(4 ) , 127.6(8 ) , 76.9 , 73.1 , 67.4 , 54.0 , 34.5 , 29.1 , 18.5 , 12.1 ; m / z ( esi - ms ) 280.2 [ m + h ] ; hplc daicel chiralcel oj - h , n - hexane / i - proh = 90/10 , flow rate = 1 ml / min , uv = 230 nm , tr = 78.2 min ( major ) and tr = 96.4 min ( minor ) , 99% ee . a solution of the amine ( 0.232 g , 1.2 mmol , 2 equiv ) and acoh ( 0.346 ml , 6 mmol , 10 equiv ) in toluene ( 6 ml ) was heated under reflux . 4-nitrobutyraldehyde 18 ( 0.167 g , 0.6 mmol , 0.12 m solution in toluene ) was delivered through the top of the reflux condenser over 15 h via syringe pump . the reaction was then kept under reflux for a further 0.51 h at which time the aldehyde was consumed as judged by tlc analysis . subsequently , the reaction mixture was allowed to cool to room temperature , diluted with etoac ( 20 ml ) , and washed with saturated aqueous nahco3 ( 3 20 ml ) . the combined aqueous layers were extracted with etoac ( 2 10 ml ) , and the combined organic layers were dried over anhydrous na2so4 . solvent was removed under reduced pressure and the residue purified by silica gel chromatography . products 19 , 19 , and 19 were obtained in a 2:2:1 ratio , 61% combined yield . the relative stereochemistry was determined by gcosy and noesy nmr : brown oil ; rf = 0.45 in 50% etoac / hex ; [ ]d 16.4 ( c 0.25 , chcl3 ) ; ir ( kbr ) 3061 , 2959 , 2865 , 2831 , 1741 , 1682 , 1631 , 1539 , 1519 , 1467 , 1356 , 1262 , 1225 , 1143 , 1111 , 740 cm ; h nmr ( 500 mhz , cdcl3 ) 7.387.19 ( comp , 5h ) , 6.58 ( s , 1h ) , 6.34 ( s , 1h ) , 4.884.77 ( m , 1h ) , 4.47 ( d , j = 12.0 hz , 1h ) , 4.43 ( d , j = 12.0 hz , 1h ) , 4.09 ( d , j = 9.8 hz , 1h ) , 3.83 ( s , 3h ) , 3.76 ( s , 3h ) , 3.48 ( t , j = 6.6 hz , 2h ) , 3.13 ( dd , j = 13.5 , 4.0 hz , 1h ) , 3.022.97 ( m , 1h ) , 2.922.85 ( m , 1h ) , 2.812.62 ( comp , 3h ) , 2.252.18 ( m , 1h ) , 1.891.82 ( m , 1h ) , 1.801.58 ( comp , 3h ) , 1.231.09 ( m , 1h ) , 0.90 ( t , j = 7.4 hz , 3h ) ; c nmr ( 125 mhz , cdcl3 ) 148.7 , 147.2 , 138.4 , 128.6 , 127.7(7 ) , 127.7(5 ) , 126.4 , 125.4 , 111.8 , 109.8 , 91.2 , 73.3 , 67.0 , 62.8 , 59.0 , 56.1 , 56.0 , 46.1 , 43.6 , 33.8 , 29.4 , 28.2 , 23.3 , 11.0 ; m / z ( esi - ms ) 455.1 [ m + h ] . the relative stereochemistry was determined by gcosy and noesy nmr : brown oil ; rf = 0.42 in 20% etoac / hex ; [ ]d 69.9 ( c 0.25 , chcl3 ) ; ir ( kbr ) 3026 , 3009 , 2937 , 2865 , 2806 , 1726 , 1608 , 1551 , 1462 , 1363 , 1269 , 1225 , 1096,1010 , 876 , 743 , 698 cm ; h nmr ( 500 mhz , cdcl3 ) 7.387.26 ( comp , 5h ) , 6.56 ( s , 1h ) , 6.41 ( s , 1h ) , 4.52 ( d , j = 11.9 hz , 1h ) , 4.44 ( d , j = 11.9 hz , 1h ) , 4.394.30 ( m , 1h ) , 4.01 ( d , j = 8.9 hz , 1h ) , 3.83 ( s , 3h ) , 3.75 ( s , 3h ) , 3.503.43 ( comp , 2h ) , 3.002.94 ( comp , 2h ) , 2.88 ( ddd , j = 11.0 , 4.9 , 3.0 hz , 1h ) , 2.792.68 ( comp , 2h ) , 2.592,54 ( comp , 2h ) , 1.741.53 ( comp , 4h ) , 1.521.42 ( m , 1h ) , 1.421.31 ( m , 1h ) , 0.88 ( t , j = 7.3 hz , 3h ) ; c nmr ( 125 mhz , cdcl3 ) 148.1 , 147.4 , 138.4 , 128.6 , 128.4 , 127.9(2 ) , 127.8(5 ) , 125.8 , 111.8 , 108.8 , 92.4 , 73.1 , 67.0 , 65.0 , 57.0 , 56.0 , 51.3 , 42.7 , 37.2 , 30.4 , 28.2 , 18.9 , 12.5 ; m / z ( esi - ms ) 455.1 [ m + h ] . brown oil ; rf = 0.55 in 50% etoac / hex ; [ ]d + 49.4 ( c 0.25 , chcl3 ) ; ir ( kbr ) 3063 , 3033 , 2959 , 2932 , 2860 , 2801 , 2754 , 1734 , 1615 , 1541 , 1519 , 1363 , 1264 , 1104 , 740 , 696 cm ; h nmr ( 500 mhz , cdcl3 ) 7.407.26 ( comp , 5h ) , 6.56 ( s , 1h ) , 6.50 ( s , 1h ) , 5.53 ( app t , j = 2.1 hz , 1h ) , 4.62 ( d , j = 12.0 hz , 1h ) , 4.56 ( d , j = 12.0 hz , 1h ) , 3.83 ( s , 3h ) , 3.80 ( s , 1h ) , 3.803.73 ( m , 1h ) , 3.723.67 ( m , 1h ) , 3.55 ( s , 3h ) , 3.203.07 ( m , 1h ) , 3.072.95 ( m , 1h ) , 2.86 ( dd , j = 10.8 , 3.0 hz , 1h ) , 2.812.70 ( m , 1h ) , 2.592.46 ( comp , 2h ) , 2.392.19 ( comp , 2h ) , 1.921.74 ( comp , 2h ) , 1.351.23 ( comp , 2h ) , 0.94 ( t , j = 7.4 hz , 3h ) ; c nmr ( 125 mhz , cdcl3 ) 147.6 , 147.2 , 137.8 , 128.5 , 127.9 , 127.8 , 127.5 , 125.5 , 111.6 , 107.2 , 86.0 , 73.5 , 70.1 , 59.8 , 56.9 , 55.8 , 55.7 , 52.7 , 39.8 , 36.3 , 28.9 , 25.1 , 23.1 , 11.5 ; m / z ( esi - ms ) 455.1 [ m + h ] . to a stirred solution of starting material 19 ( 91 mg , 0.2 mmol ) in etoh ( 4 ml ) the reaction mixture was purged twice with hydrogen gas and then placed under 1 bar of hydrogen gas at 70 c for 30 h. after being cooled to room temperature , the mixture was filtered through a celite pad and washed with ethyl acetate . the solvent was removed under reduced pressure and the crude material purified by flash chromatography to give the desired product as a yellow oil in 64% yield . ( )-protoemetinol was previously reported , and its published characterization data matched our own in all respects . yellow oil ; rf = 0.45 in 30% meoh / etoac ; [ ]d 46.5 ( c 0.6 , chcl3 ) ; ir ( kbr ) 3422 , 2956 , 2931 , 2874 , 2868 , 2751 , 1610 , 1510 , 1463 , 1365 , 1330 , 1253 , 1230 , 1148 , 1100 , 1040 , 1000 , 867 , 767 , 732 ; h nmr ( 500 mhz , cdcl3 ) 6.67 ( s , 1h ) , 6.56 ( s , 1h ) , 3.84 ( s , 3h ) , 3.83 ( s , 3h ) , 3.793.69 ( comp , 2h ) , 3.143.00 ( comp , 3h ) , 2.992.93 ( m , 1h ) , 2.61 ( app d , j = 15.9 hz , 1h ) , 2.47 ( app td , j = 11.5 , 3.9 hz , 1h ) , 2.33 ( app d , j = 12.8 hz , 1h ) , 2.051.97 ( m , 1h ) , 1.93 ( app dt , j = 11.1 , 7.8 hz , 1h ) , 1.701.63 ( m , 1h ) , 1.491.36 ( comp , 3h ) , 1.281.18 ( m , 1h ) , 1.161.06 ( m , 1h ) , 0.91 ( t , j = 7.5 hz , 3h ) ; c nmr ( 125 mhz , cdcl3 ) 147.4 , 147.1 , 130.0 , 126.7 , 111.5 , 108.3 , 62.7 , 61.4 , 60.6 , 56.1 , 55.8 , 52.5 , 41.3 , 37.6 , 37.3 , 35.9 , 29.1 , 23.5 , 11.1 ; hrms ( esi ) m / z calcd for c19h30no3 [ m + h ] 320.2220 , found 320.2225 .

cyclic amines such as 1,2,3,4-tetrahydroisoquinoline undergo regiodivergent annulation reactions with 4-nitrobutyraldehydes . these redox - neutral transformations enable the asymmetric synthesis of highly substituted polycyclic ring systems in just two steps from commercial materials . the utility of this process is illustrated in a rapid synthesis of ( )-protoemetinol . computational studies provide mechanistic insights and implicate the elimination of acetic acid from an ammonium nitronate intermediate as the rate - determining step .

chronic myeloid leukemia ( cml ) is the most common leukemia seen at regional cancer center , indira gandhi institute of medical sciences , sheikhpura , patna and bihar , india . cml cases constitute about 70% of total leukemia cases seen at our institution , whereas in india , cml accounts for 30% to 60% of all adult leukemia . in india , its incidence varies from 0.8 to 2.2 per hundred thousand population in males and 0.6 - 1.6/1 lakh population in females . the introduction of imatinib mysalate in 2002 has virtually changed the prognosis of patients with cml . majority of our patients were from the various districts of bihar , with a large number of cases coming from districts of samastipur , patna and dharbhanga . furthermore , many of these patients came from neighboring states such as jharkhand , uttar pradesh , especially from the eastern part , delhi , west - bengal , madhya pradesh , orissa and chhattisgarh . approximately , 85% of patients were from a low to middle socio - economic strata and hence were unable to afford imatinib . they extremely benefited from glivec international patient assistance program as our center is the only approved center in the entire state of bihar . we are thankful to max foundation and novartis oncology access program for their wholehearted support to the poor and needy patients . here , we report our experience of patients of cml undergoing treatment in limited resource set - up . this is a retrospective analysis and the data was extracted from clinical case files of patients registered at indira gandhi institute of medical sciences since january 2002 to december 2009 . all patients were philadelphia chromosome positive ( examined by fluorescence in situ hybridization method on bone marrow sample ) and were treated with imatinib mesylate . the aims and objective of this retrospective analysis were to document the clinical profile of patients of cml patients , response and compliance to imatinib and to evaluate its toxicity profile . the total number of patients registered and treated with imatinib mesylate during this period was 205 , out of which females were 54 ( 26% ) versus males who were 151 ( 74% ) . it was observed that 6 ( 3% ) of patients were less than 20 years of age ; whereas 135 ( 66% ) were between 20 and 40 and 64 ( 31% ) were above 40 years of age . clinical characteristics majority presented to us in chronic phase except 5 ( 2.4% ) who presented initially in blast crises / accelerated phase . total of 189 ( 92% ) of patients had fatigue and weakness at the time of diagnosis . splenomegaly a leading manifestation was associated in 166 ( 81% ) of patients , whereas lymphadenopathy was present in 4 ( 2% ) . at the time of diagnosis 84 ( 41% ) patients had fever . anemia was present in 75 ( 37% ) of patients and in 29 ( 14% ) cases had less than 8 g / dl of hemoglobin . the median time from diagnosis to start of imatinib therapy was 2.6 months ( ranged from 24 days to 4 years ) in our subset of patients . patients were monitored on a 3 monthly basis with hemogram , liver and kidney profile , a complete physical examination as far as possible . analysis for bcr - abl was repeated after 6 month 2 years depending upon affordability of the patients . cytogenetic response was assessed in 127 ( 62% ) of patients at one point of time after initiating therapy . one of them while on treatment for cml underwent a medical termination of pregnancy ; while the other was diagnosed with cml during the 7 month of pregnancy and imatinib was started only after the uneventful delivery of a full - term normal baby boy . she has been on follow - up and is in complete molecular remission at the end of 1 year of treatment . responses to treatment 117 ( 57% ) patient presented with non - hematological toxicities in the form of hypopigmentation 87 ( 42% ) , rashes 49 ( 12% ) . pigmentation over malar area was seen in the majority of cases while on imatinib therapy . fluid retention 13 ( 6% ) , weight gain 35 ( 17% ) , musculoskeletal pain was also associated in 31 ( 15% ) as shown in table 3 . common adverse event to imatinib mesylate hematological toxicity in the form of anemia was seen in 13 ( 6% ) , which required blood transfusion during treatment . liver toxicity was associated in the form of deranged enzyme levels in 17 ( 8% ) , which however did not require treatment interruption . ototoxicity was surprisingly seen in 5 ( 2.4% ) patients , one whom presented just within 3 days of start of imatinib . a total of 197 ( 96% ) patients were alive at the time of analysis of which 179 ( 87% ) were still in chronic phase with hematological remission . about 6 patients were in accelerated phase and were being treated with higher dose of imatinib . over all 182 ( 89% ) of patients said they had a better quality - of - life with treatment . analysis for bcr - abl was repeated after 6 month 2 years depending upon affordability of the patients . cytogenetic response was assessed in 127 ( 62% ) of patients at one point of time after initiating therapy . one of them while on treatment for cml underwent a medical termination of pregnancy ; while the other was diagnosed with cml during the 7 month of pregnancy and imatinib was started only after the uneventful delivery of a full - term normal baby boy . she has been on follow - up and is in complete molecular remission at the end of 1 year of treatment . 117 ( 57% ) patient presented with non - hematological toxicities in the form of hypopigmentation 87 ( 42% ) , rashes 49 ( 12% ) . pigmentation over malar area was seen in the majority of cases while on imatinib therapy . fluid retention 13 ( 6% ) , weight gain 35 ( 17% ) , musculoskeletal pain was also associated in 31 ( 15% ) as shown in table 3 . common adverse event to imatinib mesylate hematological toxicity in the form of anemia was seen in 13 ( 6% ) , which required blood transfusion during treatment . liver toxicity was associated in the form of deranged enzyme levels in 17 ( 8% ) , which however did not require treatment interruption . ototoxicity was surprisingly seen in 5 ( 2.4% ) patients , one whom presented just within 3 days of start of imatinib . a total of 197 ( 96% ) patients were alive at the time of analysis of which 179 ( 87% ) were still in chronic phase with hematological remission . about 6 patients were in accelerated phase and were being treated with higher dose of imatinib . over all 182 ( 89% ) of patients said they had a better quality - of - life with treatment . bihar is a poor state with the per capita income being one of the lowest in the country . it has been shown in a number of studies that cml tends to afflict people at a younger age in asia than in western nations . instead of being diagnosed in their 40s and 50s , patients in india are more likely to be in their 20s and 30s . our analysis confirmed that the relative age of patient presenting to us was younger than western population . the male : female ratio was 3:1 , which is more than that documented in the literature suggesting a gender bias . rashes and hypo pigmentation was more in our patients sub population as related to western data . monitoring responses was difficult because of outstation visits and financial implications and despite these constrains majority of the patients have done well . many of the patients who were largely asymptomatic had difficulty in accepting that they have a potentially lethal disease . single institution data is unlikely to give a complete picture indicative of all indian patients . it is proposed to have collation of data from different institutions in indian treating cml in india to have a larger picture of cml patients in india .

indira gandhi institute of medical sciences , regional cancer center was established in 1993 . it 's one of the main health - care institution in the state of bihar . the data of 205 patients was presented in the icon meeting and 98% of patients were diagnosed in chronic phase . complete hematological response was seen in 91% of patients in 3 months . a total of 197 ( 96% ) patients were alive at the time of analysis of which 179 ( 87% ) were still in chronic phase with hematological remission .

the hh - signaling pathway . ( a ) a diagram of the hh - signaling pathway , showing the site of action of the agonists ( green ) and antagonists ( red ) discussed in the text , as well as many additional factors that affect the pathway . abbreviations : ck1 , casein kinase 1 ; cos2 , costal2 ; dyrk1 , dual - specificity yak1-related kinase 1 ; gsk3 , glycogen synthase kinase 3 ; fu , fused ; gas1 , growth arrest specific 1 ; hh , hedgehog ; hip , hedgehog - interacting protein 1 ; rab23 , a rab - family ras - like gtpase associated with vesicle traffic ; ptc , patched1 ; pka , protein kinase a ; smo , smoothened ; sufu , suppressor of fused . ( b ) a schematic generalized view of the regulation of gli activator ( gli - act ) and gli repressor ( gli - rep ) forms by hh signaling . see [ 2 - 4 ] for further details . examples of diseases caused by loss of or ectopic function of the hh - gli signaling pathway , and the possible agents that could , in principle , be used as therapeutics * hh - ag is the hh agonist described by frank - kamenetsky et al ..

the discovery of small molecules that act as agonists and antagonists of the hedgehog - gli signaling pathway , which plays important roles in the embryo and adult , opens a new avenue for the treatment of diseases caused by aberrant suppression or activation of this complex pathway .

for visual function measurement , a 10-month - old domestic male pig ( sus scrofa domesticus ) , weighing approximately 30 kg , was used . the animal was treated according to the arvo statement for the use of animals in ophthalmic and vision research . the animal was sedated with an intramuscular injection ( ketamine , 22 mg / kg ; xylazine , 2 mg / kg ; and glycopyrrolate , 0.01 mg / kg ) , masked with n2o , intubated , and maintained on n2o gas anesthesia . the pig was kept on intravenous maintenance fluids ( lactated ringer 's solution 10 ml / kg / hr ) , and the heart rate and systemic arterial blood pressures were monitored throughout the procedures . after finishing the experiment , 1.2 g pentobarbital sodium and 150 mg phenytoin sodium were injected through a catheter in the central ear vein to euthanize the animals . visual stimuli were generated using a goggle type of flash stimulator ( sls-3100 ; nihon kohden , new york , ny , usa ) . at 5 to 20 hz placed in front of the eyes of the animal . visual acuity , in response to the flash stimulus , was assessed with veps . for electrical stimulation , the frontal lobe was removed and linearly configured platinum wire electrodes were implanted into the optic nerve , the optic tract and lgb of the right eye . electric stimulations were delivered through an isolated biphasic stimulator ( grass 12 ; astro - med , west warwick , ri , usa ) . the current stimulus consisted of a 0.3 ms negative square pulse was followed by 0.3 ms positive pulse and the amplitude of the current stimuli was varied between 0.5 and 5 ma . the vep and eep were recorded with a 2-channel data acquisition system using data collected on the scalp over the visual cortex at position oz , referenced with position fz . we used a domestic pig as the experiment model , and the feasibility of visual prostheses based on penetrating electrical stimulations were investigated ( fig . the globe was removed for exposure , a platinum wire electrode was implanted , and optic nerve eep was done ( fig . the frontal skull including right orbital wall was removed to visualize the right side intracranial optic nerve and optic chiasm . vep was performed by implantation of the platinum wire electrode to the optic tract near the optic chiasm . subsequently , the right frontal lobe and part of the temporal lobe were removed , and deep optic tract and lgb responses were recorded after direct electrical stimulation . left eye vep was done to compare with the right - side eep . for visual function measurement , a 10-month - old domestic male pig ( sus scrofa domesticus ) , weighing approximately 30 kg , was used . the animal was treated according to the arvo statement for the use of animals in ophthalmic and vision research . the animal was sedated with an intramuscular injection ( ketamine , 22 mg / kg ; xylazine , 2 mg / kg ; and glycopyrrolate , 0.01 mg / kg ) , masked with n2o , intubated , and maintained on n2o gas anesthesia . the pig was kept on intravenous maintenance fluids ( lactated ringer 's solution 10 ml / kg / hr ) , and the heart rate and systemic arterial blood pressures were monitored throughout the procedures . after finishing the experiment , 1.2 g pentobarbital sodium and 150 mg phenytoin sodium were injected through a catheter in the central ear vein to euthanize the animals . visual stimuli were generated using a goggle type of flash stimulator ( sls-3100 ; nihon kohden , new york , ny , usa ) . at 5 to 20 hz placed in front of the eyes of the animal . flash stimuli effectiveness was defined by trials and error using different grades of filters . visual acuity , in response to the flash stimulus , the frontal lobe was removed and linearly configured platinum wire electrodes were implanted into the optic nerve , the optic tract and lgb of the right eye . electric stimulations were delivered through an isolated biphasic stimulator ( grass 12 ; astro - med , west warwick , ri , usa ) . the current stimulus consisted of a 0.3 ms negative square pulse was followed by 0.3 ms positive pulse and the amplitude of the current stimuli was varied between 0.5 and 5 ma . the vep and eep were recorded with a 2-channel data acquisition system using data collected on the scalp over the visual cortex at position oz , referenced with position fz . we used a domestic pig as the experiment model , and the feasibility of visual prostheses based on penetrating electrical stimulations were investigated ( fig . the globe was removed for exposure , a platinum wire electrode was implanted , and optic nerve eep was done ( fig . the frontal skull including right orbital wall was removed to visualize the right side intracranial optic nerve and optic chiasm . vep was performed by implantation of the platinum wire electrode to the optic tract near the optic chiasm . subsequently , the right frontal lobe and part of the temporal lobe were removed , and deep optic tract and lgb responses were recorded after direct electrical stimulation . left eye vep was done to compare with the right - side eep . in the domestic pig , the locations of the optic nerve , optic tract and lgb are analogous to those of human . data were collected from the animal and consistent positive potentials were obtained for vep ( p75 ) and for eeps of the intraorbital optic nerve ( p70 ) , optic tract ( p37 ) , and lgb ( p36 ) . for a control , we performed vep in a normal left eye ( fig . 3a ) . an anesthesia effect was observed on veps after anesthesia in the right eye ( fig . the administration of anesthesia may modify the response to flash stimuli in comparison with a sedated animal . characteristic peaks appeared in the early part of the waves n1 ( 40 msec ) and p1 ( 70 msec ) and with vep on the optic nerve ( fig . however , when the optic nerve was stimulated with two electrodes ( about 1.0 mm apart ) the characteristic eep patterns of each were different ( fig . eeps on the optic tract and lgb were measured , and each showed short latency ( p39 and p33 , respectively ) compared with p70 of vep ( fig . finally , the skull of the animal was opened from the right frontal lobe to part of the temporal lobe , while vep to flash stimuli on the left eye were recorded to compare with the preoperative vep ( fig . the lgn is the first area to receive synaptic input from the axon of ganglion cells . studies in primate visual cortex indicate that the functional magnetic resonance imaging ( fmri ) response is roughly correlated with local field potential activity when both are measured simultaneously . from fmri studies , a 3 retinal area of central vision is magnified anatomically in the lgn to occupy more than 60% of the lgn volume . this enlarged lgn foveal representation could facilitate stimulation of the central visual area by an lgn - stimulated prosthetic device . even when there is substantial damage to the eye and possible atrophy of the optic nerve , the lgn remains largely intact . therefore , stimulation of the lgn has the potential to be a viable treatment approach for many causes of blindness , including the most prevalent one as well as blindness caused by trauma to the eye or surgical enucleation . approximately 10 mm away from the lgn are structures such as the subthalamic nucleus and substantia nigra that are accessed using surgical techniques from deep brain stimulation ( dbs ) for electrical stimulation treatment of movement disorders . this suggests that the same dbs techniques could be used to place stimulating electrodes in the lgn . many studies based on electrical stimulation of the human visual system promise to offer blind people the ability to see . up to now , problems ( in the stimulation of the visual cortex ) exist in obtaining a useful correlation between a stimulus applied and the actual vision obtained . in stimulating the retina , other problems may occur because of the difficulty in pinpoint visual stimulation generating any useful degree of resolution . unlike the non - homogeneous retinal visual field , the visual field of the lgb is homogeneous . image - view mapping may be designed to take advantage of this as all of the currently known implants are based on a trial - and - error approach because little is known regarding exactly how the brain processes artificially - stimulated neurons . experimental applications on humans proceed with extreme caution because of the potential risks involved . in our attempt to refine the basic reference ranges of eeps on the optic nerve , optic tract and lgb , a model system was employed because of the ocular morphology and physiology is very similar to the human eye . it was difficult to define the magnocellular , parvocellular and koniocellular layers in the lgb of the pig , due to the varying exactness of and occasionally conflicting characterizations of the different functional cortex areas . a limitation of this work is that as the number of subject in this study is small , and the result does not lead to a definite conclusion . in addition , further analysis as to whether wave - forms generated by lgn electrical stimulation can be processed in the brain and applied to movements is needed since actual stimuli responses such as walking and sitting were difficult to assess properly . furthermore , no comprehensive functional mapping of the cortical areas has yet been defined in the animal . the results of the present study showed that a characteristic peak ( p1 ) of eep emerged earlier than that of the vep and this may correlate with the different transmission between the electrical line and natural optical pathway including retina . eep has similar peaks and amplitude to vep , enabling us to conclude that electrical stimulation of lgb could mimic natural optical stimulation . our findings support the idea that electrical microstimulation of the lgb creates visual percepts , or phosphenes , which could be interpreted as normal visual events .

purposeblind individuals who have photoreceptor loss are known to perceive phosphenes with electrical stimulation of their remaining retinal ganglion cells . we proposed that implantable lateral geniculate body ( lgb ) stimulus electrode arrays could be used to generate phosphene vision . we attempted to refine the basic reference of the electrical evoked potentials ( eeps ) elicited by microelectrical stimulations of the optic nerve , optic tract and lgb of a domestic pig , and then compared it to visual evoked potentials ( veps ) elicited by short - flash stimuli.methodsfor visual function measurement , veps in response to short - flash stimuli on the left eye of the domestic pig were assessed over the visual cortex at position oz with the reference electrode at fz . after anesthesia , linearly configured platinum wire electrodes were inserted into the optic nerve , optic track and lgb . to determine the optimal stimulus current , eeps were recorded repeatedly with controlling the pulse and power . the threshold of current and charge density to elicit eeps at 0.3 ms pulse duration was about 10 a.resultsour experimental results showed that visual cortex activity can be effectively evoked by stimulation of the optic nerve , optic tract and lgb using penetrating electrodes . the latency of p1 was more shortened as the electrical stimulation was closer to lgb . the eeps of two - channel in the visual cortex demonstrated a similar pattern with stimulation of different spots of the stimulating electrodes . we found that the lgb - stimulated eep pattern was very similar to the simultaneously generated vep on the control side , although implicit time deferred.conclusionseeps and veps derived from visual - system stimulation were compared . the lgb - stimulated eep wave demonstrated a similar pattern to the vep waveform except implicit time , indicating prosthetic - based electrical stimulation of the lgb could be utilized for the blind to perceive vision of phosphenes .

sepsis , a systemic inflammatory response to infection , is one of the most challenging clinical problems worldwide and the leading cause of death in noncoronary icus . recent studies have shown that both pro- and anti - inflammatory mechanisms may occur early and simultaneously in sepsis . during the proinflammatory phase , septic patients also enter a stage of protracted immunosuppression . patients in the immunosuppressive phase of sepsis are often unable to control the primary infection and may also acquire a secondary infection during hospitalization . our previous study showed that the proportion of cd4 cd25foxp3 t cells ( tregs ) was significantly elevated in the peripheral blood from early - stage septic shock patients . tregs are involved in the regulation of peripheral tolerance which demonstrated again that immunosuppression was a key factor in the development of sepsis [ 5 , 6 ] . all these suggested that understanding the mechanism underlying the immunosuppression will help to identify the novel targets in the treatment of sepsis . recently , more and more lines of evidence have revealed the important role of programmed cell death 1 ( pd-1 ) and programmed death - ligand 1 ( pd - l1 ) in the pathogenesis of sepsis . the pd-1 , a surface receptor , belongs to the cd28 family and is mainly expressed on activated t cells , natural killer t cells , and myeloid cells . pd-1 interacts with two b7 family ligands , pd - l1 and pd - l2 . pd - l1 is widely expressed on antigen presenting cells ( apcs ) and hematopoietic cells . the interaction between pd-1 and pd - l1 activates an inhibitory pathway that leads to abrogation of cellular response and causes cd8 t cell exhaustion during chronic viral infection [ 9 , 10 ] . zhang and colleagues found that in vitro blockade of the pd-1/pd - l1 pathway decreased apoptosis and improved immune cell function in septic patients . there are also three independent groups which showed that blockade of the pd-1 pathway improved survival in clinically relevant animal models of bacterial and fungal sepsis [ 7 , 11 , 12 ] . these studies strongly suggest that pd-1 pathway may become a potential immune - modulatory therapy for sepsis - induced immunosuppression . therefore , a better understanding of the molecular events controlling pd-1 expression is essential for sepsis . yet very little is known on the regulation of pd-1 expression during sepsis - induced immunosuppression . in this study , first we investigated the expressions of pd-1/pd - l1 on both cd4 t cells and monocytes from peripheral blood in the early - stage septic shock patients and showed the relationship between pd-1/pd - l1 and clinical outcomes . then , we evaluated the notch signaling pathway , which contributes to the regulation of the peripheral immune cells tolerance through its ability to influence cell survival and growth , in the peripheral blood mononuclear cells ( pbmcs ) from the septic shock patients . finally , we explored the regulation of pd-1 expression by inhibiting the notch signaling pathway in a lps - tolerant thp1 cells model . here , for the first time , we demonstrated that notch signaling pathway was activated in the early - stage septic shock patients and involved in the regulation of pd-1 expression . the patients enrolled in this observational / clinical study were recruited from the surgical , emergency , and respiratory icus of ruijin hospital , shanghai jiaotong university ( shanghai , china ) . the study protocol was approved by the ruijin hospital ethics committee , shanghai jiaotong university school of medicine , china . the individuals in this paper sepsis was diagnosed according to criteria established by the american college of chest physicians / society of critical care medicine . the following information was collected : demographic characteristics ( age and gender ) , apache ii score ( acute physiology and chronic health evaluation ii ) at inclusion , white blood cell count at inclusion , site of infection , microbiological findings , and the outcome after 28 days ( nonsurvival or survival ) . study exclusion criteria included patients aged < 18 years ; more than 24 hours from sepsis diagnosis to blood collection ; preexisting cancer ; an absence of circulating leukocytes ; the presence of preexisting hematological or immunological disease ; patients with hiv or hbv . peripheral vein blood samples were obtained from each patient , diagnosed with septic shock within 24 hours , and the healthy individuals . the blood samples were transported to the clinical research center at 4c within 1 hour . to detect the expressions of pd-1 and pd - l1 on the immune cells by flow cytometry , peripheral blood mononuclear cells ( pbmcs ) were isolated by ficoll density gradient centrifugation . after lysing red cells with facs - lysing solution ( bd biosciences ; san jose , ca , usa ) , the immune cells were stained with fitc - labeled anti - cd4 ( clone a161a1 ) or fitc - labeled anti - cd14 ( clone hcd14 ) and pe - labeled anti - pd-1 ( clone eh12.2h7 ) or pe - labeled anti - pd - l1 antibodies ( clone 29e.2a3 ) ( biolegend ; san diego , ca , usa ) . the stained cells were analyzed by using a facs calibur flow cytometer ( becton - dickinson , bd biosciences ) and cellquest software . thp-1 cells were purchased from the type culture collection of the chinese academy of sciences , shanghai , china . cells were maintained under conditions of 37c and 5% co2 in rpmi-1640 media supplemented with penicillin g ( 10 u / ml ) , streptomycin ( 10 g / ml ) , l - glutamine ( 2 mm ) , and 10% fetal bovine serum ( fbs ) ( hyclone laboratories , logan , ut , usa ) . thp1 cells were pretreated with 1.0 g / ml gram - negative lps ( escherichia coli 0111:b4 ; sigma - aldrich , st . louis , mo , usa ) for 16 h. then , nontreated and pretreated thp-1 cells were pelleted , washed once in pbs , and resuspended in culture media at 5.010.0 10 cells / ml . finally , cells of both groups were stimulated with lps ( 1.0 g / ml ) for another 6 hours to make lps - nontolerant and lps - tolerant models . after above treatment , cells were washed for three times and total rna was extracted . reduced proinflammatory cytokines ( mainly tnf- ) and increased immunosuppression mediators ( mainly il-10 ) are always used to evaluate whether the endotoxin tolerant model is set up successfully . in our study , the tnf- and il-10 expressions were examined by using real - time pcr to verify the in vitro model of endotoxin tolerance . additionally , in lps - tolerant group , gamma secretase inhibitor n-[n-(3,5-difluorophenacetyl)-l - alanyl]-s - phenyl glycinet - butyl ester ( dapt ) ( sigma , st . louis , mo , usa , 40 m ) was added into the culture at the beginning of the treatment of lps to inhibit the notch signaling pathway . total rna was extracted from patient pbmcs and thp1 cells by using trizol reagent ( invitrogen ; grand island , nt , usa ) according to the manufacturer 's instructions . rna ( 2 g ) was reverse transcribed by using reverse transcriptase with random hexamers as primers ( primescript tm rt - pcr kit , takara , kyoto , japan ) . the resulting complementary dnas ( cdnas ) were diluted 2-fold with dnase / rnase - free water . real - time quantitative pcr was performed by using a 7500 fast real - time pcr system ( applied biosystems ; foster city , ca , usa ) and sybr premix ex taq ( takara , kyoto , japan ) under the following conditions : denaturation at 95c for 30 sec , followed by 40 cycles at 95c for 5 sec , 60c for 34 sec , and then a dissociation phase . -actin was used as an endogenous control to normalize for rna levels and efficiency of the reverse - transcription reaction . the clinical and biological parameters of patients are presented as frequencies , percentages , medians , or interquartile ranges ( iqrs ) . experiment data are presented as the median value with interquartile ranges or as the mean sem . normally distributed data were analyzed by using two - tailed student 's t - test , and nonnormally distributed data were analyzed by using the mann - whitney u test . all statistical analyses were conducted by using prism 4.0 software ( graphpad software , la jolla , ca , usa ) . fifty - six patients at early stage of septic shock ( 24 females and 32 males ) were included in this study . the median age of the patients was 59 years ( range 54 to 63 years ) , and the median apache ii score was 16 ( range 14 to 23 ) . patients with septic shock showed increased numbers of white blood cells compared to healthy volunteers . 29 septic shock patients were infected with gram - negative bacilli , 22 patients were infected with gram - positive cocci , and 5 patients were infected with fungi . twenty - five of the 56 patients died within 28 days after diagnosis of septic shock . expressions of both pd-1 and pd - l1 on cd4 t cells and monocytes were measured in each patient diagnosed with septic shock within 24 hours ( figure 1 ) . cd4 t cells obtained from patients showed significantly greater expressions of both pd-1 and pd - l1 compared to cd4 t cells obtained from healthy volunteers ( 21.59% versus 6.37% , p < 0.001 , and 13.67% versus 4.97% , p < 0.01 , resp . ) ( figures 1(a ) and 1(b ) ) . additionally , pd-1 and pd - l1 expressions on monocytes ( figures 1(c ) and 1(d ) ) were also much higher in patients compared to healthy volunteers ( for pd-1 : 14.91% versus 6.89% , p < 0.01 ; for pd - l1 : 51.82% versus 17.12% , p < 0.01 ) . here , we assessed the association between pd-1/pd - l1 expressions and illness severity and found that patients with an apache ii score > 20 showed significantly greater pd-1 expression on cd4 t cells and monocytes compared to patients with an apache ii score < 20 ( for cd4 t cells : 28.68% versus 19.67% , p < 0.01 , figure 1(e ) ; for monocytes : 22.55% versus 11.73% , p < 0.01 , figure 1(f ) ) . however , there were no differences in pd - l1 expression on cd4 t cells and monocytes obtained from patients with different levels of physical dysfunction ( data not shown ) . then , we analyzed the association between pd-1/pd - l1 expression and patient prognosis by comparing expression levels of pd-1 and pd - li on cd4 t cells and monocytes obtained from sepsis survivors and nonsurvivors . nonsurvivors showed higher levels of both pd-1 and pd - l1 expressions on monocytes ( for pd-1 : 19.13% versus 13.79% , p < 0.05 , figure 1(g ) ; for pd - l1 : 60.21% versus 45.06% , p < 0.01 , figure 1(h ) ) . however , survivors and nonsurvivors showed similar levels of pd-1 and pd - l1 expressions on cd4 t cells ( data not shown ) . notch signaling is a key pathway to control cell - fate choice in a large number of cell types including immune cells . the role of the notch signaling pathway in peripheral tolerance leads us to evaluate its expression in endotoxin tolerance , which is a hallmark of sepsis - induced immunosuppression . the decreased mrna level of proinflammatory cytokine tnf- and increased mrna level of anti - inflammatory cytokine il-10 were proved in a lps - tolerant thp1 cell model ( figures 2(a ) and 2(b ) ) . the pd-1 mrna levels were markedly increased in lps - tolerant cells compared to those in lps - nontolerant cells ( p < 0.05 , figure 2(c ) ) . we also measured the mrna levels of notch signaling proteins including jagged1 and hes1 and found that , compared to lps - nontolerant cells , jagged1 and hes1 expressions were much higher in lps - tolerant cells ( p < 0.05 , figures 2(d ) and 2(e ) ) . these data suggest that mrna levels of pd-1 and notch signals were increased and there may be an association between the notch signaling pathway and pd-1 expression in the endotoxin tolerance . in addition , the results of pd-1 were proved in protein levels ( p < 0.05 , figure 2(f ) ) . to approve the activation of the notch signaling pathway during sepsis , we measured the mrna levels of notch signal proteins in pbmcs obtained from patients at early stage of septic shock . we found that mrna levels of jagged1 and hes1 in patients were much higher compared to those in healthy volunteers ( p < 0.05 ) ( figures 3(a ) and 3(b ) , resp . ) . additionally , among the septic shock patients , mrna levels of jagged1 in nonsurvivors were higher than those in survivors ( figure 3(c ) ) . to explore the role of the notch signaling pathway in pd-1 expression , after inhibition of the notch signaling by dapt , both mrna and protein levels of pd-1 were significantly decreased in the lps - tolerant cells ( both p values < 0.05 , figures 4(a ) and 4(b ) ) . and mrna levels of il10 were also decreased when notch signaling pathway was blocked in the lps - tolerant cells ( p values < 0.05 , figure 4(c ) ) . tnf- mrna levels were increased after blockading the notch signaling pathway ( p values > 0.05 , figure 4(d ) ) . previous studies suggested that both proinflammatory and anti - inflammatory processes begin promptly after sepsis initiation . it has been reported that pd-1 and its ligand pd - l1 deliver inhibitory signals capable of inducing immune tolerance and prolonged overexpression of pd-1 might result in t cell dysfunction in sepsis patients [ 17 , 18 ] . here , we have shown overexpressions of pd-1 and pd - l1 on monocytes and cd4 lymphocytes obtained from patients at early stage of septic shock . it not only demonstrated the role of pd-1 and pd - l1 in the pathogenesis of sepsis but also suggested that the early upregulated pd-1 might be a useful marker to evaluate the immune state of sepsis patients . furthermore , the present work showed that pd-1 expression on immune cells was much higher in nonsurvival septic shock patients than survivors . recent studies suggested that , in a murine model of sepsis , administration of pd-1 antibody markedly diminished sepsis - induced apoptosis and improved survival and it also showed an enhanced bacterial clearance capacity after induction of sepsis by cecal ligation and puncture ( clp ) in pd-1 mice [ 7 , 19 ] . all above lines of evidence suggested that pd-1 could assess illness severity , predict the prognosis of sepsis , and be a potential target for the treatment of sepsis - induced immunosuppression . therefore , it is important to find out the regulation mechanism for this novel approach during sepsis . recently , notch signaling pathway was identified to induce pd-1 expression on t cells maintaining them in an exhausted state during chronic viral infection . in mammals , four notch receptors ( notch 1notch 4 ) and five ligands ( jagged1 , jagged2 , delta - like-1 , delta - like-3 , and delta - like-4 ) notch downstream target genes include members of the basic helix - loop - helix family ( hes1 and hes5 ) and the hairy and enhancer of split - related ( hesr ) family ( hey1 and hey2 ) [ 21 , 22 ] . during the peripheral tolerance , jagged1 and hes1 have been reported to be involved in inhibiting primary and secondary immune responses [ 23 , 24 ] . in our experiment , the decreased tnf- and increased il-10 transcriptomic responses to two subsequent lps challenges demonstrated that endotoxin tolerance thp1 cell model was established successfully . we found that the expressions of jagged1 and hes1 in lps - tolerant cells were significantly higher than lps - nontolerant cells . , we demonstrated that notch signaling pathway was activated in both early septic shock patients and thp1 cells response to two subsequent lps challenges . the specific notch ligands can be induced by pathogen - derived signals on the differentiation or function of cd4 t cells and haematopoietic cells . previous study suggested that the notch signaling jagged1 was induced by lps occurring in a jnk - dependent manner . therefore , the septic shock patients infected with bacteria , especially g - bacteria , may activate the notch signaling pathway via jnk - dependent manner induced by lps . it has been reported that , during chronic virus infection , the inhibition of pd-1 expression by the highly specific inhibitor sahm1 showed that notch signaling directly controls pd-1 via pdcd1 transcription in activated cd8 t cells . here , the inhibition of notch signaling pathway induced significantly decreased pd-1 expression in the lps - tolerant cell model . we hypothesized that the activated notch signaling pathway may be involved in the expression of pd-1 during sepsis . the mechanism leading to the regulation of pd-1 by notch signaling pathway in lps - tolerant thp1 cells may be the similar directly regulated by varied transcription factors involved in thp1 monocytes . in current experiment , we also found that both pd-1 and il-10 were increased in lps - tolerant cells . recent studies noted the interaction between them : pd-1 could enhance il-10 production and il-10 also induced pd-1 expression in monocytes and cd4 t cells . additionally , previous research showed that dc matured by jagged1 ( jgd1-conditioned dc ) produced the high level of il-10 through tlr ( via lps ) signaling . however , whether the notch signaling pathway in regulation of pd-1 expression is through the il-10 during sepsis - induced immunosuppression is unclear . second , our findings concerning the role of notch signaling in pd-1 and il-10 expressions under conditions of lps tolerance are mainly at the gene transcription level and should be further confirmed at the protein level . third , our current study was very preliminary and exploratory in nature , and the mechanisms responsible for notch regulation of pd-1 expression on pbmc under conditions of endotoxin tolerance require further investigation in a dedicated study . in summary , our data showed that the enhanced pd-1 could be an early biomarker to assess not only the proper immune phase but also the illness severity and prognosis for the septic shock patients . noticeably , we also found that notch signaling pathway was activated during the early stage of the septic shock and involved in regulation of pd-1 expression . thus it revealed a possible regulation mechanism of pd-1 , which may be a promising target for the therapeutic intervention in sepsis and in the field of infectious disease .

programmed cell death 1 ( pd-1 ) plays an important pathologic role in sepsis - induced immunosuppression . however , whether pd-1 overexpression occurs early during septic shock is unknown and its regulation mechanism is also unknown . our study investigated the expressions of pd-1/programmed death - ligand 1 ( pd - l1 ) on immune cells in peripheral blood from the early - stage septic shock patients . we found that both pd-1 and pd - l1 showed increased expressions on the cd4 + t cells and monocytes . it indicated that pd-1 expression might be an early biomarker to assess illness severity and predict the prognosis of septic shock . then , we further investigated the mechanism underlying the regulation of pd-1 expression . our data showed that notch signaling pathway was activated in both septic shock patients and lipopolysaccharide- ( lps- ) tolerant thp1 cells and both interleukin-10 ( il-10 ) and pd-1 were increased in the thp1 cells . inhibition of notch signaling by n-[n-(3,5-difluorophenacetyl)-l - alanyl]-s - phenyl glycinet - butyl ester ( dapt ) induced significantly decreased expressions of pd-1 and il-10 in the lps - tolerant cell model . our work suggested that notch signaling pathway was involved in the regulation of pd-1 expression .

the functional response of a predator is a key factor regulating the population dynamics of predator - prey systems . it describes the rate at which a predator kills its prey at different prey densities and can thus determine the efficiency of a predator in regulating prey populations ( murdoch and oaten 1975 ) . this is further supported by plotting the number of prey killed against the number of prey available and analyzing a continuum of patterns , which ecologists have delimited into three types ( holling 1959 , 1965 ) . the functional response curves may represent an increasing linear relationship ( type i ) , a decelerating curve ( type ii ) , or a sigmoidal relationship ( type iii ) . that is , they result in a constant ( i ) , decreasing ( ii ) and increasing ( iii ) rate of prey killing and yield density - dependent , negatively density dependent and positively density dependent prey mortality , respectively . predators or parasitoids that impose positively density dependence prey mortality ( type iii ) are supposed to potentially manage the prey population and could be considered as efficient biocontrol agents ( fernndez - arhex and corley 2003 ) . however , certain predators and parasitoids exhibiting type ii response have been successfully established and managed prey populations ( hughes et al . usually , a type iii response does not exhibit positive density dependence throughout the range , as only a portion , i.e. at the initial level , shows a sigmoidal increase ; later on , it also exhibits negative density dependence due to satiation . hence , it is important to differentiate type ii and iii at relatively low prey values . coefficient of attack rate and handling time ( time spent by predator in attacking , killing , subduing , and digesting the prey ) . the coefficient of attack rate estimates the steepness of the increase in predation with increasing prey density , and handling time helps estimate the satiation threshold . ecologists normally face difficulties in determining functional response when the curve lies between type ii and iii . hence , a suitable analysis that can best determine the functional response is highly needed , as it is of great practical relevance in estimating the bio - efficacy of predatory insects ( trexler et al . coccinellids are one of the important groups of predatory insects , that have immense biocontrol potential ( omkar and pervez 2003a ) with all three types of functional responses reported for the group ( hodek and honek 1996 ) . the multi - colored asian coccinellid , harmonia axyridis is reported to exhibit all three types , i.e. type i on the aphid , rhopalosiphum prunifoliae ) ( lou 1987 ) , type ii on the aphid , lipaphis erysimi ( he et al . 1994 ) , and type iii on the aphid , cinara sp . interestingly , on a single prey species , eggs of the monarch butterfly , danaus plexippus , third instars and adults of h. axyridis exhibited type ii and type i responses , respectively ( koch et al . 2003 ) . a single predator can therefore respond differentially to various prey - types and it seems likely that a predator 's response to single prey type differs within and between species . an hypothesis was framed that a single predator can exhibit different responses to various prey species . cheilomenes sexmaculata ( fabricius ) , coccinella transversalis fabricius and propylea dissecta ( mulsant ) are aphidophagous coccinellids , abundant in the agricultural fields of lucknow , india . they co - occur in bean ( dolichos lablab ) and deadly nightshade ( solanum nigrum ) fields infested with the aphids , aphis craccivora koch and myzus persicae ( sulzer ) , respectively . both c. sexmaculata and c. transversalis are exhaustively studied coccinellids in the subcontinent as they are highly voracious and fecund , and have a wide prey range , which includes , aphids , coccids , diaspids , aleyrodids , etc . ( agarwala and yasuda 2000 ; omkar and bind 2004 ; omkar and james 2004 ; omkar and pervez 2004a ) . p. dissecta , co - occurring with the above two coccinellid species , is little studied coccinellid . however , recent research has revealed it to be a potential predator of certain aphid species ( pervez and omkar 2004a ) . being ecologically plastic , it can withstand the stresses of prey deprivation and temperature changes ( omkar and pervez 2003b ; pervez and omkar 2003 ; pervez and omkar 2004b ; pervez et al . the present study was thus designed to compare functional responses of the three coccinellids on two aphid species . we also tried to answer the following questions : ( i ) are functional responses different among predators when they feed on same prey ? ( ii ) are the functional responses for each predator different when they feed on different prey ? ( iii ) what is the potential role of these predators for managing a. craccivora and m. persicae populations ? adults of c. sexmaculata , c. transversalis , and p. dissecta were collected from d. lablab and s. nigrum fields infested by a. craccivora and m. persicae , respectively , and brought to the laboratory . ten conspecific coccinellids ( sex - ratio 1:1 ) were kept in glass jars ( 15 10 cm ) containing moist filter paper for females to oviposit and held at 27 1 c , 65 5% rh and ld 14:10 . the glass jars were covered with muslin and predators fed daily on ad libitum food ( prey and host as above ) . the eggs were collected from the filter paper and reared individually from egg hatch to adult eclosion in glass beakers ( 8 11 cm ) on the above prey to obtain 10-day - old virgin adult females to be used in experiments . 10-day - old adult females of the three predatory species were kept separately without food for 12 hours in different glass beakers ( size as above ) in order to standardize their hunger . 25 , 50 , 100 , 200 , 300 , and 400 of above prey ( each prey species in separate beaker ) with host plant twigs in ten replicates ( n = 10 ) . the beakers were covered with muslin and kept in an environmental test chamber maintained at 27 c and 65 5% rh . usually , it is difficult to discriminate between type ii and type iii functional responses as mentioned by many workers ( trexler et al . 1988 ; casas and hulliger 1994 ) . hence , prior to fitting the data to a particular holling 's equation , a logistic regression model ( 1 ) was used . this model is used only to determine the shape of functional response by taking into consideration the proportion of prey eaten ( na / no ) as a function of prey offered ( no ) ( juliano 2001 ) . the data were fitted to a polynomial function that describes the relationship between na / no and no : with po , p1 , p2 , and p3 being the intercept , linear , quadratic and cubic coefficients , respectively , estimated using the method of maximum likelihood . if p1 > 0 and p2 < 0 , the proportion of prey consumed is positively density dependent , thus describing a type iii functional response . if p1 < 0 , the proportion of prey consumed declines monotonically with the initial number of prey offered , thus describing a type ii functional response ( juliano 2001 ) . in the above , we used a cubic expression to determine the correctness of the functional response curves because a cubic expression will often provide a good fit to a type iii response ( trexler et al . 1988 ) and will provide a good starting point for fitting a logistic regression ( trexler and travis 1993 ) . higher order expression will , of course , fit even better , but this improved fit is usually the result of a better fit to points at higher values of no . after the determination of the shape of the curve , the handling times and attack coefficients of a type ii response were estimated using holling 's disc equation modified by reciprocal linear transformation ( livdahl and stiven 1983 ) . this method is preferred because of its simplicity ( veeravel and baskaran 1997 ; pervez and omkar 2003 ; omkar and pervez 2004 ) . the simplified holling 's equation , modified by reciprocal linear transformation ( livdahl and stiven 1983 ) , is as follows : where na is the number of prey consumed by predator , no is the initial prey density , a the attack rate , t the time that predator and prey are exposed to each other , and th the handling time associated with each prey consumed . the parameters were obtained by fitting the data to the least square regression . for a type iii response , model ( 3 ) ( 1977 ) , which is as follows : where , b , c , and d are constants from the function that relate a and no in type iii functional response : a = ( d + bno ) / ( 1 + cno ) . parameters were obtained by fitting observed data to the models above using non - linear least square regression . to compare type ii response of two groups of predators , the following equation was used : where z = an indicator variable that takes on the value 0 for predator species one and 1 for predator two . the parameters a and th estimate the differences between the individual parameters of a and th , respectively of the two coccinellid species . if these parameters are significantly different from 0 then the two predators differ significantly in the corresponding parameters . for predator one , and t1h are the estimates of the parameters a1 and th1 . for predator 2 , + a and t1h + th , the performance in terms of a and th of all three predators tested were compared between and within the species when fed on two prey . this was done by student t - test followed by bonferroni 's post hoc test of significance using a statistical software minitab on personal computer . adults of c. sexmaculata , c. transversalis , and p. dissecta were collected from d. lablab and s. nigrum fields infested by a. craccivora and m. persicae , respectively , and brought to the laboratory . ten conspecific coccinellids ( sex - ratio 1:1 ) were kept in glass jars ( 15 10 cm ) containing moist filter paper for females to oviposit and held at 27 1 c , 65 5% rh and ld 14:10 . the glass jars were covered with muslin and predators fed daily on ad libitum food ( prey and host as above ) . the eggs were collected from the filter paper and reared individually from egg hatch to adult eclosion in glass beakers ( 8 11 cm ) on the above prey to obtain 10-day - old virgin adult females to be used in experiments . 10-day - old adult females of the three predatory species were kept separately without food for 12 hours in different glass beakers ( size as above ) in order to standardize their hunger . 25 , 50 , 100 , 200 , 300 , and 400 of above prey ( each prey species in separate beaker ) with host plant twigs in ten replicates ( n = 10 ) . the beakers were covered with muslin and kept in an environmental test chamber maintained at 27 c and 65 5% rh . usually , it is difficult to discriminate between type ii and type iii functional responses as mentioned by many workers ( trexler et al . hence , prior to fitting the data to a particular holling 's equation , a logistic regression model ( 1 ) was used . this model is used only to determine the shape of functional response by taking into consideration the proportion of prey eaten ( na / no ) as a function of prey offered ( no ) ( juliano 2001 ) . the data were fitted to a polynomial function that describes the relationship between na / no and no : with po , p1 , p2 , and p3 being the intercept , linear , quadratic and cubic coefficients , respectively , estimated using the method of maximum likelihood . if p1 > 0 and p2 < 0 , the proportion of prey consumed is positively density dependent , thus describing a type iii functional response . if p1 < 0 , the proportion of prey consumed declines monotonically with the initial number of prey offered , thus describing a type ii functional response ( juliano 2001 ) . in the above , we used a cubic expression to determine the correctness of the functional response curves because a cubic expression will often provide a good fit to a type iii response ( trexler et al . 1988 ) and will provide a good starting point for fitting a logistic regression ( trexler and travis 1993 ) . higher order expression will , of course , fit even better , but this improved fit is usually the result of a better fit to points at higher values of no . after the determination of the shape of the curve , the handling times and attack coefficients of a type ii response were estimated using holling 's disc equation modified by reciprocal linear transformation ( livdahl and stiven 1983 ) . this method is preferred because of its simplicity ( veeravel and baskaran 1997 ; pervez and omkar 2003 ; omkar and pervez 2004 ) . the simplified holling 's equation , modified by reciprocal linear transformation ( livdahl and stiven 1983 ) , is as follows : where na is the number of prey consumed by predator , no is the initial prey density , a the attack rate , t the time that predator and prey are exposed to each other , and th the handling time associated with each prey consumed . the parameters were obtained by fitting the data to the least square regression . for a type iii response , model ( 3 ) ( 1977 ) , which is as follows : where , b , c , and d are constants from the function that relate a and no in type iii functional response : a = ( d + bno ) / ( 1 + cno ) . parameters were obtained by fitting observed data to the models above using non - linear least square regression . to compare type ii response of two groups of predators , the following equation was used : where z = an indicator variable that takes on the value 0 for predator species one and 1 for predator two . the parameters a and th estimate the differences between the individual parameters of a and th , respectively of the two coccinellid species . if these parameters are significantly different from 0 then the two predators differ significantly in the corresponding parameters . for predator one , and t1h are the estimates of the parameters a1 and th1 . for predator 2 , + a and t1h + th , are the estimates of parameters a2 and th2 ( juliano 2001 ) . the performance in terms of a and th of all three predators tested were compared between and within the species when fed on two prey . this was done by student t - test followed by bonferroni 's post hoc test of significance using a statistical software minitab on personal computer . the logistic regression for all three predatory species had a significant linear parameter p1 < 0 ( table 1 ) and the proportion of prey consumed by all predators declined with increasing prey density ( figs 1 and 2 ) . their handling times and coefficient of attack rates are presented in table 2 , which shows that c. transversalis had the shortest handling time , followed by c. sexmaculata and p. dissecta using a. craccivora as prey . however , when provided with m. persicae , c. sexmaculata spent less time in handling the prey , followed by c. transversalis and p. dissecta . all three predatory species spent relatively more time in handling when m. persicae was the prey . both handling time and coefficient of attack rate were dependent on the prey species with optimal values on a. craccivora , followed by m. persicae ( table 2 ) . comparison of functional response curves revealed that c. transversalis responded more vigorously at lower densities of both aphid species with an elevation in the curve over c. sexmaculata . however , there was a decline in the consumption rate at higher densities , which resulted in an increase in the functional response curve of c. sexmaculata . the functional response curve of p. dissecta was significantly lower than the other two predatory species on both prey species ( figs . 1 and 2 ) . the differences in the attack coefficients and handling times of the three predators on two prey species were compared within and between the species ( tables 3 and 4 ) . prey species significantly affected the attack coefficients of c. sexmaculata and p. dissecta , as those obtained on a. craccivora were significantly higher than those on m. persicae . the differences in the attack coefficients of different predatory species are presented in table 3 , which were largely unaffected by prey species ( table 3 ) , while the handling times mostly showed significant variations both within and between the species ( table 4 ) . this indicates that predators responded differentially in terms of handling time to different prey species . a logistic regression model was further used to determine the correctness of the shapes , as in such studies ecologists normally face difficulties in curve - fitting when the data set of type ii response shows inclination towards type iii response . this can lead to drawing misleading inferences ( as one shows negative density dependence while other shows positive density dependence ) , which further might lead to erroneous predictions about the fate of prey - predator interactions . the negative values for the linear parameters ( p1 < 0 ) obtained in the present study confirm the type ii response for all three predators used . the logistic regression model thus can be recommended as a tool for further analyzing functional response curves . a type iii functional response shows a positive density dependence of the proportion of prey killed as the density increases , hence a polynomial fit to a type iii should have a linear term that is positive . conversely , for a type ii response , there should be a decline in the proportion killed as the density increases , so that the linear term should be negative . however , very rarely , it is possible to get a type ii response with a positive linear term , if negative quadratic or cubic terms are sufficiently large ( juliano personal communication ) . although the logistic model ( juliano 2001 ) easily illuminates the subtle differences in the type ii and iii responses , it fails to discriminate them from type i ( linear ) . the empirical data , especially c. sexmaculata preying on a. craccivora at lower prey densities , appears to support a type i functional response . however , the type i equation ( holling 1959 ) does not give the best fit to the data . the differential response of predator at lower and higher prey densities might lead to erroneous predictions . hence , efforts are needed to make a similar logistic model to differentiate type i from ii and iii . the functional response parameters obtained by reciprocal transformation ( livdahl and stiven 1983 ) gave the best fit to the data , and can be used to determine the simulated na value at any prey density . thus , minimizing the efforts needed to generate voluminous empirical data at different aphid densities in the laboratory . the coefficient of attack rate and handling time were the parameters used to find out the magnitude of these responses . their values differed significantly within and between the predatory species when exposed to two prey species , which indicate that they have different abilities to respond to increasing prey densities . this also indicates that predators exhibiting similar functional response curves can not be deemed to respond similarly . the differences in parametric values might be due to the variation in size , voracity , satiation time , hunger levels , digestive ability , walking speed , etc . ( mills 1982 ; ofuya and akinbohungbe 1988 ; omkar and pervez 2004b ) . amongst the three predators , c. sexmaculata responded maximally to increasing densities of a. craccivora , followed by c. sexmaculata and p. dissecta . this inference is strongly supported by the empirical evidence of the reproductive biology of c. sexmaculata , which obtains higher values in fitness and reproduction using this prey species ( omkar and bind 2004 ) . as is evident from the functional response curve , the relative rate of prey consumption by c. transversalis was higher at lower densities on both prey species , indicating that it could be more effective at lower prey densities . it exhibited a significant decline in consumption rate at higher prey densities , which might be due to the attainment of satiation ( mills 1982 ) . the elevated functional response curve that c. sexmaculata attained over that of c. transversalis indicates a possible delayed satiation and/or a possible faster digestive rate . a high rate of prey consumption at higher densities is not a feature of aphidophagous coccinellid predators ( pervez and omkar 2003b ) , which is a major reason for the failure in aphid biocontrol programs using predatory coccinellids . the differences in the coefficients of attack rates did not vary significantly in most of the treatments , which indicates that this parameter is least affected by the change in predator and/or prey species . similar insignificant effects of predator and prey types were reported in four heteropteran predators preying on whiteflies and thrips ( montserrat et al . the handling time estimate is the cumulative effect of time taken during capturing , killing , subduing , and digesting the prey ( veeravel and baskaran 1997 ) . thus , significant variation in the estimates of handling times of a single predator on different prey species indicates that any one of these integral components of the handling time might have been negatively affected when m. persicae was used as prey rather than a. craccivora . this leads to the inference that though these predators co - occur in m. persicae colonies , they will be more efficient if employed against a. craccivora infestations . functional response , though an important tool , can not only be attributed to reported success and failures in biocontrol programs . for instance , other factors , such as intrinsic growth rates , host patchiness , predation and competition , host traits , and environmental complexities ( abiotic and biotic factors ) also have a major influence on the efficiency of predator in managing the prey population . our laboratory data provide information as to how these predators will respond to increasing prey density under simplified experimental conditions . for conclusive estimations of their biocontrol potential , further field based studies are needed . however , from the evidence of functional response curves , coefficient of attack rates and handling time , we can predict that among the three co - occurring predatory species , c. sexmaculata could be an efficient biocontrol agent , supporting our first hypothesis that predators respond differentially . the differential functional response of predators on a. craccivora and m. persicae obtaining higher parametric values using former prey supports our second hypothesis that predators respond differentially to various prey species . it can be concluded that the preceding logistic regression model should be encouraged for the conclusive analysis of functional responses . all three predators exhibited type ii response , which varied differentially within and between the species on two aphid species used . maximal values for coefficient of attack rate and handling time were obtained by c. sexmaculata , followed by c. transversalis and p. dissecta . c. sexmaculata has the potential to be exploited successfully as a biocontrol agent for the management of a. craccivora infestations ; however , further field - based studies are needed . type ii functional response of ladybirds , c. sexmaculata ( cs ) c. transversalis ( ct ) , and p. dissecta ( pd ) at different densities of a. craccivora ( derived using livdahl & stiven model ( n=1 ) ) . type ii functional response of ladybirds , c. sexmaculata ( cs ) c. transversalis ( ct ) , and p. dissecta ( pd ) at different densities of m. persicae ( derived using livdahl & stiven model ( n=1 ) ) . maximum likelihood estimates from logistic regression of proportion of prey eaten as a function of initial prey densities by adult females of c. sexmaculata , c. transversalis and p. dissecta ( n=10 ) . coefficient of attack rate ( a ) and handling time ( th ) of c. sexmaculata , p. dissecta and c. transversalis derived from livdahl and stiven model using a. craccivora and m. persicae as prey ( t=1day ) . da values when comparing coefficient of attack rates of three predatory species on two aphid prey , a. craccivora and m. persicae both between and within the predatory species . dth values when comparing handling times of three predatory species on two aphid prey , a. craccivora and m. persicae both between and within the predatory species .

the functional response parameters and patterns of three coccinellid predators , cheilomenes sexmaculata , propylea dissecta , and coccinella transversalis ( coleoptera : coccinellidae ) were evaluated to find out how these predators respond at two different prey species across various prey densities levels . all three predators exhibited a decelerating curve type ii response determined by a logistic regression model . the linear reciprocal transformation of holling 's disc equation was used to further evaluate the parametric values . c. sexmaculata responded maximally , followed by c. transversalis and p. dissecta , in terms of consumption of the aphids , aphis craccivora and myzus persicae , with suitable values of coefficient of attack rates and handling times on these prey species . differences in handling times were found to be significant within and between the predatory species on both prey species indicating that predators respond differentially to prey species . differences in coefficients of attack rates , however , did not vary significantly in most of the treatments . the potential role of these predators in biocontrol of a. craccivora and m. persicae suggests that c. sexmaculata is the best predator for the management of both prey species , particularly a. craccivora . however , further field based studies are needed to confirm this hypothesis .

although ct coronary angiography ( ctca ) can reliably rule out coronary artery stenosis in symptomatic angina patients , the specificity is limited in these patients [ 13 ] . therefore , present guidelines discourage the use of ctca in symptomatic angina patients , as the percentage of conventional coronary angiograms ( cca ) that would be replaced by ctca is considered too limited to justify the health risk and costs associated with ctca [ 4 , 5 ] . we speculate that the influence of coronary calcification on the diagnostic performance of ctca may in part explain the limited specificity of ctca in symptomatic angina patients . first , the prevalence of coronary artery disease ( cad ) increases with coronary cs . second , blooming artifacts caused by coronary calcification resulting in over - estimation of lesion severity may result in false positive findings [ 7 , 8 ] . previous studies on the influence of cs on the diagnostic performance of ctca have provided contradictory evidence . as mentioned , some previous studies have reported a high number of false positive findings in patients with a high cs [ 7 , 8 ] . also , a recent large study reported that the specificity of ctca was 86% in subjects with a cs 400 , while it was reduced to 53% in subjects with a cs > 400 . however , others have reported no significant or only a limited impact of coronary calcification on the diagnostic accuracy of ctca [ 9 , 10 ] . in order to understand under which conditions ctca might be a gate - keeper to cca in patients with stable angina and unstable angina , we examined the relation between cs and the performance of ccta in these patients . this cross sectional study was designed to prospectively include symptomatic angina patients who presented with stable and unstable anginal syndromes who were referred for clinically indicated cca irrespective of cs . the study protocol was approved by the institutional review board of the erasmus university medical center . as described previously , for this multi - center multi - vendor study conducted from october 2004 until june 2006 360 patients between 50 and 70 years of age with stable angina pectoris or non - st segment elevation acute coronary syndrome underwent a non - enhanced ct scan to determine the cs followed by a ctca scan in addition to cca . no patients , vessels or segments were excluded from the analysis , even if image quality was poor due to extensive calcification , coronary motion or breathing artifacts . no patients with previous history of percutaneous transluminal coronary angioplasty , coronary artery bypass surgery , impaired renal function ( serum creatinine > 120 mol / l ) , persistent arrhythmias , inability to perform a breath hold of 15 s or known allergy to iodinated contrast material , were included . patients with a heart rate exceeding 65 beats per minute ( bpm ) received additional beta - blockers ( up to 100 mg metoprolol p.o . or up to 20 mg metoprolol i.v . ) . all scans were performed with 64-slice ct scanners in center a ( sensation 64 , siemens , forchheim , germany ) , center b ( brilliance 64 , philips medical systems , best , the netherlands ) and center c ( toshiba multi - slice aquilion 64 system , toshiba medical systems , tokyo , japan ) . a non - enhanced scan to calculate the total cs was performed prior to ctca . a bolus - tracking technique was used to synchronize the start of image acquisition with the arrival of contrast agent in the coronary arteries . the effective dose of the nonenhanced scan and the ctca was estimated from the product of the dose - length product and a conversion coefficient ( k = 0.017 msv/[mgy cm ] ) for the chest as the investigated anatomical region .table 1scan parameterssensation 64 , siemensbrilliance 64 , philipsaquilion 64 , toshibact coronary angiography gantry rotation time ( ms)330420400 slices per rotation32 264 164 1 individual detector width ( mm)0.60.6250.5 table feed ( mm / rotation)3.885.76 tube voltage ( kv)120120120 tube current ( ma s)850960900670710 retrospective gatingyesyesyes ecg x - ray tube modulationoffoffoff contrast materialiomeron 400ultravist 300370iomeron 400 volume ( ml)9510014080110 iodine flux ( g / s)2.01.62.02.0 estimated effective dose ( msv)15.5 2.218.4 3.216.0 2.3calcium score tube current ( ma s)150150150 ecg - synchronizationretrospective gatingretrospective gatingprospective triggering ecg x - ray tube modulationonon estimated radiation exposure1.7 0.81.8 0.91.2 0.5 multiple datasets were reconstructed separately with retrospective ecg - gating in order to obtain optimal image quality for all coronary segments . the agatston cs was calculated using dedicated software ( heartbeat - cs , extended brilliance workspace , philips medical system , best , the netherlands ) . ctca scans were evaluated by two experienced readers , blinded for the cca results , for lumen stenosis > 50% based on a visual estimate , using the axial images and multiplanar reformatted images . in case of disagreement , a third reader was consulted . the kappa - value for inter- and intraobserver variability was 0.70 and 0.72 , respectively . all ccas , which were used as the standard of reference , were evaluated by a core lab of experienced cardiologists , who were unaware of the results of the ctca . stenoses were evaluated in the cca projection with the worst degree of stenosis , and classified as significant if the lumen diameter reduction exceeded 50% as measured by qca . descriptive statistics were used to evaluate the diagnostic performance of cs and ctca to detect patients with significant cad , including sensitivity , specificity , positive and negative predictive values , and positive and negative likelihood ratios . the likelihood ratio incorporates both the sensitivity and specificity of a test and provides a direct estimate of how much a test result will change the odds of having a disease . these diagnostic parameters were expressed with a 95% confidence interval ( ci ) calculated with binomial expansion . the diagnostic performance of ctca was calculated for all patients combined , and for pre - defined subgroups of patients with an agatston cs < 10 , between 10 and 100 , between 100 and 400 and > 400 . a chi - square test was performed to test for statistical significance ( p < 0.05 ) . prevalence of significant cad was based on the presence of at least one significant stenosis as determined by qca , which was considered the standard of reference . two - hundred and forty - five ( 68% ) of 360 study participants were male , and 233 ( 65% ) presented with stable chest pain syndromes . the mean age was 60 6 years , the mean body mass index was 27.3 3.8 kg / m and the median agatston cs was 213 ( inter - quartile range 42553 ) . no significant differences in patient demographics were seen in patients presenting with stable and unstable anginal syndromes , except for a higher incidence of smokers and a higher prevalence and extent of significant cad in the unstable angina patients.table 2patient characteristicsvariableall patients(n : 360 , 100%)stable anginal syndromes(n : 233 , 65%)unstable anginal syndrome(n : 127 , 35%)ptypical angina pectoris151 ( 42%)151 ( 65%)atypical angina pectoris82 ( 23%)82 ( 35%)unstable angina pectoris127 ( 35%)127 ( 100%)men245 ( 68%)156 ( 67%)89 ( 70%)0.56age ( years)60 660 660 60.72body mass index ( kg / m)27.3 3.827.6 3.926.8 3.50.06heart rate ( bpm)59 959 1059 80.99risk factors hypertension219 ( 61%)149 ( 64%)70 ( 55%)0.11 hypercholesterolemia228 ( 63%)151 ( 65%)77 ( 61%)0.49 diabetes mellitus63 ( 18%)47 ( 20%)16 ( 13%)0.08 smoker137 ( 38%)74 ( 32%)63 ( 50%)0.001 family history of cad183 ( 51%)113 ( 48%)70 ( 55%)0.27 body mass index 30 kg / m85 ( 24%)59 ( 25%)26 ( 20%)0.36 previous myocardial infarction53 ( 15%)36 ( 15%)17 ( 13%)0.64calcium score ( agatston score)213 ( 42553)211 ( 31639)216 ( 44478)0.59conventional coronary angiography prevalence of obstructive cad246 ( 68%)146 ( 63%)100 ( 79%)0.002 absence of significant cad114 ( 32%)87 ( 37%)27 ( 21%)0.009 single vessel disease141 ( 39%)88 ( 38%)53 ( 42% ) two vessel disease78 ( 22%)46 ( 20%)32 ( 25% ) three vessel disease21 ( 6%)10 ( 4%)11 ( 9%)left main coronary artery disease6 ( 2%)2 ( 1%)4 ( 3%)mean and standard deviationblood pressure 140/90 mm hg or treatment for hypertensiontotal cholesterol > 180 mg / dl or treatment for hypercholesterolemiatreatment with oral anti - diabetic medication or insulinmedian and quartiles . p - values are significant if values < 0.05 patient characteristics mean and standard deviation blood pressure 140/90 mm hg or treatment for hypertension total cholesterol > 180 mg / dl or treatment for hypercholesterolemia treatment with oral anti - diabetic medication or insulin median and quartiles . p - values are significant if values < 0.05 as shown in table 3 , for all patients combined the sensitivity of ctca to detect significant cad was 99% ( 95%ci 97100% ) , the specificity was 64% ( 95%ci 5573% ) , positive and negative predictive value were 86% ( 95%ci 8189% ) and 97% ( 95%ci 90100% ) , respectively . positive and negative likelihood ratios to detect or exclude significant cad were 2.8 ( 95%ci 2.23.5 ) and 0.01 ( 95%ci 0.000.05 ) for ctca . six of 37 ( 16% ) subjects with a cs of 0 , and 15 of 62 ( 24% ) subjects with a cs < 10 had significant cad on cca . detection of significant cad by ctca was successful in all these subjects.table 3diagnostic performance of ctca in all patientsprevalence of disease ( % ) ntptnfpfnsensitivity ( % ) specificity ( % ) ppv ( % ) npv ( % ) lr+lrdiagnostic accuracy ( % ) all patients683602447341299 ( 97100)64 ( 5573)86 ( 8189)97 ( 90100)2.8 ( 2.23.5)0.13 ( 0.000.05)88 ( 8591)a : cs < 102462154250100 ( 75100)89 ( 7696)75 ( 5190)100 ( 90100)9.4 ( 4.122)0.00 ( 0.00n / an)92 ( 8599)b : cs 10 to < 1006563411390100 ( 89100)59 ( 3779)82 ( 6891)100 ( 721002.4 ( 1.54.0)0.00 ( 0.00n / an)86 ( 7794)c : cs 100 to < 40075118861515298 ( 91100)50 ( 3268)85 ( 7691)88 ( 6298)2.0 ( 1.42.8)0.12 ( 0.020.38)86 ( 7992)d : cs 40087114993120100 ( 95100)20 ( 549)89 ( 8294)100 ( 31100)1.3 ( 1.01.6)0.00 ( 0.00n / an)89 ( 8495)a vs. dnsp = 0.017nsnsp < 0.0001n / annsdiagnostic performance of ctca in all patients combined , and in subgroups per agatston cs ( a : cs less than 10 , b : cs between 10 and 100 , c : cs between 100 and 400 , and d : cs greater than 400 ) . specificity and positive likelihood ratio decreased with increasing cs ( p - value for the comparison by chi - squared test across groups a and d)abbreviations : n , number of patients ; tp , true positive ; at least one significant stenosis in a patient detected by ctca and cca regardless of location of stenosis ; tn , true negative ; no significant stenosis in a patient detected either by ctca or cca ; fp , false positive ; significant stenosis detected by ctca and no significant stenosis detected by cca ; fn , false negative , no significant stenosis detected by ctca and at least one significant stenosis detected by cca ; lr+ , positive likelihood ratio ; lr , negative likelihood ratio ; n / an , not analyzed ( division by zero ) diagnostic performance of ctca in all patients diagnostic performance of ctca in all patients combined , and in subgroups per agatston cs ( a : cs less than 10 , b : cs between 10 and 100 , c : cs between 100 and 400 , and d : cs greater than 400 ) . specificity and positive likelihood ratio decreased with increasing cs ( p - value for the comparison by chi - squared test across groups a and d ) abbreviations : n , number of patients ; tp , true positive ; at least one significant stenosis in a patient detected by ctca and cca regardless of location of stenosis ; tn , true negative ; no significant stenosis in a patient detected either by ctca or cca ; fp , false positive ; significant stenosis detected by ctca and no significant stenosis detected by cca ; fn , false negative , no significant stenosis detected by ctca and at least one significant stenosis detected by cca ; lr+ , positive likelihood ratio ; lr , negative likelihood ratio ; n / an , not analyzed ( division by zero ) the estimated average effective radiation exposure was 1.2 0.5 to 1.8 0.9 msv for cs and 15.5 2.2 to 18.4 3.2 msv for ctca . with increasing cs the prevalence of significant cad increased steeply ( table 3 ) . whereas for patients with a cs < 10 the prevalence of significant cad was 24% , in those with a cs > 400 it was 87% . negative ctca scans were associated with a negative likelihood ratio of 0.00.1 independent of cs ( table 3 ) . a positive ctca was associated with a high positive likelihood ratio of 9.4 ( 95%ci 4.122 ) if cs was < 10 . however , for higher cs the positive likelihood ratio never exceeded 3.0 and for cs > 400 it decreased to 1.3 ( 95%ci 1.01.6 ) . also , the specificity of ctca diminished significantly with increasing cs ( table 3 ) . in patients with a cs < 10 specificity was 89% ( 95%ci 7696% ) . for higher cs specificity never exceeded 59% and for cs > 400 decreased to 20% ( 95%ci 549% ) . only 3 of 15 patients with a cs > 400 and no significant cad on cca were evaluated as negative by ctca . as shown in table 4 , the impact of cs on the diagnostic performance of ctca is similar for patients with stable and unstable anginal syndromes . a cs < 10 was present in 43 of 233 ( 18% ) stable angina patients and in 19 of 127 ( 15% ) unstable angina patients . in 43 stable angina patients and 19 unstable angina patients with cs < 10 , ctca correctly identified 32 ( 74% ) stable angina patients and 10 ( 53% ) unstable angina patients in whom no significant cad was present on cca , at no false negative ctca scans.table 4diagnostic performance of ctca in patients with ( a ) stable angina and ( b ) unstable anginaprevalence of disease ( % ) ntptnfpfnsensitivity ( % ) specificity ( % ) ppv ( % ) npv ( % ) lr+lrdiagnostic accuracy ( % ) ( a ) stable angina632331455631199 ( 96100)64 ( 5374)83 ( 7688)98 ( 89100)2.8 ( 2.13.7)0.01 ( 0.000.08)86 ( 8291)a : cs < 10164373240100 ( 56100)89 ( 7396)64 ( 3288)100 ( 87100)9.0 ( 3.622.7)0.00 ( 0.00n / an)91 ( 8299)b : cs 10 to < 100543519970100 ( 79100)56 ( 3179)73 ( 5288)100 ( 63100)2.3 ( 1.34.0)0.00 ( 0.00n / an)80 ( 6793)c : cs 100 to < 4007076521310198 ( 89100)57 ( 3576)84 ( 7292)93 ( 64100)2.3 ( 1.43.6)0.03 ( 0.000.25)86 ( 7893)d : cs 4008477652100100 ( 93100)17 ( 349)86 ( 7693)100 ( 20100)1.2 ( 0.91.5(0.00 ( 0.00n / an)97 ( 8095)a vs. dnsp = 0.023nsnsp < 0.0001n / anns(b ) unstable angina79127991710199 ( 94100)63 ( 4280)91 ( 8395)94 ( 71100)2.7 ( 1.64.4)0.02 ( 0.000.12)91 ( 8696)a : cs < 10421981010100 ( 60100)91 ( 57100)89 ( 51100)100 ( 66100)11.0 ( 1.771.3)0.00 ( 0.00n / an)95 ( 85100)b : cs 10 to < 100792822420100 ( 82100)67 ( 2494)92 ( 7299)100 ( 40100)3.0 ( 1.09.3)0.00 ( 0.00n / an)93 ( 83100)c : cs 100 to < 40083423425197 ( 93100)29 ( 570)87 ( 7295)67 ( 1398)1.4 ( 0.92.2)0.10 ( 0.011.39)86 ( 7596)d : cs 400923734120100 ( 87100)33 ( 287)94 ( 8099)100 ( 5100)1.5 ( 0.73.3)0.00 ( 0.00n / an)95 ( 87100)a vs. dnsnsnsnsp < 0.0001n / annsdiagnostic performance of ctca in all patients with ( a ) stable angina and ( b ) unstable angina combined , and in subgroups per agatston cs . specificity and positive likelihood ratio decreased with increasing cs ( p - value for the comparison by chi - squared test across groups a and d)abbreviations as for table 3 diagnostic performance of ctca in patients with ( a ) stable angina and ( b ) unstable angina diagnostic performance of ctca in all patients with ( a ) stable angina and ( b ) unstable angina combined , and in subgroups per agatston cs . specificity and positive likelihood ratio decreased with increasing cs ( p - value for the comparison by chi - squared test across groups a and d ) abbreviations as for table 3 in this study we set out to determine the relation between cs and the performance of ccta in symptomatic stable and unstable angina patients in order to understand under which conditions ctca might be a gate - keeper to cca in such patients . we report that , in symptomatic angina patients , a negative ctca reliably excludes significant cad independent of cs but that the specificity and the positive likelihood ratio of ctca decrease sharply with cs > 10 , and especially with cs > 400 . thus , in symptomatic angina patients with a cs > 10 , and especially in those with a cs > 400 , ctca has limited additional value in the diagnostic work - up . in the 17% of symptomatic angina patients with a cs < 10 , ctca provides excellent negative and positive likelihood ratios for the detection or ruling out of significant cad . ctca correctly identifies the 74% of stable angina patients and 53% of unstable angina patients with a cs < 10 without significant cad in whom no cca would be necessary , at no false negative scans . the negative influence of cs on the diagnostic performance of ctca was especially apparent in patients with a cs > 400 , in whom the specificity was only 20% ( 95%ci 549% ) and the positive likelihood ratio was only 1.3 ( 95%ci 1.01.6 ) . ninety - nine of 114 ( 87% ) patients with a cs > 400 had significant cad on cca . of the remaining 15 patients , i.e. those with a cs > 400 and no significant cad on cca , only 3 were evaluated as negative by ctca . the 87% prevalence of significant cad in patients with a cs > 400 is probably the most important factor . in addition , calcium related blooming artifacts due to the limited spatial resolution of ctca may have caused overestimation of lesion size , and thus false positive results [ 7 , 8 ] . these findings are well in line with a previous study in which ctca was of limited additional value in the diagnostic work - up of symptomatic patients with a high prevalence of significant cad . as mentioned above , previous studies on the impact of cs on the diagnostic impact of ctca are contradictory . a previous large study reported that the specificity of ctca was 86% in subjects with a cs 400 , while it was 53% in subjects with a cs > 400 . unfortunately , no data on the diagnostic performance of ctca in subcategories of cs < 400 were reported . also , two other previous studies found that coronary calcification was one of the main factors leading to false positive results [ 7 , 8 ] . however , pundziute et al . reported no significant impact of a cs > 400 on the diagnostic performance of ctca in 110 patients clinically referred for cca . divided 120 patients clinically referred for cca in groups with a cs below and above the median agatston cs of 55 . he reported that the diagnostic performance of ctca was affected only to a small extent by the cs . the prevalence of significant cad and the cs of our population are similar to the previous studies mentioned , yet our study is a multicenter multivendor study and it has included substantially more subjects . interestingly , in a recent study in 664 asymptomatic individuals the prevalence of at least one significant stenosis on ctca increased from 7.9% for patients with a cs between 1 and 100 to 14.5% for patients with a cs between 400 and 1,000 . thus , due to the absence of a standard of reference the diagnostic performance of ctca for the detection of significant cad could not be determined . the prevalence of significant cad in the highest cs categories was significantly lower than in our study . this could be explained by the fact that the former study included asymptomatic subjects whereas we studied symptomatic angina patients . importantly however , cca merely provides information on vessel lumen while ctca also gives information on stenosis location and size , plaque burden , and plaque composition . future research is needed to determine the clinical value of this information , and specifically so in stable and unstable patients with a high cs in whom ctca seems to have limited additional value if only the detection of significant cad is taken into consideration . when compared to most previous studies , the present study has included a larger number of subjects in a multicenter multivendor setting , allowing for a more reliable subgroup analysis . most importantly , in line with most previous studies , our subjects were derived from a selected population of symptomatic stable and unstable angina patients who were referred for cca for clinical reasons . future studies will need to address the value of ctca in the diagnostic work - up of asymptomatic patients with a high cs . future research will need to determine whether improvements in spatial and temporal resolution with new generation scanners using dual source techniques , dual energy techniques or more detector rows may improve the diagnostic performance of ctca in patients with a high cs . the median cs for patients under 50 years of age is likely to be lower , and that of patients over 70 years of age is likely to be higher than the median cs of our population [ 17 , 18 ] . as the association between cs and significant cad varies with age [ 17 , 18 ] , our findings may not fully apply to subjects outside this age range . fourthly , in line with previous studies , we excluded patients who were not in stable sinus rhythm , or did have renal insufficiency . additional research should focus on the value of the combination of cs , ctca and functional tests such as positron emission tomography and single photon emission computed tomography in the diagnostic work - up of angina patients referred for cca .

present guidelines discourage the use of ct coronary angiography ( ctca ) in symptomatic angina patients . we examined the relation between coronary calcium score ( cs ) and the performance of ctca in patients with stable and unstable angina in order to understand under which conditions ctca might be a gate - keeper to conventional coronary angiography ( cca ) in such patients . we included 360 patients between 50 and 70 years old with stable and unstable angina who were clinically referred for cca irrespective of cs . patients received cs and ccta on 64-slice scanners in a multicenter cross - sectional trial . the institutional review board approved the study . diagnostic performance of ctca to detect or rule out significant coronary artery disease was calculated on a per patient level in pre - defined cs categories . the prevalence of significant coronary artery disease strongly increased with cs . negative ctca were associated with a negative likelihood ratio of < 0.1 independent of cs . positive ctca was associated with a high positive likelihood ratio of 9.4 if cs was < 10 . however , for higher cs the positive likelihood ratio never exceeded 3.0 and for cs > 400 it decreased to 1.3 . in the 62 ( 17% ) patients with cs < 10 , ctca reliably identified the 42 ( 68% ) of these patients without significant cad , at no false negative ctca scans . in symptomatic angina patients , a negative ctca reliably excludes significant cad but the additional value of ctca decreases sharply with cs > 10 and especially with cs > 400 . in patients with cs < 10 , ctca provides excellent diagnostic performance .

however , tumor recurrence occurs in approximately 50% of cases . circulating tumor cells ( ctcs ) are assumed to be the predominant cause of metastatic dissemination , and evidence supports the tumor - initiating properties of ( a subset of ) ctcs.1 , 2 the cellsearch platform ( janssen diagnostics llc , raritan , nj ) exploits expression of cell surface epithelial markers to capture , enrich , and enumerate ctcs . the detection of ctcs in peripheral blood is associated with a worse prognosis in nsclc.3 , 4 however , the total number of ctcs detected by cellsearch in early - stage nsclc is low . proximity to primary tumor and blood draw upstream of capillary bed filtering implies that pulmonary vein sampling may be advantageous to assess the association of ctc prevalence with disease outcome . to test this hypothesis , we performed a pilot study of cellsearch ctc enumeration , comparing blood sampling from peripheral and tumor - draining pulmonary veins at the time of tumor resection . we hypothesized that patients with detectable pulmonary vein ctcs would have a higher risk for disease recurrence . this was a single - center , prospective study of patients undergoing a curative - intent operation for stage i to iiia nsclc ( university hospital of south manchester , manchester , uk ) . blood samples ( 10 ml ) were taken immediately before surgery from a peripheral vein and intraoperatively ( 10 ml ) from the cancer - draining pulmonary vein before tumor resection or vessel ligation to minimize artifactual elevation in ctc number . cellsearch processing and analysis followed previously published protocols.6 , 7 ctc enumeration was expressed as number of cells per 7.5 ml of blood . circulating tumor microemboli ( ctms ) were defined as three or more contiguous ctcs in a cluster . primary and secondary outcome measures were disease - free survival ( dfs ) and overall survival ( os ) , respectively . patients found to have unresectable disease during the operation were not included in the survival analysis . dfs and os were measured from the day of the operation until either the diagnosis of lung cancer recurrence or death . in patients with no evidence of recurrence peripheral vein ctc status was predefined as positive at one or more ctcs per 7.5 ml of blood . pulmonary vein ctc status was an exploratory end point , and high was defined according to the upper quartile of the ctc count . kaplan - meier curves were used to demonstrate dfs and os for each ctc category and compared using log - rank tests . a cox proportional hazard regression analysis with a backward conditional regression approach was used to define the optimal multivariate model to determine independent risk factors associated with dfs and os . the study was granted ethical approval by the north west 7 research ethics committee ( reference no . the clinical parameters of the study cohort ( n = 30 ) are detailed in table 1 . lung cancer recurrence affected 10 of 30 patients ( 33% ) , most commonly ( in seven of 10 cases ) at distant sites . almost half of the study population died ( 13 of 30 patients [ 43% ] ) ; one patient who died within 30 days of having the operation was excluded . cause of death was not recorded ; however , nine of 10 patients with recurrent disease ( 90% ) died compared with three of 19 ( 16% ) patients without disease recurrence ( p = 0.0002 ) , suggesting that lung cancer was the predominant cause . peripheral blood was taken from 27 of 30 patients ( 90% ) and a pulmonary vein sample was obtained from the whole study cohort . ctcs ( 1 ctc per 7.5 ml of blood ) were more commonly detected in the pulmonary vein ( in 13 of 30 patients [ 43% ] , range 13093 ctcs per 7.5 ml of blood ) than in the peripheral blood ( in six of 27 patients [ 22% ] , range 14 ctcs per 7.5 ml of blood ) ( p = 0.018 ) and total ctc number was greater in the pulmonary vein ( p = 0.002 ) . only two of 27 patients ( 7% ) had ctcs detected in the peripheral and pulmonary vein samples ; both died after lung cancer recurrence within 8 months of having their operation . ctms were detected only in pulmonary vein blood ( six of 30 patients ( 20% ) , range 137 ctms per 7.5 ml of blood ) . the numbers of pulmonary vein ctcs and ctms were significantly correlated ( r = 0.51 , p = 0.002 ) . patients were stratified according to high ( 18 ctcs per 7.5 ml of blood ) or low ( < 18 ctcs per 7.5 ml of blood ) pulmonary vein ctc groups . differences approaching significance in dfs ( p = 0.055 ) and 3-year os ( p = 0.082 ) were observed ( fig . 1a and b ) . clinical characteristics stratified according to these groups are detailed in table 1 . ctms were identified in six of seven of those in the high - ctc group ( 85.7% ) compared with in none of 23 of the low - ctc group ( p = 0.00001 ) . the presence of ctms ( 1 ctm per 7.5 ml of blood ) was associated with a significant reduction in dfs ( p = 0.011 ) and os ( p = 0.024 ) . the detection of any ctcs ( 1 ctc per 7.5 ml of blood ) in peripheral blood was associated with reduced dfs ( p = 0.011 ) and os ( p = 0.037 ) ( fig . variables included in the model were age , pathological stage , and log2-transformed ctc and ctm numbers in pulmonary and/or peripheral vein blood as continuous variables . both peripheral vein ( p = 0.01 ) and pulmonary vein ctc number ( p = 0.001 ) remained independent risk factors ( after bonferroni correction ) for lung cancer recurrence along with pathological stage ( p = 0.00006 ) . the hazard ratio ( hr ) for lung cancer recurrence for every doubling of ctc count was 1.51 ( 95% confidence interval [ ci ] : 1.171.94 ) for pulmonary vein and 5.26 ( 95% ci : 1.4818.68 ) for peripheral vein ctcs . pulmonary vein ctc count ( p = 0.01 ) and pathological stage ( p = 0.001 ) were independent risk factors for death ; the association with peripheral vein ctc number was of borderline significance ( p = 0.026 ) after bonferroni correction . the hr for death for every doubling of ctc count was 1.33 ( 95% ci : 1.061.67 ) for pulmonary and 3.73 ( 95% ci : 1.1711.93 ) for peripheral blood . patients were categorized in a high - risk group if they had ctms detected ( 1 ctm per 7.5 ml of blood ) or two or more ctcs per 7.5 ml of peripheral blood . these measures were chosen to define the highest - risk group because of increased specificity for lung cancer recurrence ( in six of nine patients [ 66.7% ] ) compared with cutoff values of one or more ctc per 7.5 ml of peripheral blood or 18 or more ctcs per 7.5 ml of pulmonary vein blood ( in six of 11 patients [ 54.5% ] ) . lung cancer recurred in five of seven of these high - risk patients ( 71.4% ) compared with in five of 23 low - risk patients ( 21.7% ) ( p = 0.026 ) . deaths occurred in five of seven high - risk patients ( 71.4% ) and eight of 22 low - risk patients ( 36.4% ) ( p = 0.19 ) . kaplan - meier analysis demonstrated a significant difference in dfs ( p = 0.001 ) and os ( p = 0.002 ) ( fig . the hr for lung cancer recurrence was 8.44 ( 95% ci : 2.0934.05 ) ( p = 0.003 ) and that for death was 7.53 ( 95% ci : 1.7732.04 ) ( p = 0.006 ) in the high - risk group after multivariate analysis . overall , these data show that pulmonary vein ctc / ctm enumeration provides additional information on risk for disease recurrence compared with ctc enumeration from the peripheral circulation alone . this study assessed the prognostic significance of ctcs and the feasibility of pulmonary vein sampling at the time of surgical resection nsclc . significantly more cellsearch - enriched ctcs were detected in pulmonary blood than in matched peripheral blood , and the presence of pulmonary vein ctcs was an independent risk factor for lung cancer recurrence and death . ctms were also detected in samples of pulmonary vein blood , and patients with detectable ctms or two or more peripheral vein ctcs represented a high - risk subgroup with an eightfold increased risk for disease recurrence and sevenfold increased risk for death . blood was taken before tumor manipulation , vessel ligation , or lung resection to avoid as much as possible artifactual elevation of ctc number . the association of pulmonary vein ctc count with disease recurrence and reduced os suggests that this may be a biologically important cell population reflecting tumor biology rather than being a consequence of surgery alone . the detection of ctms has been reported to increase the risk for lung cancer recurrence after resection . we have previously described the prognostic significance in sclc of cellsearch - detected ctms and postulate that ctms have an increased ability to survive in the circulation . in vivo models also support evidence that ctms have increased malignant potential.10 , 11 three patients suffered disease recurrence despite negative ctc detection by cellsearch . coexpression of epithelial cell adhesion molecules and cytokeratins is a requirement for ctc capture and assignment using the cellsearch platform . although this subpopulation of ctcs is biologically important as evidenced by the association between cellsearch ctc detection and prognosis in numerous studies,3 , 4 , 12 , 13 , 14 it may not provide a comprehensive assessment of total ctc burden . subpopulations of ctcs that lose their epithelial phenotype ( e.g. , epithelial - to - mesenchymal transition ) may become undetectable by cellsearch . to more fully assess ctc heterogeneity , the use of marker - independent platforms is required ; however , no other platform has been through the vigorous validation process required for routine use in the clinic . to confirm our findings and explore the potential for ctcs to be used in the clinical management of early - stage nsclc , a larger validation study is required . in addition to the potential role of ctcs as a prognostic / predictive biomarker , isolation and genetic analysis of individual ctcs may shed light on tumor biology and the metastatic process . whether ctcs are representative of the cells responsible for distant metastatic spread and whether they predict the genetic landscape of recurrent disease are important areas for future research , which we are addressing as part of the tracerx study .

tumor recurrence after surgical resection of nsclc obstructs long - term disease - free survival in approximately 50% of cases . our data suggest that combining circulating tumor cell enumeration ( as single cells or clusters ) in tumor - draining pulmonary vein and peripheral blood ( assessed by cellsearch ) at the time of nsclc surgery better identifies those patients at higher risk for lung cancer recurrence than does peripheral circulating tumor cell number alone .

the current classification of periodontal diseases includes accidental , iatrogenic , and factitious traumatic lesions.1 although the prevalence of traumatic gingival lesions is relatively high , there are limited reports on the diagnosis and management of these injuries . traumatic lesions , whether chemical , physical , or thermal in nature , are among the most common in the mouth . sometimes the lesions are termed gingivitis artefacta.24 self - injurious behavior affecting the gingival tissues has minor and major variants.2 gingivitis artefacta minor was recognized as being more common and thought to be provoked by a preexisting locus of irritation . this form results from rubbing or picking the gingiva using the fingernail , or perhaps from abrasive foods such as crisps.2,3,5 in this form , the habit was claimed to respond readily to simple treatment that removed the underlying source of irritation . the major form is more severe and widespread and can involve the deeper periodontal tissues.2,6 other areas of the mouth such as the lips and tongue may be involved and extraoral injuries may be found on the scalp , limbs , or face.2 this type of behavior also differed in being more resistant to conventional forms of treatment and was probably associated with an emotional disorder.2,4 self - inflicted oral injuries seem to be much more common in children than in teenagers and adults . most of the reported cases have involved female patients.7 although the gingiva is the most commonly targeted tissue , no structure in the oral cavity is immune from the effects of this type of behavior . self - inflicted gingival injuries in children and young individuals can occur as a result of accidental trauma , premeditated infliction , or chronic habits such as fingernail biting , digit sucking , or sucking on objects such as pens , pencils , toothpicks , dental floss , or pacifiers.224 most case reports suggest that the method of producing injury is by picking or scratching the gingival tissues with fingers or fingernails . the purpose of this case report was to illustrate the destructive nature of the habit and to describe the successful treatment of this case . a 14-year - old girl was referred by her dentist to the periodontology clinic of the faculty of dentistry , ataturk university , for evaluation and treatment of the gingival recession associated with the mandibular right incisor . upon questioning the patient , she admitted to have been scratching her gingiva with her fingernail for a long period of time . the patient further stated that this scratching habit occurred mainly time of exam . the patient s medical history was non - contributory and she did not take any medications . clinical evaluation revealed gingival recession on the buccal surface extending 3 mm apical to the cemento - enamel junction ( cej ) and a narrow zone of attached gingival measuring approximately 1 mm ( figure 1 ) . there was loss of papilla height on the mesial aspect of the incisor and moderate gingival overgrowth on the distal aspect . written informed consent was obtained from the patient s parent after all treatment procedures had been fully explained . rendering treatment in this case would help the dental problem but total treatment would require discovery and elimination of the intrinsic emotional factors and extrinsic contributing factors . thus the patient was referred to a psychiatrist for evaluation and treatment . the damage caused by scratching was pointed out to the patient and she was convinced that she should discontinue this habit . before surgical treatment of the gingival recession , the patient underwent scaling , root planning , crown polishing , oral hygiene instruction , and psychological support . the goal of surgical periodontal treatment was to restore harmonious appearance of the gingiva by covering the root surface to an equal level of the neighboring teeth and , at the same time ; to increase the zone of attached gingiva . it was decided to treat this problem with a free gingival graft ( fgg ) . the area was gently irrigated with sterilized physiological saline solution . before surgery , extraoral antisepsis was performed with 10% povidone - iodine solution . the defect was treated by approach described by miller.25 a local anesthetic was administered to donor and recipient sites to achieve anesthesia . following local anesthesia , for preparing a recipient bed for the fgg , a horizontal incision was made at the level of the cej in correspondence to the base of adjacent papilla to the line angles of the neighboring teeth from which vertical incisions were realized to the apical extent of the recipient bed , 3 mm apical to the recession , such delimited tissue was then removed by a partial thickness incision . the graft of adequate size and of about 1.5 mm thickness was then harvested from the palate in correspondence to the bicuspid area by a partial thickness incision with a no.15 blade ; within 1 min of removal , the graft was then sutured to the recipient bed by two lateral 4 - 0 silk sutures and was then stabilized by a tooth - suspended 4 - 0 silk crossed suture ( figure 2 ) ; an attempt was made to place the coronal margin of the graft in correspondence to the cej . the grafted tissue was then compressed for about 5 min onto the recipient bed . the patient was prescribed analgesics and instructed to rinse twice daily with 0.12% chlorhexidine rinse for 2 weeks postoperatively and to avoid trauma or pressure at the surgical site . the dressing and sutures were removed 10 days after surgery , home care instructions were given . healing was uneventful . at 4 weeks , her plaque control was not good and the gingiva at the surgical site was edematous and reddish ( figure 3 ) . normal appearance was established at 4 months postoperatively and the amount of attached gingiva was approximately 3 mm . postoperatively , probing depth at the midbuccal site was less than 1 mm , and the free gingival margin was located less than 0.5 mm apically to the cej ( figure 5 ) . the patient was followed for approximately 1 year postoperatively and complete tissue healing was achieved . the patient reported at her postoperative appointment that she had stopped her fingernail scratching habit . rendering treatment in this case would help the dental problem but total treatment would require discovery and elimination of the intrinsic emotional factors and extrinsic contributing factors . thus the patient was referred to a psychiatrist for evaluation and treatment . the damage caused by scratching was pointed out to the patient and she was convinced that she should discontinue this habit . before surgical treatment of the gingival recession , the patient underwent scaling , root planning , crown polishing , oral hygiene instruction , and psychological support . the goal of surgical periodontal treatment was to restore harmonious appearance of the gingiva by covering the root surface to an equal level of the neighboring teeth and , at the same time ; to increase the zone of attached gingiva . it was decided to treat this problem with a free gingival graft ( fgg ) . the area was gently irrigated with sterilized physiological saline solution . before surgery , extraoral antisepsis was performed with 10% povidone - iodine solution . the defect was treated by approach described by miller.25 a local anesthetic was administered to donor and recipient sites to achieve anesthesia . following local anesthesia , for preparing a recipient bed for the fgg , a horizontal incision was made at the level of the cej in correspondence to the base of adjacent papilla to the line angles of the neighboring teeth from which vertical incisions were realized to the apical extent of the recipient bed , 3 mm apical to the recession , such delimited tissue was then removed by a partial thickness incision . the graft of adequate size and of about 1.5 mm thickness was then harvested from the palate in correspondence to the bicuspid area by a partial thickness incision with a no.15 blade ; within 1 min of removal , the graft was then sutured to the recipient bed by two lateral 4 - 0 silk sutures and was then stabilized by a tooth - suspended 4 - 0 silk crossed suture ( figure 2 ) ; an attempt was made to place the coronal margin of the graft in correspondence to the cej . the patient was prescribed analgesics and instructed to rinse twice daily with 0.12% chlorhexidine rinse for 2 weeks postoperatively and to avoid trauma or pressure at the surgical site . the dressing and sutures were removed 10 days after surgery , home care instructions were given . healing was uneventful . at 4 weeks , her plaque control was not good and the gingiva at the surgical site was edematous and reddish ( figure 3 ) . normal appearance was established at 4 months postoperatively and the amount of attached gingiva was approximately 3 mm . postoperatively , probing depth at the midbuccal site was less than 1 mm , and the free gingival margin was located less than 0.5 mm apically to the cej ( figure 5 ) . the patient was followed for approximately 1 year postoperatively and complete tissue healing was achieved . the patient reported at her postoperative appointment that she had stopped her fingernail scratching habit . self - inflicted oral injuries can be premeditated or accidental or can result from an uncommon habit . these injuries usually results from a foreign object or a patient s fingernail that habitually causes an erosion of the gingival tissue in a specific area.19 there are varying degrees of self - injurious behavior from simple fingernail biting to extremes in self - mutilation.8,12,15,18,24 in the present case , the mechanical trauma caused by the almost constant self - injurious behavior is considered to have been the primary etiologic factor . this case serves as another opportunity to emphasize the necessity of a comprehensive history which obtains the more subtle information relative to etiology . habitual fingernail scratching is a common behavior among children.19 this is probably true but such injuries are not limited to children , diagnosed adolescents and adults.4,7,26 in this case , a teenage patient has a habitual fingernail scratching . dentists need to be cognizant of the potential ramifications of fingernail scratching including not only physical injury but also gingival recession , potential bacterial contamination ( infection ) , inflammation , attachment loss , bone loss , and even tooth loss . the case described here demonstrates that scratching is a potential cause of localized gingival recession , attachment loss , edema and ulceration . the etiology of self - inflicted oral injuries in adolescent and adult includes some emotional disturbance.20,22 in the case presented here , the anxiety and stress of exam are believed to have been the main reason behind the patient s behavior . management of patients with self - inflicted injuries is usually complicated by their lack of compliance and communication . in cases of self - inflicted injury sedation , behavior modification and restraints are usually utilized to control the destructive behavior.7,2224 our patient was referred to a psychiatrist for evaluation and she was convinced that she should discontinue this habit . in the case presented here , treatment of the gingival recession was done surgical periodontal treatment . fggs have been utilized to increase amounts of keratinized tissue and obtain root coverage , considered necessary to improve the marginal adaptation of soft tissue to the root surfaces and to inhibit further apically - directed loss of soft tissues and bone.27 therefore , it was decided to treat this problem with a fgg . the successful root coverage was obtained % s ranging from 90 to 100% in class 1 and 2 gingival recession,2628 as was demonstrated in this case . this case report shows that it is possible to treat gingival injury and maintain the periodontal health of a patient with destructive habit . patient compliance , regular dental follow - ups , and psychologic support may be useful in stabilizing the periodontal condition of these patients . dentists must be aware that self - inflicted gingival injury , although thought to be uncommon , is quite widespread .

injuries to oral soft - tissues can occur due to accidental , iatrogenic , and factitious traumas . traumatic lesions , whether chemical , physical , or thermal in nature , are among the most common in the mouth . a type of physical injury to the gingival tissues is self - inflicted . sometimes the lesions are termed gingivitis artefacta . self - inflicted gingival injuries in children and adolescents can occur as a result of accidental trauma , premeditated infliction , or chronic habits such as fingernail biting , digit sucking , or sucking on objects such as pens , pencils or pacifiers . the purpose of this case report was to illustrate the destructive nature of the habit and to describe the successful treatment of this case . a 14-year - old girl with moderate pain , gingival bleeding and recession in the anterior mandibulary region was admitted to periodontology clinic . upon questioning , the patient readily admitted traumatizing her gingiva with her fingernail . treatment consisted of oral hygiene instruction , mechanical debridement , psychological support and surgical periodontal treatment . postoperatively , complete root coverage , gains in clinical attachment levels , and highly significant increases in the width of keratinized gingiva were observed . this case report shows that it is possible to treat gingival injury and maintain the periodontal health of a patient with destructive habit . patient compliance , regular dental follow - ups , and psychological support may be useful in stabilizing the periodontal condition of these patients . dentists must be aware that self - inflicted gingival injury , although thought to be uncommon , is quite widespread .

, blood pressure is measured using a sphygmomanometer ( manual or automatic ) over the brachial artery . the vast majority of the evidence supporting the benefits of blood pressure reduction is based on brachial blood pressure measurements . over the last 10 years , several antihypertensive drug treatment trials have shown an unexpected discrepancy between reduction in brachial blood pressure and observed clinical outcomes . in the heart outcomes prevention evaluation ( hope ) , the losartan intervention for endpoint reduction ( life ) in hypertension , and the australian national blood pressure 2 ( anbp2 ) trials , the observed clinical benefits tended to be greater than those expected from the decrease in brachial blood pressure . this may be explained by the pleiotropic effects of the antihypertensive drugs used in these trials . in particular , the beneficial effects of these agents on endothelial function and its vascular manifestations , such as changes in central aortic pressure and atheroma development . it has been proposed that central aortic pressure may have more pathophysiological relevance than peripheral blood pressure as a marker for the development of cardiovascular disease [ 6 , 7 ] . central aortic pressure is determined by the combination of cardiac output and peripheral vascular resistance that is modulated by arterial stiffness along with the timing and magnitude of pressure wave reflections in the arterial tree . the pressure wave generated by the left ventricle during cardiac systole travels through the vessels until it reaches the small muscular arteries and arterioles where it is reflected . the pressure waveform at any point in the arterial tree is therefore the sum of both forward and backward waveforms . when the large arteries are healthy and compliant , the reflected wave merges with the forward wave in the proximal aorta during diastole , augmenting diastolic blood pressure and aiding coronary perfusion . however , when the large arteries are stiff , pulse wave velocity increases , accelerating both incident and reflected waves . this results in the reflected wave merging with the incident wave in systole , thus augmenting central aortic systolic rather than diastolic pressure . thus , central aortic stiffness contributes directly to a wide pulse pressure with higher systolic and lower diastolic blood pressure . furthermore , the state of the vasculature in the peripheral circulation also affects the proportion of the incident wave that is reflected , thus affecting central pressure . observational studies have shown that the difference between brachial and central arterial pressures can vary by between 2 and 33 mm hg [ 9 , 10 ] . furthermore , different antihypertensive drugs have been shown to have similar effects on brachial blood pressure , but different effects on central aortic pressure [ 6 , 1113 ] . this may , in part , explain why central aortic pressure has been shown to have superior prognostic value with respect to cardiovascular events than brachial blood pressure in clinical studies [ 6 , 1113 ] . the conduit artery function evaluation ( cafe ) study [ 6 ] , was a substudy of the anglo - scandinavian cardiac outcomes trial ( ascot ) . in ascot , patients with hypertension and at least three additional cardiovascular risk factors were randomized to an atenolol plus bendroflumethiazide - based treatment regimen or an amlodipine plus perindopril - based regimen . the cafe study recruited 2,199 ascot patients after the first year of ascot follow - up ( mean age of the cohort at baseline , 63 years ) and followed them for up to 4 years . radial artery applanation tonometry and pulse wave analysis were used to derive central aortic pressures and hemodynamic indices . throughout follow up , derived central aortic systolic pressure was substantially lower in the amlodipine - based treatment group compared with the atenolol - based treatment group ( area under the curve [ auc ] difference , 4.3 mm hg ; 95 % confidence interval [ ci ] , 3.3 to 5.4 ; p < 0.0001 ) , whereas brachial systolic blood pressure was similar between the two treatment arms ( auc difference , 0.7 mm hg ; 95 % ci , 0.4 to 1.7 ; p = 0.2 ) . furthermore , central pulse pressure ( pp ) was associated with total cardiovascular events and procedures and the development of renal impairment ( unadjusted p < 0.0001 ; adjusted for baseline values p < 0.05 ) . the cafe study demonstrated that antihypertensive drugs can have different effects on central aortic pressure despite similar brachial blood pressure measurements . the authors of the cafe study proposed that their study elucidated a plausible mechanism to explain the superior clinical outcomes observed in the amlodipine - based treatment arm of ascot . they also speculated that central aortic pressure measurements may provide an explanation for the differences observed in other major outcome trials including life and hope . the findings of the cafe study [ 6 ] supported those of the preterax in regression of arterial stiffness in a controlled double - blind study ( reason ) , which reported that peripheral blood pressure measurements did not accurately reflect changes in central aortic pressure following treatment with different antihypertensive drugs . the finding that central pressure and wave reflection indices are strong independent predictors of all - cause and cardiovascular mortality has also been demonstrated in high - risk patient groups , including those with end - stage renal failure ( table 1 ) [ 11 , 12].table 1studies reporting differences in measured brachial and central blood pressure that may explain cardiovascular outcomesstudymeasurementspatient populationkey resultstrong heart study ( 2007 , 2009 ) [ 7 , 14]sphygmomanometer to measure brachial bp . radial applanation tonometry to determine central bppopulation - based , longitudinal study among 3,520 american indians followed for a mean of 4.8 yearscentral pp was strongly associated with carotid intima - media thickness , plaque score , and vascular mass . central pp was an independent predictor of cv outcomescafe ( 2006 ) [ 6]semiautomated oscillometric device to measure brachial bp . radial artery applanation tonometry and pulse wave velocity analysis to derive central aortic pressure and hemodynamic indexessubstudy of the ascot study . 2,199 patients with hypertension and 3 additional cv risk factors previously randomized to an amlodipine or atenolol - based regimen followed for up to 4 yearscentral aortic systolic bp was lower in the amlodipine versus atenolol arm throughout follow - up ( auc difference 4.3 mm hg , 3.3 to 5.4 , p < 0.0001 ) . brachial systolic bp similar ( auc difference 0.7 mm hg , 0.4 , 1.7 , p = 0.2 ) . may explain lower cv events in amlodipine armreason ( 2004 ) sphygmomanometer to measure brachial bp . pulse wave velocity analysis and pattern of wave reflections to derive central aortic pressure375 patients with hypertension randomized to atenolol or perindopril + indapamide , followed for 1 yeartreatment with perindopril + indapamide decreased brachial and central systolic bp significantly more than atenolol . in the perindopril + indapamide group the difference between brachial and central systolic bp was 8.28 1.53 mm hg versus 0.29 1.61 mm hg in the atenolol groupsafar ( 2002 ) , london ( 2001 ) aortic pulse wave velocity measurement and determination of arterial wave reflection by applanation tonometry on the common carotid artery180 patients with end - stage renal failure followed for a mean of 4.3 years . 40 cv and 30 non - cv events occurredaortic pulse wave velocity , increased augmentation index and carotid pp , were independent predictors of all - cause and cv mortalityascot anglo - scandinavian cardiac outcomes trial ; auc area under the curve ; bp blood pressure ; cafe conduit artery function evaluation ; cv cardiovascular ; pp pulse pressure ; reason preterax in regression of arterial stiffness in a controlled double - blind study studies reporting differences in measured brachial and central blood pressure that may explain cardiovascular outcomes ascot anglo - scandinavian cardiac outcomes trial ; auc area under the curve ; bp blood pressure ; cafe conduit artery function evaluation ; cv cardiovascular ; pp pulse pressure ; reason preterax in regression of arterial stiffness in a controlled double - blind study these findings are complemented by the results of the strong heart study [ 7 , 14 ] , a population - based , longitudinal study among 3,520 american indians ( mean age 58 years ) followed for a mean of 4.8 years . this study reported that a central pp greater than 50 mm hg and not brachial pp was an independent predictor of cardiovascular outcomes , regardless of age , sex , or diabetes . furthermore , central pp was strongly associated with carotid intima - media thickness , plaque score , and vascular mass , and was a stronger predictor of cardiovascular events than brachial blood pressure . together , these studies provide evidence to support the hypothesis that central aortic pressure may more accurately reflect the load on the central vasculature than brachial blood pressure ( table 1 ) [ 1113 , 6 , 7 ] . it is therefore a reasonable proposition that central pressure relates more directly to target organ damage and clinical cardiovascular disease . this has led to the suggestion that central and not brachial blood pressure should be a treatment target for cardiovascular disease risk reduction strategies . the mechanisms by which some antihypertensive drugs , including calcium channel blockers , affect the vasculature and lead to differential lowering of central and brachial blood pressure may be because of their effects on endothelial function , as will now be discussed in more detail . endothelial dysfunction , a key feature of hypertension , is primarily caused by enhanced oxidative stress , but other important contributors include age , vascular injury , metabolic disorders , deficiencies in essential substrates ( e.g. , l - arginine ) , and enzyme cofactors ( e.g. , tetrahydrobiopterin [ bh4 ] ) . endothelial dysfunction is characterized by reduced nitric oxide ( no ) bioavailability resulting in increased vascular resistance and reduced sensitivity to normal stimuli of vasodilation , such as shear stress and acetylcholine . the signaling molecule no is produced by the endothelium and has a key role in regulating vasomotor tone . in addition , no has atheroprotective effects by reducing smooth muscle cell proliferation and migration , adhesion of leukocytes to the endothelium , and platelet aggregation . no is derived from the conversion of l - arginine to l - citrulline by the enzymatic activity of endothelial no synthase ( enos ) . the activity of this electron transport enzyme requires calcium / calmodulin , flavin adenine dinucleotide , flavin mononucleotide , and bh4 as cofactors . normal physiologic levels of no increase vasodilation and interfere with the atherothrombotic process , thereby helping to maintain a healthy circulatory system . the net concentration of no in the circulation is dependent on a balance between the enzymatic production of no through the activity of enos and the production of superoxide ( o2 ) . many factors can influence enos activity , but studies have shown that its enzymatic cofactor bh4 has a particularly important role [ 1820 ] . when levels of bh4 are insufficient enos can not couple the reduction of molecular oxygen with the oxidation of l - arginine . oxidative modification of bh4 by various oxidases is a leading reason for abnormal low levels in the cell.fig . enos , endothelial nitric oxide synthase ; bh4 , tetrahydrobiopterin ; fad , flavin adenine dinucleotide ; fmn , flavin mononucleotide ; ca , calcium ; o2 , oxygen ; o2 , superoxide , no , nitric oxide ; onoo , peroxynitrite targeting mechanisms a global approach to cardiovascular risk management . enos , endothelial nitric oxide synthase ; bh4 , tetrahydrobiopterin ; fad , flavin adenine dinucleotide ; fmn , flavin mononucleotide ; ca , calcium ; o2 , oxygen ; o2 , superoxide , no , nitric oxide ; onoo , peroxynitrite reactive oxygen species ( ros ) are also generated in the vasculature by oxidases such as nad(p)h oxidase that contribute to oxidative stress . in the presence of excessive levels of o2 , no is rapidly converted to peroxynitrite ( onoo ) resulting in decreased no bioavailability and further impairment of endothelium - mediated vasodilation . furthermore , onoo molecules themselves are highly reactive and oxidize lipids , cause cellular injury , and enhanced arterial contraction ( fig . 1 ) [ 21 ] . animal models have shown that mice deficient in ros - generating enzymes have lower blood pressure levels compared with wild - type animals . further studies found that , compared with normotensive rats , spontaneously hypertensive rats had lower no bioavailability despite increased levels of enos . the effects were even more pronounced after induction of diabetes among the hypertensive animals [ 21 ] . the findings of reduced no bioavailability , despite increased levels of enos observed in these studies , may be explained by the increased production of o2 by uncoupled enos . glucose intolerance is also believed to impair enos activity directly through enhanced oxidative stress . in a human tissue study comparing endothelial cells from healthy african american and caucasian donors , those of african american origin this paradox was attributed to excessive o2 generation by nad(p)h oxidase , which contributes to increased onoo formation and uncoupled enos activity . these results are consistent with studies reporting differences in endothelial - dependent vasodilation in african american subjects compared with age- and gender - matched caucasians . reduced no bioavailability may partly explain the higher rates of hypertension observed among african americans compared with caucasians in various surveys . this has clinical importance as drug treatments that enhance endothelial no production may be of particular benefit in these populations . the finding of a relationship between reduced endothelial - derived no and increased oxidative stress led to the design of the a - heft study ( african american heart failure trial ) . that trial showed that the addition of isosorbide dinitrate and hydralazine to conventional therapy reduced relative 1-year mortality by 43 % among african american subjects with advanced heart failure . isosorbide dinitrate is an organic nitrate that directly increases vascular no levels , whereas hydralazine is a vasodilator with antioxidant activity that may scavenge oxyradical species , including o2. this adds further support to the theory that agents that enhance no bioavailability while reducing nitroxidative stress may have important benefits in this population . in summary , the relationship between no and ros is strongly implicated in the etiology of hypertension , and drugs that have both antihypertensive and antioxidant properties may be more effective at reducing blood pressure and subsequent pathology . indeed , improving no bioavailability may be an important treatment goal in the management of hypertension ( fig . 1 ) . dihydropyridine ( dhp)-type calcium channel blockers ( ccbs ) reversibly inhibit calcium entry into cardiac and vascular smooth muscle cells by binding to l - type voltage - sensitive calcium channels . of particular importance , certain dhp - type ccbs have been shown to modify endothelial function by enhancing enos activity , resulting in increased no production [ 29 , 30 ] . studies have also suggested that some of these drugs increase the antioxidant capacity of the endothelium by scavenging o2 [ 29 , 30 ] . this further protects the endothelium by reducing the availability of free radicals to react with no . the antioxidant activity is attributed to ccbs high lipophilicity , and a chemical structure that facilitates proton - donation and resonance - stabilization mechanisms that inhibit the free - radical reaction [ 30 , 31 ] . in animal models , the ccb amlodipine has been shown to increase enos activity and its mrna level in hypertensive rats . in coronary microvessels isolated from canine cardiac tissue , amlodipine caused a dose - dependent release of nitrite , the hydration product of no [ 33 ] . the effects of amlodipine on both nitrite release and the no - dependent regulation of cardiac oxygen consumption were inhibited with specific antagonists of enos such as l - n - monomethylarginine ( l - nmma ) . under identical conditions , other dhp and non - dhp - type ccbs , including nifedipine and diltiazem , failed to reproduce these effects . in clinical studies among patients with essential hypertension , nifedipine has been shown to attenuate circulating plasma levels of lipoperoxides and isoprostanes , increase plasma antioxidant capacity , and restore no bioavailability . in the elevation of nifedipine and cerivastatin on recovery of endothelial function ( encore ) i and encore ii studies , nifedipine significantly improved no - mediated coronary endothelial function in patients with coronary artery disease [ 35 , 36 ] . the safety and efficacy of ccb therapy and its role in reducing cardiovascular events and procedures has been demonstrated in several large clinical studies among patients with and without established cardiovascular disease [ 3746 , 47 , 48 ] . key findings from these studies are summarized in table 2 for both dhp - type ccbs and other members of this drug class.table 2outcome trials using calcium channel blockersstudydesign / drugpatient populationkey resultaccomplish ( 2008 ) [ 48]double - blind , randomized trial . mean follow - up 36 months.11,506 patients with hypertension at high risk of cv events.compared with benazepril + hydrochlorothiazide , fewer individuals on benazepril + amlodipine had a primary endpoint ( death from cv causes , nonfatal mi , nonfatal stroke , hospitalization for angina , resuscitation after sudden cardiac arrest , and coronary revascularization ) hr , 0.80 ; 95 % ci , 0.72 to 0.90 ; p < 0.001 . for the secondary endpoint of death from cv causes , nonfatal mi , and nonfatal stroke , hr 0.79 ; 95 % ci , 0.67 to 0.92 ; p = 0.002.ascot-bpla ( 2005 ) [ 47]open - label , randomized trial . mean 5.5 year follow - up.19,257 patients with hypertension and 3 additional cv risk factors.compared with the atenolol - based regimen , fewer individuals on the amlodipine - based regimen had a primary endpoint ( nonfatal mi and fatal chd ) hr , 0.90 ; 95 % ci , 0.79 to 1.02 ; p = 0.1052 ; fatal and nonfatal stroke , hr , 0.77 ; 95 % ci , 0.66 to 0.89 ; p = 0.0003 ; total cv events and procedures , hr , 0.84 ; 95 % ci , 0.78 to 0.90 ; p < 0.0001 ; all - cause mortality , hr , 0.89 ; 95 % ci , 0.81 to 0.99 ; p = 0.025.camelot ( 2004 ) double - blind , placebo - controlled , randomized trial . 24 month follow - up.1,991 patients with cad and dbp < 100 mm hg.compared with placebo , there was a 31 % reduction in cv events in the amlodipine group ( p = 0.003 ) and a 15 % reduction in the enalapril group ( p = 0.16 ) . in the amlodipine group , ivus showed evidence of slowing atherosclerosis progression.value ( 2004)double - blind , parallel - group , randomized trial . mean follow - up 4.2 years.15,245 patients with hypertension at high risk of cardiac events.no difference in the primary outcome ( cardiac mortality and morbidity ) between treatment groups , hr 1.04 ; 95 % ci , 0.94 to 1.15 ; p = 0.49 . bp reduced by both treatments , but amlodipine had greater effect especially in the early period . amlodipine was superior to valsartan at preventing mi and angina.invest ( 2003)open - label , blinded endpoint , randomized trial . . mean follow - up 2.7 years.22,576 patients with hypertension and cad.no difference in the primary outcome ( first occurrence of all - cause mortality , nonfatal mi or nonfatal stroke ) between treatment groups , rr 0.98 ; 95 % ci 0.90-1.06.convince ( 2003)double - blind , randomized trial . . mean follow - up 3 years.16,602 patients with hypertension and 1 additional cv risk factor.no difference in the primary outcome ( first occurrence of stroke , mi , or cv - related death ) between treatment groups , hr 1.02 ; 95 % ci , 0.88 to 1.18 ; p = 0.77.allhat ( 2002)double - blind , randomized trial . 3 treatment groups : chlorthalidone ; amlodipine ; lisinopril . mean follow - up 4.9 years.33,357 patients with hypertension and 1 additional chd risk factor.no difference in the primary outcome ( fatal chd or nonfatal mi ) between treatment groups . compared with chlorthalidone : rr for amlodipine 0.98 ; 95 % ci , 0.90 to 1.07 ; lisinopril 0.99 ; 95 % ci , 0.91 to 1.08.nordil ( 2000)open - label , blinded endpoint , randomized trial . mean follow - up 4.5 years.10,881 patients with dbp 100 mm hg.no difference in the primary outcome ( fatal and non - fatal stroke , mi , cv death ) between the 2 groups , rr 1.00 ; 95 % ci , 0.87 to 1.15 ; p = 0.97.insight ( 2000)double - blind , randomized trial . follow - up 3 years after recruitment of the last patient.6,321 patients with hypertension and 1 additional cv risk factor.no difference in the primary outcome ( cv death , mi , heart failure , or stroke ) between the 2 groups , rr , 1.10 ; 95 % ci , 0.91 to 1.34 ; p = 0.35.prevent ( 2000 ) double - blind , placebo - controlled , randomized trial . 36-month follow - up.825 patients with cad.no difference in coronary stenosis between the amlodipine and placebo group . amlodipine slowed the progression of carotid artery atherosclerosis ( imt : amlodipine 0.0126 versus placebo + 0.033 ; p = 0.007 ) and was associated with fewer hospitalizations for unstable angina and coronary revascularization.syst-eur ( 1997 ) double - blind , placebo - controlled , randomized trial . median follow - up 2 years.4695 patients with isolated systolic hypertension ( sbp 160 mmhg and dbp < 95 mmhg).compared with placebo , nitrendipine reduced the total rate of stroke by 42 % ( p = 0.003 ) ; nonfatal stroke by 44 % ( p = 0.007 ) and all fatal and nonfatal cardiac events by 26 % ( p = 0.03).midas ( 1996 ) double - blind , randomized trial . isradipine or hydrochlorothiazide . 3 year follow - up.883 patients with hypertensionno difference in the rate of progression of mean maximum imt ( p = 0.68 ) between treatment groups . higher ( but non - significant , p = 0.07 ) incidence of major vascular events ( mi , stroke , heart failure , angina , sudden death ) in the isradipine versus the hydrochlorothiazide group ( 25 vs 14 events).accomplish avoiding cardiovascular events through combination therapy in patients living with systolic hypertension study ; ascot - bpla anglo - scandinavian cardiac outcomes trial blood pressure lowering arm ; camelot comparison of amlodipine versus enalapril to limit occurrences of thrombosis study ; value , valsartan antihypertensive long - term use evaluation ; invest , the international verapamil - trandolapril study ; convince , controlled onset verapamil investigation of cardiovascular end points ; allhat , the antihypertensive and lipid - lowering treatment to prevent heart attack trial ; nordil , the nordic diltiazem study ; insight , intervention as a goal in hypertension treatment ; prevent prospective randomized evaluation of the vascular effects of norvasc trial ; sys - eur , systolic hypertension in europe ; midas , multicenter isradipine diuretic atherosclerosis study.cad coronary artery disease ; chd coronary heart disease ; ci confidence interval ; cv cardiovascular ; dbp diastolic blood pressure ; hr hazard ratio ; imt intimal - media thickness ; ivus intravascular ultrasound ; mi myocardial infarction ; sbp , systolic blood pressure outcome trials using calcium channel blockers accomplish avoiding cardiovascular events through combination therapy in patients living with systolic hypertension study ; ascot - bpla anglo - scandinavian cardiac outcomes trial blood pressure lowering arm ; camelot comparison of amlodipine versus enalapril to limit occurrences of thrombosis study ; value , valsartan antihypertensive long - term use evaluation ; invest , the international verapamil - trandolapril study ; convince , controlled onset verapamil investigation of cardiovascular end points ; allhat , the antihypertensive and lipid - lowering treatment to prevent heart attack trial ; nordil , the nordic diltiazem study ; insight , intervention as a goal in hypertension treatment ; prevent prospective randomized evaluation of the vascular effects of norvasc trial ; sys - eur , systolic hypertension in europe ; midas , multicenter isradipine diuretic atherosclerosis study . cad coronary artery disease ; chd coronary heart disease ; ci confidence interval ; cv cardiovascular ; dbp diastolic blood pressure ; hr hazard ratio ; imt intimal - media thickness ; ivus intravascular ultrasound ; mi myocardial infarction ; sbp , systolic blood pressure hypertension commonly clusters with other cardiovascular risk factors , including dyslipidemia and diabetes , greatly increasing an individual s risk of an event [ 49 , 50 ] . from a mechanistic viewpoint , these risk factors act synergistically to exacerbate endothelial dysfunction , oxidative stress , and inflammation , thereby accelerating the atherosclerotic process . although controversy remains over the optimal choice of antihypertensive drug therapy , hypertension management guidelines agree that most patients may require at least two antihypertensive drugs to reach the currently recommended blood pressure targets [ 5254 ] . in addition , many patients with hypertension also benefit from concurrent statin therapy [ 5254 ] . thus most patients with hypertension require multiple drug treatment regimens to manage their cardiovascular risk . a greater understanding of the mechanisms of actions of antihypertensive and lipid - lowering drugs may lead to more effective drug combinations with respect to clinical outcomes . this review focuses on the combination of ccbs with statins and ccbs in combination with drugs that affect the renin - angiotensin system ( ras ) . both ccbs and statins have been shown to reduce cardiovascular events in large clinical outcome trials [ 45 , 46 , 55 , 56 , 47 , 48 ] . ascot highlighted a potential synergy between co - administered amlodipine and atorvastatin in terms of the cardio - protective effect of this drug combination . in ascot , those randomized to amlodipine plus atorvastatin had a 53 % reduction in coronary heart disease events compared with a 16 % reduction among those randomized to atenolol plus atorvastatin . this could not be explained by differences in blood pressure and lipid parameters between the two treatment regimens . furthermore , the significant benefits of the amlodipine plus atorvastatin combination were observed within the first 3 months of treatment ( p = 0.02 ) . the findings from ascot may be explained , in part , by observations from the avalon arterial wall compliance ( awc ) trial . in the awc trial , 668 patients ( 61 % male , mean age 55 years ) with concomitant hypertension and dyslipidemia were randomized to one of four treatment groups ( placebo , amlodipine 5 mg , atorvastatin 10 mg , co - administered amlodipine 5 mg and atorvastatin 10 mg ) . after 8 weeks of treatment , there was a 19.3 % improvement in small artery compliance in the co - administered amlodipine and atorvastatin group compared with 11.7 % in the amlodipine alone group ( p = 0.03 ) , 3.1 % in the atorvastatin alone group ( p < 0.001 ) , and 1.3 % in the placebo group ( p < 0.0001 ) . after 28 weeks of treatment , the greatest improvement in small artery compliance remained among those taking co - administered amlodipine and atorvastatin ( p < 0.05 ) . the observation that combination therapy had a more than additive effect on small artery compliance is consistent with an improvement in the mechanisms that control vascular function [ 59 ] . indeed , the authors of the awc trial proposed that their study provided evidence that the synergistic effects of co - administered amlodipine and atorvastatin may be mediated by endothelial function . this hypothesis is supported by the results of the encore i study that compared no - mediated endothelial function among 343 patients with coronary artery disease . patients were randomized to 6 months treatment with placebo , cerivastatin 0.4 mg / day , nifedipine 30 to 60 mg / day , or their combination . endothelial function was assessed by infusing acetylcholine into a coronary segment and measuring luminal diameter by quantitative angiography . in the most constricted segment , nifedipine but not cerivastatin , significantly reduced acetylcholine - induced vasoconstriction by 18.8 % ( p < 0.05 , compared with placebo ) . the combination of nifedipine and cerivastatin also reduced acetylcholine - induced vasoconstriction by 11 % , but this only reached statistical significance when all coronary segments were analyzed together . after cerivastatin was withdrawn from the market in 2001 , the encore study design was modified to compare nifedipine with placebo . in encore ii , all patients were eligible for statin therapy at the discretion of their physician . in addition to measuring changes in luminal diameter , intravascular ultrasound ( ivus ) was used to assess change in plaque volume . overall , 454 patients were randomized and followed for 1824 months . compared with placebo , nifedipine plus background statin therapy significantly improved coronary endothelial function in the most constricted segment ( difference in luminal diameter 6.3 % , 95 % ci 1.6 to 10.9 ; p = 0.0088 ) . however , compared with placebo , nifedipine plus background statin therapy did not have a significant effect on coronary plaque volume as measured by ivus . together the encore studies add to the evidence that ccb plus statin therapy improves no - mediated endothelial function [ 35 , 36 ] . from a mechanistic perspective , synergy between ccbs and statins is plausible , as these drugs have complementary chemical structures . atorvastatin has negative polarity associated with its heptanoic side chain , whereas amlodipine is distinct among the dhps in having a positive charge on its aminoethoxy side chain [ 31 , 60 , 61 ] . indeed , studies have demonstrated that the concentration of these drugs is much higher in the cell membrane than in the surrounding aqueous environment [ 31 , 60 , 61 ] . these properties may facilitate interactions with novel receptor sites in vascular cell membranes , and contribute to explaining the superior clinical outcomes observed when these drugs are co - administered . this hypothesis was evaluated in a study using human umbilical vein endothelial cells ( huvec ) [ 59 ] . the combination of amlodipine and atorvastatin directly stimulated no release , which was about twofold greater than the sum of their separate effects ( p < 0.05 ) . this was attributed to enhanced enos function and expression along with decreased levels of cytotoxic onoo . following low density lipoprotein ( ldl ) enrichment there was a 60 % reduction in no production in the huvec and an almost twofold increase in onoo . treatment with the combination of amlodipine and atorvastatin partially reversed the adverse effects of ldl , including a 90 % increase in no and a 50 % reduction in onoo . small angle x - ray diffraction analysis indicated that both amlodipine and atorvastatin are lipophilic and share a common membrane location [ 59 ] . this study provides evidence to suggest the observed synergy between these drugs may be explained by electron transport mechanisms that facilitate antioxidant activity in the cell membrane related to their complementary locations . the ras has an important role in blood pressure control by regulating blood volume and peripheral vascular resistance . in brief , in response to the release of renin from the kidneys , the circulating substrate angiotensinogen is converted to angiotensin i. the angiotensin - converting enzyme ( ace ) in the vascular endothelium cleaves off two amino acids to form the octapeptide , angiotensin ii ( ang ii ) . ang ii binds to angiotensin ii type-1 ( at1 ) receptors in vascular smooth muscle cells , promoting vasoconstriction and increasing peripheral vascular resistance . ang ii also stimulates the release of various hormones including aldosterone and vasopressin , which act on the kidneys to increase sodium and fluid retention . ang ii also facilitates norepinephrine release from sympathetic nerve endings and inhibits its reuptake , thereby enhancing sympathetic adrenergic function . the interaction between ang ii and at1 receptors also activates signal transduction mechanisms that promote oxidative stress , inflammation , cell proliferation , and fibrosis . studies have shown that ang ii activates nad(p)h oxidase in endothelial and vascular smooth muscle cells . this results in the production of o2 and other ros , and contributes directly to reduced no bioavailability and endothelial dysfunction . blocking the ras with ace inhibitors and angiotensin receptor blockers ( arbs ) has been demonstrated to improve endothelial function and increase no bioavailability in mechanistic [ 6467 ] and clinical [ 6870 ] studies . in an animal model using infarcted adult male sprague dawley rats , treatment with the arb candesartan , enhanced vasorelaxation by increasing no bioavailability via an at2 receptor mediated upregulation of enos . a complementary in vitro study , using the arbs losartan and valsartan , stimulated no release from both platelets and human umbilical vein endothelial cells in a dose - dependent manner . however , there was more than 70 % greater potency in no release in platelets than endothelial cells . furthermore , the degree of inhibition of platelet adhesion and aggregation by losartan and valsartan was closely correlated with the level of no production . in an animal model using isolated coronary arterioles from pigs , ang ii was shown to evoke at1 receptor - mediated vasoconstriction and at2 receptor - mediated vasodilation . at the vascular level , ang ii was shown to impair endothelium - dependent no - mediated dilation attributable to elevated o2 production via at1 receptor activation of nad(p)h oxidase . the authors of this study commented that their results may partly explain why impaired coronary flow is associated with upregulation of the ras . the long - term effect of the arb valsartan on endothelial function and vascular structural changes in the aorta was explored in hypercholesterolemic rabbits . treatments with 3 and 10 mg / kg per day of valsartan reduced intimal lesion to 2.4 0.7 % and 2.7 0.9 % , respectively ( p < 0.05 ) and increased lumen area . the authors suggested that at1 receptor antagonists , besides their antihypertensive effects , could also have a role in reducing the development of atherosclerosis . the results of these mechanistic studies were consistent with the results from clinical studies [ 6870 ] . in an active - controlled , randomized trial , 35 patients with coronary artery disease received 4 weeks of treatment with either an ace inhibitor ( ramipril 10 mg / day ) or arb ( losartan 100 mg / day ) . no - mediated vasodilation of the radial artery was determined before and after intra - arterial l - nmma infusion . no - mediated vasodilation was increased by more than 75 % after treatment with ramipril and losartan ( each group p < 0.01 ) . these results were consistent with a double - blind , placebo - controlled trial among 60 patients with essential hypertension who received 6 weeks of treatment with either an arb ( valsartan 80 mg / day ) , a diuretic ( hydrochlorothiazide 25 mg / day ) , or placebo . there were similar reductions in brachial blood pressure in both active treatment arms ( p < 0.001 ) . however , patients in the arb group had significantly improved vasoconstrictive response to l - nmma , whereas no effect was seen in the diuretic and placebo groups . together , these clinical studies suggest that agents that block the ras may improve endothelial function by increasing no bioavailability . a further study , among 53 hypertensive patients with and without type 2 diabetes , showed that treatment with valsartan 80 mg / day for 8 weeks significantly decreased monocyte and endothelial cell activation markers among those with diabetes , but not among those without diabetes . this study highlights the importance of using complex disease models to explore mechanisms of actions , because focusing on single - risk - factor models may miss important biological pathways . there is a growing body of evidence to suggest that dhp - type ccbs and ras inhibitors have additional beneficial effects when used in combination . for example , in an animal model using dahl salt - sensitive rats , the combination of ccb plus ace inhibitor was more effective than either monotherapy in normalizing systolic blood pressure and proteinuria . this study was complimented by a rat myocardial infarction model that showed that treatment with the ccb amlodipine plus ace inhibitor benazepril increased no production and decreased inflammatory markers . in contrast , treatment with the diuretic hydrochlorothiazide had no effect on these indicators of endothelial dysfunction . in clinical studies , the combination of ccbs with an ace inhibitor versus a ccb alone has been shown to increase flow - mediated dilation , a surrogate biomarker of endothelial function . as previously described , the cafe study [ 6 ] demonstrated that the benefits of the ccb plus ace inhibitor combination may extend beyond brachial blood pressure lowering and have a positive impact on central blood pressure hemodynamics [ 6 ] . furthermore , outcome trials have reported that these beneficial effects may translate into reduced cardiovascular endpoints . the ascot - bpla study showed that treatment with an amlodipine plus perindopril - based regimen prevented more major cardiovascular events than an atenolol plus bendroflumethiazide - based regimen [ 47 ] . the accomplish study showed that treatment with benazepril plus amlodipine was more effective at preventing cardiovascular events than treatment with benazepril plus hydrochlorothiazide [ 48 ] . together , these mechanistic and clinical studies demonstrate the pleiotropic effects of the combination of ccb plus ras blockade on enhanced no bioavailability and reduced oxidative stress . understanding the complementary mechanisms of actions of these drugs forms the rationale for combining different antihypertensive agents to enhance their therapeutic effects . this review has explored the pleiotropic effects of ccbs alone and in combination with statins and ras inhibitors . the mechanisms by which these drugs exert their beneficial effects on endothelial and vascular function have been described , with a particular focus on the effects on no - mediated vasodilation and oxidative stress . by further elucidating the biological pathways by which these drugs exert their effects , we considered the rationale for choosing optimal drug combinations that will benefit patients at risk . the differential effect of ccbs on brachial and central blood pressure has been explored , along with the future role of central blood pressure as a prognostic marker for cardiovascular disease events . indeed , clinical hypertension trials have demonstrated the importance of incorporating measurements of central aortic pressure in their study designs . another important aspect of future hypertension research is the shift towards studying complex disease models . from a mechanistic viewpoint , multiple cardiovascular risk factors act synergistically to exacerbate endothelial dysfunction , oxidative stress and inflammation , thereby accelerating the atherosclerotic process . it is therefore logical that they should be considered together in global risk assessment . by further elucidating the underlying biological pathways and mechanisms that lead to target organ damage in multiple cardiovascular risk factor models such approaches will continue to require the scientific community to work together to carry out large - scale studies and to link the findings from experimental models with human populations .

clinical trials have reported reduced cardiovascular events with certain antihypertensive agents at a rate that could not be predicted by changes in brachial arterial pressure alone . these findings may be explained , in part , by pleiotropic effects of these agents and modulation of central blood pressures . this review focuses on the mechanisms by which calcium channel blockers exert pleiotropic effects , both alone and in combination with statins and inhibitors of the renin - angiotensin system . the essential role of nitric oxide ( no ) in maintaining endothelial function and the relationship between no and reactive oxygen species are discussed in the context of the etiology of hypertension . the importance of managing global cardiovascular risk is emphasized , as hypertension commonly clusters with dyslipidemia and loss of glucose control . from a mechanistic viewpoint , these risk factors contribute to endothelial dysfunction , oxidative stress , and inflammation in a synergistic fashion . a greater understanding of the mechanisms of actions of these cardiovascular agents may lead to more effective drug combinations , to the benefit of individual patients . furthermore , by elucidating the biological mechanisms by which cardiovascular risk factors lead to vascular injury , we may highlight common pathways and identify novel therapeutic targets .

compared to advanced gastric cancer , early gastric cancer ( egc ) shows a favorable prognosis , with 5-year survival rates exceeding 94%.1 lymph node ( ln ) status is the most important prognostic factor . even in egc , the incidence of ln metastasis exceeds 10% ; is reported to be 14.1% overall and appears in 4.8 to 23.6% of cases depending on cancer depth.2 it is important to evaluate ln status preoperatively for proper treatment strategy ; however , sufficient results are not being obtained using various evaluation modalities . according to japanese staging guideline , n3 level node was regarded as distant metastasis.3 it is possible to cure local disease without distant metastasis by gastrectomy and ln dissection . however , there is no survival benefit from surgery for systemic disease with distant metastasis such as para - aortic ln metastasis.4 therefore , whether the disease is local or systemic is an important prognostic indicator for gastric cancer , and the debate continues over the importance of extended lymphadenectomy for gastric cancer . a 72-year - old woman , with no history of neurofibromatosis or other systemic disease , was referred to chosun university hospital ( gwangju , korea ) for egc . abdominal computed tomography did not show the gastric tumor lesion but revealed a well - defined , 1.6 cm sized ovoid retroperitoneal mass located posterior to the duodenum ( fig . an operator and scopist stood to the right side of the patient , and an assistant surgeon stood to the left of the patient . a total of 5 trocars ( two 12-mm trocars and three 5-mm trocars ) were used . the first dissection began with the mobilization of the duodenum from hepatoduodenal ligament and right gastrocolic ligament . an assistant retracted the duodenum left laterally , and an operator dissected between the pancreaticoduodenal unit and inferior vena cava ( ivc ) and right renal vein . 2 ) . several vessels from the mass to the ivc were ligated by the ligasure vessel sealing system ( valleylab , boulder , co , usa ) . the final pathological diagnosis was a gastric adenocarcinoma that invaded a mucosa ( t1a ) without nodal metastasis ( 0/20 , n0 ) and neurofibroma with immunohistochemical stains positive for s-100 protein ( fig . about 15% of egc have ln metastases.2 this makes the prognosis of egc better than advanced gastric cancer . nevertheless , while there is little chance of nodal or distant metastasis in egc , some case reports revealed stage iv egc accompanied by extensive ln and distant metastasis.5 - 8 accurate assessment of ln status is of crucial importance for appropriate treatment planning and determining prognosis in gastric cancer . but abdominal ultrasonography ( aus ) , endoscopic ultrasonography , multidetector - row computed tomography ( mdct ) , magnetic resonance imaging ( mri ) , and 18f - fluoro-2-deoxyglucose positron emission tomography can not reliably be used to confirm or exclude the presence of ln metastasis.9 in this case , preoperatively the patient was diagnosed as egc with distant metastasis by mdct . but a pathological report indicated mucosa - confined adenocarcinoma ( t1a ) without nodal metastasis ( n0 ) and solitary neurofibroma . typically , neurofibromas grow slowly and have minimal potential for malignant transformation unless they are associated with neurofibromatosis type 1 ( formerly known as von recklinghausen disease ) . specifically , solitary retroperitoneal neurofibromas were studied in several case reports.10 - 12 neurofibroma is more common in men , particularly in the 20~40 year age group.13 but in this case , patient was a 72-year old aged woman . retroperitoneal masses not arising from major solid organs are uncommon . because the treatment options vary , it is useful to be able to differentiate these masses by using imaging criteria.14 currently , imaging modalities such as mdct , aus and mri show progress technically . but there is still a considerable overlap of imaging findings for these masses , and histological examination is often required for definitive diagnosis.14 additionally , in gastrointestinal tract malignancy , a retroperitoneal mass is easily confused with ln metastasis . in conclusion , staging laparoscopy with frozen biopsy might be helpful in treating a preoperatively egc with distant metastasis .

neurofibromas are benign tumors that originate from the peripheral nerves , including neurites and fibroblasts . generally , a solitary neurofibroma is located in the skin and rarely in other places . a 72-year - old female suffered from epigastric discomfort for 2 months . endoscopic findings showed an early gastric cancer type iic at the antrum . abdominal computed tomography revealed early gastric cancer with a 1.6 cm - sized metastatic node posterior to the duodenum . laparoscopic assisted distal gastrectomy and retro - pancreatic dissection were performed uneventfully . histological examination revealed gastric adenocarcinoma , invading the mucosa without nodal metastasis , and a neurofibroma . herein , we present a case of a gastric cancer patient with a solitary retroperitoneal neurofibroma which mimicked a distant metastatic node .

stroke is the second most common cause of death after ischemic heart disease in the world with 4.38 million deaths in developed countries and almost 3 million in developing countries.1 stroke causes 10% to 12% of deaths in industrialized countries . almost 90% of deaths caused by stroke occur in people over 65 years of age.2 the framingham study showed that af is an independent risk factor for development of stroke , particularly in the elderly.3 given that af is a known risk factor for stroke , it seems appealing that a drug with antiarrhythmic effect can reduce the incidence of stroke in patients with af . however , previous studies point in the opposite direction : rhythm control versus rate control for atrial fibrillation and heart failure , atrial fibrillation follow - up investigation of rhythm management ( affirm ) showed that antiarrhythmic drugs reduced the recurrence of af , but did not show any reduction in stroke.4 in a meta - analysis of randomized trials of rate vs rhythm control for af from 2005 there was an insignificant increase in stroke in the group of patients who received rhythm control.5 the incidence of stroke has declined due to treatment with oral anticoagulant therapy , and is around 1.4%/year in patients with af.6,7 however , af is often associated with advanced age and concomitant disease that disposes to stroke : hypertension , hypercholesterolemia , diabetes and heart failure . thus , it may not be surprising that antiarrhythmic therapy have not been able to prevent stroke in randomized clinical trials . dronedarone has been developed by sanofi - aventis for treatment of af or atrial flutter ( afl ) . dronedarone is a benzofuran derivative and primarily a class iii antiarrhythmic drug but with properties from all four classes of the vaughan - williams classification . it is an amiodarone analogue but noniodinized and without many of the adverse effects associated with amiodarone . it has antiadrenergic properties and inhibits multiple transmembrane potassium currents , including the delayed rectifier current ( both the rapid and slow components ( ikr and iks , respectively ) , the ultrarapid rectifier current ( ikur ) , the inward rectifier current ( ik1 ) , and the transient outward current ( ito ) , as well as sodium and l - type calcium currents.8,9 dronedarone is approximately 100 times more potent than amiodarone and sotalol on the acetylcholine receptor - operated k current which is important when vagal tone plays a role in the genesis of af.10 the removal of iodine is thought to be the reason for the lack of typical amiodarone side effects such as discoloration of skin , and affects on of lung , liver , and thyroid gland . dronedarone is less lipophilic and has a much shorter half - life than amiodarone because of a methyl - sulfonamide group . dronedarone interferes with the metabolism of digoxin , but not with the metabolism of warfarin.11 it is hepatically metabolized and excreted with feces . there are 7 important studies with dronedarone , of which two were powered to show effects on mortality and morbidity . the dafne trial ( dronedarone atrial fibrillation study after electrical cardioversion ) studied the most appropriate dose of dronedarone to maintain sinus rhythm after cardioversion.12 the study included 199 patients with persistent af . patients with severe heart failure or left ventricular ejection fraction ( lvef ) < 35% were excluded . the patients were randomized to 4 different groups and received either placebo or dronedarone in doses of 400 mg , 600 mg or 800 mg twice daily . dafne showed that the most appropriate dose of dronedarone was 400 mg twice daily for prevention of af relapses in patients after cardioversion . it was somewhat puzzling that higher doses of dronedarone were less efficient than 400 mg twice daily . the most likely explanation is that higher dosages were limited by adverse events resulting in discontinuation of the drug . there was no report of stroke in any of the dronedarone groups or in the placebo group . there was one case of thrombosis ( 1.3% ) in the group that received 800 mg twice daily and none in the other groups . euridis ( european trial in af or afl patients receiving dronedarone for the maintenance of sinus rhythm ) and adonis ( american - australian - african trial with dronedarone in af or afl patients for the maintenance of sinus rhythm ) were two identical studies of which one was european and one non - european.13 all included patients had a history of af but did not have af at the time of enrolment . the purpose of the studies was to prevent recurrence of af with dronedarone ( n = 828 ) compared to placebo ( n = 409 ) . in both euridis and adonis dronedarone increased the time to recurrence of af significantly compared to the placebo : 96 days in 41 days ( p value = 0.01 ) and 158 days vs 59 days ( p = 0.002 ) , respectively . in the european trial 67% of the patients had had a recurrence of af vs 77.5% in the placebo group ( hazard ratio [ hr ] 0.78 , 95% confidence interval [ ci ] 0.64 to 0.96 , p = 0.01 ) . in the non - european trial 61.1% of patients in the dronedarone group had had a recurrence of af vs 72.8% in the placebo group ( hr 0.73 , 95% ci 0.59 to 0.89 , p = 0.002).these high rates of recurrence of af in euridis and adonis could lead to the assumption that dronedarone is less efficient in maintaining sinus rhythm than other antiarrhythmic drugs . however , the populations of different studies vary considerably , which makes direct comparison impossible . there was no significant difference in the number of strokes reported as there were 4 strokes ( 0.5% ) in the dronedarone group and 3 strokes ( 0.7% ) in the placebo group ( p = 0.69 ) . stroke included cerebral - artery embolism , cerebrovascular accident , cerebral infarction , and transient ischemic attack . the efficacy and safety of dronedarone for the control of ventricular rate during af ( erato ) study assessed the efficacy of dronedarone in the control of ventricular rate in patients with permanent af.14 the study enrolled 85 patients in the dronedarone group receiving 400 mg dronedarone twice daily and 89 in the placebo group . all patients had a history of symptomatic , permanent af for minimum 6 months and resting ventricular rate of at least 80 beats per minute . erato demonstrated a highly significant rate reduction in patients with permanent af . at day 14 the mean ventricular rate was measured to be 11.7 beats less in the dronedarone group than at day 0 ( p < 0.0001 ) . during submaximal and maximal exercise the mean ventricular rate at day 14 was reduced by 25.6 and 27.4 beats , respectively , vs a 2.2 and 2.9 beats per minute reduction in the placebo group ( both p < 0.0001 ) . the serious cardiovascular events included 3 myocardial infarctions ( 1 in dronedarone vs 2 in placebo group ) and 1 heart failure ( in the dronedarone group ) and 1 unstable angina ( in the placebo group).14 a comparison of dronedarone and amiodarone was made in the efficacy and safety of dronedarone vs amiodarone for the maintenance of sinus rhythm in patients with af ( dionysos ) trial . the 504 patients scheduled for cardioversion for af or afl were randomized to amiodarone or dronedarone ( 400 mg twice daily ) , and the primary endpoint was the combined endpoint of recurrence of af or premature discontinuation . dionysos has not been published , but a press release ( http://en.sanofi-aventis.com/binaries/20081223_dionysos_fe_en_en_tcm28-23624.pdf ) reveals that dronedarone has a higher rate of recurrence of af or premature discontinuation after a year than amiodarone ( hr approximately 1.6 ; p < 0.01 ) . recurrence of af occurred for 158 patients in the dronedarone group vs 107 in the placebo group . the significant reduction in recurrence of af by amiodarone is consistent with an indirect meta - analysis based on 4 studies of amiodarone and 4 studies of dronedarone.15 the antiarrhythmic trial with dronedarone in moderate to severe congestive heart failure evaluating morbidity decrease ( andromeda ) trial was a placebo - controlled multicenter study performed in patients admitted to hospital with moderate to severe heart failure and reduced left ventricular ejection fraction ( lvef).16 the study was performed in order to show a reduction in morbidity and mortality in patients with severe heart failure . the study was planned to include 1000 patients and last for a minimum of 2 years . the primary end point was death from any cause or hospitalization for worsening heart failure . after inclusion of 627 patients ( 310 in the dronedarone group and 317 in the placebo group ) , the study was discontinued , when the data , safety and management board ( dsmb ) found that dronedarone was associated with a significantly higher mortality . after a median 2-month follow - up , 25 patients in the dronedarone group and 12 patients in the control group had died , respectively . based on the small number of events it is difficult to know if this decision was correct . however , the increased mortality in the dronedarone was discovered at the first look at data , and repeated 1 month later . the steering committee discussed whether the advice of the dsmb should be followed , and decided to stop the study . subgroup analyses could not identify a specific group of patients to which the increased risk was confined , but the risk seemed to be associated with the sickest patients . in the dronedarone group it was suggested that dronedarone might decrease renal function but this suggestion was later rejected . on the contrary , a study among 12 healthy males showed that dronedarone increases serum creatinine ( increasing tubular secretion ) without decreasing renal function.17 in andromeda , 4 ( 5.6% ) in the dronedarone group and 3 ( 6.0% ) in the placebo group suffered from stroke requiring a first hospitalization . as the population in andromeda consisted of patients with moderate to severe heart failure , a large study in patients of medium risk the study was done in patients with current or recent af in order to show if dronedarone could affect mortality or morbidity . a placebo - controlled , double - blind , parallel - arm trial to assess the efficacy of dronedarone 400 mg twice daily for the prevention of cardiovascular hospitalization or death from any cause in patients with af / afl ( athena ) was a placebo controlled multicenter study including 4628 patients . inclusion criteria were a history of paroxysmal or persistent af and at least one of following risk factors : age of minimum 70 years , arterial hypertension ( with ongoing therapy involving at least two antihypertensive drugs of different classes ) , diabetes mellitus , previous stroke , transient ischemic attack , systemic embolism , minimum left atrial diameter of 50 mm or lvef of 40% or less . at the time of enrollment 1176 ( 25% ) of the patients had either af or afl . a history of nyha class ii or iii heart failure was present in 979 patients . the overall mortality figures were lower than expected , so the steering committee changed the inclusion criteria to enrich the risk profile of the overall study population . with the revised criteria patients aged 75 years or older could be included whether or not they had any of the specified risk factors . patients aged 70 years or older could be included if they had at least one of the risk factors . patients included in the study had an intermediate to high risk for stroke and other cardiovascular events . in total , 60% received oral anticoagulant therapy ( oac ) and one - fourth of these received both oac and an antiplatelet agent . exclusion criteria included permanent af , severe heart failure ( nyha class iv ) , unstable hemodynamic condition , bradycardia , planned major surgery and glomerular filtration rate of less than 10 ml per minute . the population in athena therefore varied considerably from the population in andromeda as the athena patients were all clinically stable and had non - permanent af . in contrast , patients in the andromeda study were hospitalized and the majority did not have af . thus , in the athena study investigators excluded patients similar to those participating in andromeda , and included only patients for whom dronedarone was developed . in athena , 734 patients ( 31.9% ) in the dronedarone group reached the primary end point of either death from any cause or hospitalization for cardiovascular reasons vs 917 ( 39.4% ) in the placebo group ( hr 0.76 ; 95% ci 0.69 to 0.84 ; p < 0.001 ) , respectively . death from any cause did not differ significantly between the two groups , as 116 ( 5% ) in the dronedarone group vs 139 ( 6.0% ) in the placebo group died , respectively ( hr 0.84 , 95% ci , 0.66 to 1.08 ; p = 0.18 ) . hospitalization for cardiovascular reasons occurred in 675 ( 29.3% ) in the dronedarone group vs 859 ( 36.9% ) in the placebo group , respectively ( hr 0.74 ; 95% ci 0.67 to 0.82 ; p < 0.001 ) . this difference in hospitalizations between the two groups was mainly caused by a reduction in the number of hospitalizations for af . however , hospitalizations for af were often related to other cardiovascular events ( ie , incompensation , stroke or myocardial infarction ) . death from cardiovascular reasons occurred in 63 ( 2.7% ) in the dronedarone group vs 90 ( 3.9% ) in the placebo group ( hr = 0.71 ; 95% ci 0.51 to 0.98 ; p = 0.03 ) , respectively . also , there were 26 deaths from cardiac arrhythmia ( 1.1% of patients ) in the dronedarone group and 48 ( 2.1% ) in the placebo group ( hr , 0.55 ; 95% ci , 0.34 to 0.88 , p = 0.01 ) , respectively . any hospitalization due to any cardiovascular event or death from any cause occurred in 1253 ( 54.5% ) cases in the dronedarone group vs 1668 ( 71.7% ) in the placebo group ( hr 0.76 , 95% ( ci 0.68 to 0.84 ) , p < 0.001 ) . overall , the athena study demonstrated that in elderly patients with nonpermanent af and additional risk factors , dronedarone was safe and reduced cardiovascular morbidity / mortality . the positive outcome in the athena trial was mainly driven by a reduction in cardiovascular hospitalizations . from the published data it is difficult to see how many of these events were related to an effect on af , and to see if dronedarone has additional effects not previously anticipated . in a post hoc analysis deaths were categorized into four subgroups : cardiac , arrhythmic ; cardiac , nonarrhythmic ; vascular , noncardiac ; and nonvascular . all incidents of preferred terms for adverse effects that contained the word stroke , cerebrovascular accident or the total number of strokes was 46 ( annual event rate of 1.2% ) in the dronedarone group and 70 ( annual event rate of 1.8% ) in the placebo group , respectively ( hr 0.66 , ci 0.46 to 0.96 , p = 0.027 ) . figure 1 shows the cumulative risk of stroke and the composite outcome of stroke , acute coronary syndrome or cardiovascular death . ischemic strokes accounted for 33 ( annual event rate 0.9% ) in the dronedarone group and 49 ( annual event rate 1.3% ) in the placebo group , respectively ( hr 0.68 , ci 0.44 to 1.05 , p = 0.08 ) . hemorrhagic strokes were 6 in both groups ( annual event rate 0.2 ; p = 0.99 ) . subgroup analysis to investigate what baseline characteristics were predictive of a response to dronedarone in response to stroke showed that patients with a chads2 score 2 had a significantly greater effect of dronedarone than patients with a chads2 score of 1.18 in euridis and adonis the overall incidence of stroke was 0.3% ( n = 4 ) in the dronedarone vs 0.5% ( n = 3 ) in the placebo group . erato and dafne , which were short - term studies , did not report any cases of stroke , and data from dionysos are not available . in the andromeda trial stroke occurred in 4 ( 1.3% ) in the dronedarone group and 3 ( 0.9% ) in the placebo group , respectively . in total , 54 cases of stroke occurred in 3439 patients ( crude rate 1.6% ) receiving dronedarone compared to 76 strokes in 3048 patients on placebo ( crude rate 2.5% ) , respectively . the information on the overall risk of stroke associated with dronedarone originates mainly from the athena trial . this effect has never been demonstrated with any other antiarrhythmic drug , but the mechanism is uncertain . suppression of af is the most obvious explanation , but the details of the information collected during the athena trial do not allow us to conclude this . secondly , patients with af that persisted during the entire study also had a reduction ( nonsignificant ) in stroke . dronedarone had at least two other effects that could have resulted in a reduction in stroke . heart rate is reduced with dronedarone and there was a significant reduction in blood pressure in the dronedarone group ( approximately 3 mmhg in both systolic and diastolic blood pressure ) compared to the placebo group . these changes in blood pressure are modest , but may have importance in a group of patients with many risk factors for stroke . the most important limitation of the findings of the post hoc analysis of athena is that the reduction in stroke was not anticipated and therefore not prespecified , but was found retrospectively . the results of the post hoc analysis do not provide any information on why antiarrhythmic drugs might be able to reduce the risk of stroke , but they do provide reasons for future trials to investigate if similar results can be repeated and what mechanisms lie behind . until now the reasons for pursuing rhythm control in af have been to improve quality of life and reduce symptoms . however , if stoke can be prevented this will open a new scenario for treatment of patients at higher risk of stroke . in this context it is important to note that the effect on stroke was only seen in patients with a chads2 score 2 ( p = 0.03 ) . the fda approval of dronedarone suggests that it may be used in patients with paroxysmal or persistent af or afl who are in sinus rhythm or will be cardioverted . patients should have additional risk factors defined as age above 70 years , diabetes , hypertension , prior stroke , increased size of the left atrium or depressed lvef ( < 40% ) . contraindications to dronedarone are nyha class iv , or class ii or iii with a recent episode of hospitalization , ie , the andromeda population . this reflects the available data , but physicians may speculate if patients for whom rate control is a target could be candidates for dronedarone treatment . the available studies have mainly focused on rhythm control , except for the erato study that showed a significant reduction in heart rate with dronedarone . whether the beneficial effect of dronedarone found in the athena trial can be extrapolated to patients with permanent af , if patients develop permanent af on dronedarone this does not imply that treatment necessarily has to be discontinued . dronedarone is developed as an antiarrhythmic drug and the euridis / adonis studies found that dronedarone can decrease the recurrence of af . in the athena trial it was demonstrated that dronedarone may reduce cardiovascular mortality and morbidity . importantly , in a post hoc analysis stroke was observed to be significantly reduced in elderly patients with af . since af is a known risk factor for stroke , this result may not be surprising , but previous studies have not found any decreased risk in patients treated with antiarrhythmic drugs compared to patients treated with rate - controlling drugs . thus , dronedarone may be useful for treatment of elderly patients with non - permanent af .

background : dronedarone has been developed for treatment of atrial fibrillation ( af ) or atrial flutter ( afl ) . it is an amiodarone analogue but noniodinized and without the same adverse effects as amiodarone.objective and methods : this is a review of 7 studies ( dafne , adonis , euridis , athena , andromeda , erato and dionysos ) on dronedarone focusing on efficacy , safety and prevention of stroke . there was a dose - finding study ( dafne ) , 3 studies focusing on maintenance of sinus rhythm ( adonis , euridis and dionysos ) , 1 study focusing on rate control ( erato ) and 2 studies investigating mortality and morbidity ( andromeda and athena).results : the target dose for dronedarone was established in the dafne study to be 400 mg twice daily . both euridis and adonis studies demonstrated that dronedarone was superior to placebo for maintaining sinus rhythm . however , dionysos found that dronedarone is less efficient at maintaining sinus rhythm than amiodarone . erato concluded that dronedarone reduces ventricular rate in patients with chronic af . the andromeda study in patients with severe heart failure was discontinued because of increased mortality in dronedarone group . dronedarone reduced cardiovascular hospitalizations and mortality in patients with af or afl in the athena trial . secondly , according to a post hoc analysis a significant reduction in stroke was observed ( annual rate 1.2% on dronedarone vs 1.8% on placebo , respectively [ hazard ratio 0.66 , confidence interval 0.46 to 0.96 , p = 0.027 ] ) . in total , 54 cases of stroke occurred in 3439 patients ( crude rate 1.6% ) receiving dronedarone compared to 76 strokes in 3048 patients on placebo ( crude rate 2.5% ) , respectively.conclusion:dronedarone can be used for maintenance of sinus rhythm and can reduce stroke in patients with af who receive usual care , which includes antithrombotic therapy and heart rate control .

the epithelium that lines our digestive tract harbors 100 trillion bacteria1 and mediates our relationship to the world outside . our intestine is constantly exposed to a wide variety of immunogenic particles and microorganism - derived antigens . its surface area is large enough to process over 50 tons of food in a lifetime . intestinal and microbial components are strongly linked to each other and represent the most important barrier to limit luminal antigens from travelling across and reach the mucosal - associated immune system . they also play a critical role in training and modulating our immune system , helping it to distinguish between friend and foe.2,3 the absence of constructive engagement between microbes and intestinal mucosa may render epithelial barrier more permeable , allowing it to be breached . when this happens , resident cells become over - activated , and a cascade of pro - inflammatory signals is initiated.4,5 altered intestinal permeability has large implications for human health , being involved in the origin and development of many digestive and non - digestive diseases . however , up to date , it is not clear whether intestinal barrier dysfunction is a primary event , an epiphenomenon or a consequence in the pathogenesis of these disorders . the regulation of intestinal permeability involves terminals from ascending and descending pathways from the autonomic nervous system and the central nervous system , the hypothalamic pituitary - adrenal axis , and the enteric nervous system as well . the vast and versatile array of bidirectional and integrative communications between the brain and the gut allows the brain to respond to internal and external signals and , in return , to modify the autonomic and enteric nervous systems to keep intestinal permeability tight . physical and psychological stresses represent convincing evidence of the influence of the brain - gut axis on the intestinal barrier function . in fact , stress has been associated with reactivation of inflammatory and functional gastrointestinal disorders mainly through disruption of the intestinal barrier6 in both human and animal models . the stress response is vehiculated via 2 main systems : the sympatho - adrenomedullary system and the hypothalamic pituitary - adrenal axis . stress stimulates the parvocellular neurons in the paraventricular nucleus of the hypothalamus to secrete corticotropin - releasing factor ( crf ) , and arginine vasopressin . crf promotes the synthesis and release of adenocortico - tropic hormone in the anterior pituitary . the adenocorticotropic hormone , in turn , activates the adrenal cortex to induce a temporary rise in blood levels of cortisol and corticosterone7 and also the release of catecholamines by the adrenal medulla . it has been believed for a long time that central secretion of crf was the main and unique mediator of the majority of the endocrine , behavioral and gastrointestinal changes induced by stress.8 however , we will show that peripheral release of crf also plays a key role in the regulation of gastrointestinal permeability , and discuss the link to both health and pathological conditions . the intestinal mucosal barrier includes several consecutive layers , from the outermost microbiota , through the external mucus , the epithelium , and down to the innermost lamina propria.9 the epithelial layer is described as a continuous polarized monolayer of columnar cells that separates the intestinal lumen from the internal milieu . aside from epithelial cells , a variety of different cell types are also intermingled with enterocytes , including goblet cells , paneth cells , enteroendocrine cells , and m cells . within the lamina propria we can find blood and lymph vessels , and a plethora of distinct immune cells such as plasma cells , lymphocytes , macrophages , eosinophils , mast cells , dendritic cells , and a significant number of intrinsic and extrinsic nerve terminals.10 all of these components are exquisitely reactive and adaptive and display critical effector and modulatory functions . these functions are relevant for the control of inflammation , absorption and secretion , transport of macromolecules , and metabolic processes.11 moreover , most of the cellular components have been shown to express receptors for crf.12,13 but the job of the intestinal barrier now appears to be far more nuanced and complex as it communicates multidirectionally with the immune system and the microbes . communication is developed through the release of an extensive array of chemical mediators , namely neuropeptides , neurohormones , neurotransmitters , cytokines , chemokines , growth factors , and other regulatory molecules.14,15 enterocytes are tightly bonded to each other sealing the para - cellular space through the apical junctional complex , composed of tight junctions ( tjs ) , adherens junctions , and desmosomes.16 the transmembrane tj proteins occludin and claudins form complex protein systems , which interact with zonula occludens proteins that bind to the actin cytoskeleton . when actin contracts , it leads to increased permeability to electrolytes and small molecules.17 the paracellular space is not fully impermeable to molecules and antigens , allowing a controlled amount of small particles ( less than 400 daltons ) to penetrate across to reach the lamina propria,9 a phenomenon that plays a key role in the induction of immune tolerance . this passage of molecules also takes place through the transcellular pathway via endocytosis or exocytosis.18 disruption of the intestinal barrier leads to uncontrolled flux of luminal antigens and possibly microbes across the epithelium , which may trigger immune activation and sepsis , and also lead to the development of chronic inflammation in the gut.6,19,20 therefore , the tight regulation of intestinal permeability emerges as a central mechanism to prevent inflammatory diseases . numerous pathogens and toxins , hormones and neurotransmitters , and gastrointestinal and non - gastrointestinal diseases have been associated with an augmented intestinal permeability.21 among them , stress hormones and neurotransmitters have been consistently shown to modulate ion and water secretion , intestinal permeability , mucus secretion , and also intestinal flora.2225 stress represents a threat to the internal homeostasis that initiates a systemic coordinated response driven by the autonomic , endocrine , and immune systems to maintain stability . crf and other members of the crf signaling family , including urocortin ( ucn ) 1 , ucn2 , and ucn3 , are the best known and most important neuroendocrine mediators of the stress response.26,27 central release of crf and urocortins mediates autonomic , hormonal , and behavioural responses to stress and at the gastrointestinal tract , stimulates the enteric nervous system to modulate gastrointestinal motility and secretion.2830 in addition , immune cells , regional sensory and sympathetic nerves , enterochromaffin cells , and enteric cells release crf and urocortins within the gastrointestinal tract28 to modulate mucosal function and gastrointestinal motility.29 different type of stress , acute or chronic , physical or psychological , have been shown to influence properties of the intestinal barrier function , including ion and water secretion , intestinal permeability , mucus secretion , and also intestinal flora in both human and animal models.6,3135 similarly , both central and peripheral administration of exogenous crf have been shown to mimic the effects of acute stress in the gastrointestinal tract inducing mucin release,36 and increasing ion and water secretion and intestinal permeability.37,38 figure summarizes the main effects of stress on the intestinal barrier and the role of crf in gastrointestinal permeability . the intestinal mucosal surface is covered by a layer of mucus gel containing antibacterial peptides and digestive enzymes.39,40 mucus protects the epithelial lining from adhesion and invasion by attacking microorganisms , and other antigens present in the intestinal lumen.41 mucus is produced by goblet cells , which express the crf receptor 1 ( crf1).42 stress has been shown to increase mucin release in colonic explants from rats submitted to immobilization , and to enhance rat mast cell protease ii and prostaglandin e2 secretion . intravenous or intracerebral injection of crf in non - stressed rats reproduced stress - associated changes whereas administration of the crf antagonist -helical - crf941 or the mast cell stabilizer lodoxamide inhibited them.36 in addition , those changes were abolished in mast - cell deficient mice , highlighting the key role of the crf - mast cell axis in stress - mediated mucin release.43 on the contrary , rats submitted to chronic stress display mucus depletion and reduced number of goblet cells in the distal colon in association with increased bacterial adhesion and penetration into enterocytes,44,45 what could represent a step towards intestinal barrier dysfunction . ion and water secretions also help to prevent the adhesion of pathogens and noxious substances to the mucosal surface , and dilute and flush out them down the gut to minimize their penetration to the lamina propria . convergent evidences from studies in animal models indicate that both water and ion secretions increase in the intestinal tract in response to acute and chronic stress24,46 and in response to peripheral administration of crf47 whereas selective crf1 receptor agonists prevent this response to stress.48,49 increased baseline short - circuit current ( isc ) , indicative of enhanced anion secretion , was found in the jejunum of wistar - kyoto rats submitted to restraint stress or cold restraint stress when compared to non - stressed rats . this increase in isc was mediated by chloride anion secretion because replacement of the buffer by chloride free solution normalized isc values.50 later , santos et al37,51 reproduced the results using a model of repetitive exposure to water avoidance stress ( was ) and found that peripheral crf reproduced stress - induced rat jejunal and colonic epithelial barrier dysfunction via cholinergic and adrenergic nerves , and mast cells . more recently , long - term crowding exposure , a model of psychosocial stress , has been shown to increase baseline isc in both the jejunum and the colon compared to non - stressed animals.52 interestingly , colonic tissues from stressed rats exhibited a reduced isc response to the addition of cholinergic agonists and crf , suggesting an impaired secretory response to incoming stressful stimuli . mitochondrial activity was disturbed throughout the intestine , although mitochondrial response to crf was preserved . these changes were associated with an increased expression of crf1 receptor in the colon of stressed rats and colonic hyperalgesia.53 several other studies using various stress models have reproduced the elevation of isc in the colon and ileum of rodents and showed the involvement of crf receptors.24,33,45,50,5456 both the acute intraperitoneal administration of crf and chronic peripheral administration of crf , the selective crf1 receptor agonist stressin-1 and the selective crf2 receptor ligand , ucn3 , increase the basal rat colonic isc.33,57 therefore , a role for both crf receptor subtypes in this alteration is suggested , although the response is not reduced by the selective crf2 receptor antagonist antisauvagine.57 furthermore , elevated baseline isc has been also observed in the jejunum of early - weaned pigs , while activation of mast cells and crf induced elevations in isc in the pig jejunum mounted in ussing chambers . this elevation in isc was inhibited using mast cell protease inhibitors.58 experiments using selective crf receptor antagonists reveal that crf1 receptor activation mediates barrier dysfunction and hypersecretion in the porcine intestine in response to early life stress.58 apart from ex vivo and in vivo studies , convincing evidence for a direct effect of crf peptides on intestinal epithelial secretory response comes from in vitro work showing a consistent increase in isc induced by crf in the rat colon.33,37,59 interestingly , maximal isc increments were obtained with sauvagine , a crf agonist with high affinity to both crf receptors , at doses 2001000 fold lower than crf , suggesting a predominant crf2 receptor effect.59 in addition , the effect of peripheral crf on isc increase in rat colonic tissue implicates mast cells , and nicotinic and muscarinic receptors , as established by the pharmacological blockade of specific receptors and the use of mast cell deficient rats.37,57 other studies performed in the guinea - pig support the ample distribution of crf1 receptor , but not crf2 receptor throughout the myenteric and submucosal plexuses of the gastrointestinal tract.60 however , all crf and ucn1 , ucn2 , and ucn3 were able to increase the excitability of neurons from the enteric nervous system in vitro.61 experimental studies in humans reveal that acute physical or psychological stress modulate the intestinal barrier . more than 20 years ago , barclay and turnberg et al,62,63 using segmental perfusion techniques in the human jejunum , showed that acute stress reduced net water absorption or increased secretion , supporting the role of the central and the autonomic nervous system in the control of intestinal function . in particular , a reduction in water absorption coupled with net sodium / chloride secretion was observed in healthy subjects subjected to psychological stress induced by dichotomous listening.62,63 later , santos et al64 showed that cold pain stress increased jejunal water secretion in healthy subjects and in patients with food allergy . these observations have been confirmed and extended recently : using the cold press - or test . the increase in intestinal water secretion during cold pain stress was higher in healthy female volunteers with lower background stress levels , yet blunted in those with elevated background stress.65 these results suggest that chronic life stress could lead to the loss of regulatory mechanisms in healthy individuals , as previously described in the jejunum of chronic stressed rats after neural stimulation.50 crf1 receptor is expressed throughout the gastrointestinal tract in healthy subjects , and located in the lamina propria , mostly in macrophages , epithelial cells and enteric neurons , with the highest levels in the ileum and rectum and the lowest level in the colon.66 crf2 receptors are located in the lamina propria and in the epithelial cells of the distal / sigmoid biopsy samples in healthy subjects.67 in contrast to animal studies , the addition of crf to human colonic biopsies from healthy individuals mounted in ussing chambers did not show any effect on isc.68 numerous studies demonstrate the enhancement of intestinal permeability by acute and chronic stressors . in rodents , acute or subchronic stressors , including restraint stress , cold restraint stress , was , mild noise stress , and mixed restraint and acoustic stress , increased tissue conductance , and the fluxes of h mannitol,51 cr - labeled edta , horseradish peroxidase , in the jejunum , ileum , and colon.32,50,54,6972 these results indicate the ability of stress to modulate paracellular and transcellular transport of ions , and small and large macromolecules . the list includes acetylcholine release , histamine , glucagon - like peptide-2 , myosin light chain kinase , cytokines such as interferon- , interleukin ( il)-4 and il-13 , and mast cell activation , among the most relevant.32,61,71,7375 moreover , increased intestinal permeability , measured by lactulose , mannitol and sucralose ratios , was found in rats small intestine , following acute swimming stress.23 this increase was glucocorticoid - mediated because adrenalectomy and pharmacologic blockade of glucocorticoid receptors inhibited the response , and dexamethasone increased gastrointestinal permeability in control rats.23 similarly , rats models of chronic stress ( maternal deprivation,55,7779 was,45 and crowding stress52 ) also show increased ionic permeability , and macromolecular permeability throughout the intestine . the increase was mediated partly by mechanisms including , muscarinic and nicotinic pathways , activation and number of mast cells and the release of nerve growth factor.79 stress - induced enhancement of colonic permeability was mimicked by exogenous administration of crf,33 and abolished by pretreatment with the peripheral administration of the non - selective crf antagonists astressin or -helical crf941.37,55,80 likely , the selective crf1 receptor agonist , cortagine,48 the selective crf1 receptor antagonist , ssr-125543,80 and the selective crf2 receptor antagonist , antisauvagine-30,78 reduced the response , supporting the participation of both crf receptors in the modulation of colonic permeability . more recently , studies in several cultured epithelial cell lines , including intestinal human ht-29 cells , showed that crf induced the upregulation of the endotoxin receptor , toll - like receptor 4 ( tlr4 ) , and that pretreatment with the crf2 receptor antagonist , antalarmin , abrogated the response . the concomitant presence of crf and lipopolysaccharide increased permeability to horseradish peroxidase and decreased transepithelial resistance . this effect was abolished by the crf2 receptor antagonist , astressin2-b , and was mediated through the upregulation of claudin-2 . similarly , the expression of tlr4 and claudin-2 increased in the small and large intestine of pregnant mice submitted to 10 days of was , as was enhanced the permeability to horseradish peroxidase , and the effect abolished by -helical crf94181 and anti - claudin antibodies . interestingly , rats submitted to was for 10 days , showed higher sensitization to orally - delivered horse - radish peroxidase , as shown by enhanced intestinal permeability upon antigen re - exposure . this effect was prevented by crf blockade with by -helical crf941.82 another line of evidence in animal models highlights the relevance of stress - crf - mast cell axis in the regulation of intestinal permeability . elevation of intestinal permeability , in response to restraint stress or intraperitoneal administration of crf , was reduced or abolished after pretreatment with doxantrazole.59 similarly , pharmacological inhibition of mast cell activation has been shown to inhibit stress - induced increased intestinal permeability in different animal models.37,58,59,83 stress / crf - induced changes in the rat intestinal permeability were also significantly reduced in tissues from mast - cell deficient rats.45,51,59,84 teitelbaum et al57 also found crf to cause mast cell hyperplasia and abnormal bacterial attachment and/or penetration into the rat mucosa except in mast - cell deficient rats . the authors revealed that stimulation of crf1 receptor induced an elevated secretory state , while crf2 receptor stimulation was associated with permeability dysfunction.57 although it is clear that mast cell activation disrupts intestinal barrier and increases intestinal permeability , the ultimate mechanisms remain to be elucidated . in this sense , several mast cell mediators have been involved . tumor necrosis factor- ( tnf- ) has been shown to increase intestinal permeability through nuclear factor - kappa b activation in association with nuclear factor - kappa b - dependent downregulation of zona occludens protein-1 expression and alteration in junctional localization in caco-2 cells,85 and through myosin light chain kinase phosphorylation.86 more recently , using a porcine ex vivo intestinal model , overman et al87 found that crf increased intestinal paracellular permeability via mast cell dependent release of tnf- and proteases . on the other hand , the tryptase released by mast cells upon activation has been shown to induce tjs disassembly through the activation of proteinase - activated receptor-2 of the epithelial cells.8890 these receptors can modulate enteric neurotransmission , secretion , motility , epithelial permeability , and visceral sensitivity , and are also known to regulate intestinal inflammation.91 anatomical contacts between mast cells and enteric nerve fibers have been demonstrated in the human gastrointestinal mucosa and inflammation multiplies these contacts.92,93 mast cells communicate , bidirectionally , with both the enteric , autonomic and central nervous system through mast cell mediators and neuropeptides.94 in fact , human mast cells synthesize and secrete both crf and ucn in response to immunoglobulin e receptor activation.95 mast cells also express crf receptors , whose activation leads to the release of cytokines and other pro - inflammatory mediators . crf enhances the trans - cellular uptake of macromolecules in the human colonic mucosa in vitro , via crf receptor subtypes crf1 and crf2 expressed on subepithelial mast cells.58 crf1 and crf2 receptors have been described in human umbilical cord blood - derived mast cells , while only crf1 receptor has been found in the human leukemic mast cell hmc-1 line . crf stimulation induces the secretion of vascular endothelial growth factor without tryptase , histamine , il-6 , il-8 , or tnf- release through crf1 receptor.96 more recently , acute stress- induced bladder vascular permeability and vascular endothelial growth factor release have been shown to be dependent on crf2 receptor.97 it is important to note here the ability of eosinophils to alter intestinal permeability and the mechanisms and mediators involved.98 barrier function has been shown to be affected by eosinophil - derived major basic protein ( mbp ) through downregulation of occludin in a mast cell independent fashion.98 the neuropeptide substance p ( sp ) has been found to induce the release of vasoactive mediators from mast cells , macrophages , and t cells , contributing to chloride secretion , enhanced intestinal permeability , and vascular leakiness.99,100 sp increases the expression of crf1 receptor in mast cells , and in turn , crf induces the expression of the specific receptor neurokinin ( nk)-1.101 more recently , psychological stress , through sp release , mediated stress - induced crf expression in mice eosinophils and eosinophil - derived crf was responsible for mast cell activation and epithelial barrier dysfunction . in this work , a cell line of eosinophils was treated with a number of stress mediators , but only sp induced crf release via nk-1 and nk-2 . moreover , priming of eosinophils with sp resulted in mast cell activation through eosinophil - derived crf that in turn induced intestinal barrier dysfunction.102 wallon et al83 examined non - inflamed colonic mucosal biopses from patients with ulcerative colitis and found that eosinophils displayed immunoreactivity to crf . in addition , in co - culture studies , carbachol activation of eosinophils induced crf release and subsequent activation of mast cells , which increased permeability of epithelial cells to macromolecules.83 similar to mast cells , eosinophils have also been shown to localize close to airway nerve terminals in patients with asthma as well as in animal models of bronchial hyperreactivity.103,104 this proximity to nerve terminals is described in certain digestive disorders,105,106 providing the anatomical substrate to understand the relevance of the mast cell - eosinophil - stress / crf axis in the regulation of epithelial permeability and the initiation of immune and inflammatory diseases . stress - induced increase in gut paracellular permeability has also been shown to depend on crf1 receptor - mediated mast cell release of nerve growth factor ( ngf ) . maternal deprivation has been shown to enhance colon permeability in association with elevated ngf expression.76 a subsequent study from the same group showed that crf , acting through its receptor crf1 receptor , stimulated ngf release from mast cells , which in turn increased gut paracellular permeability.80 dendritic cells are relevant for the regulation of intestinal immune function and permeability through crf production , a process augmented by commensal bacteria.107 similarly , crf1 and crf2 receptor agonists exert a biphasic effect on macrophages . during the early stages of the inflammatory response , they suppress tnf- release via induction of cyclooxygenase-2/prostaglandin e2 while later on they induce tnf- transcription.108 unfortunately , the role of crf - mediated activation of macrophages and dendritic cells in stress - related intestinal barrier dysfunction is largely ignored . vanuytsel et al38 have recently shown that , both psychological stress ( public speech ) and a single intravenous bolus of crf ( 100 g ) increased small intestinal permeability in healthy humans , measured by the lactulose / mannitol ratio . two weeks pretreatment with 800 mg / day of the mast cell stabilizer , disodium cromoglycate , blocked the effect of both stress and crf , invoking the participation of the crf - mast cell axis in this response.38 we have also observed in healthy volunteers that cold pain stress enhanced both the blood - to - lumen albumin65 ratio and the blood - to - lumen mannitol and xylose permeability.109 this response was mainly observed in females with higher background stress levels , suggesting an impaired epithelial response to incoming stressful stimuli in this group . in addition , acute cold stress was shown to induce a significant release of -defensin in the jejunum in this study , supporting the possibility that stress might affect this protective pathway in the gut , as have been shown in the skin.110 furthermore , we have also found that cold stress evoked a differential gender - determined increase in human intestinal macromolecular permeability.111 this enhanced permeability could lead to excessive uptake of luminal antigens and bacterial products that may initiate an inflammatory response in the mucosa.112 other studies showing the effect of stress and crf on intestinal and extra - intestinal permeability are shown in tables 1 and 2 , respectively . the intestinal mucosal surface is covered by a layer of mucus gel containing antibacterial peptides and digestive enzymes.39,40 mucus protects the epithelial lining from adhesion and invasion by attacking microorganisms , and other antigens present in the intestinal lumen.41 mucus is produced by goblet cells , which express the crf receptor 1 ( crf1).42 stress has been shown to increase mucin release in colonic explants from rats submitted to immobilization , and to enhance rat mast cell protease ii and prostaglandin e2 secretion . intravenous or intracerebral injection of crf in non - stressed rats reproduced stress - associated changes whereas administration of the crf antagonist -helical - crf941 or the mast cell stabilizer lodoxamide inhibited them.36 in addition , those changes were abolished in mast - cell deficient mice , highlighting the key role of the crf - mast cell axis in stress - mediated mucin release.43 on the contrary , rats submitted to chronic stress display mucus depletion and reduced number of goblet cells in the distal colon in association with increased bacterial adhesion and penetration into enterocytes,44,45 what could represent a step towards intestinal barrier dysfunction . ion and water secretions also help to prevent the adhesion of pathogens and noxious substances to the mucosal surface , and dilute and flush out them down the gut to minimize their penetration to the lamina propria . convergent evidences from studies in animal models indicate that both water and ion secretions increase in the intestinal tract in response to acute and chronic stress24,46 and in response to peripheral administration of crf47 whereas selective crf1 receptor agonists prevent this response to stress.48,49 increased baseline short - circuit current ( isc ) , indicative of enhanced anion secretion , was found in the jejunum of wistar - kyoto rats submitted to restraint stress or cold restraint stress when compared to non - stressed rats . this increase in isc was mediated by chloride anion secretion because replacement of the buffer by chloride free solution normalized isc values.50 later , santos et al37,51 reproduced the results using a model of repetitive exposure to water avoidance stress ( was ) and found that peripheral crf reproduced stress - induced rat jejunal and colonic epithelial barrier dysfunction via cholinergic and adrenergic nerves , and mast cells . more recently , long - term crowding exposure , a model of psychosocial stress , has been shown to increase baseline isc in both the jejunum and the colon compared to non - stressed animals.52 interestingly , colonic tissues from stressed rats exhibited a reduced isc response to the addition of cholinergic agonists and crf , suggesting an impaired secretory response to incoming stressful stimuli . mitochondrial activity was disturbed throughout the intestine , although mitochondrial response to crf was preserved . these changes were associated with an increased expression of crf1 receptor in the colon of stressed rats and colonic hyperalgesia.53 several other studies using various stress models have reproduced the elevation of isc in the colon and ileum of rodents and showed the involvement of crf receptors.24,33,45,50,5456 both the acute intraperitoneal administration of crf and chronic peripheral administration of crf , the selective crf1 receptor agonist stressin-1 and the selective crf2 receptor ligand , ucn3 , increase the basal rat colonic isc.33,57 therefore , a role for both crf receptor subtypes in this alteration is suggested , although the response is not reduced by the selective crf2 receptor antagonist antisauvagine.57 furthermore , elevated baseline isc has been also observed in the jejunum of early - weaned pigs , while activation of mast cells and crf induced elevations in isc in the pig jejunum mounted in ussing chambers . this elevation in isc was inhibited using mast cell protease inhibitors.58 experiments using selective crf receptor antagonists reveal that crf1 receptor activation mediates barrier dysfunction and hypersecretion in the porcine intestine in response to early life stress.58 apart from ex vivo and in vivo studies , convincing evidence for a direct effect of crf peptides on intestinal epithelial secretory response comes from in vitro work showing a consistent increase in isc induced by crf in the rat colon.33,37,59 interestingly , maximal isc increments were obtained with sauvagine , a crf agonist with high affinity to both crf receptors , at doses 2001000 fold lower than crf , suggesting a predominant crf2 receptor effect.59 in addition , the effect of peripheral crf on isc increase in rat colonic tissue implicates mast cells , and nicotinic and muscarinic receptors , as established by the pharmacological blockade of specific receptors and the use of mast cell deficient rats.37,57 other studies performed in the guinea - pig support the ample distribution of crf1 receptor , but not crf2 receptor throughout the myenteric and submucosal plexuses of the gastrointestinal tract.60 however , all crf and ucn1 , ucn2 , and ucn3 were able to increase the excitability of neurons from the enteric nervous system in vitro.61 experimental studies in humans reveal that acute physical or psychological stress modulate the intestinal barrier . more than 20 years ago , barclay and turnberg et al,62,63 using segmental perfusion techniques in the human jejunum , showed that acute stress reduced net water absorption or increased secretion , supporting the role of the central and the autonomic nervous system in the control of intestinal function . in particular , a reduction in water absorption coupled with net sodium / chloride secretion was observed in healthy subjects subjected to psychological stress induced by dichotomous listening.62,63 later , santos et al64 showed that cold pain stress increased jejunal water secretion in healthy subjects and in patients with food allergy . these observations have been confirmed and extended recently : using the cold press - or test . the increase in intestinal water secretion during cold pain stress was higher in healthy female volunteers with lower background stress levels , yet blunted in those with elevated background stress.65 these results suggest that chronic life stress could lead to the loss of regulatory mechanisms in healthy individuals , as previously described in the jejunum of chronic stressed rats after neural stimulation.50 crf1 receptor is expressed throughout the gastrointestinal tract in healthy subjects , and located in the lamina propria , mostly in macrophages , epithelial cells and enteric neurons , with the highest levels in the ileum and rectum and the lowest level in the colon.66 crf2 receptors are located in the lamina propria and in the epithelial cells of the distal / sigmoid biopsy samples in healthy subjects.67 in contrast to animal studies , the addition of crf to human colonic biopsies from healthy individuals mounted in ussing chambers did not show any effect on isc.68 numerous studies demonstrate the enhancement of intestinal permeability by acute and chronic stressors . in rodents , acute or subchronic stressors , including restraint stress , cold restraint stress , was , mild noise stress , and mixed restraint and acoustic stress , increased tissue conductance , and the fluxes of h mannitol,51 cr - labeled edta , horseradish peroxidase , in the jejunum , ileum , and colon.32,50,54,6972 these results indicate the ability of stress to modulate paracellular and transcellular transport of ions , and small and large macromolecules . the list includes acetylcholine release , histamine , glucagon - like peptide-2 , myosin light chain kinase , cytokines such as interferon- , interleukin ( il)-4 and il-13 , and mast cell activation , among the most relevant.32,61,71,7375 moreover , increased intestinal permeability , measured by lactulose , mannitol and sucralose ratios , was found in rats small intestine , following acute swimming stress.23 this increase was glucocorticoid - mediated because adrenalectomy and pharmacologic blockade of glucocorticoid receptors inhibited the response , and dexamethasone increased gastrointestinal permeability in control rats.23 similarly , rats models of chronic stress ( maternal deprivation,55,7779 was,45 and crowding stress52 ) also show increased ionic permeability , and macromolecular permeability throughout the intestine . the increase was mediated partly by mechanisms including , muscarinic and nicotinic pathways , activation and number of mast cells and the release of nerve growth factor.79 stress - induced enhancement of colonic permeability was mimicked by exogenous administration of crf,33 and abolished by pretreatment with the peripheral administration of the non - selective crf antagonists astressin or -helical crf941.37,55,80 likely , the selective crf1 receptor agonist , cortagine,48 the selective crf1 receptor antagonist , ssr-125543,80 and the selective crf2 receptor antagonist , antisauvagine-30,78 reduced the response , supporting the participation of both crf receptors in the modulation of colonic permeability . more recently , studies in several cultured epithelial cell lines , including intestinal human ht-29 cells , showed that crf induced the upregulation of the endotoxin receptor , toll - like receptor 4 ( tlr4 ) , and that pretreatment with the crf2 receptor antagonist , antalarmin , abrogated the response . the concomitant presence of crf and lipopolysaccharide increased permeability to horseradish peroxidase and decreased transepithelial resistance . this effect was abolished by the crf2 receptor antagonist , astressin2-b , and was mediated through the upregulation of claudin-2 . similarly , the expression of tlr4 and claudin-2 increased in the small and large intestine of pregnant mice submitted to 10 days of was , as was enhanced the permeability to horseradish peroxidase , and the effect abolished by -helical crf94181 and anti - claudin antibodies . interestingly , rats submitted to was for 10 days , showed higher sensitization to orally - delivered horse - radish peroxidase , as shown by enhanced intestinal permeability upon antigen re - exposure . this effect was prevented by crf blockade with by -helical crf941.82 another line of evidence in animal models highlights the relevance of stress - crf - mast cell axis in the regulation of intestinal permeability . elevation of intestinal permeability , in response to restraint stress or intraperitoneal administration of crf , was reduced or abolished after pretreatment with doxantrazole.59 similarly , pharmacological inhibition of mast cell activation has been shown to inhibit stress - induced increased intestinal permeability in different animal models.37,58,59,83 stress / crf - induced changes in the rat intestinal permeability were also significantly reduced in tissues from mast - cell deficient rats.45,51,59,84 teitelbaum et al57 also found crf to cause mast cell hyperplasia and abnormal bacterial attachment and/or penetration into the rat mucosa except in mast - cell deficient rats . the authors revealed that stimulation of crf1 receptor induced an elevated secretory state , while crf2 receptor stimulation was associated with permeability dysfunction.57 although it is clear that mast cell activation disrupts intestinal barrier and increases intestinal permeability , the ultimate mechanisms remain to be elucidated . in this sense , tumor necrosis factor- ( tnf- ) has been shown to increase intestinal permeability through nuclear factor - kappa b activation in association with nuclear factor - kappa b - dependent downregulation of zona occludens protein-1 expression and alteration in junctional localization in caco-2 cells,85 and through myosin light chain kinase phosphorylation.86 more recently , using a porcine ex vivo intestinal model , overman et al87 found that crf increased intestinal paracellular permeability via mast cell dependent release of tnf- and proteases . on the other hand , the tryptase released by mast cells upon activation has been shown to induce tjs disassembly through the activation of proteinase - activated receptor-2 of the epithelial cells.8890 these receptors can modulate enteric neurotransmission , secretion , motility , epithelial permeability , and visceral sensitivity , and are also known to regulate intestinal inflammation.91 anatomical contacts between mast cells and enteric nerve fibers have been demonstrated in the human gastrointestinal mucosa and inflammation multiplies these contacts.92,93 mast cells communicate , bidirectionally , with both the enteric , autonomic and central nervous system through mast cell mediators and neuropeptides.94 in fact , human mast cells synthesize and secrete both crf and ucn in response to immunoglobulin e receptor activation.95 mast cells also express crf receptors , whose activation leads to the release of cytokines and other pro - inflammatory mediators . crf enhances the trans - cellular uptake of macromolecules in the human colonic mucosa in vitro , via crf receptor subtypes crf1 and crf2 expressed on subepithelial mast cells.58 crf1 and crf2 receptors have been described in human umbilical cord blood - derived mast cells , while only crf1 receptor has been found in the human leukemic mast cell hmc-1 line . crf stimulation induces the secretion of vascular endothelial growth factor without tryptase , histamine , il-6 , il-8 , or tnf- release through crf1 receptor.96 more recently , acute stress- induced bladder vascular permeability and vascular endothelial growth factor release have been shown to be dependent on crf2 receptor.97 it is important to note here the ability of eosinophils to alter intestinal permeability and the mechanisms and mediators involved.98 barrier function has been shown to be affected by eosinophil - derived major basic protein ( mbp ) through downregulation of occludin in a mast cell independent fashion.98 the neuropeptide substance p ( sp ) has been found to induce the release of vasoactive mediators from mast cells , macrophages , and t cells , contributing to chloride secretion , enhanced intestinal permeability , and vascular leakiness.99,100 sp increases the expression of crf1 receptor in mast cells , and in turn , crf induces the expression of the specific receptor neurokinin ( nk)-1.101 more recently , psychological stress , through sp release , mediated stress - induced crf expression in mice eosinophils and eosinophil - derived crf was responsible for mast cell activation and epithelial barrier dysfunction . in this work , a cell line of eosinophils was treated with a number of stress mediators , but only sp induced crf release via nk-1 and nk-2 . moreover , priming of eosinophils with sp resulted in mast cell activation through eosinophil - derived crf that in turn induced intestinal barrier dysfunction.102 wallon et al83 examined non - inflamed colonic mucosal biopses from patients with ulcerative colitis and found that eosinophils displayed immunoreactivity to crf . in addition , in co - culture studies , carbachol activation of eosinophils induced crf release and subsequent activation of mast cells , which increased permeability of epithelial cells to macromolecules.83 similar to mast cells , eosinophils have also been shown to localize close to airway nerve terminals in patients with asthma as well as in animal models of bronchial hyperreactivity.103,104 this proximity to nerve terminals is described in certain digestive disorders,105,106 providing the anatomical substrate to understand the relevance of the mast cell - eosinophil - stress / crf axis in the regulation of epithelial permeability and the initiation of immune and inflammatory diseases . stress - induced increase in gut paracellular permeability has also been shown to depend on crf1 receptor - mediated mast cell release of nerve growth factor ( ngf ) . maternal deprivation has been shown to enhance colon permeability in association with elevated ngf expression.76 a subsequent study from the same group showed that crf , acting through its receptor crf1 receptor , stimulated ngf release from mast cells , which in turn increased gut paracellular permeability.80 dendritic cells are relevant for the regulation of intestinal immune function and permeability through crf production , a process augmented by commensal bacteria.107 similarly , crf1 and crf2 receptor agonists exert a biphasic effect on macrophages . during the early stages of the inflammatory response , they suppress tnf- release via induction of cyclooxygenase-2/prostaglandin e2 while later on they induce tnf- transcription.108 unfortunately , the role of crf - mediated activation of macrophages and dendritic cells in stress - related intestinal barrier dysfunction is largely ignored . vanuytsel et al38 have recently shown that , both psychological stress ( public speech ) and a single intravenous bolus of crf ( 100 g ) increased small intestinal permeability in healthy humans , measured by the lactulose / mannitol ratio . two weeks pretreatment with 800 mg / day of the mast cell stabilizer , disodium cromoglycate , blocked the effect of both stress and crf , invoking the participation of the crf - mast cell axis in this response.38 we have also observed in healthy volunteers that cold pain stress enhanced both the blood - to - lumen albumin65 ratio and the blood - to - lumen mannitol and xylose permeability.109 this response was mainly observed in females with higher background stress levels , suggesting an impaired epithelial response to incoming stressful stimuli in this group . in addition , acute cold stress was shown to induce a significant release of -defensin in the jejunum in this study , supporting the possibility that stress might affect this protective pathway in the gut , as have been shown in the skin.110 furthermore , we have also found that cold stress evoked a differential gender - determined increase in human intestinal macromolecular permeability.111 this enhanced permeability could lead to excessive uptake of luminal antigens and bacterial products that may initiate an inflammatory response in the mucosa.112 other studies showing the effect of stress and crf on intestinal and extra - intestinal permeability are shown in tables 1 and 2 , respectively . the growing acknowledgment of the scientific community to the role of intestinal permeability in keeping health and well - being and its relation to the origin of digestive and extradigestive disorders is becoming more and more universal for clinicians . increased permeability and breakdown of intestinal barrier have been implicated in the origin of gastrointestinal and liver disorders , including celiac disease,113 inflammatory bowel disease,114118 food allergy,119 acute pancreatitis,120 irritable bowel syndrome ( ibs),121,122 functional dyspepsia,123 infectious diarrheal syndromes,124 primary biliary cirrhosis , and primary sclerosing cholangitis,125 liver cirrhosis,126 alcoholic liver disease,127 liver encephalopathy,128 and gastroesophageal reflux disease,129131 among others . however , whether enhanced gut permeability is an early manifestation of disease , a central step in disease pathogenesis , or a simple epiphenomenon , and its relationship with life stress in the clinical setting , remains to be elucidated . interestingly , life events may favor the clinical appearance of celiac disease,132 and cellular stress , through mhc class i chain related genes a and b and endoplasmic reticulum stress pathways , is linked to the disregulation of mucosal homeostasis.133,134 early in the 80s , celiac patients were shown to display increased intestinal permeability that normalized after several months on a gluten - free diet.113 more precise in vitro investigations revealed that although strict gluten withdrawal restored intestinal histology , a subjacent defect in mucosal permeability , measured by cellobiose / mannitol ratio , was transiently induced by short exposure to gluten , suggesting that increased intestinal permeability in celiac disease could be a primary defect.135 recently , this defect in intestinal permeability has been related to altered expression of tj genes related to permeability , polarity , and cell proliferation in active celiac disease,136 partly through the activation of the zonulin pathway in a myd88-dependent fashion.137,138 again , the majority of genes returned to normal after 2 years of gluten eviction with the exception of ppp2r3a , possibly indicating a constitutive defect in these patients.136 moreover , unlike celiac disease , gluten sensitivity is not associated with increased intestinal permeability.139 increased intestinal permeability has also been described in patients with inflammatory bowel disease . enhanced intestinal permeability in this population is considered an initial event because it is increased in first - degree relatives of crohn s disease patients.140,141 several observations link clinical relapses to the increase in intestinal permeability142,143 and to life stress,144,145 partly through mast cell activation146 and the release of crf from eosinophils83 and neighboring nerves.68 moreover , inflammatory bowel disease patients show associations with genes involved in the regulation of intestinal barrier and various alterations of the transmembrane and intracytoplasmic proteins.147,148 in addition , increased crf1 receptor66 and ucn expression , but decreased crf2 receptor67 expression have been shown in the colonic mucosa of active ulcerative colitis patients.149,150 accumulating evidence also indicates that ibs is linked to abnormal intestinal permeability,151153 crf release and life stress154157 in close association with low - grade mucosal inflammation and immune activation.158 several groups have provided preliminary evidence linking clinical manifestations of ibs to structural abnormalities of the apical junctional complex in both the small159,160 and large bowel mucosa.81,161163 furthermore , unpublished observations from our group indicate that a single intravenous bolus of crf ( 100 g ) increased intestinal permeability , measured as the blood - to - lumen albumin ratio , in healthy subjects and in ibs patients through mast cell activation.164 in the same line , increased intestinal permeability and stress / crf axis have also been involved in the pathophysiology of food allergy , as these patients display an enhancement of intestinal permeability even in the absence of food allergens165 and stress facilitates sensitization to luminal antigens.82 in fact , patients following immunosuppressive treatment have been shown to develop new - onset food allergies that may be related to the increase in intestinal permeability induced by treatment.166 many other stress - related conditions such as severe burn,167,168 hemorragic shock,169 chronic kidney disease,170 type 1 diabetes and the metabolic syndrome,171,172 neuropsychiatric disorders,173 autism,174 autoimmune thyroiditis,175 iga nephropathy,176 patients with primary immunodeficiencies,177 and sepsis178 have been associated with increased intestinal permeability . the ability of stress and peripheral crf to affect intestinal epithelial function and , particularly , intestinal permeability , is well documented in human and several animal species . interestingly , apart from the direct effects on the enterocytes , the stress - induced increase of intestinal permeability is mediated via recruitment and activation of mast cells , eosinophils , macrophages and other mononuclear cells , and implicates both crfr1 and crf2 receptors.28 there is abundant literature supporting the link between life stress and the origin and clinical course of several human disorders,179 however , the ultimate clinical relevance of stress - crf - mediated dysregulation of gastrointestinal permeability , remains at present mostly intuitive , with few exceptions . more work in this area is needed to confirm the findings . in this sense , studies with crf and related peptides and the development of new antagonists for human use , will offer the opportunity to test this hypoyhesis .

the interface between the intestinal lumen and the mucosa is the location where the majority of ingested immunogenic particles face the scrutiny of the vast gastrointestinal immune system . upon regular physiological conditions , the intestinal micro - flora and the epithelial barrier are well prepared to process daily a huge amount of food - derived antigens and non - immunogenic particles . similarly , they are ready to prevent environmental toxins and microbial antigens to penetrate further and interact with the mucosal - associated immune system . these functions promote the development of proper immune responses and oral tolerance and prevent disease and inflammation . brain - gut axis structures participate in the processing and execution of response signals to external and internal stimuli . the brain - gut axis integrates local and distant regulatory networks and super - systems that serve key housekeeping physiological functions including the balanced functioning of the intestinal barrier . disturbance of the brain - gut axis may induce intestinal barrier dysfunction , increasing the risk of uncontrolled immunological reactions , which may indeed trigger transient mucosal inflammation and gut disease . there is a large body of evidence indicating that stress , through the brain - gut axis , may cause intestinal barrier dysfunction , mainly via the systemic and peripheral release of corticotropin - releasing factor . in this review , we describe the role of stress and corticotropin - releasing factor in the regulation of gastrointestinal permeability , and discuss the link to both health and pathological conditions .

thyroid cancer is the most common malignant tumor of the endocrine system ; approximately 37,200 new cases were expected to occur in the united states in 2009.1 in the city of so paulo , brazil , 1207 and 6004 cases of thyroid cancer in males and females , respectively , were identified from 1997 to 2003.2 according to some studies , the increase in the incidence of thyroid cancer can be explained by a higher frequency of early tumor detection due to the use of more sophisticated diagnostic techniques.3,4 thyroid carcinomas that are derived from follicular epithelium can be classified into well - differentiated carcinomas ( including papillary and follicular carcinomas ) , poorly differentiated carcinomas and undifferentiated ( or anaplastic ) carcinomas , based on well - established clinical findings and histological criteria.5 however , despite standardized criteria and nomenclature , variations exist in the histological classification of thyroid tumors among different observers . this disagreement is greater for encapsulated follicular thyroid tumors ( adenoma , follicular carcinoma and some cases of the follicular variant of papillary carcinoma ) , in which the histological diagnosis is prone to subjective and discordant interpretations among pathologists.6 - 8 histological findings may not be sufficient to establish a precise diagnosis and , consequently , to predict the clinical courses of these cases . objective criteria and the identification of markers that permit better characterization of thyroid tumors are therefore required.9 n - myc downstream - regulated gene 1 ( ndrg1 ) is a member of the ndrg gene family and encodes a 43-kd protein ( ndrg1 ) with 394 amino acids.10 ndrg1 is located on human chromosome 8q24 and has been recognized as a gene whose mutation is associated with a demyelinating neuropathy called charcot - marie - tooth disease , type 4d.11 ndrg1 is ubiquitously expressed in human tissues , and its messenger rna has been detected in a variety of tissues , such as the digestive and respiratory tracts , liver , pancreas , kidneys , reproductive organs , placenta , skeletal muscle , heart , and brain.10,12 immunohistochemical studies have shown that the ndrg1 protein is mainly expressed in the epithelial cells of different tissues , whereas no expression could be demonstrated in mesenchymal or endothelial cells.12 other members of the ndrg family ( ndrg2 , ndrg3 and ndrg4 ) are homologous to the ndrg1 gene . however , in contrast to ndrg1 , these genes most likely exert tissue - specific functions , as they are only expressed in certain types of tissues.13 ndrg1 protein expression is known to be induced by a variety of physiological and pathological stimuli . expression of this protein is associated with cell growth arrest and terminal cell differentiation , as observed in squamous epithelial cells of the more superficial layers of the epidermis.12 during embryogenesis , ndrg1 expression is repressed by n - myc and c - myc during cell proliferation phases.14 conversely , ligands such as vitamin d and retinoic acid , which bind to transcription factors involved in cell differentiation , inhibit growth and induce cell differentiation and ndrg1 expression.15 other factors , such as an increase in intracellular calcium concentration , a reduction in glucose concentration , hypoxia , dna damage and neoplasms , are also associated with ndrg1 expression.16 heavy metals ( such as nickel ) induce cellular hypoxia and the expression of various genes that are responsive to hypoxia , including ndrg1.17,18 ndrg1 has also been characterized as a mediator of p53-dependent , anti - oncogenic functions . demonstrated that the p53-induced ndrg1 expression reduces cell proliferation in metastatic pulmonary cancer cells.19 however , the role of ndrg1 in tumor progression and the formation of metastases is controversial . some studies have suggested that ndrg1 overexpression reduces the metastatic potential of tumor cells,20,21 whereas others have associated its increased expression with less differentiated or more metastatic aggressive tumors and a poor prognosis.22,23 in the present study , we analyzed the quantitative immunohistochemical expression of the ndrg1 protein in normal thyroid glands and benign and malignant thyroid lesions . we also investigated the correlation between ndrg1 protein expression and clinical - pathological variables in cases of primary thyroid carcinoma . to our knowledge , this is the first study to analyze ndrg1 protein expression in benign and malignant thyroid lesions . paraffin blocks from 225 patients , including 62 cases of nodular goiter ( ng ) , 53 cases of follicular adenoma ( fa ) , 41 cases of classical papillary thyroid carcinoma ( ptc ) , 31 cases of the follicular variant of papillary thyroid carcinoma ( fv - ptc ) , and 36 cases of follicular carcinoma ( fc ) were selected from the archives of the department of pathology , university of so paulo medical school ( comprising the period from 2000 to 2006 ) and the department of pathology , a. c. camargo hospital ( comprising the period from 1984 to 2001 ) . histological diagnoses were made according to the classification of thyroid tumors proposed by the who.5 when available , paraffin blocks of lymph node metastases from thyroid carcinomas ( m - tc ) were also selected and included 17 cases of metastatic ptc , 5 cases of metastatic fv - ptc , and 1 case of metastatic fc . the patient age ranged from 15 to 88 years ( mean : 48.4 years old ) , and 184 ( 81.8% ) patients were female . data regarding the tnm stage24 and age , metastasis , and extent and size ( ames ) death risk criteria25 were obtained from the clinical records of patients with thyroid carcinoma ( ptc , fv - ptc and fc ) . the median time of clinical follow - up and the interval for recurrence for patients with thyroid carcinoma was 53.5 months ( range : 1 to 175 months ) and 18.0 months ( range : 2 to 48 months ) , respectively . no clinical follow - up data were available for eight patients ( six with fc , one with ptc and one with fv - ptc ) . during this period , 16 cases of recurrence ( 4 followed by death ) and 4 cases of death due to disseminated metastatic disease were observed . all deaths occurred in the group of patients with fc , and four patients were alive but still had diseases ( two with ptc and two with fc ) . this study was approved by the ethics committee for the analysis of research projects of the university of so paulo medical school and the research ethics committee of a. c. camargo hospital . slides were prepared for the tissue microarray ( tma ) from the selected blocks , stained with hematoxylin - eosin and examined under a binocular light microscope ( nikon , eclipse e100 ) for the selection of representative areas of the lesion . the areas of interest in the slides were stained with indian ink and compared to the corresponding areas in the paraffin blocks , which were also stained with indian ink . using a tissue microarrayer ( beecher instruments , silver springs , md , usa ) , the areas of interest in each block were punctured with a 1-mm diameter needle , and cylindrical fragments were transferred in an ordered fashion to a recipient paraffin block ( tma block ) , as described previously.26 for the detailed identification of each cylindrical fragment , a map was constructed in an excel spreadsheet to permit the exact localization of each case . the tma block contained 265 thyroid cases ( one core per case ) , corresponding to fragments of normal thyroid tissue ( nt , n = 18 ) , ng ( n = 62 ) , fa ( n = 53 ) , ptc ( n = 41 ) , fv - ptc ( n = 31 ) , fc ( n = 36 ) , and m - tc ( n = 24 , including 17 fragments of ptc metastases , 5 fv - ptc metastases , and 2 fc metastases ) . a total of 120 histological 5-m - thick sections were cut from each tma block . the slides were covered with a layer of paraffin to prevent oxidation and stored in a freezer at 20c . slides were left overnight in an oven at 56c , followed by deparaffinization in xylene , graded alcohol , tap water and distilled water . heat - induced antigen retrieval was performed using a domestic pressure cooker ( nigro , model eterna 41/2 l , brazil ) with a boiled 0.01 m sodium citrate buffered solution ( ph 6.0 ) for 4 min and cooled under running water , as described previously.27 endogenous peroxidase activity was quenched with 3% hydrogen peroxide for 20 min . the antibody used was purchased from santa cruz biotechnology ( ndrg1 n-19 , catalog sc-19464 , santa cruz , ca , usa ) . the optimal dilution was defined using a well - known positive sample that was tested before it was used on tma sections . the antibody was diluted 11000 in 0.01 m phosphate - buffered saline ( pbs ) , ph 7.4 , with 0.1% sodium azide ( sigma , catalog s8032 , st . louis , mo , usa ) and 1% bovine serum albumin ( sigma , catalog a9647 , usa ) to reduce background staining and incubated overnight at 4c in a moist chamber . the staining procedure was performed using the labeled streptavidin - biotin method ( lsab+ system - hrp , dakocytomation , catalog k0690 , carpinteria , ca , usa).28 slides were incubated for 30 min at 37c with the link , washed with pbs and incubated for another 30 min at 37c with streptavidin . the slides were developed with 60 mg 3,3-diaminobenzidine tetrahydrochloride ( sigma , catalog d5637 , st louis , mo , usa ) , 1% dimethyl sulfoxide ( sigma , catalog d5879 , st . louis , mo , usa ) and 0.06% hydrogen peroxide in pbs for 5 min and counterstained with harris 's hematoxylin , dehydrated and mounted with entellan neu ( merck , catalog 1.07961 , damstadt , germany ) . cytoplasmic ndrg1 protein expression was analyzed quantitatively using a computer image capture system ( automated cellular imaging system - acis iii , k0690 ; dako ) . next , parameters were established regarding the staining intensity of ndrg1-immunostained thyroid follicular cells and unstained stromal ( fibroblasts and endothelial cells ) cells ( blue area ) . between two and five circular areas per core first , we calculated the integrated optical density ( iod ) , which is related to the amount ( density ) of the antigen.29 a numerical value corresponding to the iod for each circular area was obtained by multiplying the staining intensity by the area of positive staining ( brown area ) . then , the iod was normalized to the tissue area : the numerical iod value was divided by the total circular area ( sum of the positively stained brown area and unstained blue area).30 for each core , the final numerical value corresponded to the mean value of the corrected iods of the two to five circular areas analyzed . cores presenting more than 50% tissue loss were excluded from the analysis . finally , the mean of the final numerical values obtained for the cores present in each of the two slides was calculated for each case . box plots were constructed for the different thyroid lesions using the results of the quantitative analysis of ndrg1 immunohistochemical expression . these groups were compared using the kruskal - wallis and mann - whitney tests . for cases with a thyroid carcinoma diagnosis ( ptc , fv - ptc or fc ) , the association between the quantitative immunohistochemical expression of ndrg1 and the clinical - pathological variables ( age , tumor size , tnm stage and ames risk classification ) was determined using a student 's t - test . paraffin blocks from 225 patients , including 62 cases of nodular goiter ( ng ) , 53 cases of follicular adenoma ( fa ) , 41 cases of classical papillary thyroid carcinoma ( ptc ) , 31 cases of the follicular variant of papillary thyroid carcinoma ( fv - ptc ) , and 36 cases of follicular carcinoma ( fc ) were selected from the archives of the department of pathology , university of so paulo medical school ( comprising the period from 2000 to 2006 ) and the department of pathology , a. c. camargo hospital ( comprising the period from 1984 to 2001 ) . histological diagnoses were made according to the classification of thyroid tumors proposed by the who.5 when available , paraffin blocks of lymph node metastases from thyroid carcinomas ( m - tc ) were also selected and included 17 cases of metastatic ptc , 5 cases of metastatic fv - ptc , and 1 case of metastatic fc . the patient age ranged from 15 to 88 years ( mean : 48.4 years old ) , and 184 ( 81.8% ) patients were female . data regarding the tnm stage24 and age , metastasis , and extent and size ( ames ) death risk criteria25 were obtained from the clinical records of patients with thyroid carcinoma ( ptc , fv - ptc and fc ) . the median time of clinical follow - up and the interval for recurrence for patients with thyroid carcinoma was 53.5 months ( range : 1 to 175 months ) and 18.0 months ( range : 2 to 48 months ) , respectively . no clinical follow - up data were available for eight patients ( six with fc , one with ptc and one with fv - ptc ) . during this period , 16 cases of recurrence ( 4 followed by death ) and 4 cases of death due to disseminated metastatic disease were observed . all deaths occurred in the group of patients with fc , and four patients were alive but still had diseases ( two with ptc and two with fc ) . this study was approved by the ethics committee for the analysis of research projects of the university of so paulo medical school and the research ethics committee of a. c. camargo hospital . slides were prepared for the tissue microarray ( tma ) from the selected blocks , stained with hematoxylin - eosin and examined under a binocular light microscope ( nikon , eclipse e100 ) for the selection of representative areas of the lesion . the areas of interest in the slides were stained with indian ink and compared to the corresponding areas in the paraffin blocks , which were also stained with indian ink . using a tissue microarrayer ( beecher instruments , silver springs , md , usa ) , the areas of interest in each block were punctured with a 1-mm diameter needle , and cylindrical fragments were transferred in an ordered fashion to a recipient paraffin block ( tma block ) , as described previously.26 for the detailed identification of each cylindrical fragment , a map was constructed in an excel spreadsheet to permit the exact localization of each case . the tma block contained 265 thyroid cases ( one core per case ) , corresponding to fragments of normal thyroid tissue ( nt , n = 18 ) , ng ( n = 62 ) , fa ( n = 53 ) , ptc ( n = 41 ) , fv - ptc ( n = 31 ) , fc ( n = 36 ) , and m - tc ( n = 24 , including 17 fragments of ptc metastases , 5 fv - ptc metastases , and 2 fc metastases ) . a total of 120 histological 5-m - thick sections were cut from each tma block . the slides were covered with a layer of paraffin to prevent oxidation and stored in a freezer at 20c . slides were left overnight in an oven at 56c , followed by deparaffinization in xylene , graded alcohol , tap water and distilled water . heat - induced antigen retrieval was performed using a domestic pressure cooker ( nigro , model eterna 41/2 l , brazil ) with a boiled 0.01 m sodium citrate buffered solution ( ph 6.0 ) for 4 min and cooled under running water , as described previously.27 endogenous peroxidase activity was quenched with 3% hydrogen peroxide for 20 min . the antibody used was purchased from santa cruz biotechnology ( ndrg1 n-19 , catalog sc-19464 , santa cruz , ca , usa ) . the optimal dilution was defined using a well - known positive sample that was tested before it was used on tma sections . the antibody was diluted 11000 in 0.01 m phosphate - buffered saline ( pbs ) , ph 7.4 , with 0.1% sodium azide ( sigma , catalog s8032 , st . louis , mo , usa ) and 1% bovine serum albumin ( sigma , catalog a9647 , usa ) to reduce background staining and incubated overnight at 4c in a moist chamber . the staining procedure was performed using the labeled streptavidin - biotin method ( lsab+ system - hrp , dakocytomation , catalog k0690 , carpinteria , ca , usa).28 slides were incubated for 30 min at 37c with the link , washed with pbs and incubated for another 30 min at 37c with streptavidin . the slides were developed with 60 mg 3,3-diaminobenzidine tetrahydrochloride ( sigma , catalog d5637 , st louis , mo , usa ) , 1% dimethyl sulfoxide ( sigma , catalog d5879 , st . louis , mo , usa ) and 0.06% hydrogen peroxide in pbs for 5 min and counterstained with harris 's hematoxylin , dehydrated and mounted with entellan neu ( merck , catalog 1.07961 , damstadt , germany ) . cytoplasmic ndrg1 protein expression was analyzed quantitatively using a computer image capture system ( automated cellular imaging system - acis iii , k0690 ; dako ) . next , parameters were established regarding the staining intensity of ndrg1-immunostained thyroid follicular cells and unstained stromal ( fibroblasts and endothelial cells ) cells ( blue area ) . between two and five circular areas per core first , we calculated the integrated optical density ( iod ) , which is related to the amount ( density ) of the antigen.29 a numerical value corresponding to the iod for each circular area was obtained by multiplying the staining intensity by the area of positive staining ( brown area ) . then , the iod was normalized to the tissue area : the numerical iod value was divided by the total circular area ( sum of the positively stained brown area and unstained blue area).30 for each core , the final numerical value corresponded to the mean value of the corrected iods of the two to five circular areas analyzed . cores presenting more than 50% tissue loss were excluded from the analysis . finally , the mean of the final numerical values obtained for the cores present in each of the two slides was calculated for each case . box plots were constructed for the different thyroid lesions using the results of the quantitative analysis of ndrg1 immunohistochemical expression . these groups were compared using the kruskal - wallis and mann - whitney tests . for cases with a thyroid carcinoma diagnosis ( ptc , fv - ptc or fc ) , the association between the quantitative immunohistochemical expression of ndrg1 and the clinical - pathological variables ( age , tumor size , tnm stage and ames risk classification ) was determined using a student 's t - test . all statistical analyses were performed using spss software , version 15.0 ( spss inc . , ndrg1 protein expression was observed in epithelial ( follicular ) cells , with no staining in stromal or endothelial cells . the positive cases showed moderate - to - strong cytoplasmic ndrg1 immunoreactivity ( figure 1 ) . for each case with a thyroid lesion and nt , the numerical values obtained by the quantitative analysis of the immunohistochemical expression of ndrg1 are reported as means and standard deviations ( table 1 ) . the quantitative expression of ndrg1 was higher in carcinomas compared to nts and ngs , with higher expression levels in ptc and m - tc ( p < 0.001 ) ( figure 2 ) . when analyzed together , the quantitative expression of ndrg1 was higher in the group of malignant lesions ( ptc , fv - ptc , fc , and m - tc ) compared to the group of benign thyroid lesions ( ng and af ) ( p = 0.043 ) ( figure 3 ) . the association between the quantitative immunohistochemical expression of ndrg1 and the clinical - pathological variables was analyzed in cases of primary thyroid carcinoma ( ptc , fv - ptc and fc ) . in this group of tumors , the quantitative expression of ndrg1 was significantly higher in patients who were aged 45 years or older ( p = 0.019 ) , at an advanced tnm stage ( stages iii and iv ) ( p = 0.007 ) , and in an ames high - risk group ( p = 0.012 ) ( table 2 ) . to our knowledge , this is the first study to analyze the immunohistochemical expression of the ndrg1 protein in a large number of benign ( ng and fa ) and malignant ( ptc , fv - ptc , fc , and m - tc ) thyroid lesions . cangul also studied the immunohistochemical expression of ndrg1 in a variety of human tumors ( including breast , kidney , lung and prostate carcinomas , and melanoma and glioblastoma multiforme ) and reported higher ndrg1 protein expression in tumors compared to normal tissue.18 in the colon , wang et al . observed increased ndrg1 expression in carcinomas compared to adenomas and normal colon mucosa.31 reis et al . analyzed 213,636 transcripts that were derived from normal and tumor tissues of the oral cavity , larynx , pharynx and thyroid using the open reading frame expressed sequence tags ( orestes ) technique . three ( zrf1 , rap140 and ndrg1 ) of the 250 potential markers selected in that study were analyzed by quantitative rt - pcr in oral cavity and laryngeal samples . the authors detected an increase in the levels of ndrg1 mrna in 50% of oral cavity tumors.32 in contrast , bandyopadhyay et al . observed lower ndrg1 immunohistochemical expression in breast carcinoma but strong expression in normal mammary lobules.33 the role of the ndrg1 protein in tumor progression and metastasis formation is still controversial and is likely to be tissue - specific . several studies have shown that a decrease in ndrg1 protein expression is associated with a more advanced tumor stage and lower survival rates . in prostate carcinomas , lower immunohistochemical expression of ndrg1 was associated with a gleason grade > 7 and poorly differentiated tumors . according to the authors , 70% of localized prostate tumors expressed ndrg1 , whereas only 25% of metastatic tumors were positive for this marker.21 in breast cancer , 60% of patients with bone metastases presented with reduced ndrg1 expression , with survival being lower in patients whose tumors did not express ndrg1.33 in esophageal squamous cell carcinoma , reduced ndrg1 expression was associated with more invasive tumors and a more advanced tnm stage . in addition , a lower survival rate was observed in patients with low ndrg1 expression compared to those with high expression.34 a less differentiated histological grade , more advanced pathological stage and lower overall survival were correlated with reduced immunohistochemical expression of ndrg1 in pancreatic ductal adenocarcinoma.35 we found that increased immunohistochemical expression of ndrg1 was correlated with a more advanced tnm stage ( stages iii and iv ) and an ames high - risk category in patients with thyroid carcinoma ( ptc , fv - ptc , and fc ) . an association between increased ndrg1 expression and more aggressive tumors and poor prognosis has been reported in other studies . chua et al . observed that increased ndrg1 expression in hepatocellular carcinoma was associated with a more advanced tumor stage ( stages iii and iv ) , larger tumors and poorly differentiated tumors , according to the edmondson - steiner histological classification . in their study , patients whose tumors presented increased ndrg1 protein expression showed lower overall survival compared to those with low ndrg1 levels.22 in cervical adenocarcinoma , increased immunohistochemical expression of ndrg1 was correlated with a larger tumor diameter , greater depth of stromal invasion , the presence of angiolymphatic invasion , a larger number of lymph node metastases , poorly or moderately differentiated tumors and poor prognosis , as demonstrated by lower progression - free and overall survival rates.23 some studies have suggested that ndrg1 is a metastasis suppressor gene . guan et al . observed lower ndrg1 expression in cell lines derived from colon cancer metastases compared to cell lines derived from primary colon cancer . inducing ndrg1 expression in metastatic cell lines , the authors observed that neoplastic cells expressed markers related to cell differentiation of the colon epithelium , such as cea and e - cadherin.20 in vivo studies have shown that cell lines derived from colon and prostate cancers that were transfected with ndrg1 and injected into mice produced a smaller number of hepatic and pulmonary metastases , respectively , than control lines.20,21 however , in human colorectal carcinoma samples , wang et al . demonstrated increased ndrg1 protein expression in primary tumors with metastases to the lymph nodes compared to those without metastases ( non - metastatic carcinoma ) . the authors concluded that higher ndrg1 expression was associated with a higher probability of lymph node metastasis . within this context , according to the authors , ndrg1 could be a gene that is involved in tumor progression and the promotion of metastases of colorectal carcinoma.31 the present study demonstrated an increase in the immunohistochemical expression of ndrg1 in primary and metastatic thyroid carcinomas compared to nt , ng , and fa . we also observed an association between ndrg1 expression and a more advanced tnm stage and an ames high - risk category in patients with thyroid carcinoma ( ptc , fv - ptc , and fc ) . more detailed studies are necessary to better define the role of ndrg1 in tumorigenesis and thyroid tumor progression , including the investigation of this protein in poorly differentiated or anaplastic thyroid carcinomas .

objectives : the aim of this study was to examine the expression of the n - myc downstream - regulated gene 1 protein in benign and malignant lesions of the thyroid gland by immunohistochemistry.introduction:n-myc downstream - regulated gene 1 encodes a protein whose expression is induced by various stimuli , including cell differentiation , exposure to heavy metals , hypoxia , and dna damage . increased n - myc downstream - regulated gene 1 expression has been detected in various types of tumors , but the role of n - myc downstream - regulated gene 1 expression in thyroid lesions remains to be determined.methods:a tissue microarray paraffin block containing 265 tissue fragments corresponding to normal thyroid , nodular goiter , follicular adenoma , papillary thyroid carcinoma ( classical pattern and follicular variant ) , follicular carcinoma , and metastases of papillary and follicular thyroid carcinomas were analyzed by immunohistochemistry using a polyclonal anti- n - myc downstream - regulated gene 1 antibody.results:the immunohistochemical expression of n - myc downstream - regulated gene 1 was higher in carcinomas compared to normal thyroid glands and nodular goiters , with higher expression in classical papillary thyroid carcinomas and metastases of thyroid carcinomas ( p < 0.001 ) . a combined analysis showed higher immunohistochemical expression of ndrg1 in malignant lesions ( classical pattern and follicular variant of papillary thyroid carcinomas , follicular carcinomas , and metastases of thyroid carcinomas ) compared to benign thyroid lesions ( goiter and follicular adenomas ) ( p = 0.043 ) . in thyroid carcinomas , n - myc downstream - regulated gene 1 expression was significantly correlated with a more advanced tnm stage ( p = 0.007 ) and age , metastasis , tumor extent , and size ( ames ) high - risk group ( p = 0.012).conclusions : thyroid carcinomas showed increased immunohistochemical n - myc downstream - regulated gene 1 expression compared to normal and benign thyroid lesions and is correlated with more advanced tumor stages .

aluminum ( al ) is abundantly distributed in our environment , and compounds containing al have been used in manufacturing ( e.g. , clays , glasses , and alum ) for centuries . despite its abundance , al was first isolated as an element in 1827 , and its use as being a silvery metal began only after 1886 . because of its beneficial characteristics such as a lightweight , nonmagnetic , malleable , and ductile element , al has a widespread and important use in industrial applications and consumer products . al is also used in cooking utensils and in pharmacological agents including antacids and antiperspirants from which the element enters the human body . , al is a well established neurotoxin and is suspected to be linked with various neurodegenerative diseases including alzheimer 's disease ( ad ) , amyotrophic lateral sclerosis ( als ) , and parkinsonism dementia in the kii peninsula and guam , and the gulf war syndrome . in particular , a possible relationship between al and the pathogenesis of ad has been discussed for several decades [ 37 ] . the pathological hallmarks of ad are the deposition of extracellular senile plaques , intracellular neurofibrillary tangles ( nfts ) , and the selective loss of synapses and neurons in the hippocampal and cerebral cortical regions . senile plaques are largely comprised of -amyloid protein ( ap ) . the hypothesis that al is an environmental contributor to the pathogenesis of ad , termed the aluminum hypothesis , was proposed in the 1960s based on various neurotoxicological , analytical , and epidemiological findings [ 911 ] . in spite of these findings , the aluminum hypothesis has been the subject of much debate and criticism for several decades . during this period , particularly , numerous studies have supported the idea termed amyloid cascade hypothesis , namely that the conformational changes of ap and its neurotoxicity play a central role in ad pathogenesis [ 12 , 13 ] . al and other metals including zn , cu , and fe influence the oligomerization and conformational changes of ap as cross - linkers , and , therefore , their implications are important in this context . furthermore , increasing evidence suggests the implication of these metals in the pathogenesis of ad [ 1416 ] . al binds to various metal - binding proteins and influences homeostasis of other metals . we review here the detailed characteristics of aluminum neurotoxicity based on our own studies and the recent literature . our aim is to update the various adverse effects of al and revisit the link between al and ad based on new findings on al - induced conformational changes and metal - metal interactions . an association between al poisoning and memory disorder in humans was first reported in 1921 . later , it was shown that the intracerebral administration of al induced epilepsy in experimental animals . as a component of dialysis solutions or al - containing pharmacological compounds , al is known to cause various dialysis - related disorders , including osteomalacia ( aluminum bone disease ) , microcytic anemia , 2-microglobulin - associated amyloidosis , and dialysis encephalopathy in hemodialysis patients . the accidental contamination of al into drinking water occurred and more than 20,000 persons were exposed to high level of al at 1988 in camelford ( cornwall , uk ) . residents exposed to contaminated al exhibited various symptoms related to cerebral impairments such as loss of concentration and short term memory in a 10-year follow - up study . martyn et al . reported a high incidence of ad in areas with a high level of al in the drinking water in england and wales . a considerable number of studies have provided evidence to support an association between ad and al in drinking water after this initial report . frecker reported on a norwegian area where high al concentrations in drinking water were linked with high dementia mortality . neri and hewitt found a positive relationship between al in drinking water and ad risk in canada . forbes and mclachlan demonstrated a greater risk of ad in canadian areas where concentrations of al are high and those of fluoride are low . rondeau et al . demonstrated that high daily intake of al was correlated with increased risk of dementia or cognitive decline in a 15-year follow - up french cohort study [ 2628 ] . these studies suggest that al has adverse effects on human memories and causes dementia when it enters the brain . despite it 's environmental abundance , al is not an essential element for living organisms , and no enzymatic reaction requires al . al is reported to influence more than 200 biologically important reactions and to cause various adverse effects on the mammalian central nervous system ( cns ) ( table 1 ) . these include crucial reactions for brain development such as the axonal transport , neurotransmitter synthesis , synaptic transmission , phosphorylation or dephosphorylation of proteins , protein degradation , gene expression , and inflammatory responses . inorganic and organic phosphates , carboxylate , and deprotonated hydroxyl groups form strong bonds with al . owing to these chemical characteristics , al binds to the phosphate groups of dna and rna , affecting dna topology and influencing the expression of various genes essential for brain functions . reported that nanomolar levels of al were sufficient to influence neuronal gene expression [ 33 , 35 ] . al also binds to the phosphate groups of nucleoside di- and triphosphates , such as atp and can thus influence energy metabolism . for example , the ligand - exchange rate of mg is 10 times faster than that of al , and therefore , al inhibits enzymes with mg cofactors . al also inhibits biological processes involving rapid ca exchange : the exchange rate for al is 10 times slower than that of ca . these properties make al useless in enzymatic reactions and increase its half - life in the human body . al has strong positive charges and a relatively small ionic radius in comparison to other metal ions such as ca , zn , and na ( figure 2 ) . thus , al firmly binds to metal - binding amino acids ( histidine ( his ) , tyrosine ( tyr ) , arginine ( arg ) etc . ) or phosphorylated amino acids and acts as a cross - linker ; this property has made it useful as a leather tanning agent . by binding to various proteins , al can cause the oligomerization of proteins , inducing conformational changes that can inhibit their degradation by proteases . strong binding of al to phosphorylated amino acids promotes the self - aggregation and accumulation of highly phosphorylated cytoskeleton proteins , including neurofilament and microtubule - associated proteins ( maps ) , and so forth . chronic administration of al impairs long - term potentiation ( ltp ) , which is a form of synaptic information storage well - known as a paradigm of memory mechanisms . al also impairs various enzymes including those related to neurotransmitter synthesis and thus affects the neurotransmitter content . al also inhibits voltage - gated ca channels and neurotransmitter receptors , and impairs synaptic transmission . finally , al causes spatial memory deficit , influences emotional reactivity , and impairs various brain functions related to learning and memory . these adverse effects may be involved in the mechanisms that underlie al - induced memory disorder . a link between al and ad is supported on many fronts , beginning in 1965 with the finding of klatzo et al . that the intracerebral administration of al to experimental animals induced neurofibrillary degeneration and the appearance of tangle - like structures that were similar to the nfts found in the brains of ad patients . crapper et al . reported an increased level of al in the brains of ad patients . in the 1970s , al in dialysis solutions or pharmacological compounds was found to cause dementia in dialysis patients ( dialysis encephalopathy ) . as noted previously , several epidemiological studies reported a high percentage of ad cases in areas with high al level in drinking water [ 11 , 19 ] . despite supporting evidence , the aluminum hypothesis of ad remains controversial and has been the subject of much debate in the past few decades . first , it has been argued that neurofibrillary changes in al - intoxicated animals ( al - nfts ) are different from those in ad patients ( ad - nfts ) . arguments cite morphological and biochemical differences such as the lack of paired helical filamental ( phf ) structures , their different distributions in nerve terminals , and the absence of immunoreactivity for tau protein , which is the main component of nfts in ad patients . second , there is no significant difference in al levels of ad patients and age - matched controls . we would like to reinvestigate these early arguments in the context of new findings in the study of ad . regarding the first argument , more recent immunohistochemical studies have indicated that depositions in the brains of al - intoxicated animals are stained with the anti - tau antibody [ 62 , 63 ] . the accumulation of tau protein was reported in patients with dialysis encephalopathy , and in al - intoxicated cultured neuronal cells [ 60 , 61 ] . furthermore , nfts in some ad patients have been shown to be composed of straight - type filaments rather than phf - type filaments as is observed in al - nft . these data indicate that attempts to discredit the aluminum hypothesis on the basis of differences between al - nfts and ad - nfts are no longer tenable . another argument cites a lack of significant difference between al levels in ad patients and age - matched controls . one reason for the controversy may be al contamination of the solutions used in the process of tissue fixation and staining . therefore , prior studies in fixed tissues can not be relied upon for precise measures of al ; quantitative analysis of nonfixed and freshly frozen tissues is necessary . one such study showed that the amount of al in whole brains of ad patients was not significantly different in comparison to controls . claimed that they could not detect al in senile plaques or nfts using nuclear microscopy . however , this failure could simply be due to low detection limits of their analytical method used highly sensitive laser microprobe mass analysis ( lamma ) with nonfixed brain samples and reported an accumulation of al in nft - bearing neurons of ad brains . an accumulation of al in both senile plaques and nfts has been reported in renal failure patients . recently , yumoto et al . analyzed al using energy - dispersive x - ray spectroscopy combined with transmission electron microscopy ( tem - edx ) , a method which yields a high - resolution and low detection limit . their detailed analysis demonstrated that al was present in cores of senile plaques at a concentration of 3550 ppm . some epidemiological studies have failed to demonstrate the relationship between al and ad [ 119 , 120 ] . however , there are a number of possible explanations for this inconsistency , particularly when considering the difficulty in making side - by - side comparisons of epidemiological studies of al ( e.g. , intake estimations , effect of move , changes in water - treatment processes , etc . ) . using strict neuropathological criteria to discriminate between ad patients and controls ( including histopathological verification ) , mclachlan et al . found an elevated risk of histopathologically verified ad to be associated with the consumption of higher concentrations of al in drinking water . more detailed analysis revealed an association between exposure to organic monomeric al and ad , even after adjustment for education level , family history and presence of the apoe4 allele . the amount of al consumed in drinking water is approximately 5% of the total daily intake . thus , it is possible that some factors that prevent or accelerate al absorption may exist in drinking water . silicate in the water was reported to interact with al and prevent al toxicity to fish [ 123 , 124 ] . therefore , the level of silicate in drinking water may also be important . in a french cohort study , the relationship between al and cognitive impairment cognitive impairment among women was correlated with low concentrations of silica in drinking water . in considering the above new lines of evidence about the neurotoxicity and epidemiology of al , it is difficult to agree with the early criticisms of the aluminum hypothesis . in the 1990s when the early arguments were claimed , al - induced alzheimer - like pathological changes were first attributed to tau proteins ( nft ) . however , numerous biochemical , toxicological , cell biological , and genetic studies have supported the amyloid cascade hypothesis , namely , that the accumulation of ap and its neurotoxicity play a central role in the pathogenesis of ad [ 12 , 13 ] . ap is a small peptide of 3943 amino acid residues , secreted by cleavage of the amyloid precursor protein ( app ) n - terminus by -app cleaving enzyme ( bace ) and intramembrane cleavage of its c - terminus by -secretase . genetic studies of early - onset cases of familial ad indicated that app mutations and ap metabolism are associated with ad . yankner et al . reported that the first 40 amino acid residues of ap ( ap(140 ) ) caused the death of cultured rat hippocampal neurons or neurodegeneration in the brains of experimental animals . ap is a hydrophobic peptide with an intrinsic tendency to self - assemble and form sds - stable oligomers in aqueous solution . neurotoxicity of ap(140 ) peptides was enhanced by the process of aging ( aggregated under incubation at 37c for several days ) compared to freshly prepared ap(140 ) in cultured neurons , and were correlated with its -sheet contents . recent approaches using size - exclusion chromatography , gel electrophoresis , and atomic force microscopy have demonstrated that the soluble oligomers are synaptotoxic and neurotoxic . considering that ap is secreted in the cerebrospinal fluid ( csf ) of young individuals as well as in aged or dementia patients , factors that accelerate or inhibit oligomerization may play essential roles in the pathogenesis of ad . several factors such as peptide concentration , ph or composition of solvents , and temperature can influence the oligomerization processes . interestingly , rodent ap exhibits less tendency to oligomerization than human ap in vitro and the accumulation of ap is rarely observed in the brains of rodents ( rats or mice ) as compared to primates ( humans or monkeys ) . as shown in figure 3 , the amino acid sequences of human and rodent ap are similar , but rodent ap differs from primate only 3 amino acids ( arg , tyr , and his ) from primate ap . all 3 amino acids have the ability to bind metals . therefore , trace elements including al are of particular interest as potential acceleratory factors and may play important roles in the accumulation of ap in the human brain . table 2 summarizes the effects of al on conformational changes of ap and other various disease - related proteins . exley et al . first demonstrated by cd spectroscopy that al induces a conformational change in ap(140 ) . al has also been shown to promote the aggregation of i - labelled ap(140 ) , with similar findings for fe and zn . demonstrated that zn and cu caused the oligomerization of ap [ 149 , 150 ] . we have demonstrated that zn blocks ap - channels formed on membranes and inhibits the neurotoxicity . we have developed a system for investigating ap oligomerization that involves immunoblotting and precipitation . using this system , we have demonstrated that al enhances the polymerization of ap(140 ) and forms sds - stable oligomers in vitro [ 64 , 133 , 134 ] . the aggregated ap(140 ) is redissolved by adding deferoxamine ( dfo ) , an al chelator . the oligomerization induced by al is more marked than that induced by other metals , including zn , fe , cu , and cd(figure 4(a ) ) . furthermore , while al - aggregated aps bind tightly to the surface of cultured neurons and form fibrillar deposits , zn - aggregated aps are rarely observed on the surface of cultured neurons ( figure 4(b ) ) . these results suggest that al - aggregated aps have a strong affinity for membrane surfaces as a result of minimal degradation by proteases . indeed , al has been shown to inhibit the degradation of ap as the result of conformational changes [ 43 , 153 ] . furthermore , ap coupled with al is more toxic than normal ap causing membrane disruption or perturbation of neural ca homeostasis and mitochondrial respiration [ 154156 ] . the chronic application of al caused the accumulation of ap in cultured neurons of rat cerebral cortex and in neuroblastoma cells . ( 2002 ) found that orally administered al caused a marked increase in the amount of ap both in its secreted and accumulated forms , and increased deposition of senile plaques in ad - model mice transfected with the human app gene ( tg 2576 ) . these results are consistent with other studies demonstrating that oral al exposure causes the accumulation of ap and impairs spatial learning memory in ad - model mice . exposure to al causes the accumulation of ap and induces adverse effects in humans as seen in the aftermath of the accidental al exposure in 1988 at camelford . the neuropathological case study of a 58 year - old woman who was exposed to al and died 15 years later with unspecified neurological symptoms demonstrated the rare form of sporadic cerebral amyloid angiopathy , which is characterized by the deposition of ap in blood vessels and has a causative link with ad . the deposition of high amounts of al in the patient 's brain was also observed . al has also been reported to bind and cause conformational changes in other ad - related proteins , including app , tau protein [ 32 , 57 ] , and phf - tau protein and in proteins related to other neurodegenerative diseases such as -synuclein ( parkinson 's disease ( pd ) and dementia with lewy bodies ; dlb ) [ 140 , 141 ] , amylin ( diabetes mellitus ) , abri ( familial british dementia ) , and ataxin 3 ( spinocerebellar ataxia type 3 ) , 2-microglobulin ( dialysis - related arthropathy ) ( table 2 ) . the evidence now suggests that the significance of al in the pathogenesis of ad should be concerned . other metals usually share the binding site of one metal ion , although their binding constants differ . the interactions between al and other metals should be considered owing to the implications of various trace elements in the pathogenesis of ad . figure 5 illustrates the modified aluminum hypothesis that accounts for the implications of al and other trace metals in ad pathology from the secretion of ap to its neurotoxicity as mentioned below . al has similar characteristics to iron ( fe ) and binds to fe - binding proteins such as ferritin , transferrin , iron regulatory protein ( irp ) or to iron chelators such as dfo . the iron responsive element / iron regulatory protein ( ire / irp ) network regulates the production of iron binding proteins which prevent the formation of free fe , which causes toxic free radicals . in iron - deficient conditions , irp binds to ire and regulates the expression of genes that contain ires in their mrna , such as ferritin or transferrin . as the concentration of free fe increases , the binding of iron to irp , expression of transferrin is downregulated and that of ferritin is upregulated , and the amount of free fe is thereby decreased . al also binds to irp [ 34 , 160 ] , and thus influences the expression of fe - binding proteins with ires in their mrna causing an elevated fe concentration . al also influences the uptake of iron into cultured neurons or glial cells [ 34 , 162 ] . thus , al affects iron homeostasis and the expression of various iron - regulated proteins with ires . important findings are that app mrna contains an ire as well as ferritin , and its expression is regulated by iron . indeed , al caused elevated expression of app in experimental animals [ 40 , 41 ] . recently , duce et al . demonstrated that app has ferroxidase activity , which converts fe to fe and regulates free pro - oxidant fe concentrations . app also possesses copper / zinc binding sites in its amino - terminal domain and in the ap domain and may be involved in homeostasis of these metals . al stimulates fe - induced membrane lipid peroxidation and causes oxidative damage in vitro and in vivo , although al does not directly affect peroxidation [ 89 , 90 ] . iron related genes such as transferrin c2 or hemochromatosis were revealed to be risk factors for ad [ 166 , 167 ] . ( 1992 ) reported that iron supplementation was effective for the recovery of cognitive functions in ad patients . an abnormal expression of app could lead to an increased secretion of ap , and then enhance its accumulation . secreted ap is usually degraded by various proteases such as neprilysin within a short period . furthermore , ap becomes oligomerized in the presence of trace metals such as al , zn , fe , and cu , could be resistant to proteases , and thus accumulates in the brain . ap oligomers could be readily incorporated into cell membranes , resulting in the formation of ion channels . a subsequent influx of ca through these amyloid channels would lead to the phosphorylation of tau , depletion of neurotrophic factors , and the formation of free radicals , and so forth , with the outcome of these effects being neuronal death . we found that al also inhibits the increase in ca levels induced by brain - derived neurotrophic factor ( bdnf ) . as described previously , al is implicated in most of these neurodegenerative pathways such as dephosphorylation of tau , depletion of neurotrophic factor , formation of free radicals , and induction of neuronal death . this working hypothesis may be useful in developing an understanding of the link between ad and trace elements including al , zn , cu , and fe . considering the implications of metals in ad pathogenesis , chelation therapy for ad treatment is of great interest . clioquinol ( quinoform ) , a chelator of cu or zn , inhibits oligomerization of ap and attenuates the accumulation of amyloid in the brains of experimental animals . dfo , a chelator of al and fe , attenuates the decline of daily living skills in ad patients . silicates , which couple with al and reduce its toxicity , are also candidates for chelation therapy in ad . in this review , we have summarized the properties associated with various aspects of al neurotoxicity . there is growing evidence for a link between al and ad , and between other metals and ad . nevertheless , because the precise mechanism of ad pathogenesis remains unknown , this issue is controversial . however , it is widely accepted that al is a recognized neurotoxin , and that it could cause cognitive deficiency and dementia when it enters the brain and may have various adverse effects on cns . in general , the absorption of metals by the gastrointestinal tract is widely variable and is influenced by various factors including an individual difference , age , ph , stomach contents . recent studies using mass spectrometry of al have demonstrated that small , but a considerable amount of al crosses the blood brain barrier , enters into the brain , and accumulates in a semipermanent manner [ 174 , 175 ] . therefore , al can cause severe health problems in particular populations , including infants , elderly people , and patients with impaired renal functions , and unnecessary exposure to al should be avoided for such patients . in 1989 , a joint fao / who expert committee on food additives ( jecfa ) recommended a provisional tolerable weekly intake ( ptwi ) of 7.0 mg / kg body weight al ; however , this was changed in 2007 to 1.0 mg / kg body weight because of potential effects on the reproductive system and the developing nervous system . the characteristics of al neurotoxity are complex , and further research is needed especially in relation to bioavailability , cellular effects , metabolism , and metal - metal interactions .

whilst being environmentally abundant , aluminum is not essential for life . on the contrary , aluminum is a widely recognized neurotoxin that inhibits more than 200 biologically important functions and causes various adverse effects in plants , animals , and humans . the relationship between aluminum exposure and neurodegenerative diseases , including dialysis encephalopathy , amyotrophic lateral sclerosis and parkinsonism dementia in the kii peninsula and guam , and alzheimer 's disease ( ad ) has been suggested . in particular , the link between aluminum and alzheimer 's disease has been the subject of scientific debate for several decades . however , the complex characteristics of aluminum bioavailability make it difficult to evaluate its toxicity and therefore , the relationship remains to be established . mounting evidence has suggested that significance of oligomerization of -amyloid protein and neurotoxicity in the molecular mechanism of ad pathogenesis . aluminum may play crucial roles as a cross - linker in -amyloid oligomerization . here , we review the detailed characteristics of aluminum neurotoxicity based on our own studies and the recent literatures . our aim is to revisit the link between aluminum and ad and to integrate aluminum and amyloid cascade hypotheses in the context of -amyloid oligomerization and the interactions with other metals .

deficits of zinc due to primary ( low dietary intakes ) , or secondary ( nutrient - nutrient , disease - nutrient , and gene - nutrient interactions ) causes , can result in an increased risk for multiple pregnancy complications including birth defects . low zinc status has been reported to affect up to 50% of the world s population . women with acrodermatitis enteropathica ( ae ) , a genetic disorder that results in a low absorption of zinc , are characterized by a high frequency of pregnancy complications unless they receive zinc supplementation . the extent to which other gene defects may contribute to the occurrence of zinc deficiency is a subject of debate . we submit that the identification of other gene abnormalities that increase the risk of maternal / conceptus zinc deficiency would be valuable as it could result in new genetic screens and therapeutic approaches that would reduce the risk for certain birth defects . we identified slc39a12 , a solute carrier and integral membrane protein , as a possible zinc transporter gene . was used to identify slc39a12 as a gene with high expression in the human brain relative to other tissues . this data set analysis is supported by expressed sequence tag ( est ) data in the national center for biotechnology information ( ncbi ) unigene database , as previously noted by eide and taylor et al . we determined that the slc39a12 gene , also designated as zinc transporter zip12 , is highly expressed in the adult brains of human and mouse and developing brain of the frog , xenopus tropicalis . given these observations , we hypothesized that zip12 has a significant role during neurulation and neuronal development . knockdown in neuro-2a cells , primary neurons , and differentiated primary neuronal precursor cells leads to impaired neurite sprouting and outgrowth . furthermore , zinc transport by zip12 affects an early stage of neuronal differentiation through creb phosphorylation , which is evident within 30 min of differentiation . while zinc transporters zip4 , zip8 , and zip14 have been shown to affect creb phosphorylation in tissues outside the nervous system , no previous studies have linked neuronal zinc transport to creb activity . mechanistically , it is unclear how perturbations to zinc homeostasis , zip12 , and creb phosphorylation influence neuronal differentiation . for example , imr-32 neuroblastoma cells incubated in zinc deficient media have impaired translocation of nfat and nf- secondary to perturbed tubulin polymerization . several kinase pathways can phosphorylate and activate creb ( fig . 1 ) , as reviewed by johannessen et al . we observed that neurite extension induced by constitutive creb activation ( expressed by viral protein 16-creb ) is not sensitive to zip12 inhibition or extracellular zinc chelation . constitutive creb activation effectively rescues impaired neurite outgrowth caused by zip12 knockdown , indicating that its impact on neuronal differentiation occurs upstream of creb , rather than affecting cre - dependent transcription activated by creb . zip12 is important for tubulin polymerization as evidenced by the observation that zip12 knockdown leads to reduced microtubule stability and increased sensitivity to depolymerization by nocodozole . disruption of tubulin polymerization in cultured neural cells decreases sprouting and reduces mean neurite length . intracellular chelation of zinc leads to die - back of axonal and dendritic neurites without increased apoptosis . also , imr-32 neuroblastoma cells show altered microtubule structure and reduced tubulin polymerization in zinc chelated media . while tubulin possesses low affinity binding sites for zinc , they are likely not relevant under physiological conditions . these low affinity sites have historically been used to produce tubulin sheets with anti - parallel protofilaments , not the parallel orientation found endogenously . zinc may indirectly affect tubulin polymerization as tubulin oxidation and altered tubulin dynamics have been found in cellular and rat models of zinc deficiency and have been linked to impaired transcription factor translocation . the link between tubulin polymerization and zinc may involve microtubule associated proteins ( maps ) assisting in microtubule stability and affecting neurite outgrowth . a question that remains to be answered is whether the multiple defects in neuronal development observed by zip12 inhibition are due to separate processes such as creb signaling and tubulin polymerization that are disrupted by reduced zinc availability or due to an interconnection between these pathways , with a single upstream process that is zinc dependent . zinc is known to be important for multiple physiological processes that span roles including cell signaling , enzyme co - factor mediation , and dna - binding structural motifs . a piece of evidence that supports the possibility of a single process is the rescue of neurite outgrowth due to zip12 inhibition by expression of a constitutively active creb . we determined that zip12 is required for neural tube closure and embryonic viability during xenopus tropicalis embryogenesis . this observation was somewhat unexpected given the substantial ab initio zinc content present within the xenopus zygote that does not seem to change through developmental stage 50 and the large number of varied zinc uptake mechanisms in vertebrates . this led us to the supposition that zip12 is functionally involved in redistribution of zinc within the xenopus embryo rather than zinc uptake from its environment . intracellular chelation of zinc during xenopus development appears to slow development during neurulation and arrests development of the cns with obvious craniofacial deformities , including microcephaly and anopia . two explanations for why zip12 is critical for neural tube closure are its possible roles in cell signaling and morphogenesis , which are processes that are disrupted by zip12 knockdown in mouse neuronal cultures . our studies indicate that creb is sensitive to intracellular zinc concentrations , and creb inhibition leads to neural tube defects in xenopus . further studies are needed to determine if neurulation requires transcription factors that are sensitive to zinc or zip12 activity . it is possible that the impaired morphogenesis observed by reduced tubulin polymerization in zip12 knockdown embryos reflects a disruption in cell signaling . additional research is needed to identify the mechanisms linking zinc to neural tube closure , and whether there are common processes between neurulation and neurite extension that are dependent upon zip12 and zinc . the identification of zip12 in regulating nervous system zinc homeostasis and development represents an important step in elucidating the connections between zinc transport mechanisms and brain function . given our findings in xenopus tropicalis and the high conservation of many pathways and processes for neurulation and brain development , we propose that slc39a12 is a candidate gene for nervous system defects during prenatal development with increased penetrance during low maternal intake of dietary zinc . we suggest that prevention of zinc deficiency , such as through dietary zinc supplementation , will reduce the risk of neural tube defects and other congenital malformations in individuals with select zip12 polymorphisms .

the essentiality of zinc for normal brain development is well established . it has been suggested that primary and secondary zinc deficiencies can contribute to the occurrence of numerous human birth defects , including many involving the central nervous system . in a recent study , we searched for zinc transporter genes that were critical for neurodevelopment . we confirmed that zip12 is a zinc transporter encoded by the gene slc39a12 that is highly expressed in the central nervous systems of human , mouse , and frog ( xenopus tropicalis).using loss - of - function methods , we determined that zip12 is required for neuronal differentiation and neurite outgrowth and necessary for neurulation and embryonic viability . these results highlight an essential need for zinc regulation during embryogenesis and nervous system development . we suggest that slc39a12 is a candidate gene for inherited neurodevelopmental defects in humans .

significant progress in our understanding of neuronal network dynamics underlying brain function requires the ability to monitor the activity of multiple neurons simultaneously . optical imaging based on voltage - sensitive dyes offers the spatio - temporal resolution necessary to fulfill this requirement ( reviewed in baker et al . , 2005 ; grinvald and hildesheim , 2004 ; grinvald et al . , 1988 ; knpfel et al . , these organic dyes have been successfully used during the past 20 years to report changes in membrane potential from single or large numbers of neurons in a variety of preparations , including mammalian brain tissue ( ferezou et al . , 2007 ; grinvald and hildesheim , 2004 ) . however , conventional voltage - sensitive dyes are generally not suitable for the labeling of specific cell populations . in the absence of targeting to a defined cell population , the optical signal is often drowned out either by background fluorescence from inactive cells or by signals from cells that are not the focus of interest . moreover , unspecific staining of brain tissue prevents the unequivocal attribution of the fluorescence output signal to a defined cell population . to overcome these limitations of classical voltage - sensitive dyes , it is greatly desirable to find a way to target specific cell populations . with the molecular cloning of green fluorescent protein ( gfp ) from aequorea victoria ( chalfie et al . , 1994 ) and subsequent generation of new and improved spectral variants derived from various sea organisms ( reviewed in shaner et al . , 2007 ; verkhusha and lukyanov , 2004 ) , the construction of genetically - encoded sensors for visualization of cellular dynamics became conceivable . a now broadly used approach to fluorescent biosensor engineering involves the molecular fusion of a gfp - based reporter protein to a second protein that undergoes conformational transitions in response to a physiological signal such as fluctuations in calcium or membrane potential ( recently reviewed in knpfel et al . , 2006 ; qiu et al . since protein - based sensors are encoded in dna , they can be expressed under the control of cell specific promoters and introduced in vivo using gene transfer techniques . in a transgenic animal , a genetically - encoded voltage sensor could be expressed in practically any cell type and would have the advantage of staining only the cell population determined by the promoter used to drive the expression . during recent years , several designs of genetically - encoded optical probes for membrane potential have been explored . flash , the first prototype , was obtained by inserting gfp within the c - terminal tail of the voltage - gated shaker potassium channel ( siegel and isacoff , 1997 ) . concomitantly , our laboratory explored a fret ( fluorescence resonance energy transfer ) design principle based on the voltage - dependent conformational change associated with the voltage - sensing domain of the kv2.1 potassium channel , resulting in a voltage sensor we named vsfp1 ( sakai et al . , 2001 ) . finally , the third prototype , sparc , was generated by introducing gfp into a reversibly nonconducting form of the rat i skeletal muscle sodium channel ( ataka and pieribone , 2002 ) . although these first generation fluorescent protein voltage sensors were shown to optically report changes in membrane potential , their application in mammalian systems is severely hindered by their poor targeting to the plasma membrane in transfected cells ( baker et al . , 2007 ) . indeed , confocal microscopy analysis revealed a prominent intracellular expression for flare ( a kv1.4 flash variant ) , vsfp1 and sparc with little , if any , fluorescence associated with the cell surface in both hek293 cells and hippocampal neurons . unfortunately , neither the mutagenesis of potential er retention sites nor the introduction of er export motifs has resulted in a significant improvement of the low plasma membrane expression displayed by the first generation fp voltage - sensitive probes ( baker et al . , 2008 ) . despite this setback , the functional concept underlying vsfp1 ( sakai et al . , 2001 ) recently , a self - contained voltage sensing domain ( vsd ) was isolated from the non - ion channel protein ciona intestinalis voltage sensor - containing phosphatase ( ci - vsp ) ( murata et al . , 2005 ) . interestingly , a single vsd was shown to be functional in ci - vsp ( kohout et al . , 2008 ) while four vsd - containing subunits are required for the gating of the kv potassium channel pore region ( bezanilla , 2000 ) . furthermore , the vsd of ci - vsp operates as a sensor by itself since robust sensing currents were shown in the absence of the enzyme region ( murata et al . , 2005 ) . in contrast , sensing or gating currents of voltage - gated ion channels have so far been elusive if the voltage sensor is separated from the pore region ( okamura et al . , we thus reasoned that the limited cell surface targeting of first generation voltage - sensitive proteins could be resolved by using the structurally much simpler ci - vsp scaffold and tested this hypothesis by exchanging the vsd of vsfp1 with that of ci - vsp ( dimitrov et al . the initial series included the variant vsfp2.1 in which a positive arginine was mutated to a neutral glutamine at position 217 ( r217q ) within the charged s4 membrane segment of the vsd . as a result of this charge neutralization , the voltage - activity curve of the native ci - vsp showed a leftward shift to the physiological range of membrane potential fluctuations in electrically active mammalian cells ( dimitrov et al . , 2007 ) . as expected from the self - contained properties of ci - vsp , vsfp2s displayed a predominant targeting to the plasma membrane in transfected pc12 cells . figure 1a illustrates the expression and clear membrane localization of vsfp2.1 , which represents a major advance when compared to the poor cell surface trafficking exhibited by the first generation fp voltage sensors ( baker et al . , 2007 ) . ( b ) voltage dependence of apparent activation and deactivation time constants of vsfp2.1 fluorescence signals in pc12 cells at 35c . vsfp2.1-expressing pc12 cells were voltage - clamped with membrane voltage traces recorded from olfactory mitral cells that were stimulated to generate a series of action potentials by direct current injection ( c ) or by electrical stimulation of the olfactory nerve ( d ) . traces in ( c ) and upper trace in ( d ) are the average of 50 and 90 sweeps , respectively . traces show membrane potential ( v ) , yellow fluorescence ( fy ) , cyan fluorescence ( fc ) and the ratio of yellow and cyan fluorescence ( fy / fc ) . fluorescence signals were digitally low pass filtered ( 0.2 khz ) and were not corrected for photobleaching . vsfp2.1 responded to depolarizing voltage pulses by a decrease in cyan fluorescence and a concomitant raise in yellow florescence , resulting from increased energy transfer from the cyan to the yellow - emitting fp chromophore following the translocation of the s4 transmembrane segment ( dimitrov et al . , 2007 ) . moreover , vsfp2.1 showed relatively fast kinetics with an apparent on - time constant of 15 ms upon a depolarization from a holding potential of 70 to + 40 mv as shown in figure 1b . to investigate whether vsfp2.1 could be a candidate for optical measurements of neuronal activity , pc12 cells were voltage - clamped with membrane voltage transients recorded from olfactory mitral cells that were stimulated either by direct current injection to generate a series of action potentials ( figure 1c ) or synaptically to induce a burst of fast action potentials ( figure 1d ) . fluorescence traces revealed that vsfp2.1 could clearly resolve individual action potentials as well as the underlying membrane depolarization ( figure 1c ) . however , vsfp2.1 mainly reported the slow components of these voltage transients , as expected from its response kinetics ( dimitrov et al . , 2007 ) . indeed , the optical readout of the fast action potentials was significantly reduced relative to the slower component of the membrane potential change . this phenomenon was also clearly noticeable when using the membrane voltage transients from a mitral cell succeeding a single shock electrical stimulation of the olfactory nerve ( figure 1d ) . most importantly , it should be noted that the responses shown in figure 1d could be resolved in single sweeps . optimizing the length of the amino acid linkers connecting either the donor chromophore to the vsd or the fret donor / acceptor pair in vsfp2.1 resulted in vsfp2.3 with both improved fluorescence response kinetics and fret efficiency ( d. dimitrov et al . , unpublished ; akemann et al . , 2009 ; cyan- and yellow - emitting variants of gfp from a. victoria are most often used as a fluorescent reporter component for fret - based sensors . however , the photophysical properties of this fp pair are less than ideal for fret imaging since both chromophores have broad emission / absorption spectra with relatively small stokes shift ( chapman et al . , 2005 ) and considerable fret donor emission within the acceptor emission band . furthermore , red - shifted emitting variants would ultimately yield a higher signal - to - noise ratio if tissue autofluorescence is an issue . indeed , red fluorescence should provide better spectral separation from the intrinsic green autofluorescence of brain tissue given that the fluorescence of flavins , vitamins and nadph is considerably lower in the red region of the spectrum than in the blue - green region . additionally , long - wavelength light is usually associated with reduced phototoxicity and deeper penetration into biological tissue . to this aim , we generated a red - shifted vsfp2.1 variant comprising a pair of yellow and far - red emitting fps that we termed vsfp2.4 ( mutoh et al . , 2009 ) . the absorption spectrum of the acceptor , mkate2 , shows considerable overlap with the emission spectrum of the donor , citrine , with a calculated frster distance of 5.82 nm ( mutoh et al . , furthermore , both spectra are well enough separated to allow independent excitation of the chromophores , limiting spectral bleed through from the donor emission into the acceptor channel . in parallel , tsutsui et al . ( 2008 ) reported another vsfp2.1 spectral variant based on fps isolated from corals ( mukg and mko ) referred to as mermaid . the conceptual design of the original series of vsfp2s and its color variants is illustrated in figure 2a . second generation voltage - sensitive fluorescent proteins . ( a ) alignment of the amino acid sequences of vsfp2.1 variants . the c - terminal residues and downstream segment ( 240254 ) of the vsd of ci - vsp are shown in gray and italic text , respectively . fps are mcerulean ( blue ) , citrine / mcitrine ( yellow ) , mkate2 ( red ) , mukg ( green ) and mko ( orange ) . ( b ) acceptor ( upper color traces ) and donor ( lower color traces ) signals of pc12 cells in response to a family of 500 ms voltage steps from a holding potential of 70 mv to test potentials of 140 to + 60 mv recorded at 35c . traces corresponding to a depolarization to + 60 mv are indicated by arrows ( from mutoh et al . , 2009 ) . quantitative comparison of these three most advanced fret - based voltage probes ( vsfp2.3 , vsfp2.4 and mermaid ) revealed relatively similar steady state spectrally - resolved maximal change in fluorescence ( r / r ) upon a depolarization from 100 to + 40 mv ( 13.3 3.4 , 12.4 1.0 and 12.9 4.8% for vsfp2.3 , vsfp2.4 and mermaid , respectively ) ( mutoh et al . , 2009 ) . acceptor and donor fluorescence signals in response to voltage steps from a holding potential of 70 mv to test potentials of 140 to + 60 mv are shown in figure 2b . likewise , vsfp2.1 spectral variants displayed comparable voltage dependencies ( v1/2 = 54.2 mv , v1/2 = 54.2 mv and v1/2 = 43.6 mv for vsfp2.3 , vsfp2.4 and mermaid , respectively ) . upon depolarization from a holding potential of 70 mv , all three sensors exhibited fluorescence signals that could be fitted with two main time constants ( table 1 ; akemann et al . , 2009 ; tsutsui et al . , 2008 ; villalba - galea et al . , 2009 ) that likely correspond to the conformational transition states of ci - vsp ( villalba - galea et al . the values for these on - time response components were very similar except that the fast on - time constant contributed to a larger fraction of the total signal in vsfp2.4 when compared to mermaid ( 40 4 and 23.5 5% at + 60 mv , respectively ) ( mutoh et al . , 2009 ) . the off - time kinetics did not differ among the vsfp2.1 variants under this assay protocol . the fluorescence response properties of the vsfp2.1 variants detailed above are summarized in table 1 . steady state spectrally - resolved maximal changes in fluorescence acquired from voltage - clamped pc12 cells . the r / r values represent the average of 1437 measurements from hyperpolarized ( 100 mv ) to depolarized ( + 40 mv ) membrane potential from five representative cells ( data are from mutoh et al . , 2009 ) . to validate the expression pattern of the latest fret - based voltage - sensitive probes in neurons , we transfected primary hippocampal neurons after 6 days of culture with vsfp2.3 , vsfp2.4 and mermaid and evaluated them by confocal fluorescence imaging 1 week later . as shown in figure 3 , vsfp2.3 , vsfp2.4 and mermaid fluorescence was distributed over the cell body , dendrites and axons of a variety of neurons including pyramidal cells . primary hippocampal cultures derived from mouse e18 embryos were transfected with either vsfp2.3 , vsfp2.4 or mermaid 6 days after plating and imaged by confocal fluorescence microscopy 1 week later . note the targeting of vsfp2.3 and vsfp2.4 to the plasma membrane and mermaid - associated intracellular aggregates in magnified views . the insert in the lower right image shows an example of a cell with clear expression of mermaid at the cell surface . scale bars are 50 and 10 m for left and right panels , respectively . in particular , vsfp2.3 showed efficient targeting to the plasma membrane as indicated by arrows in magnified views . likewise , vsfp2.4 fluorescence was mainly found at the cell surface while some fluorescence was also observed intracellularly within a juxtanuclear trans - golgi network - like structure ( arrowheads ) which is likely involved in endosome trafficking . mermaid was also in part targeted to the plasma membrane but the extent of membrane - associated fluorescence was largely overwhelmed by much stronger fluorescence derived from structures reminiscent of intracellular vesicles as indicated by arrowheads in figure 3 . these punctuate structures have previously been reported for fluorescent proteins isolated from reef coral anthozoan species which are known for their high tendency to form aggregates ( hirrlinger et al . , 2005 ; katayama et al . , indeed , bright fluorescent clusters were observed in cell somata and processes of a series of transgenic mouse lines expressing various reef coral fps within early postnatal weeks , which were shown to increase substantially with the age of the animal ( hirrlinger et al . , 2005 ) . in contrast , fluorescent aggregates were not detected in mice expressing a. victoria gfp variants even at older ages ( hirrlinger et al . . since the establishment of transgenic mice expressing vsfp2s requires long - term expression of the reporter proteins , vsfp2.3 and vsfp2.4 would likely be better candidates due to their reduced intracellular accumulation and aggregation ( figure 3 ) . accordingly , with the relatively small signal amplitude of these second generation fp voltage sensors , optimal responses are largely dependent on proper trafficking to the plasma membrane since intracellular expression principally contributes to background fluorescence , decreasing the signal - to - noise ratio ( rs / n ) significantly . in order to investigate the relationship between vsfp activation kinetics and vsfp - mediated optical report of neuronal activity , we represented vsfp2.3 and vsfp2.4 by an eight state markov process kinetic model reflecting their experimental response properties ( figures 4a1,b1 ; see akemann et al . , 2009 ) . inclusion of these kinetic models into a realistic conductance - based computational version of a rat somato - sensory layer 5 pyramidal neuron given by mainen and sejnowski ( 1996 ) enabled us to predict in silico the possible vsfp fluorescence readouts that would be obtained from neuronal activity ( akemann et al . , 2009 ) . the simulations indicated that the second generation vsfps can provide an activation mechanism sufficiently fast to track burst firing of action potentials albeit with significant attenuation as shown in figure 4 , wherein individual action potentials were resolved as narrow peaks superimposed on a slowly rising ( vsfp2.3 ; figure 4a2 ) or declining ( vsfp2.4 ; figure 4b2 ) curve representing the activation state of both voltage reporters . additionally , simulation of vsfp2.3 and vsfp2.4 optical readouts in response to a subthreshold or suprathreshold activation of a distal synaptic conductance ( causing an excitatory postsynaptic potential , epsp ) showed that both fp voltage sensors are able to readily resolve synaptic potentials in individual l5 pyramidal neurons ( figures 4a3,b3 ) . however , as the fluorescence response properties of vsfp2s to membrane potential changes consist of a slow and fast kinetic response with time constants differing by more than an order of amplitude ( lundby et al . , 2008 ; mutoh et al . , 2009 ; villalba - galea et al . , 2009 ) , the optical resolution of any membrane voltage transient rising faster than the slow kinetic component will critically depend on the relative contribution of the slow versus fast component of the vsfp activation response . computer simulations of vsfp2.3 and vsfp2.4 optical response signals in cortical layer 5 ( l5 ) pyramidal neurons . ( a1 ) family of yfp fluorescence ( f / f0 ) responses ( lower panel ; green traces ) to 500 ms voltage steps ( upper panel ) from a holding membrane voltage of 70 mv to test potentials of 140 to + 60 mv recorded from voltage - clamped pc12 cells expressing vsfp2.3 . overlaid to the experimental traces are simulated traces obtained from a vsfp2.3 model wherein the kinetics are represented as an eight state markov process as given in ( akemann et al . , 2009 ) . the simulated response to ( a2 ) predicted vsfp2.3 fluorescence signal ( f / f0 ; lower panel ) in response to an action potential burst ( middle panel ) in the cell body of a simulated l5 pyramidal neuron evoked by constant current injection ( upper panel ) . the schematic to the left depicts the neuron with a point current source attached to the cell body ( red electrode ) . the electrical response was simulated using a conductance - based model of a reconstructed rat l5 neuron given by mainen and sejnowski ( 1996 ) . vsfp2.3 was homogenously inserted within the membrane ( all 177 compartments ) at a density of 800 units/m . the fluorescence signal ( lower panel ) represents the response of vsfp2.3 in the somatic membrane with ( green ) or without ( dashed black ) photon quantum noise calculated assuming a sampling rate of 2 khz and excitation light level that bleaches gfp within 10 s of illumination . ( a3 ) vsfp2.3 fluorescence signal ( f / f0 ; lower panel ) as predicted by the simulation in response to a subthreshold ( left column ) or suprathreshold ( right column ) activation of a distal synaptic conductance ( schematically depicted to the left with the activated synapse in red ) . the voltage signal in the cell body ( upper row left : epsp ; upper row right : epsp plus evoked action potential ) together with the associated fluorescence signals ( lower row ) , with ( green ) or without ( dashed black ) photon quantum noise are shown . ( b1b3 ) same as in ( a1a3 ) , but using a model of vsfp2.4 instead of vsfp2.3 . vsfp2.4 was simulated as an eight state markov chain model analogously to vsfp2.3 . for methodological details cyan- and yellow - emitting variants of gfp from a. victoria are most often used as a fluorescent reporter component for fret - based sensors . however , the photophysical properties of this fp pair are less than ideal for fret imaging since both chromophores have broad emission / absorption spectra with relatively small stokes shift ( chapman et al . , 2005 ) and considerable fret donor emission within the acceptor emission band . furthermore , red - shifted emitting variants would ultimately yield a higher signal - to - noise ratio if tissue autofluorescence is an issue . indeed , red fluorescence should provide better spectral separation from the intrinsic green autofluorescence of brain tissue given that the fluorescence of flavins , vitamins and nadph is considerably lower in the red region of the spectrum than in the blue - green region . additionally , long - wavelength light is usually associated with reduced phototoxicity and deeper penetration into biological tissue . to this aim , we generated a red - shifted vsfp2.1 variant comprising a pair of yellow and far - red emitting fps that we termed vsfp2.4 ( mutoh et al . , 2009 ) . the absorption spectrum of the acceptor , mkate2 , shows considerable overlap with the emission spectrum of the donor , citrine , with a calculated frster distance of 5.82 nm ( mutoh et al . , 2009 ) . furthermore , both spectra are well enough separated to allow independent excitation of the chromophores , limiting spectral bleed through from the donor emission into the acceptor channel . in parallel , tsutsui et al . ( 2008 ) reported another vsfp2.1 spectral variant based on fps isolated from corals ( mukg and mko ) referred to as mermaid . the conceptual design of the original series of vsfp2s and its color variants is illustrated in figure 2a . the c - terminal residues and downstream segment ( 240254 ) of the vsd of ci - vsp are shown in gray and italic text , respectively . fps are mcerulean ( blue ) , citrine / mcitrine ( yellow ) , mkate2 ( red ) , mukg ( green ) and mko ( orange ) . ( b ) acceptor ( upper color traces ) and donor ( lower color traces ) signals of pc12 cells in response to a family of 500 ms voltage steps from a holding potential of 70 mv to test potentials of 140 to + 60 mv recorded at 35c . traces corresponding to a depolarization to + 60 mv are indicated by arrows ( from mutoh et al . , 2009 ) . quantitative comparison of these three most advanced fret - based voltage probes ( vsfp2.3 , vsfp2.4 and mermaid ) revealed relatively similar steady state spectrally - resolved maximal change in fluorescence ( r / r ) upon a depolarization from 100 to + 40 mv ( 13.3 3.4 , 12.4 1.0 and 12.9 4.8% for vsfp2.3 , vsfp2.4 and mermaid , respectively ) ( mutoh et al . , 2009 ) . acceptor and donor fluorescence signals in response to voltage steps from a holding potential of 70 mv to test potentials of 140 to + 60 mv are shown in figure 2b . likewise , vsfp2.1 spectral variants displayed comparable voltage dependencies ( v1/2 = 54.2 mv , v1/2 = 54.2 mv and v1/2 = 43.6 mv for vsfp2.3 , vsfp2.4 and mermaid , respectively ) . upon depolarization from a holding potential of 70 mv , all three sensors exhibited fluorescence signals that could be fitted with two main time constants ( table 1 ; akemann et al . , 2009 ; lundby et al . , 2008 ; mutoh et al . , 2009 ; tsutsui et al . , 2008 ; villalba - galea et al . , 2009 ) that likely correspond to the conformational transition states of ci - vsp ( villalba - galea et al . , 2008 ) . the values for these on - time response components were very similar except that the fast on - time constant contributed to a larger fraction of the total signal in vsfp2.4 when compared to mermaid ( 40 4 and 23.5 5% at + 60 mv , respectively ) ( mutoh et al . , 2009 ) . the off - time kinetics did not differ among the vsfp2.1 variants under this assay protocol . the fluorescence response properties of the vsfp2.1 variants detailed above are summarized in table 1 . steady state spectrally - resolved maximal changes in fluorescence acquired from voltage - clamped pc12 cells . the r / r values represent the average of 1437 measurements from hyperpolarized ( 100 mv ) to depolarized ( + 40 mv ) membrane potential from five representative cells ( data are from mutoh et al . , 2009 ) . to validate the expression pattern of the latest fret - based voltage - sensitive probes in neurons , we transfected primary hippocampal neurons after 6 days of culture with vsfp2.3 , vsfp2.4 and mermaid and evaluated them by confocal fluorescence imaging 1 week later . as shown in figure 3 , vsfp2.3 , vsfp2.4 and mermaid fluorescence was distributed over the cell body , dendrites and axons of a variety of neurons including pyramidal cells . primary hippocampal cultures derived from mouse e18 embryos were transfected with either vsfp2.3 , vsfp2.4 or mermaid 6 days after plating and imaged by confocal fluorescence microscopy 1 week later . note the targeting of vsfp2.3 and vsfp2.4 to the plasma membrane and mermaid - associated intracellular aggregates in magnified views . the insert in the lower right image shows an example of a cell with clear expression of mermaid at the cell surface . scale bars are 50 and 10 m for left and right panels , respectively . in particular , vsfp2.3 showed efficient targeting to the plasma membrane as indicated by arrows in magnified views . likewise , vsfp2.4 fluorescence was mainly found at the cell surface while some fluorescence was also observed intracellularly within a juxtanuclear trans - golgi network - like structure ( arrowheads ) which is likely involved in endosome trafficking . mermaid was also in part targeted to the plasma membrane but the extent of membrane - associated fluorescence was largely overwhelmed by much stronger fluorescence derived from structures reminiscent of intracellular vesicles as indicated by arrowheads in figure 3 . these punctuate structures have previously been reported for fluorescent proteins isolated from reef coral anthozoan species which are known for their high tendency to form aggregates ( hirrlinger et al . indeed , bright fluorescent clusters were observed in cell somata and processes of a series of transgenic mouse lines expressing various reef coral fps within early postnatal weeks , which were shown to increase substantially with the age of the animal ( hirrlinger et al . , 2005 ) . in contrast , fluorescent aggregates were not detected in mice expressing a. victoria gfp variants even at older ages ( hirrlinger et al . , 2005 ; nolte et al . , 2001 ) . since the establishment of transgenic mice expressing vsfp2s requires long - term expression of the reporter proteins , vsfp2.3 and vsfp2.4 would likely be better candidates due to their reduced intracellular accumulation and aggregation ( figure 3 ) . accordingly , with the relatively small signal amplitude of these second generation fp voltage sensors , optimal responses are largely dependent on proper trafficking to the plasma membrane since intracellular expression principally contributes to background fluorescence , decreasing the signal - to - noise ratio ( rs / n ) significantly . in order to investigate the relationship between vsfp activation kinetics and vsfp - mediated optical report of neuronal activity , we represented vsfp2.3 and vsfp2.4 by an eight state markov process kinetic model reflecting their experimental response properties ( figures 4a1,b1 ; see akemann et al . , 2009 ) . inclusion of these kinetic models into a realistic conductance - based computational version of a rat somato - sensory layer 5 pyramidal neuron given by mainen and sejnowski ( 1996 ) enabled us to predict in silico the possible vsfp fluorescence readouts that would be obtained from neuronal activity ( akemann et al . , 2009 ) . the simulations indicated that the second generation vsfps can provide an activation mechanism sufficiently fast to track burst firing of action potentials albeit with significant attenuation as shown in figure 4 , wherein individual action potentials were resolved as narrow peaks superimposed on a slowly rising ( vsfp2.3 ; figure 4a2 ) or declining ( vsfp2.4 ; figure 4b2 ) curve representing the activation state of both voltage reporters . additionally , simulation of vsfp2.3 and vsfp2.4 optical readouts in response to a subthreshold or suprathreshold activation of a distal synaptic conductance ( causing an excitatory postsynaptic potential , epsp ) showed that both fp voltage sensors are able to readily resolve synaptic potentials in individual l5 pyramidal neurons ( figures 4a3,b3 ) . however , as the fluorescence response properties of vsfp2s to membrane potential changes consist of a slow and fast kinetic response with time constants differing by more than an order of amplitude ( lundby et al . , 2008 ; mutoh et al . , 2009 ; villalba - galea et al . , 2009 ) , the optical resolution of any membrane voltage transient rising faster than the slow kinetic component will critically depend on the relative contribution of the slow versus fast component of the vsfp activation response . computer simulations of vsfp2.3 and vsfp2.4 optical response signals in cortical layer 5 ( l5 ) pyramidal neurons . ( a1 ) family of yfp fluorescence ( f / f0 ) responses ( lower panel ; green traces ) to 500 ms voltage steps ( upper panel ) from a holding membrane voltage of 70 mv to test potentials of 140 to + 60 mv recorded from voltage - clamped pc12 cells expressing vsfp2.3 . overlaid to the experimental traces are simulated traces obtained from a vsfp2.3 model wherein the kinetics are represented as an eight state markov process as given in ( akemann et al . , 2009 ) . the simulated response to ( a2 ) predicted vsfp2.3 fluorescence signal ( f / f0 ; lower panel ) in response to an action potential burst ( middle panel ) in the cell body of a simulated l5 pyramidal neuron evoked by constant current injection ( upper panel ) . the schematic to the left depicts the neuron with a point current source attached to the cell body ( red electrode ) . the electrical response was simulated using a conductance - based model of a reconstructed rat l5 neuron given by mainen and sejnowski ( 1996 ) . vsfp2.3 was homogenously inserted within the membrane ( all 177 compartments ) at a density of 800 units/m . the fluorescence signal ( lower panel ) represents the response of vsfp2.3 in the somatic membrane with ( green ) or without ( dashed black ) photon quantum noise calculated assuming a sampling rate of 2 khz and excitation light level that bleaches gfp within 10 s of illumination . ( a3 ) vsfp2.3 fluorescence signal ( f / f0 ; lower panel ) as predicted by the simulation in response to a subthreshold ( left column ) or suprathreshold ( right column ) activation of a distal synaptic conductance ( schematically depicted to the left with the activated synapse in red ) . the voltage signal in the cell body ( upper row left : epsp ; upper row right : epsp plus evoked action potential ) together with the associated fluorescence signals ( lower row ) , with ( green ) or without ( dashed black ) photon quantum noise are shown . ( b1b3 ) same as in ( a1a3 ) , but using a model of vsfp2.4 instead of vsfp2.3 . vsfp2.4 was simulated as an eight state markov chain model analogously to vsfp2.3 . for methodological details to address whether the relatively slow fluorescence response kinetics of the second generation fp voltage probes is due to intrinsically slow operations of ci - vsp , we measured fluorescence signals along with sensing currents ( i.e. currents resulting from the displacement of charges within the vsd ) in vsfp2.3-expressing pc12 cells and found that the voltage dependency of the fluorescence read - outs closely resembles the activation curve of the sensing currents , indicating that the fluorescence signal effectively reports the voltage - dependent conformational change of the vsd ( lundby et al . , 2008 ) . however , the sensing charge movement was found to be two orders of magnitude faster than the dominant slow component of the fluorescence response ( 1 versus 100 ms ) , suggesting a relatively weak coupling between the vsd and the vsfp2 class reporter proteins ( lundby et al . , 2008 ) . already during the analysis of our initial series of ci - vsp - based vsfp2s , we noted that the ratio between the cfp and yfp signal components increases when the length of the linker connecting the vsd to the donor chromophore is shortened ( dimitrov et al . , 2007 ) . furthermore , a short linker version of vsfp2.1 ( vsfp2a r217q ) clearly exhibited a fast on - time component in the cfp response which was not observed in the yfp channel ( cfp : 7 2 and 180 15 ms , yfp : 171 15 ms ; figure 5a ) , suggesting the presence of a fret - independent response component ( lundby et al . , 2008 ) . to test this hypothesis , we measured the response characteristics of vsfp2a(r217q ) before and after photobleaching of the acceptor chromophore ( yfp ) as illustrated in figures 5a , b , respectively . single cell spectrofluorometry confirmed the disappearance of the 530-nm peak in the emission spectrum and revealed an increase in cfp emission at 470 nm due to donor dequenching ( figure 5b ) ( lundby et al . , 2008 ) . most importantly , a significant signal remained in the cyan channel after acceptor photobleaching ( figure 5b , lower panel ) , indicating that the fast cfp response component does not require the presence of a fret acceptor . to investigate whether the removal of yfp would favor the fast intrinsic cfp response further , we generated a novel ci - vsp - based voltage sensor comprising a single cyan fluorescent reporter protein that we termed vsfp3.1 . interestingly , vsfp3.1 responded to voltage steps with a very fast initial transient ( activation time constant of 1.3 0.1 ms at + 70 mv ; figure 5c ) , indicating that truncation of the acceptor chromophore significantly improved the coupling of the vsd to the fluorescent reporter protein . indeed , the fluorescence output of vsfp3.1 showed a dominant fast response component matching closely the sensing currents of ci - vsp ( lundby et al . the membrane topology of vsfp2a(r217q ) ( a ) , vsfp2a(r217q ) after acceptor photobleaching ( b ) and vsfp3.1 ( c ) is illustrated in top panels . the lower panel shows the depolarization - induced fluorescence signals recorded in the yellow and cyan channels . for vsfp2a(r217q ) , a scaled mirror image of the cyan signal is shown aligned with the yellow signal ; note the fast cfp component . for vsfp3.1 , the top trace represents the onset of the fluorescence signal at a magnified time scale ( from lundby et al . , 2008 ) . as shown in figure 6 , vsfp3.1 was efficiently targeted to the plasma membrane of transfected hippocampal neurons with a pattern similar to that of vsfp2.3 and vsfp2.4 ( figure 3 ) . we anticipate that vsfp3.1 and its color variants will facilitate improved optical measurements of fast neuronal signals primary hippocampal cultures were transfected with vsfp3.1 6 days after plating and confocal images were taken 6 days later . scale bar is 50 m ( upper left and lower panel ) and 10 m ( upper right ) . in this review , we have focused on the most advanced genetically - encoded voltage sensors reported thus far based on a fusion between the voltage - sensing domain of ci - vsp and a fp reporter module comprising either a fluorescent protein fret pair or a single fp . despite comparable amplitude and kinetics of the voltage - dependent fluorescence response of the vsfp2.1 spectral variants described in this review , each of these fluorescent probes has particular advantages when considering potential applications . for instance , vsfp2.3 is a suitable sensor for instrumentation with standard optical components since it is based on the most commonly used fret pair . on the other hand , the spectral properties of vsfp2.4 are strongly preferable for either in vivo imaging due to the elimination of green / yellow autofluorescence or deep tissue imaging using two - photon excitation fluorescence microscopy . finally , mermaid is , in principle , a good candidate for ratiometric measurements due to the good dynamic range of the donor and acceptor fluorescence responses . however , the tendency of the fps used in mermaid to form bright fluorescent aggregates may severely limit the usefulness of this variant . to our knowledge , vsfp3.1 is the fastest fp voltage sensor reported to date , exhibiting an activation time constant matching that of fast neuronal signals , which makes this single - fp voltage sensor a promising candidate for the generation of transgenic animals . however , vsfp2-type sensors have practical advantages over single color variants of the vsfp3 class . indeed , fret - based voltage sensors provide signals at two different colors with opposite polarity . this feature enables ratiometric measurements and thereby , at least theoretically , absolute calibration of membrane voltage . versions like vsfp2.3 that exhibit a large baseline fret efficacy may also be used monochromatically by exciting cfp and recording yfp fluorescence , in which case the larger separation of excitation and emission wavelengths can have practical advantages over single color variants of the vsfp3 class . current work aims at characterizing the performance of these voltage - sensitive fluorescent protein probes in differentiated neurons , which will undeniably constitute an important step towards the realization of an optical sensor for neuronal circuit activity . furthermore , expression of voltage - sensitive fluorescent proteins under cell - specific promoters will allow the labeling of defined neuronal populations within assemblies composed of diverse cell types ( diez - garcia et al . , 2005 , 2007 ; metzger et al . , 2002 ; qiu et al . , 2008 ) the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

over the last decade , optical neuroimaging methods have been enriched by engineered biosensors derived from fluorescent protein ( fp ) reporters fused to protein detectors that convert physiological signals into changes of intrinsic fp fluorescence . these fp - based indicators are genetically encoded , and hence targetable to specific cell populations within networks of heterologous cell types . among this class of biosensors , the development of optical probes for membrane potential is both highly desirable and challenging . a suitable fp voltage sensor would indeed be a valuable tool for monitoring the activity of thousands of individual neurons simultaneously in a non - invasive manner . previous prototypic genetically - encoded fp voltage indicators achieved a proof of principle but also highlighted several difficulties such as poor cell surface targeting and slow kinetics . recently , we developed a new series of fret - based voltage - sensitive fluorescent proteins ( vsfps ) , referred to as vsfp2s , with efficient targeting to the plasma membrane and high responsiveness to membrane potential signaling in excitable cells . in addition to these fret - based voltage sensors , we also generated a third series of probes consisting of single fps with response kinetics suitable for the optical imaging of fast neuronal signals . these newly available genetically - encoded reporters for membrane potential will be instrumental for future experimental approaches directed toward the understanding of neuronal network dynamics and information processing in the brain . here , we review the development and current status of these novel fluorescent probes .

breast cancer is the common cancer in women and the leading cause of death due to tumors . breast cancer is detected relatively early due to the development of screening ; however , distant metastases or local recurrences are noted in many patients even after radical treatment . the common lesions of breast cancer metastasis include the liver , lungs , brain , and bone , while metastasis to the thyroid gland is quite rare . indeed , despite the fact that the thyroid is highly vascularized , metastasis of malignant tumors to the thyroid in general is rare . recently , diagnoses of metastatic thyroid glands have increased due to the development of diagnostic techniques such as thyroid ultrasound and fine - needle aspiration . common primary sites of metastatic thyroid tumors are breast and lung cancer in autopsy series , while renal cell carcinoma is the most common site identified clinically . here , we report a case of thyroid metastasis from breast cancer accompanying primary thyroid cancer . a 51-year - old female patient presented with a palpable lymph node on her left lateral neck in december 2012 . the patient had undergone a left modified radical mastectomy for t1n1 invasive ductal carcinoma in april 2002 and received adjuvant chemotherapy ( cyclophosphamide , methotrexate , 5-fluorouracil chemotherapy , 10 cycles ) and hormonal therapy ( tamoxifen ) . ultrasonography of the neck identified a 0.9-cm nodule of the left thyroid ( fig . 1 ) and 1-cm lymphadenopathy at the right level vi , both of which appeared to be malignant . using fine - needle aspiration , a diagnosis of left thyroid nodule with papillary thyroid carcinoma was made , and the right level vi lymph node was diagnosed as hurthle cell lesion . complete blood count , serum chemistry tests , and thyroid function tests revealed no abnormal findings . the level of tumor marker ca 15 - 3 ( 19.52 u / ml ) and thyroglobulin measurements in fine - needle aspiration ( 5.07 ng / ml ) were in the normal range . the patient underwent a total thyroidectomy with a selective dissection of the left neck node . in the surgical field , solid masses on both thyroid lobe and isthmus were evaluated ( fig . the mass of the right thyroid lobe was characterized histologically as metastatic carcinoma , most likely originating from the breast carcinoma , and was confined within the thyroid capsule and abutted on by the carcinoma itself on both the right and left lobes of the thyroid gland ( fig . in addition , the patient had a 6.0 4.0 mm sized primary papillary carcinoma just beyond the thyroid capsule in the left lobe . all 7 of the cervical lymph nodes around the thyroid exhibited metastatic carcinoma . following surgery , treatment of the patient with docetaxel and doxorubicin chemotherapy breast cancer accompanies autoimmune thyroid diseases as well as non - autoimmune thyroid diseases such as thyroid nodules with a high frequency , and the incidence of thyroid cancer is a known risk factor for breast cancer . the association of thyroid cancer and breast cancer was first reported by chalstrey and benjamin in 1996 after observing that 8 of 92 thyroid cancer patients were subsequently diagnosed with breast cancer . several studies have shown that the incidence of thyroid cancer in breast cancer patients is increased compared to the general population by approximately 2030% . similarly , the incidence of breast cancer is increased during median follow - up of patients with thyroid cancer [ 7 , 8 ] . the link between breast cancer and thyroid cancer is supposedly associated with genetic and environmental factors . however , unlike cases where breast cancer is diagnosed following thyroid cancer , reports of breast cancer metastasizing to a thyroid gland with primary papillary cancer are very rare . a total of 33% of patients with breast cancer experience hematogenous metastasis of the lungs , liver , bone , and gastrointestinal tract . liver and lung metastases are common in invasive ductal carcinoma , whereas gastrointestinal tract , peritoneum , and retroperitoneum metastases are common in invasive lobular carcinoma . estrogen and progesterone receptor status is well correlated between metastasized organs and primary cancerous tissue . positive immunohistochemical staining of the estrogen receptor is observed in approximately 5080% of cases , whereas positive progesterone receptor expression is observed in 3859% of cases ; estrogen receptor - negative cases are rare [ 10 , 11 ] . estrogen receptor - positive cases tend to metastasize primarily to the thyroid and parathyroid glands , while progesterone receptor - positive cases tend to metastasize to the myocardium or the epithelium of the gastrointestinal and urinary tracts . in the present study , expression of estrogen receptor the thyroid gland is highly vascularized ; however , metastasis of malignant tumors to the thyroid is relatively rare . the exact mechanism for the lack of metastasis to the thyroid is unknown , but it is thought to be associated with immunological mechanisms . the incidence of metastasis to the thyroid gland in autopsy series varies from 0.5 to 24.2% [ 13 , 14 ] , the variation of which is likely due to the precision of investigation during the autopsy . in autopsy series , the common primary sites of metastatic thyroid tumors are breast and lung cancer , while renal cell carcinoma is the most common site clinically . in a clinical setting , metastasis of the thyroid gland is detected at a low frequency , and metastasis of the thyroid gland is most commonly detected at the same time as the diagnosis of the primary site or shortly thereafter . in some cases , these differences are thought to be related to the characteristics of the primary cancer , and it is well known that primary cancers such as renal cell carcinoma , breast cancer , cervical cancer , and melanoma take a long time to metastasize to the thyroid . diagnosis of thyroid metastasis is difficult , and in general , most cases are asymptomatic and identified due to the appearance of solitary or multiple masses . in our study , fine - needle aspiration cytology did not help with the diagnosis of metastasis of thyroid gland ; the accuracy of fine - needle aspiration cytology is heavily reliant on the experience of the clinician performing the analysis and their ability to apply strict diagnostic criteria . specifically , it is difficult to establish a correct differential diagnosis for primary anaplastic carcinoma of the thyroid gland and thyroid metastasis of poorly differentiated primary tumors . additionally , the usefulness of immunohistochemical staining for thyroglobulin is limited , because it is detected in only 2030% of anaplastic carcinomas . therefore , fine - needle aspiration cytology should be considered as a secondary means of diagnosis rather than a definitive diagnostic tool [ 2 , 14 , 15 ] . in our case , the thyroid nodules were diagnosed as papillary carcinomas based on thyroid ultrasound , because thyroid metastasis from the primary breast cancer was not observed by specific findings in imaging studies . thyroid metastasis of a primary cancer is usually considered as a systemic disseminated disease with a generally poor prognosis . survival following a diagnosis of thyroid metastasis varies depending on the site of the primary cancer . the overall survival of breast cancer , renal cell carcinoma and uterine cancer ranges from 21 to 66 months , while that of lung cancer and melanoma is 112 months [ 3 , 13 ] . there are no definitive conclusions regarding the clinical effect of surgical resection in patients with thyroid metastasis . in the absence of metastasis to other organs excluding thyroid metastasis , thyroid resection is indicated , and early diagnosis and aggressive treatment appears to improve survival . in our case , the patient also had a primary papillary thyroid cancer , for which thyroid surgery was performed . the patient received chemotherapy with docetaxel plus doxorubicin according to the following regimen : doxorubicin 50 mg / m and docetaxel 60 mg / m , repeated every 3 weeks for a total of 6 cycles along with radiotherapy . although the thyroid gland is an uncommon site of metastasis , physicians should be aware of the possibility of metastatic disease in patients with a previous history of malignancy .

abstractmetastasis to the thyroid gland is very rare . recently , we experienced a case of thyroid metastasis from breast cancer accompanying a papillary thyroid . a 51-year - old female patient presented with a palpated lymph node on her left lateral neck . the patient had undergone a left modified radical mastectomy followed by chemotherapy and hormonal therapy 12 years prior . ultrasonography of the neck revealed a malignant looking nodule at the left thyroid lobe , measuring 0.9 0.9 cm , and several cystic nodules at the right thyroid lobe . ultrasonography of the neck additionally revealed a malignant looking lymph node at the right level vi . fine - needle aspiration of the left thyroid lobe resulted in a diagnosis of papillary thyroid carcinoma and that of the right level vi in hurthle cell lesion . the patient had a total thyroidectomy with selective dissection of the left neck node . pathologic assessment of the specimen revealed metastatic carcinoma from the breast carcinoma and papillary thyroid carcinoma . although the thyroid gland is highly vascularized , metastasis of malignant tumors to the thyroid is relatively rare and detection of metastasis shows a low frequency . so a careful evaluation of thyroid tumor should be considered in a patient with a history of other malignancy .

nestin;nf1;z / eg , nestin;nf1;z / eg , and nestin;z / eg mice were generated by crossing nestin driver with z / eg reporter ( tg(cag - bgeo / gfp)21lbe / j , jackson labs ) and nf1 mice , where applicable . gli1;nf1;z / eg and gli1;z / eg mice were generated in a similar fashion , but with gli1 mice . all mice in the study were backcrossed to the c57bl/6 background for at least six generations . animals were housed in a 14 hour light/10 hour dark cycle and had free access to food and water . tamoxifen ( 66.67 mg / ml , sigma , t5648 ) was prepared in a 5:1 corn oil ( sigma ) to ethanol mixture . due to differences in creer mouse generation , different tamoxifen injection paradigms were required to enable sparse labeling for clonal analysis ( ~ 816 clones per hemisphere ) . a single 62.5 mg / kg dose of tamoxifen ( for nestin ) or a 125 mg / kg dose every 12 hours for 4 total doses ( for gli1 ) was intraperitoneally injected into adult 810 week - old male and female mice ( supplementary table 4 ) . for population cell labeling in nestin , animals were injected with 125 mg / kg every 12 hours 4 times ( supplementary table 4 ) . animals were analyzed at 2 dpi , 14 dpi , or 12 mpi . for animals with multiple injections , dpi and mpi all animal procedures were performed in accordance to institutional guidelines of johns hopkins university school of medicine . animals were transcardiac perfused with cold 4% paraformaldehyde ( wt / vol , in 0.1 m phosphate buffer , pb , ph 7.4 ) , and cryoprotected with 30% sucrose ( wt / vol ) . serial 40 m - thick coronal brain sections were cut on a frozen sliding microtome ( leica , sm2010r ) for immunohistology as previously described . antibodies diluted in tbs with 0.05% triton x-100 and 3% donkey serum , were used against gfp ( aves labs , gfp-1020 , chicken , 1:500 ; rockland , 600 - 101 - 215 , goat , 1:500 ; abd serotec , 4745 - 1051 , sheep , 1:500 ) , tbr2 ( abcam , ab23345 , rabbit , 1:250 ) , gfap ( millipore , mab360 , mouse , 1:1000 ; dako , z033401 - 2 , rabbit , 1:1000 ) , nestin ( aves labs , nes , chicken , 1:500 ) , prox1 ( millipore , mab5654 , mouse , 1:500 ) , ng2 ( millipore , ab5320 , rabbit , 1:200 ) , olig2 ( a generous gift from dr . nestin antigen was retrieved by incubating brain sections in 1x dako target retrieval solution ( dako ) at 68c for 20 minutes , followed by 10 minutes cooling to room temperature . serial sections from the entire dentate gyrus were first immunostained for gfp to allow identification of prospective clone - containing sections on an epifluorescence microscope ( zeiss , axiovert 200 m ) . labeled clone - containing sections were taken for further processing and confocal imaging at 40x on a zeiss lsm 710 confocal microscope using multi - track or spectral linear unmixing for population cell labeling experiments , cells were counted manually by visualizing the serial dentate gyrus sections on an epifluorescence microscope . due to the difficulty in reliably quantifying small , densely packed transient - amplifying neural progenitors , only cells with mature neuron morphology for clonal cell labeling experiments , identification of clones amongst gfp - labeled clusters of cells was performed as previously described . all aligned images were exported at full resolution for 3d visualization into imaris ( bitplane ) and analyzed . clones were analyzed in a dentate gyrus volume that included the molecular layer , granule cell layer , subgranular zone , and hilus , excluding the polymorphic layer ( ca4 ) protruding into the posterior dentate gyrus . numbers of cells and animals counted and used , respectively , are shown in supplementary tables 13 . no statistical methods were used to pre - determine sample sizes ; sample sizes were similar to those reported in previous studies due to the binary nature of the reported phenomenon , randomization and blinding were not employed . a two - sample kolmogorov a two - sample unpaired student s t - test was used to statistically compare the percentage of mcm2 rgls upon nf1 inactivation at 2 or 14 dpi . nestin;nf1;z / eg , nestin;nf1;z / eg , and nestin;z / eg mice were generated by crossing nestin driver with z / eg reporter ( tg(cag - bgeo / gfp)21lbe / j , jackson labs ) and nf1 mice , where applicable . gli1;nf1;z / eg and gli1;z / eg mice were generated in a similar fashion , but with gli1 mice . all mice in the study were backcrossed to the c57bl/6 background for at least six generations . animals were housed in a 14 hour light/10 hour dark cycle and had free access to food and water . tamoxifen ( 66.67 mg / ml , sigma , t5648 ) was prepared in a 5:1 corn oil ( sigma ) to ethanol mixture . due to differences in creer mouse generation , different tamoxifen injection paradigms were required to enable sparse labeling for clonal analysis ( ~ 816 clones per hemisphere ) . a single 62.5 mg / kg dose of tamoxifen ( for nestin ) or a 125 mg / kg dose every 12 hours for 4 total doses ( for gli1 ) was intraperitoneally injected into adult 810 week - old male and female mice ( supplementary table 4 ) . for population cell labeling in nestin , animals were injected with 125 mg / kg every 12 hours 4 times ( supplementary table 4 ) . animals were analyzed at 2 dpi , 14 dpi , or 12 mpi . for animals with multiple injections , dpi and mpi all animal procedures were performed in accordance to institutional guidelines of johns hopkins university school of medicine . animals were transcardiac perfused with cold 4% paraformaldehyde ( wt / vol , in 0.1 m phosphate buffer , pb , ph 7.4 ) , and cryoprotected with 30% sucrose ( wt / vol ) . serial 40 m - thick coronal brain sections were cut on a frozen sliding microtome ( leica , sm2010r ) for immunohistology as previously described . antibodies diluted in tbs with 0.05% triton x-100 and 3% donkey serum , were used against gfp ( aves labs , gfp-1020 , chicken , 1:500 ; rockland , 600 - 101 - 215 , goat , 1:500 ; abd serotec , 4745 - 1051 , sheep , 1:500 ) , tbr2 ( abcam , ab23345 , rabbit , 1:250 ) , gfap ( millipore , mab360 , mouse , 1:1000 ; dako , z033401 - 2 , rabbit , 1:1000 ) , nestin ( aves labs , nes , chicken , 1:500 ) , prox1 ( millipore , mab5654 , mouse , 1:500 ) , ng2 ( millipore , ab5320 , rabbit , 1:200 ) , olig2 ( a generous gift from dr . nestin antigen was retrieved by incubating brain sections in 1x dako target retrieval solution ( dako ) at 68c for 20 minutes , followed by 10 minutes cooling to room temperature . serial sections from the entire dentate gyrus were first immunostained for gfp to allow identification of prospective clone - containing sections on an epifluorescence microscope ( zeiss , axiovert 200 m ) . labeled clone - containing sections were taken for further processing and confocal imaging at 40x on a zeiss lsm 710 confocal microscope using multi - track or spectral linear unmixing online fingerprinting configurations ( carl zeiss ) . for population cell labeling experiments , cells were counted manually by visualizing the serial dentate gyrus sections on an epifluorescence microscope . due to the difficulty in reliably quantifying small , densely packed transient - amplifying neural progenitors , only cells with mature neuron morphology for clonal cell labeling experiments , identification of clones amongst gfp - labeled clusters of cells was performed as previously described . all aligned images were exported at full resolution for 3d visualization into imaris ( bitplane ) and analyzed . clones were analyzed in a dentate gyrus volume that included the molecular layer , granule cell layer , subgranular zone , and hilus , excluding the polymorphic layer ( ca4 ) protruding into the posterior dentate gyrus . numbers of cells and animals counted and used , respectively , are shown in supplementary tables 13 . no statistical methods were used to pre - determine sample sizes ; sample sizes were similar to those reported in previous studies due to the binary nature of the reported phenomenon , randomization and blinding were not employed . a two - sample kolmogorov a two - sample unpaired student s t - test was used to statistically compare the percentage of mcm2 rgls upon nf1 inactivation at 2 or 14 dpi .

endogenous neural stem cells ( nscs ) in the adult hippocampus are considered bi - potent , as they only produce neurons and astrocytes in vivo . here we show in mouse that inactivation of neurofibromin 1 , a gene mutated in neurofibromatosis type 1 , unlocks a latent oligodendrocyte lineage potential to produce all three lineages from nscs in vivo . our results suggest an avenue to promote stem cell plasticity by targeting barriers of latent lineage potential .

a low ankle - brachial index ( abi ) is deemed a feasible , valid , and noninvasive indicator of atherosclerosis . a low abi suggests the development and severity of lower - limb peripheral arterial disease and can play a crucial role as an independent risk indicator of cardiovascular events and mortality . a reclassification of the cardiovascular risk assessment model to include the abi led to the modification of the management of cardiovascular diseases in women ( almost 36% ) and in men ( 19% ) . the early detection and prevention of cardiovascular and cerebrovascular events require a precise identification of individual risks through the use of accurate indicators . a low abi ( 0.90 ) has been demonstrated to correlate with a twofold increase in cardiovascular mortality , ten - year mortality , and coronary events rate . a low abi is considered a potential factor in the detection of symptomatic and asymptomatic patients afflicted with peripheral arterial disease and could , as such , be an accurate and effective screening test for peripheral arterial disease and coronary artery disease ( cad ) . the abi might also provide more information regarding the prognosis of the long - term survival of patients with peripheral arterial disease . the abi , alongside type - d personality , has been shown to predict the quality of life and depressive symptoms in patients with peripheral arterial disease . both resting and post - exercise measures of the abi were strong independent predictors of mortality in a cohort study of 3209 patients4 and increased risk of stroke or transient ischemic attack in the elderly in another study . there has been other evidence for a significant decline in the abi after modifying the risk factors of cad among a cohort of 5888 participants over 6 years of follow - up . nonetheless , the reported evidence regarding the accuracy of the abi suffers from a lack of consistency . this prospective investigation was , therefore , performed to evaluate whether the abi could provide evidence for the prediction of cad , and if so , to determine the accuracy of this indicator compared with coronary angiography . a population of 699 was recruited in this case - control study with incidence cases ( 362 ) and controls ( 337 ) between 2010 and 2011 in tehran heart center . the cad cases were confirmed by angiographic findings , and the controls comprised patients with normal angiographic findings . the definition of obstructive cad was decided as stenosis of 50% or more of the diameter of any coronary artery . for the measurement of risk factors , such well - known risk factors as age , gender , hypertension , diabetes , dyslipidemia , cigarette smoking , physical activity , body mass index , family history of cad , and abi were assessed via standardized methods . the abi measurements were done using a standard sphygmomanometer and a doppler device , after 30 minutes of rest in a supine position . the detection of both upper- and lower- limb blood pressures was done by placing the blood pressure cuffs over the brachial arteries and malleoluses . for each right and left brachial artery and right and left dorsalis pedis artery , the cuff was inflated to 20 mmhg over the detectable systolic blood pressure and deflated appropriately . the abi was calculated in accordance with the american heart association definition as the ratio of the higher systolic blood pressure of each ankle - brachial artery . all the abi measurements were performed by 2 well trained physicians . in this study , the abi was treated as a categorical variable of an abi 0.9 and an abi > 0.9 . univariate analysis was performed by applying the pearson chi - squared test and the independent sample t - test as well as the estimation of the odds ratio and 95% confidence interval ( 95%ci ) and measures of accuracy . the odds ratio , 95%ci , and likelihood ratio were calculated for different endpoints of cad . bivariate analysis was carried out by performing age- and sex - adjusted mantel - haenszel odds ratio and 95%ci . multivariate analysis was conducted via the logistic regression model to evaluate multivariate adjusted relationships between cad and independent variables and for adjusting potential confounders . measure of validity was done to assess the accuracy of the abi ( 0.90 ) compared to angiographic results through the calculation of the sensitivity , specificity , and predictive values . table 1 compares the different risk factors and systolic blood pressures of both extremities between the two groups of cases and controls . cad , coronary artery disease ; ldl - c , low - density lipoprotein ; hdl - c , high - density lipoprotein ; abi , ankle brachial index ; sbp , systolic blood pressure the calculated measures of accuracy for the abi ( 0.9 ) compared to the results of coronary angiography revealed sensitivity of 64% , specificity of 99.7% , positive predictive value of 95.8% , and negative predictive value of 49.8% . the crude or and 95%ci of the relationship between the studied risk factors and cad associated with the likelihood ratio based on the likelihood ratio , we could strongly predict angiographically confirmed cad with the presence of gender differences , cigarette smoking , age , and low abi . comparison between case and controls based on predictive factors cad , coronary artery disease ; abi , ankle brachial index ; or , odds ratio ; ci , confidence interval , fbs , fasting blood sugar , hdl - c , high - density lipoprotein crude and adjusted mantel - haenszel odds ratio and 95%ci [ or ( 95% ci ) ] for age- and sex - matched comparisons were as : for age under 60 years to age above 60 years , the comparison crude estimate was 0.07 ( 0.010.45 ) and the common adjusted parameter estimate was 2.65 ( 0.974.86 ) . the sex - matched estimate of the parameters demonstrated that the crude odds ratio for the male - to - female comparison was 3.15 ( 2.324.30 ) and that the common adjusted odds ratio was 3.69 ( 2.435.58 ) . a low abi did not significantly predict the unstable angina pectoris in terms of an odds ratio and 95%ci of 2.94 ( 0.2929.23 ) and a likelihood ratio of 0.97 ( p value = 0.32 ) . acute myocardial infarction could not be predicted by a low abi with an odds ratio and 95%ci of 1.11 ( 0.971.28 ) and a likelihood ratio of 0.19 ( p value = 0.65 ) . non - st - segment elevation myocardial infarction was of a prognostic value of 1.42 ( 1.051.96 ) and a likelihood ratio of 1.30 ( p value = 0.25 ) with a low abi . a low abi predicted stable angina pectoris with an odds ratio and 95%ci of 2.43 ( 2.172.70 ) and a likelihood ratio of 20.65 ( p value = 0.001 ) . table 3 shows the adjusted odds ratios and 95%cis for the different variables derived from a logistic regression model that controlled the potential confounders , when the abi was treated as a categorical variable and was considered abnormal at 0.9 . according to the above multivariate results , an abi 0.9 had a strong and significant prognostic value for predicting cad ( odds ratio = 13.86 ) . adjusted or and 95% ci for the relationship between different factors with cad when abi is categorical cad , coronary artery disease ; abi , ankle - brachial index ; or , odds ratio ; ci , confidence interval the results of our investigation demonstrated that the measures of the accuracy of a low abi ( 0.90 ) had very high specificity ( 99.7% ) and a positive predictive value of 95.8% but low sensitivity ( 64% ) and a low negative predictive value of 49.8% . furthermore , the abi provided strongly significant information on the prediction of cad [ or ( 95%ci ) : 13.86 ( 1.7817.62 ) ] after multivariate adjustment for the potential confounding variables . there is no doubt that a low abi ( 0.90 ) increases the risk of overall mortality , proportional cardiovascular mortality , and coronary events by about twofold . cardiovascular risk assessment based on traditional cad risk factors is deemed difficult , and the introduction of new noninvasive , feasible , and valid prognostic factors seems to be necessary . therefore , in accordance with the guidelines published by the american heart association/ american college of cardiology , the present study showed that the abi had the potential to be implemented for the risk assessment of cad . future cardiovascular events in people with traditional and novel risk factors , as well as in individuals with asymptomatic atherosclerosis , may be easily predicted by the abi , which is feasible , noninvasive , and accurate . the abi has been traditionally applied for the screening of peripheral arterial disease , nowadays given that a resting attenuated abi of 0.90 is associated with an increased risk of overall mortality of about 27 fold and cardiovascular mortality of 24 fold , it can be considered as a feasible , noninvasive , and accurate tool for the screening of cardiovascular events for generalized atherosclerosis . there is also reported evidence that supports the role of a low abi in increasing the risk of cerebrovascular events , especially in the elderly . our results revealed that a resting attenuated abi had low sensitivity ( 64% ) but enormous specificity ( 99.7% ) and positive predictive value ( 95.8% ) . this measure of high specificity implies that a low abi , as a valid and trustworthy sensitive screening tool , might rule out the presence of cad . sensitivity of 64% demonstrates that a low abi at rest , when considering the higher systolic blood pressure of the limbs , should improve . considering other major cad risk factors combined with a low abi is suggested to have the potential to improve the sensitivity of this index . the second definition and calculation of the abi based on the measurement of the lower systolic blood pressures of both dorsalis pedis and brachial arteries may also augment the ability of a low abi to predict the existence of cad . the difference between our findings and those of a study previously published in 2008 could be explained by the discrepancy in the applied gold standard test for comparison . in our research , we considered angiographic findings as a gold standard in comparison with the abi , whereas the older study compared the results of the abi with single - photon emission computed tomography and electrocardiography . a low abi has been found to be associated with other cad risk factors , and it seems that the modification of modifiable cad risk factors and declining abi may cause a statistically significant decrease in the lower extremity arterial diseases . in order to evaluate the risk factors of a declining abi , the cardiovascular health study cohort was conducted . the frequency of a decrease in the abi over a follow - up period of 6.5 years was estimated at 9.5% . predictive factors for a declining abi included age , cigarette smoking , hypertension , diabetes , and high density lipoprotein cholesterol ( hdl - c ) level . we found out more features of prognostic factors . in the present study beyond the result of the previous research , we showed that gender , age , cigarette smoking , family history of cad , and abi 0.9 , played a statistically significant role in the prediction of future cad . a low abi accurately predicts the presence of peripheral arterial disease in both symptomatic and asymptomatic individuals and is also associated with the severity and progression of peripheral arterial disease . these findings demonstrate that abi has the potential to detect the peripheral arterial disease at an early stage . since a low abi shows a strong positive correlation with peripheral arterial disease , it can predict cardiovascular and cerebrovascular mortality . also , in concord with our findings , a low abi could be considered as a predictive factor for such events . a valid and reliable model of risk factor assessment guides the management strategies , in both primary and secondary prevention . a low abi has almost perfect specificity ( 99.7% ) and positive predictive value of 95.8% but low sensitivity ( 64% ) . therefore , in order for a low abi to be considered a firstline screening test for the early detection of cad , we need to factor in other cad risk factors associated with the abi . our multivariate adjusted relationships provided strongly accurate information regarding the prediction of cad with an odds ratio of 13.86 with a low abi ( 0.90 ) .

abstractbackground : given the lack of consistency in the literature regarding the reliability of the ankle - brachial index ( abi ) as a valid screening tool and an independent risk indicator of cardiovascular events and mortality , we compared it with angiography as a reference standard test.methods:this case - control study , conducted between 2010 and 2011 in tehran heart center , recruited 362 angiographically confirmed cases of coronary artery disease ( cad ) and 337 controls . a standard protocol was used to measure the abi and different cad risk factors.results:a low abi had specificity of 99.7% , positive predictive value of 95.8% , negative predictive value of 49.8% , sensitivity of 64% , likelihood ratio of 24.07 , and odds ratio ( or ) of 22.79 ( 95%ci : 3.0669.76 ) . the role of the associated risk factors was evaluated with or ( 95%ci ) , with the variables including gender 3.15 ( 2.304.30 ) , cigarette smoking 2.72 ( 1.863.99 ) , family history 1.72 ( 1.172.51 ) , diabetes 1.66 ( 1.152.4 ) , and dyslipidemia 1.38 ( 1.021.88 ) . in a multivariate model , the following variables remained statistically significantly correlated with cad [ or ( 95%ci ) ] : abi 13.86 ( 1.7817.62 ) ; gender 3.69 ( 2.435.58 ) ; family history of cad 2.18 ( 1.413.37 ) ; smoking 1.69 ( 1.082.64 ) ; age 1.04 ( 1.021.06).conclusions : a low abi had specificity of 99.7% ; however , because of its low sensitivity ( 64% ) , we should consider cad risk factors associated with a low abi in order to use it as a first - line screening test .

there is growing evidence from randomized controlled trials ( rcts ) and meta - analyses that acupuncture had clinically significant effects ( efficacy over placebo controls ) . the results have proved that acupuncture may also have clinically significant effects on objective functional outcome . according to rcts with subjective outcomes , compared with waiting list controls or treatment as usual , both placebo acupuncture and real acupuncture might play important roles . the difference between acupuncture and no acupuncture in effectiveness is often much greater than the difference between acupuncture and so - called placebo acupuncture . thus , psychosocial factors other than needle placement might be relevant to subjective outcomes of acupuncture . in particular , patients beliefs about acupuncture could predict treatment outcomes in acupuncture for pain to a significant extent . according to the traditional chinese acupuncture theory , two critical principles that are of the same importance in ensuring the effectiveness of acupuncture zhi shenis gaining full control over the spirits of the patients and the acupuncturists . the other one is named de qi , a composite of unique sensations induced by needle insertion into the acupoint which has been widely investigated in recent years . to ensure the effectiveness of acupuncture , during the whole acupuncture process including before needling , performing needling , and after needling , the acupuncturists were required to attend closely to the spiritual state of both acupuncturists and patients . the acupuncturists administrated the insertion until de qi was achieved by their patients . however , this important concept zhi shen in traditional chinese acupuncture theory is hardly mentioned in western literature and emphasized in clinical practice in western countries . what 's more , among various kinds of needle sensations , what kind of needle sensation can predict outcomes of acupuncture treatment also remains undetermined . thus , we analyzed the data on psychosocial factors from the above mentioned rct to examine whether some quantifiable psychosocial measurements could improve the therapeutic effects of acupuncture treatment . through rating different kinds of needle sensations by a visual analog scale , this study attempted to explore the relationship between each needle sensation and therapeutic effect of acupuncture . the study was approved by the ethics committee of tongji medical college , huazhong university of science and technology , and was conducted in accordance with the provisions of the declaration of helsinki ( 1975 , amended 1983 ) and good clinical practice guidelines . written informed consent was obtained from each participant . we analyzed data from a rct designed to compare efficacy of acupuncture with either strong or weak stimulation among patients with bell 's palsy . the patients were included as the following criteria : ( 1 ) must be unilateral facial nerve weakness without an identifiable cause within 168 h after the onset of symptoms ; ( 2 ) aged from 18 to 65 years ; and ( 3 ) have not received any treatment before randomization . we excluded the patients who were illiterate , had facial paralysis caused by herpes zoster , had recurrent facial paralysis , had noticeable asymmetry of the face before the onset of illness ( which may affect evaluation ) , or had a history of peptic ulcer disease , severe hypertension , uncontrolled diabetes , liver and kidney dysfunction , mental illness , or serious systemic diseases that might affect the treatment . the flowchart of this study is shown in figure 1 and other study information is shown in supplementary material 1 . the primary outcome was facial nerve function measured by the criteria of the house - brackmann ( hb ) score . the endpoint was grade 1 on the hb score ( complete recovery ) at month 6 . the hb scale is based on a six - grade score : grade 1 indicates normal function and grade 6 indicates complete paralysis . patients were asked to show four standard facial expressions : at rest , raised eyebrows , eyes tightly closed , and showing teeth , which were recorded by a digital camcorder . all digital data were assessed and graded independently by three neurologists of tongji hospital ; the neurologists were unaware of the study - group assignments and the stage of assessment . we considered that several baseline psychological factors might be potentially correlated with the hb score at the end of treatment , including participants gender , age , education status , occupation , personality traits , attention , and the score of the pretreatment mediator questionnaire ( pmq ) . on the right day of randomization , all patients were asked to fill in pmq designed for assessing pretreatment mediators including prior belief in acupuncture , experience of acupuncture treatment , and influence of people around the patients before acupuncture treatment , which consists of five related questions [ supplementary material 2 ] . patients personality traits were assessed by the cattell 's 16 personality factor questionnaire ( 16pf ) . consort 2010 checklist of information to include when reporting a randomized trial click here for additional data file . all tests were two - tailed and the differences were considered to be statistically significant when p < 0.05 . data extraction and statistical analyses were conducted using sas version 9.2 ( sas institute inc . , cary , nc , usa ) and the r statistical environment , package grplasso . for each group , we used the least absolute shrinkage and selection operator ( lasso ) algorithm for variable selection and refit the model using a standard logistic regression . candidate variables of the model included center , gender , age ( divided by 10 years ) , education status , occupation , side of palsy , time between onset of palsy and start of treatment ( divided by 72 h ) , the hb score on day 1 , attention ( 1 : correctness ratio 97% , 2 : > 97% correctness ratio 99% , 3 : correctness ratio > 99% ) , and the scores of pmq and 16pf . to examine the association of feeling factors with hb score at month 6 , we used multivariable logistic regression analysis after controlling for all confounding variables , including gender , age , treatment , interval between the onset of palsy and the start of treatment , and hb score on day 1 . hb scores of patients at month 6 were grouped into two categories in our logistic regression : 1 for normal function and 0 for others . to prevent the model from multicollinearity and overfitting , 10-fold cross - validation with minimax concave penalty ( mcp)-penalized method was used for variable selection ( r : package ncvreg ) . after variable selection , path analysis was used to test the causal relations among the variables specialized in the model . it allowed for the simultaneous conduction of multiple linear regression analyses and could determine and illustrate whether the data were consistent with the researcher 's casual hypothesis . in our previous research , variables such as treatment , hb score on day 1 , and vigilance and tension scores in 16pf may influence the value of hb score at month 6 . all these variables were selected as the path analytic model 's exogenous variables . besides , all the feelings which selected by the cross - validation process and hb score at month 6 were selected as the path analytic model 's endogenous variables . note the dependent ( endogenous ) variable hb score at month 6 was a binary variable for logistic regression but a continuous variable for path analysis ( raw data : score 16 ) . the study was approved by the ethics committee of tongji medical college , huazhong university of science and technology , and was conducted in accordance with the provisions of the declaration of helsinki ( 1975 , amended 1983 ) and good clinical practice guidelines . written informed consent was obtained from each participant . we analyzed data from a rct designed to compare efficacy of acupuncture with either strong or weak stimulation among patients with bell 's palsy . the patients were included as the following criteria : ( 1 ) must be unilateral facial nerve weakness without an identifiable cause within 168 h after the onset of symptoms ; ( 2 ) aged from 18 to 65 years ; and ( 3 ) have not received any treatment before randomization . we excluded the patients who were illiterate , had facial paralysis caused by herpes zoster , had recurrent facial paralysis , had noticeable asymmetry of the face before the onset of illness ( which may affect evaluation ) , or had a history of peptic ulcer disease , severe hypertension , uncontrolled diabetes , liver and kidney dysfunction , mental illness , or serious systemic diseases that might affect the treatment . the flowchart of this study is shown in figure 1 and other study information is shown in supplementary material 1 . the primary outcome was facial nerve function measured by the criteria of the house - brackmann ( hb ) score . the endpoint was grade 1 on the hb score ( complete recovery ) at month 6 . the hb scale is based on a six - grade score : grade 1 indicates normal function and grade 6 indicates complete paralysis . patients were asked to show four standard facial expressions : at rest , raised eyebrows , eyes tightly closed , and showing teeth , which were recorded by a digital camcorder . all digital data were assessed and graded independently by three neurologists of tongji hospital ; the neurologists were unaware of the study - group assignments and the stage of assessment . we considered that several baseline psychological factors might be potentially correlated with the hb score at the end of treatment , including participants gender , age , education status , occupation , personality traits , attention , and the score of the pretreatment mediator questionnaire ( pmq ) . on the right day of randomization , all patients were asked to fill in pmq designed for assessing pretreatment mediators including prior belief in acupuncture , experience of acupuncture treatment , and influence of people around the patients before acupuncture treatment , which consists of five related questions [ supplementary material 2 ] . patients personality traits were assessed by the cattell 's 16 personality factor questionnaire ( 16pf ) . consort 2010 checklist of information to include when reporting a randomized trial click here for additional data file . all tests were two - tailed and the differences were considered to be statistically significant when p < 0.05 . data extraction and statistical analyses were conducted using sas version 9.2 ( sas institute inc . , cary , nc , usa ) and the r statistical environment , package grplasso . for each group , we used the least absolute shrinkage and selection operator ( lasso ) algorithm for variable selection and refit the model using a standard logistic regression . candidate variables of the model included center , gender , age ( divided by 10 years ) , education status , occupation , side of palsy , time between onset of palsy and start of treatment ( divided by 72 h ) , the hb score on day 1 , attention ( 1 : correctness ratio 97% , 2 : > 97% correctness ratio 99% , 3 : correctness ratio > 99% ) , and the scores of pmq and 16pf . to examine the association of feeling factors with hb score at month 6 , we used multivariable logistic regression analysis after controlling for all confounding variables , including gender , age , treatment , interval between the onset of palsy and the start of treatment , and hb score on day 1 . hb scores of patients at month 6 were grouped into two categories in our logistic regression : 1 for normal function and 0 for others . to prevent the model from multicollinearity and overfitting , 10-fold cross - validation with minimax concave penalty ( mcp)-penalized method was used for variable selection ( r : package ncvreg ) . after variable selection , path analysis was used to test the causal relations among the variables specialized in the model . it allowed for the simultaneous conduction of multiple linear regression analyses and could determine and illustrate whether the data were consistent with the researcher 's casual hypothesis . in our previous research , variables such as treatment , hb score on day 1 , and vigilance and tension scores in 16pf may influence the value of hb score at month 6 . all these variables were selected as the path analytic model 's exogenous variables . besides , all the feelings which selected by the cross - validation process and hb score at month 6 were selected as the path analytic model 's endogenous variables . note the dependent ( endogenous ) variable hb score at month 6 was a binary variable for logistic regression but a continuous variable for path analysis ( raw data : score 16 ) . totally , 316 subjects with complete variable records were analyzed and the patients were divided into two groups : de qi group ( n = 159 ) and control group ( n = 157 ) . after 6 months , more patients had complete recovery in de qi group ( 89.8% ) than control group ( 70.8% ) ( adjusted for age , gender , treatment center , interval between onset of palsy and start of treatment , hb score on day 1 ; adjusted odds ratio [ or ] : 4.16 , 95% confidence interval [ ci ] : 2.237.78 ) . when the lasso algorithm was applied to determine whether the patient had a complete recovery , we found several factors [ table 1 ] . in de qi group , low hb score on day 1 ( or : 0.13 , 95% ci : 0.030.45 ) and low social boldness score ( or : 0.63 , 95% ci : 0.410.97 ) in 16pf were associated with the better facial function . in control group , low hb score on day 1 ( or : 0.25 , 95% ci : 0.130.50 ) and low vigilance score ( or : 0.66 , 95% ci : 0.500.88 ) and high tension score ( or : 1.41 , 95% ci : 1.121.77 ) in 16pf were associated with better facial function . analyses of factors associated with complete recovery for bell 's palsy ci : confidence interval ; or : odds ratio ; 16pf : 16 personality factor questionnaire ; hb : house - brackmann . of the 316 patients who completed the 6-month follow - up visit , 262 ( 82.9% ) rated de qi on the visual analog scale and the other 54 ( 17.1% ) stated that they could not understand or differentiate between the 8 elements of needle sensations and , thus , did not complete the scale . of these 54 patients , 24 were in the de qi group and 30 were in the control group . it was found that in both groups , the score of experience and belief in acupuncture questionnaire , gender , age , education status , occupation , and attention had no prominent effects on objective outcome after 6 months . for logistic regression , the binomial deviance was chosen as cross - validation error . to minimize the cross - validation error , however , it has revealed that only the needle sensation of fullness was associated with hb score at month 6 . path coefficients via two multiple regression analyses based on the hypothesized model predictor variables with a significant level < 0.05 were retained in the final model . the path coefficients were standardized coefficients . or : odds ratio ; 16pf : 16 personality factor questionnaire ; hb : house - brackmann . according to the model [ figure 3 ] , the path coefficients showed that hb score on day 1 , needle sensation of fullness , vigilance score in 16pf , and tension score in 16pf had direct effects on hb score at month 6 . if hb score on day 1 raised 1 unit , the hb score at month 6 would increase 0.27 unit . inversely , if the score of needle sensation of fullness raised 1 unit , the hb score at month 6 would decrease 0.55 unit . meanwhile , if the vigilance score in 16pf raised 1 unit , the hb score at month 6 would increase 0.19 unit , and if the tension score in 16pf raised 1 unit , the hb score at month 6 would decrease 0.12 unit . furthermore , the treatment has a direct effect on needle sensation of fullness and an indirect effect on hb score at month 6 via the needle sensation of fullness . the de qi ( treatment ) group 's score of needle sensation of fullness was 0.58 , more than the control group , and the de qi group 's hb score at month 6 was 0.32 , less than the control group . path analysis for hb score at month 6 . hb score on day 1 , needle sensation of fullness , vigilance score in 16pf , and tension score in 16pf had direct effects on hb score at 6 months . meanwhile , treatment had a direct effect on needle sensation of fullness and an indirect effect on hb score at month 6 via the needle sensation of fullness . the findings to date suggested that a number of psychosocial factors , in particular , patients beliefs about acupuncture , predict treatment outcomes in acupuncture for pain to a significant extent . as for objective outcomes in acupuncture treatment , there is almost no report in literature exploring in this regard . our findings suggested that different personality traits played the different roles on the objective outcome when using acupuncture treating patients with bell 's palsy . the score of experience and belief in acupuncture questionnaire , gender , age , education status , occupation , and attention had no prominent effects on objective outcome after 6 months . after 6 months , patients in the de qi group had a better facial function ( adjusted or : 4.16 , 95% ci : 2.237.78 ) . in control group , recovery of facial nerve function was generally attributed to spontaneous recovery and prednisone . moreover , in de qi group , it is attributed to acupuncture , in addition to spontaneous recovery and prednisone . we found that low social boldness score in 16pf was associated with better facial nerve function in de qi group , which was distinguished from the control group . according to cattell 's 16pf , the patients with low social boldness score in 16pf tend to be shy and hesitant , showed more obedience to their acupuncturists . therefore , low social boldness score in 16pf might strengthen therapeutic effects of acupuncture with de qi - related techniques . that was contrary to common belief that introversion usually led to more physical health problems . de qi , an internal compound sensation of soreness , tingling , fullness , aching , cool , warmth , heaviness , and a radiating sensation at and around acupoints , is elicited by manipulation of the needles ( rotated as well as being moved upward and downward ) . the explanation is most likely that the patients with low social boldness score would be more easily to believe the words of the acupuncturists and pay more attention to their body and their sensations elicited by needles , thereby gaining more benefit from de qi acupuncture treatment . this interesting phenomenon calls for further investigations , which ought to shed light on the underlying mechanism of acupuncture . as expected , low hb score on day 1 was associated with better recovery in both groups , such that those with worse facial nerve function at baseline would have worse facial nerve function at month 6 . however , it was found that low vigilance score in 16pf was associated with better outcome in the control group . in contrast to the patients with high vigilance score who were tense and frustrated , those with low vigilance score were more relaxed and tranquil , they could concentrate their attention to the whole acupuncture process and response to their acupuncturists instructions optimistically . it is implied that people who were trusting , unsuspecting , and accepting might have better therapeutic effects mainly from spontaneous recovery and medication . . it might be that they were more inclined to worry about their states as to ask for assistance and be more confident in acupuncturists and their therapeutic schedules . it has been corroborated by systematic reviews that acupuncture is an effective treatment in a wide variety of diseases . nevertheless , few studies have investigated the relationship between different kinds of needle sensations and outcomes of acupuncture . we found that the intensity of needle sensation fullness might predict outcomes in bell 's palsy , which suggested that the intensity of different needle sensations should be paid more attention in the future practice and research . the previous studies found that high expectation for acupuncture might be positive to outcome of pain , and it was more significant in patients receiving verum acupuncture than in patients receiving minimal acupuncture or validated sham acupuncture . that was , positive pretreatment mediators might not have effects on objective outcome during acupuncture treatment . to the best of our knowledge , no rcts have been conducted to investigate the effects of personality traits and attention on the objective functional outcome of acupuncture treatment . we found that some certain personality traits could strengthen the therapeutic effect of acupuncture and attention might not be a significant predictor of outcome after acupuncture treatment . first , personality traits are so complex that we were unable to fully assess one 's personality traits with only one questionnaire . however , we finally chose the 16pf because it was easier to administrate and less expensive . second , the insufficient sample size could have influenced our results . in conclusion , among various kinds of needle sensations , only the needle sensation of fullness could predict better facial function . the needle sensation of fullness claims more of our attention in acupuncture practice and research . personality traits may have different effects on the objective outcome when different acupuncture techniques are used to treat patients with bell 's palsy . among patients treated with the strong stimulation ( de qi ) acupuncture , low social boldness score in 16pf may strengthen the therapeutic effect of acupuncture . in acupuncture treatment and research , fostering such positive patient attitudes ( e.g. , realistic and ethical ) might be one way in which clinicians could further improve their effectiveness . 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 national science fund for distinguished young scholars ( no . 30725019 ) , the national natural science foundation of china ( no . 81030021 ) , and the national basic research program of china ( no . this work was supported by the grants from the national science fund for distinguished young scholars ( no . 30725019 ) , the national natural science foundation of china ( no . 81030021 ) , and the national basic research program of china ( no .

background : it has not been solved what kind of needle sensation might influence outcomes of acupuncture treatment . effects of personality factors on the therapeutic effect of acupuncture have not been investigated . this study aimed to find the effects of the traits of personality on the objective outcome when different acupuncture techniques were used in treating patients with bell 's palsy.methods:we performed a secondary analysis of a prospective multicenter randomized controlled trial of acupuncture for bell 's palsy . patients were randomly assigned to the de qi and control groups , respectively . the primary outcome was facial nerve function at month 6 . the intensity of each needle sensation was rated by a visual analog scale . psychosocial factors were assessed by the pretreatment mediator questionnaire ; 16 personality factor questionnaire ( 16pf ) was used for assessing personality factors and digit cancellation test for assessing attention.results:after 6 months , patients in the de qi group had better facial function ( adjusted odds ratio [ or ] : 4.16 , 95% confidence interval [ ci ] : 2.237.78 ) . path analysis showed that intensity of needle sensation of fullness had direct effect on house - brackmann ( hb ) score at month 6 . in de qi group , the low hb score on day 1 ( or : 0.13 , 95% ci : 0.030.45 ) and the low social boldness score ( or : 0.63 , 95% ci : 0.410.97 ) in 16pf were associated with better facial function . in control group , low hb score on day 1 ( or : 0.25 , 95% ci : 0.130.50 ) , low vigilance score ( or : 0.66 , 95% ci : 0.500.88 ) , and high tension score ( or : 1.41 , 95% ci : 1.121.77 ) in 16pf were related to better facial function.conclusions:the needle sensation of fullness could predict better facial function and personality traits might influence outcomes of acupuncture treatment . both of them should be considered seriously in acupuncture treatment and research .

credited by many for first detailing the lesion in a 1984 case series , oreck described std as a complete disruption of the scapulothoracic articulation with lateral scapular displacement and intact skin . patients often present with soft - tissue swelling of the shoulder , lateral displacement of the scapula , injury to bone ( an acromioclavicular separation , a displaced clavicular fracture , or a sternoclavicular disruption ) and neurovascular injury . when thrown from a motorcycle , a rider may reflexively hold onto the handle bar , leading to a distraction / sheer force strong enough to dislocate the acromioclavicular joint or tear the shoulder girdle musculature . patients with an std injury often have poor outcomes , mostly stemming from other associated injuries resulting from the causative high - energy trauma . flail extremity has been reported in 52% of patients and early amputation required in 21% . approximately 10% of patients with std die from their concomitant traumatic injuries rather then std itself . the following case describes a 25-year - old female who suffered an std injury associated with a debilitating head injury ; a complete axillary artery , axillary vein , and brachial plexus avulsion with significant blood loss . the presented patient is an intoxicated 25-year - old female who was brought to our trauma bay after an auto collision in which the patient was a restrained driver . on arrival , her glasgow coma scale was four and she required immediate intubation . she was transfused two units of packed red blood cells . due to persistent hypotension and multiple injuries initial chest and pelvis radiography revealed left clavicle , left scapula , left humerus fractures along with a left pelvic fracture [ figures 1 and 2 ] . note proximal 1/3 left clavicular fracture in 100% displacement with posteroinferior angulation left midshaft humerus fracture the patient was otherwise healthy prior to the accident with no history of medical problems . she has a history of a cesarean section 3 years prior and bilateral tubal ligation . initial laboratory values on admission include the following : white blood cell count 6.3 10/mm , hemoglobin 5.8 g / dl , hematocrit 19.9% , platelets 274 10l , sodium 140 meq / l , potassium 3.5 meq / l , chloride 108 meq / l , carbon dioxide 15 meq / l , urea nitrogen 10 mg / dl , creatinine 0.9 mg / dl , glucose 255 mg/ dl and blood alcohol level 0.23 . she was acidotic , with a ph of 6.98 and carbon dioxide tension 42 mmhg , oxygen tension 258 mmhg , bicarbonate 10 meq / l and base deficit -20 meq / l with lactate of 6.8 intraoperative evaluation revealed a left shoulder that was completely dissociated from the musculoskeletal structures and held in place only by overlying skin . given the continuously expanding left shoulder hematoma ; surgical control of the proximal subclavian artery was obtained via a left thoracotomy . once vascular control was obtained the subpectoral space was explored and contained a large hematoma , which was initially packed and later re - explored . the proximal and distal arterial stumps were identified and found to be recoiled in the proximal arm and controlled . further dissection and irrigation revealed that the left brachial plexus was completely avulsed , and that only dozens of frayed , stretched filaments remained . once hemostasis was obtained , a left chest tube was placed and the skin closed . an exploratory laparotomy was also performed which was grossly negative . in anticipation of a large volume resuscitation requirement , the patient 's abdominal fascia was left open , and the abdominal contents contained within a bogota bag allowing for a future second look . the patient was then taken to the surgical intensive care unit ( sicu ) for observation and continued resuscitation . once stabilized the patient was taken to the ct scanner to complete the trauma evaluation . she was found to have bilateral parieto - occipital and cerebellar low - density changes consistent with ischemia . a ventriculostomy was placed by neurosurgery ; however , due to increasing intracranial pressures , the patient required emergent craniectomy . on postoperative day number one , the patient was taken back to the operating room for re - exploration of the left shoulder incision . on postoperative day number two , the left upper extremity was subsequently amputated to the level of the humeral fracture near the deltoid insertion and the abdomen closed . after 5 days of observation in the sicu , the patient was brought back into the or for amputation revision and glenohumeral disarticulation , in which the distal end of the fractured clavicle and free - floating segment of the left acromion were resected . the remaining brachial plexus components were transected and allowed to retract into their medial stumps , and the left pectoralis major was brought up to the distal end of the medial clavicle to control the bone . full - thickness skin flaps were raised anteriorly and posteriorly to the level of the glenohumeral joint facilitating a tension free closure . on hospital day number seven , the patient was considered stable for transfer and taken to her hmo - approved hospital . at the time of this writing she is currently at a skilled nursing facility and requires full care . the above case describes the tragic injuries of a 25-year - old female involved in a rollover motor vehicle accident . this patient presented with the classic findings commonly associated with std injuries , involving a violent lateral rotational displacement of the shoulder girdle , likely caused by the vehicular rollover . the patient 's presentation included massive swelling , palpable hematoma , gross shoulder subluxation , weakness and tenderness highly suggestive of std . the diagnosis of std is based primarily on history , physical exam and radiographic findings . a high index of suspicion for std the frequently associated distracting injuries in severely injured patients make this a frequently " missed injury " . these patients often present with decreased mental status or require intubation on arrival , which can limit the amount of information obtained from the patient , and further delay the diagnosis . also , initial chest radiographs are often not properly aligned in a true anteroposterior position , making it difficult to identify the lateral deviation of the scapula , one of the hallmark signs of this condition . on primary survey , massive soft tissue swelling caused by hematoma and edema may be the only visible signs . additionally , the patient may present with shoulder instability , tenderness to palpation , pulselessness , weakness and numbness of the upper extremity . a common mistake is to attribute pulselessness to a more distal injury , when in fact a more proximal injury may exist . frequently , patients will maintain distal pulses due to the extensive collateral network around the shoulder girdle and its protection against limb ischemia . the scapula - index is calculated by measuring the distance from vertebral spinous processes to the medial border of the scapula bilaterally and dividing the value of the injured side by the value on the non - injured side . 0.04 . when std is suspected , emergent subclavian , axillary and brachial arteriography is essential to evaluate the vascular structures , especially if the patient has a pulseless upper extremity or is unstable . in 1977 , damschen described a classification system for std based on clinical presentation type i std is classified as musculoskeletal injury alone ; iia musculoskeletal injury with vascular disruption ; iib musculoskeletal injury with neurological impairment ; and iii musculoskeletal injury with neurologic and vascular injury . they found in their case series that the presence of a complete brachial plexus avulsion is predictive of a poor functional outcome . therefore , a fourth type was added to the damschen classification of std ; those patients with complete brachial plexus avulsion . initial management of traumatized patients should focus on cardiopulmonary resuscitation and stabilization of the patient . given the high likelihood of associated life - threatening injuries with std , these injuries must first be evaluated . care must be taken not to overlook a potential std injury , and in the acute setting , the control of vascular injuries and ischemic complications should be a priority in patients with std . vascular injury should prompt immediate surgical intervention with repair performed within 4 - 6 hours of injury . autologous saphenous vein graft is the preferred graft ; however , ptfe is acceptable if there is a significant size mismatch or prolonged ischemia is present making repair technically difficult . venous injuries can be ligated in most cases with little consequence , though rare reports of upper extremity edema have been reported . the subacute phase , 24 hours to 2 weeks after the injury , focuses on management of the sequelae of the initial injury and consideration for amputation . if a complete injury to the brachial plexus exists , a patient may benefit from glenohumeral disarticulation or above - elbow amputation . complete brachial plexus avulsion has occurred , a primary above - elbow amputation is preferred . delay in amputation is associated with myloglobinuria , hyperkalemia , vascular thrombosis , and increased risk for pressure ulcers in the flail extremity . families are also more likely to refuse amputation if it is suggested late . the type iv std injury that we present above was catastrophic for this patient and led to an upper extremity amputation . this 25-year - old female suffered complete brachial plexus avulsion and complete arterial and venous vascular compromise . given the complexity of this patient and the lack of specific guidelines in the literature , this patient was treated with ligation of vascular injuries and a two - staged upper extremity amputation . as increasing numbers of std injuries present , the body of literature will help clarify the most ideal treatment modalities .

scapulothoracic dissociation ( std ) is a devastating consequence of high - energy trauma sustained by the shoulder girdle that can easily result in rapid mortality . since described by oreck et al . in 1984 , std has been reported in a handful of journals and individual case series , though is still considered a rare occurrence in the context of shoulder injuries . in this report , we examine the case of a 25-year - old female involved in a high - speed rollover auto accident . unique to this case was the discovery of a completely transected axillary artery and vein with intracorporeal bleeding and complete avulsion of the ipsilateral brachial plexus requiring immediate ligation of the vessels followed by interval above - elbow - amputation and later glenohumeral disarticulation .

in today 's healthcare system , as the environment for care shifts to maintaining older adults in the community , more families are assuming responsibility for the care of elderly family members at home . the importance of families as a healthcare resource is predicted to significantly increase during the next century . families often take on the responsibilities of caring for sick or disabled elderly family members at home with minimal or no preparation or knowledge about the person 's health problems . caring for a person with alzheimer 's disease ( ad ) is particularly demanding as the needs for care escalate as the disease progresses . family caregivers may also experience stress because their family roles are changing ; they face the prospect of losing a family member ; and they may feel unprepared to meet caregiving demands . studies have indicated that family caregivers of persons with dementia are at risk for depression , poor health , diminished quality of life , and increased mortality [ 46 ] . family caregivers often feel powerless about their ability to influence their family member 's recovery because ad is debilitating and fatal . despite these potential adverse effects , some family caregivers view certain aspects of caregiving in a positive light and handle the stresses associated with caring for a family member with dementia with little difficulty [ 710 ] . some families report that being involved in the care of a person with dementia results in an increased sense of purpose . researchers have shown that family caregivers of persons with dementia have multiple needs , which include but are not limited to acquiring information about the health issues of the person with dementia ; developing problem - solving skills so that they may help to achieve positive outcomes for the care recipient as the disease progresses ; learning strategies to prevent and manage behavioral symptoms ; learning to communicate effectively with the person with dementia and others ; and planning for the future . interventions to alleviate distress among family caregivers of persons with dementia include three overarching , empirically based treatments : ( 1 ) psychoeducational skill building that focuses on increasing caregiver knowledge of a specific disorder and/or teaching the caregiver specific skills ; ( 2 ) specific forms of individual or group therapy or counseling ( e.g. , cognitive - behavioral or psychodynamic ) ; and ( 3 ) two or more conceptually different approaches integrated in a single intervention package , such as combining skill training with a support group or with family counseling . implemented a one - to - one home health nurse - delivered instructional program teaching behavior management to family caregivers in the home setting using the progressively lowered stress threshold theoretical framework . gitlin and associates developed an occupational therapy - based skill - building program for home caregivers that demonstrated sustained positive effects on caregiver skills and affect . ostwald and associates have described a group psychoeducational program that resulted in reduced depression and burden among caregivers . hepburn et al . have reported on a psychoeducational program on decision - making training that showed reduced caregiver distress . they also reported on the savvy caregiver program , a psychoeducational program delivered in different sites in the united states , which increased caregiver competence and mastery and reduced distress . interventions that combine education and an individualized counseling program have shown success in delaying institutionalization of persons with ad . a national multisite program ( resources for enhancing alzheimer 's caregiver health ii ) examined the effects of a multicomponent intervention using skills training , education , and telephone support , in a study including 600 black , hispanic , and white / non - hispanic white participants . this study showed significant benefit to most caregivers , but not all caregivers achieved the same results . specifically , nonspouse black / african american caregivers did not benefit as much as other groups . this finding indicates that additional study is needed to determine who benefits most from which type of intervention . most earlier intervention studies in caregiving have focused on stress and caregiver deficits . this study was designed to address the gap in the current state of intervention research with family caregivers of persons with dementia . specifically , the present study targets caregivers ' resourcefulness , a protective factor that may buffer the effects of chronic stress on caregivers ' health . rosenbaum defined learned resourcefulness as a repertoire of coping strategies to control and manage internal cognitive and emotional responses to perceived stressors . resourcefulness is linked to self - control skills , which are related to cognitive - behavioral strategies designed to manage stressful circumstances . techniques include cognitive reframing , problem solving , and the regulation of emotions and cognitions . interventions to enhance resourcefulness in family caregivers have multiple components involving psychoeducational skill building to increase knowledge about ad ; teaching specific skills such as problem solving ; teaching skills to manage stress and deal with the patient 's behavioral problems ; and group support . the development of the intervention reported on here was guided by three theoretical approaches : rosenbaum 's conceptual model of learned resourcefulness , johnson 's self - regulation theory , and burr 's interactional role theory . self - regulation theory postulates that an individual 's cognitive schema of an impending stressful event is instrumental to processing information as the event unfolds and to guiding responses ( emotional outcomes ) and behaviors ( functional outcomes ) during the event . the cognitive schema is defined as an image or picture in the mind of an event or situation that includes knowledge about what is happening in the event , as well as plans on how to deal with the event in order to achieve the desired outcomes . providing information about dementia will facilitate the caregiver 's forming a clear , unambiguous schema about what to expect as the disease advances in the person with dementia . anticipatory preparation for new roles is a key concept in burr and colleagues ' interactional role theory related to family processes . roles are defined as goal - oriented patterns of behavior , and interaction between role partners is emphasized . archbold et al . applied burr 's work to family caregiving for elders in the home setting and developed a nursing intervention program ( prep , for preparedness , enrichment , and predictability ) that uses a mutual agreement method . in this program , caregivers sign a contract with the nurse to work on two problem areas of greatest concern , caused by the patient 's condition . then specific information for each problem was provided and list of suggested care activities that caregivers could select based on their preference . building on the work of archbold and associates , a mutual agreement method was developed based on role theory 's assumptions that how well a caregiver performs in a role is associated with clarity of role expectation . an easier role adaptation is associated with well - defined role tasks . in summary , based on self - regulation theory , we postulated that providing educational information about ad to family caregivers would facilitate the formation of a clear cognitive schema that would strengthen caregivers ' understanding about disease - associated changes as well as their ability to interpret these changes and adapt to them . subsequently , understanding disease - related changes , cognitively reframing issues , and learning problem - solving skills might help caregivers moderate their responses , leading to less frustration , anxiety , or depression . we also postulated that as a result of improved emotional outcomes and strengthened role outcomes for caregivers , outcomes for the person with dementia , such as frequency of behavior problems , would be reduced . the purpose of this study was to evaluate the effects of a multicomponent intervention that included teaching resourcefulness and providing group support on outcomes of both persons with ad and their family caregivers . we examined the following research questions : what are the effects of the intervention program on family caregivers ' emotional outcomes ( anxiety , depression ) immediately ( 6 weeks ) after the intervention and 12 weeks after intervention ? what are the effects on caregivers ' role outcomes ( reward , strain , mutuality , and preparedness ) immediately ( 6 weeks ) after the intervention and 12 weeks after the intervention ? finally , what are the effects on frequency of behavior problems in the care recipient , immediately ( 6 weeks ) after the intervention and 12 weeks after the intervention ? we conducted a randomized clinical trial using the stratification variables of race ( white versus african american ) and resourcefulness at baseline ( high or low ) . stratification by race was based on previous study that showed african american caregivers are more resourceful than white caregivers . outcomes were measured at baseline , 6 weeks ( intervention endpoint ) , and 12 weeks after the intervention . the stratified random sample included 102 family caregivers ( 50 experimental and 52 control subjects ) who lived with and provided care to a person with ad . to be included , women family caregivers had to be living with a person diagnosed with ad and providing at least 8 hours of care for the person weekly ; had to be able to read , understand , and speak english ; and had to be cognitively intact , as determined by a score of at least 7 of 10 on the short portable mental status questionnaire . potential participants with family members diagnosed with probable ad were referred to the study by a neurologist research team member ( cfl ) and called the principal investigator ( ewg ) if they were interested in participating . a neurologist ( cfl ) who isa coinvestigator verified that patients had a probable diagnosis of ad using the nincds / adrd criteria for probable ad . patients were excluded if they were bed bound or had parkinson 's disease , multi - infarct dementia as a primary diagnosis , schizophrenia , or bipolar disorder . following approval for protection of human subjects and informed consent procedures data were collected during individual , face - to - face , structured interviews conducted by trained data collectors in a private setting at a mutually agreed upon time . during the initial interview , information on demographic characteristics ( age , gender of patient , and race ) and type of health conditions was obtained for both caregivers and care recipients , and the caregivers completed structured measures of the study variables . baseline data on level of and race ( african americans ; whites ) were used to randomly assign caregivers to the control ( standard care ) or the intervention group . beginning within 2 weeks of the initial interview , subjects met in groups of five or seven for 2 hours weekly , over the course of 6 weeks . within a week after the intervention concluded , participants were interviewed a second time , and a third interview took place 12 weeks after the intervention concluded . the study examined the effects of enhancing resourcefulness skills on family caregivers ' emotional outcomes ( decreased anxiety , depression ) , role outcomes ( increased preparedness , reward , mutuality ; decreased role strain ) , and care recipient outcomes ( decreased frequency of behavior problems ) . demographic information including age , gender , ethnicity , type of relationship ( spouse versus nonspouse ) , education , employment status , marital status , annual household income , and health conditions for family caregivers and care recipients , and clinical variables were obtained . the instruments used to measure the study variables secondary outcome measures include caregiver preparedness , role reward , caregiver strain , quality of caregiver - care recipient relationship ( mutuality ) , and frequency in occurrence of behavior problems . the state - trait anxiety inventory , a - state ( stai ) , a 20-item tool , was used to measure family caregivers ' current anxiety level . for this sample , internal consistency reliability for the scale was 0.93 . depression was measured using the center for epidemiologic studies depression scale ( cesd ) , a 20-item self - report scale that represents a symptom cluster consisting of negative affect , positive affect , interpersonal problems , and somatic activity . caregivers respond to 20 statements ( based on feelings during the past week ) using the response categories 0 ( rarely or none ) , 1 ( some or a little ) , 2 ( occasionally or moderate ) , and 3 ( most or all ) . the ces - d scale has been widely used with older adults , demonstrating good internal consistency with cronbach 's alphas in the 0.86 to 0.92 range in caregiving samples . for this sample , internal consistency reliability for participants indicate the degree to which each item describes their behavior on a 6-point scale , ranging from 3 ( very much like me ) to 3 ( not at all like me ) . scores may range from 180 to + 180 ; higher composite scores , after reverse scoring of 11 items negatively phrased , indicate greater resourcefulness . internal consistency estimates ranging from 0.75 to 0.85 have been reported in studies with older adults [ 29 , 34 , 35 ] . for this sample , internal consistency reliability for preparedness , defined as a caregiver 's perceived readiness to provide care , was measured using the preparedness for caregiving scale ( stewart & archbold , 1994 , family caregiving inventory , unpublished ) . the family caregiver rates each of the nine items on a 5-point scale that ranges from 0 ( not at all prepared ) to 4 ( very well prepared ) . the summed scored has a possible range from 0 to 36 , with higher scores indicating higher preparedness levels . an example of an item on the preparedness for caregiving scale is how well prepared do you think you are to take care of your family member 's emotional needs ? internal consistency reliability for the scale was 0.84 in this sample . family caregivers ' role reward was measured using the family role reward scale ( frrs ) ( stewart & archbold , 1994 ) . family caregivers rate each of the items on a 5-point scale that ranges from 0 ( not at all ) to 4 ( a great deal ) . an example of an item on the frrs is does caring for your family member allow you to preserve his / her integrity ? cronbach 's alpha was 0.93 in this study . family caregiver strain was measured using the caregiver role strain global strain subscale ( crs ) ( stewart & archbold , 1994 ) . family caregivers rate each item on a 5-point scale that ranges from 0 ( not at all ) to 4 ( a great deal ) . an example of an item on the crs is how often would you say that taking care of your family member is very difficult ? cronbach 's alpha was 0.86 in this study . the quality of the relationship between the caregiver and the care recipient was measured using the mutuality scale ( ms ) ( stewart & archbold , 1994 ) . family caregivers rate each of the 14 items on a 5-point scale that ranges from 0 ( not at all ) to 4 ( a great deal ) . an example of an item on the ms is to what extent do the two of you laugh together ? behavior problems were measured using the 24-item revised memory and behavior problem checklist ( rmbpc ) . this instrument measures the frequency of the care recipient 's problem behaviors in the following domains : disruptive behavior ( wandering , aggression ) , memory - related behavior ( repeating questions and stories ) , and depression . family caregivers participated in six group sessions of resourcefulness training , in groups of five to seven caregivers who met for 2 hours weekly . a modified version of the resourcefulness training developed by zauszniewski ( personal communication ) and the problem - solving training developed by kurylo and associates were used . the training was a short - term , structured , time - limited intervention that taught and reinforced the cognitive - behavioral skills constituting problem identification , coping strategies , problem solving , priority setting , and decision making . the training includes six modules using focus as an acronym for ease in recalling the components ( focus , optimism , creativity , understanding , and solution ) . the first component involves description of a problem identified in a card - sorting procedure based on the revised memory problem checklist . the caregiver is asked to rank - order concerns or problem from the most important to the least important . family caregivers are asked to select two problem areas to focus on in providing care based on their own preferences and on the abilities of the person with dementia . the caregiver is then assisted in articulating a specific attainable goal for the most important problem . the second component is to assist the caregiver in developing a sense of optimism ( o in focus ) regarding her abilities to problem solve by instilling a belief that she is sufficiently skilled to solve the problem and instilling a sense of motivation . the training stresses the importance of realistic expectations about the time and effort that will be necessary to identify and use chosen strategies . ( c in focus ) by helping her brainstorm multiple solutions to the identified problem . caregivers are instructed to think of as many solutions to the problem as possible and to write each solution down on a worksheet . the fourth component involves understanding ( u in focus ) the patient 's preference in considering which solutions to implement . before deciding on a solution , the caregiver is encouraged to consider the potential outcomes of the chosen solutions and weigh the cost and benefits of each . the caregiver rates the likelihood that she will implement each solution as 0 ( not at all likely ) , 1 ( somewhat likely ) , or 3 ( very likely ) . the fifth component is the implementation of the solution ( s in focus ) and evaluation of how effective the solution was in solving the problem . this self - monitoring component is important to promote understanding about what made the chosen solution effective or ineffective and how to implement similar or alternative solutions in future problem situations . the intervention was delivered by a registered nurse , whose own training was based on a training manual . the nurse was trained by the principal investigator using a one - on - one review of the intervention and was required to demonstrate the ability to deliver the intervention in a group of three caregivers who were not included in the study . the nurse and the principal investigator met monthly to discuss the intervention process and activities , in order to prevent intervention drift . overall , the nurse completed the modules in 48 weeks ( range , 130 to 150 minutes per module ) . although the intervention module was intended to last for 105 minutes per module , the average amount of time spent to deliver the intervention was 138 minutes ( s.d . treatment fidelity related to receipt of the training intervention was determined by comparing baseline scores on a measure of resourcefulness , the scs , with scores obtained immediately after intervention and 12 weeks after the intervention in family caregivers who participated in the intervention ( n = 46 ) . among those who received training , mean scores on the scs showed an increase in resourcefulness . paired t - tests showed significant improvements in resourcefulness from baseline to 6 weeks ( t(1 , 49 ) = 3.62 , p < 0.001 ) , although no significant increase was found from baseline at 12 weeks ( ( t(1 , 46 ) = 1.17 , p = 0.25 ) . similar improvements were not seen in the caregivers who received the standard care at 6 weeks t(1 , 44 ) = 1.50 , p = 0.14 ) or at 12 weeks ( t(1 , 36 ) = 1.023 , p = 0.31 ) . thus , the fidelity of the resourcefulness training intervention was demonstrated immediately after the training , although the improvement in resourcefulness was not sustained at 12 weeks after the intervention . after they completed the baseline data collection , these caregivers received a binder that included information on community resources , handouts about ad , and new scientific findings . data were collected 6 weeks after baseline and 12 weeks after the second data collection period . with these families , there was no discussion on health - related matters or coping strategies before , during , or after the data collection . descriptive statistics , including means and standard deviations , were examined for resourcefulness as well as for outcome variables of interest to determine the shape of distribution at baseline and after intervention . correlations among the study variables were examined to determine relationships among these variables . between group differences for primary and secondary outcome measures were analyzed . the baseline value corresponding to each response variable was used as a covariate in univariate analyses . effect sizes were calculated to assess the magnitude of the effects of the intervention on the dependent variables . effect size was calculated by subtracting the mean of the control group from the mean of the intervention group and then dividing by the pooled standard deviation . values of 0.2 , 0.5 , and 0.8 were defined as small , medium , and large effects , respectively . a meaningful positive effect size was defined as one that was both greater than 0.20 and in favor of the intervention . of the 138 family caregivers screened , 19 ( 14% ) were ineligible and 14 ( 10% ) did not want to participate . of the remaining 105 who underwent baseline assessment , three changed their minds and decided not to participate and 102 ( 97% ) were randomly assigned to the intervention group ( 50 ) or to the control group ( 52 ) . of the 50 family caregivers assigned to the intervention group , 49 ( 98% ) completed the resourcefulness training and the second data collection interview ; 46 ( 92% ) completed the third interview . one caregiver died , one experienced declines in health , and two institutionalized the person with dementia . in the control group , only 44 ( 84% ) of 52 family caregivers completed the second data collection interview ; 36 ( 69% ) of family caregivers completed the third interview . five caregivers lost interest in the study , four institutionalized the person with dementia , three relocated out of state , and four did not continue caregiving . the attrition rate was significantly higher in the control group than in the intervention group . there were no differences between those who stayed in the study and those who did not with regard to caregiver demographic characteristics such as race , age , and perceived health . however , there were differences in caregivers ' years of education and relationship to the person with dementia . caregivers with less than high school education were more likely to drop out of the study than were caregivers with high school or higher education . additionally , nonspouse caregivers had a higher dropout rate than spouse caregivers . family caregivers in the resourcefulness training and the usual - care group did not differ from each other at baseline in race ( = 1.18 , p = 0.31 ) , age ( t(1,100 ) = 1.44 , p = 0.15 ) , household income ( = 1.33 , p = 0.57 ) , number of hours per week spent in caregiving ( t(1 , 100 ) = 1.69 , p = 0.09 ) , length of time in caregiving ( t(1 , 100 ) = 0.136 , p = 0.89 ) , relationship to the care recipient ( = 3.65 , p = 0.09 ) , or number of chronic conditions reported ( t = 51 , p = 0.61 ) . the sample was composed of 102 female caregivers ( 97% ) including 58 african americans ( 57% ) and 44 whites ( 43% ) . their ages ranged from 35 to 85 years , with a mean age of 60 years . persons with dementia ranged in age from 54 to 95 years , with a mean age of 80 years ( standard deviation = 9.02 years ) . on average , the most frequently reported conditions were high blood pressure ( n = 41 ; 40% ) , arthritis ( n = 26 ; 25% ) , diabetes ( n = 16 ; 16% ) , emotional problems ( n = 16 ; 16% ) , asthma ( n = 11 ; 11% ) , circulatory conditions ( n = 8 ; 8% ) , and cancer ( n = 6 ; 6% ) . despite their health problems , the majority of family caregivers ( n = 79 ; 77% ) perceived their health to be good or excellent . = 0.80 ) indicated that they were fairly well prepared to care for the person with dementia . table 2 presents correlations on resourcefulness and outcome measures : depression , anxiety , preparedness , strain , mutuality , reward , and frequency of behavior problems . resourcefulness was inversely related to anxiety and depression ( p < 0.00 ) , positively related to role reward ( p < 0.00 ) and preparedness ( p < 0.002 ) . the groups did not differ in their baseline measures on primary outcomes of caregivers ' learned resourcefulness ( t = 1.01 , p = 0.37 ) and depression ( t = 1.61 , p = 0.07 ) . however , there was a significant difference in anxiety ( t = 2.56 , p < 0.03 ) between the groups . specifically , at baseline , family caregivers in the control group reported higher anxiety symptoms compared with caregivers in the intervention group . the groups did not differ in their baseline measures on secondary outcomes of preparedness ( t = 1.36 , p = 0.19 ) , mutuality ( t = 0.33 , p = 0.74 ) , role reward ( t = 0.078 , p = 0.93 ) , or role strain ( t = 0.066 , p = 0.94 ) or in the frequency of behavior problems in the person with dementia ( t = 0.10 , p = 0.92 ) . table 4 presents adjusted means , standard deviations , and effect sizes for primary and secondary outcomes at weeks 6 and 12 . at week 6 ( immediately after intervention ) , family caregivers in the intervention group reported significantly more resourcefulness skills ( f(1 , 91 ) = 13.54 , p < 0.001 ) with a medium effect ( 0.54 ) compared with family caregivers in the control group . family caregivers in the intervention group showed significantly higher anxiety symptoms at 6 weeks ( immediately after intervention ) compared with caregivers in the control group ( f(1,91 ) = 4.90 , p < 0.042 ) , with an effect size of 0.44 . family caregivers in the intervention group did not differ significantly on caregiving preparedness from the control group ( f(1,91 ) = 0.817 , p = 0.14 ) , with an effect size of 0.26 . there were no immediate effects on mutuality , reward , strain , or frequency in occurrence of behavior problems . at 12 weeks after intervention , there was a significant difference between groups on resourcefulness ( f(1 , 80 ) = 3.58 , p = 0.048 ) although the effect size was small ( 0.36 ) . anxiety decreased in family caregivers in the intervention group ( mean = 36.9 ) whereas it increased in the control group ( mean = 39.7 ) , although the difference was not significant ( ( f = 1.713 , 80 , p = 0.194 ) and the effect size was small ( 0.29 ) . caregivers in the intervention group showed higher scores in preparedness compared with the control group ( f(1 , 80 ) = 3.37 , p = 0.041 ) , with an effect size of 0.41 . there were no treatment effects and effect sizes were less than 0.2 for secondary outcomes on mutuality , reward , role strain , and frequency in occurrence of behavior problems . all family caregivers in the intervention program reported that the intervention was helpful for them and for the person with dementia . as one caregiver reported , it helped me to understand how alzheimer 's disease affects my husband 's behavior . another caregiver said , you would all be so proud of me , i called the alzheimer 's association and talked to the staff to ask about resources in my community . i would never have done this if not for this program because i did not know a thing about alzheimer disease . many participants in the control group reported that they benefited some from participating in the study . this finding may indicate that even the task of responding to questionnaires during the data collection at baseline , 6 weeks , and 12 weeks had positive effects on caregivers , perhaps because they felt they were being helpful by participating in research . this study demonstrated that positive effects on role outcome ( preparedness ) and anxiety can be achieved in family caregivers of persons with dementia through use of the interventions described here . the group intervention was successful in increasing resourcefulness in caregivers immediately at 6 weeks and in decreasing anxiety in caregivers and increasing caregiver preparedness at 12 weeks after the intervention . the findings also showed that for resourcefulness to be sustained , a booster dose in training may be needed . this study represents the first attempt to examine the immediate and extended ( over 12 weeks ) effects of teaching resourcefulness on caregiver role outcomes ( preparedness and mutuality ) , emotional outcomes ( anxiety and depression ) , and patient outcome ( frequency of behavior problems ) . although baseline data revealed that greater resourcefulness was significantly associated with lower anxiety and depression , the intervention was found to have no effect in reducing depression . this lack of effect may be due to the caregivers ' relatively low scores on depression upon study enrollment . because they were not depressed caregivers in the intervention group reported increased anxiety symptoms compared with control - group caregivers at 6 weeks . this outcome is surprising because we expected that anxiety would decrease as resourcefulness was enhanced . the increase in caregivers ' anxiety in the intervention group may have resulted from their newly acquired knowledge about the disease and the uncertainty of the condition . this new knowledge may heighten the caregiver 's anticipation and feelings of loss as they understand more about the disease and the course of decline that it will follow . this result is consistent with the findings of graham et al . and proctor et al . , indicating that knowledge about ad increases anxiety . another possible reason for the increase in anxiety in caregivers in the intervention group at 6 weeks may be the timing of data collection , which coincided with the completion of the intervention program . family caregivers who participated in the intervention group developed a sense of belonging and support . the termination of the program may have triggered anxiety and a sense that they now had to move forward by themselves . although a session was devoted to stress management , relaxation exercises need to be reinforced in each session . however , at 12 weeks after intervention , family caregivers ' anxiety decreased , while it increased in the control group . the findings also showed a significant difference in preparedness between the groups at 3 months after the intervention . the delayed effects on caregiver preparedness at 12 weeks after the intervention suggest that preparedness skills may take time to be fully integrated into a caregiver 's repertoire . as a consequence , when caregiver preparedness increased 12 weeks after the intervention , their feelings of anxiety decreased . the findings support the self - regulation theory that providing educational information about ad to family caregivers facilitated the formation of a cognitive schema that strengthened caregivers ' understanding about disease - associated changes , how to interpret these changes , and how to adapt to them . additionally , the findings partially support rosenbaum 's conceptual model of learned resourcefulness , suggesting that the skills constituting resourcefulness can be taught in interactions with others . in addition , the positive effects of learned resourcefulness training found in this study are consistent with rosenbaum 's view of the beneficial effects of learned resourcefulness on the regulation of emotions . first , study results demonstrate that family caregivers can be taught resourcefulness successfully in a small group in the community . the study suggests that knowing more about ad may lead to anxiety . providing educational program about alzheimer 's disease resourcefulness as a training program was delivered in a group form that provided support to family caregivers and involves problem solving , cognitive reframing , and learning self - regulation of emotional response ( depression and anxiety ) . this training will benefit family caregivers who are anxious , depressed , less prepared , or less resourceful . second , there is a need to assess family caregiver 's preparedness when they assume the role of caregivers for person with dementia . the delayed effects on caregiver preparedness at 12 weeks after the intervention suggest that caregiver preparedness may take time to be fully integrated into a caregiver 's repertoire . several limitations of this pilot study include the small sample size and the exclusion of male caregivers . the small sample size limited the power to detect significant differences between the study groups on some of the patient and caregiver outcomes . attrition is a potential problem in studies in which outcome measures are obtained over time . we addressed this challenge by recruiting more participants than needed for the desired statistical analysis . in addition , we may have reduced the attrition rate by calling caregivers on the phone as a reminder for the upcoming intervention session and conducting home visits rather than mailing questionnaires in the follow - up phase . in this study , we used ano - treatment condition as a control , which included provision of standard materials because that is the only intervention that the vast majority of caregivers in the population receive . this lack of provision of treatment may account for more attrition in this group . in future larger studies , it will be appropriate to include a credible attention - placebo control . despite these limitations , the results of this study provide support for the effectiveness of the intervention program and its theoretical framework . findings from this study hold the promise that persons with dementia and their family caregivers can benefit from a theoretically driven , reproducible intervention aimed at improving the caregivers ' resourcefulness .

this pilot randomized trial tested an intervention aimed at enhancing resourcefulness in family caregivers of persons with dementia , postulating that caregivers ' emotional outcomes ( anxiety and depression ) and role outcomes ( reward , strain , mutuality , and preparedness ) would be improved , and problem behaviors in the care recipients ( persons with dementia ) would be reduced as a result of the intervention . subjects were stratified by race ( white or african american ) and by baseline resourcefulness ( high or low ) . family caregivers were randomly assigned to an intervention group in which subjects attended six resourcefulness training sessions , meeting for 2 hours weekly over 6 weeks , or to a control group that received no treatment . small to medium effects were shown for the intervention program on resourcefulness , anxiety , and preparedness of the caregivers and on frequency of behavior problems in the care recipients . caregivers in the intervention group reported significantly more resourcefulness skills , with a medium effect at week 6 and a small effect 12 weeks later , compared with the control group . persons with dementia had fewer behavior problems in the intervention group compared with control , although the difference was not significant . caregivers ' anxiety was reduced in the intervention group at 12 weeks .

alopecia areata ( aa ) is one among the causes of non - cicatricial alopecia in children which comes next only to non - inflammatory tinea capitis and telogen effluvium . though it affects all age groups , authentic statistical data regarding the condition in pediatric age group are lacking . aa is considered to be of autoimmune etiology affecting hair follicles and nails in genetically predisposed individuals . a rare occurrence of concomitant aa in two siblings is presented in this article along with the special aspects of aa in children . two siblings aged three and five years were presented to the outpatient department with complaints of asymptomatic patchy alopecia of the scalp of six- and three - month duration , respectively . their father was a daily laborer and the family has recently migrated from north india . there was no history of similar illness among the family members . the physical examination suggested grade three malnutrition in the girl and grade one in the boy . scalp examination of the girl showed multiple circular patches of non - scarring alopecia [ figure 1a ] with exclamation mark - shaped hairs near the periphery . the male sibling was having two diffuse patches of decreased hair density with ill - defined borders along with easy pluckability of hair [ figure 1b ] . a provisional diagnosis of patchy and reticulate type of aa was made for the girl and the boy , respectively . light microscopy of the hair roots showed predominantly dystrophic hair showing tapered roots with absent root sheath . thyroid function tests were done for both the kids including autoantibody profile for thyroid which were found to be normal . histopathological examination of the scalp confirmed the diagnosis which showed peribulbar lymphocytic infiltration with reduction in the number of hair follicles . both of them are under follow - up for past three months showing progressive growth of hair in the affected areas . ( a ) multiple circular patches of alopecia areata in the girl ; ( b ) reticulate pattern of alopecia areata in the boy aa is an autoimmune disease which often gets triggered by certain environmental factors in genetically predisposed individuals . the loss of hair in aa is thought to be due to collapse of the normal immune privilege of anagen hair bulb and subsequent attack by cd4 + and cd8 + t lymphocytes . this is responsible for the lymphocytic infiltrate seen around the anagen hair bulbs in histopathological specimens . the immune - mediated attack can also involve follicular keratinocytes , melanocytes , or dermal papillae . till date , there are only very few reports of concomitant presentation of aa in siblings in the literature . concordance rate of 55% was reported among monozygotic twins for aa , whereas such an association is not seen among dizygotic twins or siblings . this high concordance among identical twins is by virtue of sharing the same hla types . the strong hla links , especially with class ii alleles , drb1 - 1104 and dqb1 - 0301 , suggest the importance of hereditary factors in aa . several studies have suggested that the gene loci for aa are possibly on 2 , 6 , 10 , 14 , 16 , 18 , and 21 chromosomes . the significant association to down syndrome is linked to the presence of aa gene locus in chromosome 21 . direct association of malnutrition and aa is not reported yet even though malnutrition can produce hair changes like diffuse alopecia , hypopigmentation , thinning , and brittleness of hair . the probable triggering factors for this rare presentation in the siblings may be the stress involved in adjusting with the new environment as well as the social and financial reasons responsible for malnutrition . the major differential diagnoses for our cases include non - inflammatory tinea capitis , trichotillomania , and telogen effluvium . tinea capitis is associated with severe itching and scaling of the scalp along with positive fungal elements in potassium hydroxide mount of scalp scrapings . trichotillomania has localized short hairs of uneven length with perifollicular erythema following compulsive or habitual pulling by an emotionally disturbed child . telogen effluvium has a history of major physical or psychological stress which leads to significant shift to telogen phase leading to diffuse hair loss . in contrast to all these , scalp in aa is soft , smooth , and non - pruritic . the different morphological patterns of aa of the scalp are patchy , reticulate , diffuse , ophiasis , ophiasis inversa ( sisapho ) and totalis . among these , the diffuse variety and the reticulate pattern need histopathological confirmation to rule out telogen effluvium and malnutrition induced diffuse alopecia . the bad prognostic features include early onset , recurrent episodes , extensive nail involvement , ophiasis pattern , and association with atopy , down syndrome , and autoimmune diseases . pattern extends from the hair margin into the scalp which is more commonly seen in children . among children , aa seems to be more common in primary school children , the eldest child , and children who are under psychological stress . the association of stress and psychiatric symptoms in children with aa is a subject of debate . however , it seems advisable to subject the affected children to psychiatric evaluation by an expert . routine thyroid screening is also recommended for all children with long - standing aa as the incidence of autoimmune thyroiditis is found to be higher among them . most of the affected children require only reassurance as spontaneous remission occurs in majority of cases . treatment options are topical , intralesional , and systemic corticosteroids according to the severity and chronicity of the disease , topical immunomodulators , and topical irritants like dithranol , topical immunotherapy , puva therapy , and minoxidil . even though the topical immunotherapy and intralesional steroids carry better outcome , it is not routinely used in children . care should be taken to try a particular treatment for at least 3 to 6 months before declaring it as ineffective . the role of environmental factors in precipitating a concomitant attack in both the siblings is highlighted along with the special features of aa in children .

alopecia areata ( aa ) is one among the many causes of non - scarring alopecia in children . family history has been noted in 10 - 20% of cases , but concomitant presentation in siblings is extremely rare . the patterns and associations of childhood aa are similar to adults ; however , there are some differences which are being highlighted in this article .

happiness as a concept was attended by most of scientists and philosophers during the time . most of philosophers and historians believe that , at the first , happiness was focused on good chance , but now , happiness defined as an emotion that is under control of human and persuaded as a goal . centuries before christ , the word eudemonia was used for well - being and it is translated in english as happiness . socrates believed that happiness is a content that is briefly in hands of people ( 1 ) . many terms are used as a synonym of happiness up to now : life satisfaction , quality of life , well - being , positive emotions , interesting and so on ( 2 ) . today , the word subjective well - being is the common term for happiness ( 3 ) . from the mid of twenty century , scientific researchers have been performed about the role of happiness in social sciences ( 46 ) . happiness is correlated with factors like cognitive abilities , personality , behaviors , biology and emotions ( 711 ) . human being bringing in a complex system up and several aspects of environment influence on him / her . therefore , it can be said that parents have influence on children and vice versa . so , several studies accomplished in this area ( 15 , 16 ) . in recent decades , studies are directed to exceptional children family . by using these researches , our knowledge about the family of children with disabilities is promoted ( 17 ) . although primary studies were focused on negative effects of parents limitations , recent studies include positive effects of parents behaviors ( 18 ) . recent research models are investigating both strengths and distresses in the family of children with mental retardation . this studies respect to effects of parents on children and effects of child on parents ( 18 , 19 ) . it is clear that family structure has an important impact on forming child s skills ( 20 , 21 ) . an important factor that should be investigated in child - parent interaction is the effect of parents emotions on cognitive and affective abilities of their children . parents emotions like happiness can play an important role in child s development , because parents emotions affect both child s performance and their relation with family members ( 23 ) . therefore , such effects should be studied in children with mental retardation . among mentally retarded children , down syndrome has a special place : first , the prevalence of this syndrome is high . third , with existing facilities and learning methods , the ability of children with down syndrome can be improved . down syndrome is a chromosomal disorder that influence on emotional , cognitive and physical abilities . the important factor for training to this group is the realization of factors that affected emotional and cognitive abilities ( 24 ) . for example among cognitive abilities , cognitive - executive functions play an important role in learning process . cognitive - executive functions are under control of prefrontal of brain and it is responsible for planning , control and managing behavior ( 25 ) . important indicators of cognitive - executive functions are as follow : attention , working memory , inhibition planning ( 26 ) . children with down syndrome have problem in attention , working memory , inhibition and planning . in addition , among emotional abilities , interpreting the facial emotions is a necessary skill that capable people to direct their social environment ( 31 ) . emotions recognition from the face is the first sign for understanding feelings and making empathy relations . facial expressions recognition is common in all people in the world and it is not related to special culture . normal children ( after 4 yr ) are able to recognize emotions like happy , fear , anger , and sadness ( 32 ) . people with mental retardation like down syndrome have deficits in facial emotional recognition ( 31 , 33 ) . in one hand , the family is an effective system in the life of human and in another hand ; happiness influences emotional and cognitive abilities of individuals . in the scope of exceptional children , the relation between parent s happiness and emotional - cognitive abilities of children with mental retardation is on top . among emotional abilities , cognitive - executive functions are the most effective factors that affect learning and social adaptation of mentally retarded children . down syndrome is attended as an important and wide group among all groups with mental retardation . in this study , we discuss the relation between maternal happiness level with cognitive - executive function and facial emotional recognition in children with down syndrome . population is included all school children with down syndrome ( 912 yrs ) that come from tehran . according to their iq ( 5070 ) , they are placed in experimental group . among all , 30 students with mean age 10.5 yrs selected as an in access sample from resalat , touhid and pirouzi schools . after selection and agreement of parents , the wisc - r is performed to determine the student s iq . it should be noted that children with epilepsy were missed from experiment because using computerized tests can trigger the onset in epileptic children . the wechsler intelligence scale for children - revised ( wisc - r ) , is a general test of intelligence . the wisc - r consist of 12 subtests that it is divided into two parts , verbal and non - verbal . reliability of the test ( in iran ) through test- retest is 0.79 to 0.96 . the ohi comprises 29 items , each involving the selection of one of four points ( likert scale ) that are different for each item . the highest score in this scale is 87 , which shows the highest point of happiness . normal and mean range score in this scale is 40 to 42.reliability of the ohi is 0.91 and internal correlation of items is about 0.04 to 0.67 . in addition , reliability of the test in iran by test - retest is 0.79 . this test is computerized and it will be used to evaluate attention disorder . in this research , the persian form of cpt is used . the test included 150 stimulus , 30 stimulus are goal that the subject has to answer to it with pressing the button . reliability of the test in iran is 0.59 . in current study , for assessing the validity of test , experts rate is used . in addition , reliability of the test assessed in a group ( n=15 ) with down syndrome ( 0.68 ) . n - back is a task for assessing working memory as a subscale of cognitive - executive function . in current study , reliability of the test assessed in several studies and it is reported about 0.79 , 0.81 and 0.84 . authors used experts rate for validity and assessed reliability on a group of children ( n=15 ) with down syndrome ( 0.76 ) . this task assesses the ability to inhibit a predominant response . in this task , participants were asked to say night when they saw a picture of the sun drawn on a white card , and day when they saw a picture of moon and stars drawn on a black card . after being given a brief practice trial , in addition , reliability ( test - retest ) of the test is assessed by authors on a group of children ( n=15 ) with down syndrome and it is obtained 0.78 . this test was used for planning abilities of patients with frontal lobe disorder . in current study , computerized version of test is used . each set has three bases with three colored balls ( green , blue and red ) . in addition , reliability ( test - retest ) of the test was assessed by authors on a group of children ( n=15 ) with down syndrome and it is obtained 0.81 . this test is common and reliable in emotion facial recognition . in the current study , from 110 pictures of original test , 36 pictures of 3 males and 3 females were selected . pictures included 6 basic emotions ( happy , anger , sad , hate , fear and surprise ) . in addition , reliability of the test assessed by authors through test- retest , on a group of children ( n=15 ) with down syndrome and it is obtained 0.79 . in this study , after selection of sample and agreement of family to take part in research , schoolchildren iq is assessed by wisc - r . then , mothers were asked to fill the oxford happiness inventory . for assessing abilities of schoolchildren with down syndrome the following tests is used : cpt , n - back , stroop test ( day / night ) , london tower test and emotion facial expression test . these tests performed individually . for data analysis , the descriptive and interfering statistics was used . first , data were categorized in tables and figures then by using pearson correlation coefficient , the relation between variables was assessed . the wechsler intelligence scale for children - revised ( wisc - r ) , is a general test of intelligence . the wisc - r consist of 12 subtests that it is divided into two parts , verbal and non - verbal . reliability of the test ( in iran ) through test- retest is 0.79 to 0.96 . the ohi comprises 29 items , each involving the selection of one of four points ( likert scale ) that are different for each item . the highest score in this scale is 87 , which shows the highest point of happiness . normal and mean range score in this scale is 40 to 42.reliability of the ohi is 0.91 and internal correlation of items is about 0.04 to 0.67 . in addition , reliability of the test in iran by test - retest is 0.79 . this test is computerized and it will be used to evaluate attention disorder . in this research , the persian form of cpt is used . the test included 150 stimulus , 30 stimulus are goal that the subject has to answer to it with pressing the button . reliability of the test in iran is 0.59 . in current study , for assessing the validity of test , experts rate is used . in addition , reliability of the test assessed in a group ( n=15 ) with down syndrome ( 0.68 ) . n - back is a task for assessing working memory as a subscale of cognitive - executive function . in current study , reliability of the test assessed in several studies and it is reported about 0.79 , 0.81 and 0.84 . authors used experts rate for validity and assessed reliability on a group of children ( n=15 ) with down syndrome ( 0.76 ) . this task assesses the ability to inhibit a predominant response . in this task , participants were asked to say night when they saw a picture of the sun drawn on a white card , and day when they saw a picture of moon and stars drawn on a black card . after being given a brief practice trial , in addition , reliability ( test - retest ) of the test is assessed by authors on a group of children ( n=15 ) with down syndrome and it is obtained 0.78 . this test was used for planning abilities of patients with frontal lobe disorder . in current study , computerized version of test is used . each set has three bases with three colored balls ( green , blue and red ) . in addition , reliability ( test - retest ) of the test was assessed by authors on a group of children ( n=15 ) with down syndrome and it is obtained 0.81 . this test is common and reliable in emotion facial recognition . in the current study , from 110 pictures of original test pictures included 6 basic emotions ( happy , anger , sad , hate , fear and surprise ) . in addition , reliability of the test assessed by authors through test- retest , on a group of children ( n=15 ) with down syndrome and it is obtained 0.79 . in this study , after selection of sample and agreement of family to take part in research , schoolchildren iq is assessed by wisc - r . then , mothers were asked to fill the oxford happiness inventory . for assessing abilities of schoolchildren with down syndrome the following tests is used : cpt , n - back , stroop test ( day / night ) , london tower test and emotion facial expression test . these tests performed individually . for data analysis , the descriptive and interfering statistics was used . first , data were categorized in tables and figures then by using pearson correlation coefficient , the relation between variables was assessed . obtained data were categorized in order and by using descriptive mathematics , mean and standard deviation were assessed for each variable . findings are presented in table 1 . mean and standard deviation of subjects for each variable for assessing the relation between mothers happiness level as an independent variable , with 5 dependent variable in research ( attention , working memory , inhibition , planning and facial emotional recognition ) , the pearson correlation coefficient was used . correlation coefficient between mothers scores in ohi and their children ( with down syndrome ) scores in cognitive - executive functions and facial emotional recognition in table 2 , the correlation analysis between mothers scores in ohi and their children ( with down syndrome ) abilities in cognitive - executive functions and facial emotional recognition is indicated . the mathematical model used for analyzing research hypothesis relation between happiness and each variable- is the t distribution . in comparing with critical value of t ( df . = 28 , =0.05 , t=2.04 ) , obtained t value for each variable is high , then zero hypothesis is rejected and research hypothesis is accepted . moreover , as observed correlation coefficient between mothers happiness with each variables is significantly higher than table critical value ( =0.05 , p=0.36 ) , zero hypothesis is rejected and it is concluded that there is a positive and significant relation between mothers happiness with their children abilities ( attention , working memory , inhibition , planning and facial emotional recognition ) . results show that happiness of mothers of children with down syndrome has a positive and significant relation with cognitive executive functions and facial emotional recognition ability in their children . according to the authors search these supportive findings are divided into two categories:- in the scope of classical psychology ; studies that evaluating the relationship between mothers negative mood ( opposite of happiness ) , with cognitive - executive functions and facial emotional recognition of their children.- in the scope of positive psychology ; studies that evaluating the relationship between happiness and its synonyms in mothers , with cognitive - executive functions and facial emotional recognition of their children . - in the scope of classical psychology ; studies that evaluating the relationship between mothers negative mood ( opposite of happiness ) , with cognitive - executive functions and facial emotional recognition of their children . - in the scope of positive psychology ; studies that evaluating the relationship between happiness and its synonyms in mothers , with cognitive - executive functions and facial emotional recognition of their children . a group of studies indicate that emotional and mental characteristics of mothers are the important factors that have influence on cognitive - emotional abilities of their children . depression ( opposite form of happiness ) as an emotional factor has a negative and significant impact on cognitive - executive function abilities of children . it means that mothers negative emotion is a predictor of decreasing the abilities of children ( 39 ) . forty one percent of children with attention deficit , affective disorder and anxiety have a depressed mother ( 42 ) . mothers depression can influence on brain and its functions like memory ( 43 , 44 ) . its biological reason is increasing of production of stress hormone during pregnancy that leads to problems for brain growth ( 45 ) . children with depressed mother have more problems in the activity of frontal and pre - frontal than children with non - depressed mother ( 47 ) . in another study , they conclude that mothers depression has a negative and significant effect on inhibition ability of children ( 44 ) . in a classical view , the effect of mothers depression during pregnancy is investigated and they figured the negative and significant effect of mothers depression on planning ability of children ( 39 , 48 ) . a group of research in the scope of positive psychology investigates the relationship between happiness and life - satisfaction in mothers of children with attention deficit . findings figure a positive and significant relation between subjective - wellbeing in mothers and attention ability in their children . parents of children with attention deficit ( generally ) , and their mothers ( specially ) express a lower life satisfaction and lower happiness in comparing with normal population ( 49 ) . family environment is an important factor that affects children s ability . when parents are not preparing to take new responsibilities , then stress and negative emotions cover their interactions . moreover , life satisfaction and happiness , in this family , are in low level ( 50 ) . mother s positive mood likely leads to positive interaction with children and cognitive - emotion will be affected by this atmosphere , so improvement in abilities of children will be seen ( 51 ) . in the scope of positive psychology , it is focused on the relation of happiness and cognitive abilities . several studies indicate the effect of mothers happiness on cognitive abilities of children ( 51 , 52 ) . findings indicate that mothers happiness leads to improvement in cognitive abilities of children ( 23 ) . increasing of life satisfaction in parents leads to increasing the ability of inhibition ( 53 ) . subjective well - being in mothers leads to the optimal planning function in their children ( 49 ) . primary experiences of children have a significant effect on their facial emotional recognition ability ( 54 ) . in a classical view , the effect of parents depression on regulation of emotion is investigated ( 55 ) . if mothers are affected by depression , it is more likely that their child has a problem in emotional recognition ( 56 ) . mothers severe depression is a powerful predictor for emotional problems in their children ( 57 , 58 ) . effects of mothers depression on children behaviors are summarized as follow : - low adjustment- low awareness toward environment- decrease of interaction behaviors- finally , decrease of emotional recognition and empathy ( 42 ) . - low awareness toward environment - decrease of interaction behaviors - finally , decrease of emotional recognition and empathy ( 42 ) . a group of studies indicate that emotional and mental characteristics of mothers are the important factors that have influence on cognitive - emotional abilities of their children . depression ( opposite form of happiness ) as an emotional factor has a negative and significant impact on cognitive - executive function abilities of children . it means that mothers negative emotion is a predictor of decreasing the abilities of children ( 39 ) . forty one percent of children with attention deficit , affective disorder and anxiety have a depressed mother ( 42 ) . mothers depression can influence on brain and its functions like memory ( 43 , 44 ) . its biological reason is increasing of production of stress hormone during pregnancy that leads to problems for brain growth ( 45 ) . children with depressed mother have more problems in the activity of frontal and pre - frontal than children with non - depressed mother ( 47 ) . in another study , the effect of mothers depression on cognitive - executive functions of children was evaluated . they conclude that mothers depression has a negative and significant effect on inhibition ability of children ( 44 ) . in a classical view , the effect of mothers depression during pregnancy is investigated and they figured the negative and significant effect of mothers depression on planning ability of children ( 39 , 48 ) . a group of research in the scope of positive psychology investigates the relationship between happiness and life - satisfaction in mothers of children with attention deficit . findings figure a positive and significant relation between subjective - wellbeing in mothers and attention ability in their children . parents of children with attention deficit ( generally ) , and their mothers ( specially ) express a lower life satisfaction and lower happiness in comparing with normal population ( 49 ) . family environment is an important factor that affects children s ability . when parents are not preparing to take new responsibilities , then stress and negative emotions cover their interactions . moreover , life satisfaction and happiness , in this family , are in low level ( 50 ) . mother s positive mood likely leads to positive interaction with children and cognitive - emotion will be affected by this atmosphere , so improvement in abilities of children will be seen ( 51 ) . in the scope of positive psychology , it is focused on the relation of happiness and cognitive abilities . several studies indicate the effect of mothers happiness on cognitive abilities of children ( 51 , 52 ) . findings indicate that mothers happiness leads to improvement in cognitive abilities of children ( 23 ) . increasing of life satisfaction in parents leads to increasing the ability of inhibition ( 53 ) . subjective well - being in mothers leads to the optimal planning function in their children ( 49 ) . primary experiences of children have a significant effect on their facial emotional recognition ability ( 54 ) . in a classical view , the effect of parents depression on regulation of emotion is investigated ( 55 ) . if mothers are affected by depression , it is more likely that their child has a problem in emotional recognition ( 56 ) . mothers severe depression is a powerful predictor for emotional problems in their children ( 57 , 58 ) . effects of mothers depression on children behaviors are summarized as follow : - low adjustment- low awareness toward environment- decrease of interaction behaviors- finally , decrease of emotional recognition and empathy ( 42 ) . - low awareness toward environment - decrease of interaction behaviors - finally , decrease of emotional recognition and empathy ( 42 ) . mothers happiness level as an important factor is presented in cognitive abilities of children , especially mentally retarded children . mentally retarded children with happier mothers show significant performance on cognitive executive functions ( memory , attention , inhibition and planning ) and facial emotional recognition abilities . parents agreements received before starting the research . in addition , special codes assigned instead the name of schoolchildren . 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 : according to the mother s key roles in bringing up emotional and cognitive abilities of mentally retarded children and respect to positive psychology in recent decades , this research is administered to assess the relation between mother s happiness level with cognitive- executive functions ( i.e. attention , working memory , inhibition and planning ) and facial emotional recognition ability as two factors in learning and adjustment skills in mentally retarded children with down syndrome.methods:this study was an applied research and data were analyzed by pearson correlation procedure . population is included all school children with down syndrome ( 912 yr ) that come from tehran , iran . overall , 30 children were selected as an in access sample . after selection and agreement of parents , the wechsler intelligence scale for children - revised ( wisc - r ) was performed to determine the student s iq , and then mothers were invited to fill out the oxford happiness inventory ( ohi ) . cognitive - executive functions were evaluated by tests as followed : continues performance test ( cpt ) , n - back , stroop test ( day and night version ) and tower of london . ekman emotion facial expression test was also accomplished for assessing facial emotional recognition in children with down syndrome , individually.results:mothers happiness level had a positive relation with cognitive - executive functions ( attention , working memory , inhibition and planning ) and facial emotional recognition in her children with down syndrome , significantly.conclusion : parents happiness ( especially mothers ) is a powerful predictor for cognitive and emotional abilities of their children .

infection with hepatitis b virus ( hbv ) is a well - known and an important cause of liver disease in patients on hemodialysis ( hd ) . recombinant hepatitis b vaccines are commonly utilized in hd patients for prophylaxis against hbv infections . the presently available vaccines contain purified recombinant hepatitis b surface antigen ( hbsag ) obtained by culturing genetically engineered saccharomyces cerevisae , which carry the surface antigen gene of hbv . an 83-year - old iranian woman was commenced on hd in march 2010 for chronic kidney disease stage 5 secondary to hypertensive nephrosclerosis via a temporary right internal jugular catheter . viral serologies ( hiv ag / ab , hbsag , hepatitis c virus ab ) done prior to initiation of dialysis were negative . in accordance with our unit protocol , where viral screening is done every 3 months hbsag was weakly positive and the same repeated after 3 days was again reported as positive . a separate hd machine , in a separate room , hepatitis b surface antibody ( hbsab ) , hbeag and hbv dna were all negative . in further discussion with the patient s relatives , it was determined that she had received her second dose of engerix b vaccination ( 40 g intramuscular ) 5 days prior to serological testing ( 27 may 2010 ) . the first dose of the vaccine had been administered on 4 april 2010 . on 16 june 2010 , 3 weeks after the second hepatitis b vaccination , hbsag was repeated and was negative . hepatitis b core ( hbc ) igm antibody was negative , ruling out recent hbv infection . hence , it is most likely that the hbsag was falsely positive in the wake of recent hbv vaccination . the practice of extensive infection control measures , the decrease in the need for blood transfusions after the introduction of erythropoietin , the development of an effective hbv vaccine and segregation of hbsag - positive patients during hd have significantly contributed to the progressive reduction of hbv prevalence among hd patients [ 1 , 2 ] . hbsag is the most common serological marker used to identify acute or chronic hbv infections . hd patients with hbsag positivity are considered to have hbv and are segregated to limit transmission . hepatitis b vaccination is administered to hbsag - negative patients who lack or have low levels of anti - hbsab ( < 10 u / l ) , which is the protective antibody against hbv . it was initially thought that hepatitis b surface antigenemia did not occur after hepatitis b vaccination ; however , the study used only the plasma - derived hbv vaccine . there have been several reports of detectable hepatitis b surface antigenemia after vaccination , especially in neonates , children and blood donors [ 46 ] . transient hepatitis b antigenemia in hd patients following hbv vaccination was reported as early as 1996 [ 2 , 7 ] . the duration of hepatitis b surface antigenemia after vaccination is brief , usually 17 days , however , this may be prolonged up to 20 days in patients on hd . performed a prospective study of de novo hbv infection in > 2400 hd patients who were screened monthly for hbsag . they concluded that hbv immunization was the most common cause of detectable hbsag in hd patients . in their opinion , hd patients should not be screened for hbv within a week of immunization and caution should be exercised when interpreting hbsag seropositivity within 4 weeks of hbv immunization . the transient hbsag positivity may result from early , slow , variable and non - reproducible absorption of vaccine from the muscle . are tested for hbsag by a third - generation microparticle enzyme immunoassay ( axsym ; abbott laboratories , chicago , il , usa ) . the amount of vaccine injected ( 40 g ) and the sensitivity of the axsym monoclonal assay ( 0.10.6 ng / ml ) calculates to a vd ( volume of distribution ) of 70400 l. therefore , if the injected vaccine is distributed in total body water ( 42 l ) or if the antigen is circulating in a smaller space , i.e. extracellular fluid ( 12 l ) or plasma volume ( 3 l ) , this would account for the vaccine antigen being detectable . in such circumstances , the surface antigenemia is caused by a passive transfer of antigen by vaccination and not by viral replication ; hence , there is no risk for vaccination - induced infection . an important implication of this phenomenon is that the results of hbsag assays should be interpreted according to the time elapsed since the last administration of a vaccine against hepatitis b . the period of incidental hbsag ( false ) positivity , albeit brief , generated significant concern and anxiety among the patients family and our dialysis unit staff . the patient had to be isolated on a separate hd machine , in a separate room , until further hbv- and hbsag - negativity reports were obtained . although , the cdc guidelines with regard to hepatitis b screening , published in april 2001 , recommend that susceptible new hd patients ( those with anti - hbsab < 10 u / l ) be screened every month for hbsag until their anti - hbsab is > 100 u / l , most hd centers carry out hepatitis b screening with testing of hbsag once in 3 months [ 10 , 11 ] . in either case , we recommend that hbsag testing be done only after 4 weeks of having administered a vaccine dose , to avoid false positivity , as noticed in our patient . this would mean new hd patients who are being vaccinated have an individualized hbsag testing schedule , so as to maintain a gap of 4 weeks between the vaccination dose and hbsag testing . in conclusion , based on our experience with this patient and review of existing literature , we reemphasize previous recommendations that hbsag screening this applies , all the more , in hd units where hepatitis b screening is carried out routinely throughout the year and hepatitis b vaccines are frequently administered . a history of recent hepatitis b vaccination should be meticulously elicited when a previously negative patient is detected as hbsag positive. hepatitis b vaccines are clearly noninfectious and do not pose a risk of transfusion - transmitted disease . the occurrence of transient post - vaccination hbsag positivity needs to be fully acknowledged to prevent misdiagnosis , anxiety and confusion .

hepatitis b vaccination is mandatory for all hepatitis b surface antigen ( hbsag)-negative hemodialysis ( hd ) patients with low hepatitis b surface antibody titers . we report a case of transient hepatitis b surface antigenemia , detected incidentally , in an hd patient after the second dose of hepatitis b vaccine . the patient had to be isolated on a separate hd machine , in a separate room , until further investigations ruled out a true hepatitis b virus infection . this led to further unnecessary testing and generated great concern and anxiety for the patient . we present this case to emphasize that hd units should be aware of this phenomenon and that hbsag testing be deferred until 4 weeks after vaccination to avoid hbsag false positivity .

receiving long - term care ( ltc ) in old age is still widely associated with a definite and dead - end situation . for this reason preventive and rehabilitative measures for older people already in need of continuous care have not yet been perceived or treated as health policy issues of great importance . the data for this presentation and case description are based on national as well as international reports on the provision and quality of long - term care services that were undertaken during the project interlinks ( 20082011 ) financed by the 7th framework programme by the eu commission . special attention in this project is given to the coordination and transition processes between preventive , rehabilitative , informal , health and social care services in different european countries . analyse concepts of prevention and rehabilitation within the ltc provision for older people and discuss in this respect good practice as well as inherent incentives or disincentives across an ltc pathway example in austria . well - coordinated and comprehensive concepts of prevention and rehabilitation , which might well go beyond our conventional and often one - dimensional understanding of such measures , represent potential elements for sustainable ltc systems and will require a lot more attention and integration in the future . analysing quality of services as well as transition processes between these services with the help of ltc pathway examples allows for better insight into system - related preventive or rehabilitative potential and respective incentives and disincentives .

introduction and backgroundreceiving long - term care ( ltc ) in old age is still widely associated with a definite and dead - end situation . for this reason preventive and rehabilitative measures for older people already in need of continuous care have not yet been perceived or treated as health policy issues of great importance . the data for this presentation and case description are based on national as well as international reports on the provision and quality of long - term care services that were undertaken during the project interlinks ( 20082011 ) financed by the 7th framework programme by the eu commission . special attention in this project is given to the coordination and transition processes between preventive , rehabilitative , informal , health and social care services in different european countries.aimsanalyse concepts of prevention and rehabilitation within the ltc provision for older people and discuss in this respect good practice as well as inherent incentives or disincentives across an ltc pathway example in austria.interim result / conclusionwell - coordinated and comprehensive concepts of prevention and rehabilitation , which might well go beyond our conventional and often one - dimensional understanding of such measures , represent potential elements for sustainable ltc systems and will require a lot more attention and integration in the future . analysing quality of services as well as transition processes between these services with the help of ltc pathway examples allows for better insight into system - related preventive or rehabilitative potential and respective incentives and disincentives .

it is an important cause of morbidity and mortality , characterized by slow , progressive , and irreversible loss of kidney function1,2,3,4 . in brazil , the number of patients with chronic kidney failure in outpatient dialysis programs increases by 8% per year1 . dialysis can prolong the lives of patients , but does not prevent setbacks caused by the basic pathological condition and the treatment itself5 . the accumulation of toxic substances6 in the blood causes easy fatigue , mental impairment , peripheral circulatory deficits , changes in sensation , and muscle dysfunction7 , 8 . the muscle dysfunction manifests as changes in structure and function9 , which may present as atrophy , proximal muscle weakness , predominantly in the lower limbs , and results in anemia and peripheral neuropathy10 , 11 . exercise during the intradialytic period can help minimize these conditions , by promoting increased blood flow to peripheral tissues and improved perfusion of muscle cells11 , 12 . exercise programs have been proposed , and are aimed not only at the clinical signs of the disease , but also the repercussions on function and quality of life , as these individuals tend to have a sedentary lifestyle and functional limitations5 , 7 , 13 . aerobic exercise , with or without muscle strengthening , use of a bicycle ergometer , calisthenics , resistance and/or strengthening exercises , and stretching , are examples of therapeutic modalities used by physiotherapy7 , 10 , 14 . however , exercise implementation during hemodialysis ( hd ) is not yet a reality in brazil15 . although the national kidney foundation ( nkf)2 , through the clinical practice guidelines for chronic kidney disease , ( kdoqi ) ( 2002 ) advises nephrologists to prescribe routine physical activity for hd patients16 , the implementation of physical exercise programs during hd in the usa and other countries is not common17 . some studies have described physical exercise programs in other countries , but the extent to which these programs are part of the treatment of these patients during hd is unknown18,19,20 . although some studies show the benefits of regular exercise programs in hd patients12 , 21 , 22 , the assessment of physical functioning and the incentive to increase physical activity and/or regular participation in exercise programs , specifically during the hd period , are not a routine component of medical care23 . thus , this study aimed to assess the opinions of nephrologists at dialysis services in the city of recife about the importance of routine therapeutic exercise during the intradialytic period . this was an analytical , cross - sectional study , and the population consisted of nephrologists who practiced in professional groups at hd centers in the city of recife , brazil , and provided care for chronic uremic patients undergoing dialysis . the instrument used for data collection was devised specifically for this study , and consisted of a questionnaire containing 15 semi - structured questions pertinent to nephrologists . the study was conducted in accordance with the ethical standards of the declaration of helsinki . all subjects provided written informed consent prior to the study , which was approved by the ethics committee of universidade federal de pernambuco . the first phase of the research was characterized by the identification of dialysis services in the city with a query to the national register of health care facilities through the datasus website ( http://cnes.datasus.gov.br/ ) . twelve public and private hd centers were identified in the city of recife . in a second phase , consultation was conducted through the website of the regional medical council of pernambuco , to determine the number of nephrologists active in the city , and 56 physicians were identified . of the dialysis services queried , 11 could be contacted , but only 8 allowed data collection . the sample calculation was performed using a total of 56 nephrologists . with consideration of bilateral statistics at a significance level of p = 0.05 and confidence interval of 95% , the data collection instrument consisted of information about age , gender , academic background , and residency ( place and time of graduation ; training in nephrology ; hd experience ) , and knowledge about exercise ( intradialytic therapeutic exercise , level of physical activity and exercise prescription for chronic kidney disease patients , and indications for intradialytic exercise ) . for purposes of analysis in this study , therapeutic physical exercise was considered to be physical activity that was planned and aimed at the maintenance or improvement of the components of physical fitness , that promoted minimum energy expenditure above the resting state , and which should be repeated at least 3 times per week24 . to evaluate personal data , academic profile , perception of exercise during the intradialytic period , and knowledge about the physical status of patients , we calculated the percentage frequencies and constructed frequency distributions . to evaluate which factors significantly influence the practice of prescribing of physical activity , contingency tables were created , and the test was applied for independence . where the test assumptions were not met , we applied fisher s exact test . for the factors determining prescribing of physical exercise , the prevalence ratio among the levels evaluated was calculated . a database was created in a microsoft excel spreadsheet , which was exported to spss software ( ibm corporation , armonk , ny , usa ) version 18.0 for the analysis . 49 nephrologists at public and private institutions in the city of recife were evaluated . among those evaluated , 51% were females , 38% , 29% and 33% had an average of 35 , between 36 and 45 and above 45 years respectively 69.4% were engaged in regular physical activity ( p=0.007 ) , and 87.5% were working in private institutions ( p<0.001 ) . table 1table 1.characteristics of the professional profile of nephrologists in the city of recifeevaluated factor n%place of graduation * ufpe2755.1upe1836.7others48.2training in nephrology * ufpe2653.1imip612.2hbl48.2others1326.5actuation time as a nephrologist * up to 2 years612.2more than 2 to 5 years1122.4more than 5 to 10 years714.3more than 10 years2551.0time experience with hemodialysis * up to 2 years48.2more than 2 to 5 years1020.4more than 5 to 10 anos816.3more than 10 years2755.1received information on use of therapeutic exercisesin intradialytic period during residency in nephrology * * yes1530.6no3469.4ufpe : universidade federal de pernambuco ( federal university of pernambuco ) ; upe : universidade de pernambuco ( university of pernambuco ) ; imip : instituto materno infantil de pernambuco ( maternal child institute of pernambuco ) ; hbl : hospital baro de lucena ( baro de lucena hospital ) . fisher s exact test . * n = number of nephrologists shows the characteristics of medical training in nephrology . a total 69.4% of physicians surveyed did not have information on the use of physical exercise for patients on hd during their residency training ( p=0.007 ) . ufpe : universidade federal de pernambuco ( federal university of pernambuco ) ; upe : universidade de pernambuco ( university of pernambuco ) ; imip : instituto materno infantil de pernambuco ( maternal child institute of pernambuco ) ; hbl : hospital baro de lucena ( baro de lucena hospital ) . fisher s exact test . * n = number of nephrologists table 2table 2.opinion of nephrologists about the practice of therapeutic exercise during the intradialytic periodevaluated factorn%do you indicate intradialytic exercises for your patient?yes2346.9no2653.1who guides these exercises ? * p < 0.001 shows characteristics concerning prescribing , guidance , and opinions about the practice of physical exercise during the intradialytic period . although 53.1% of the interviewed nephrologists do not prescribe physical exercises for their patients , 81.6% consider therapeutic exercise to be important during the intradialytic period ( p<0.001 ) . only 8.7% advise their patient about physical exercises ( p<0.001 ) , leaving the responsibility to the physiotherapist . * p < 0.001 regarding knowledge about the level of physical activity of their patients , 61.2% of nephrologists consider it to be poor ( p<0.001 ) , 65.4% ask very little about the level of physical activity ( p<0.001 ) , and only 10.2% of hd patients perform some type of physical exercise ( p<0.001 ) ( table 3table 3.knowledge of nephrologists about the level of physical activity among hemodialysis patients in recife , brazil , 2014evaluated factorn%what level of physical activity among your patients ? * i do nt know816.3bad3061.2reasonable1020.4excellent12.0do you consider your patients active ? yes1836.7no3163.3your patients do physical exercises ? * yes510.2no4489.8how often do you ask your patient about their level of physical activity or exercise ? * p < 0.001 with regard to the association with the length of professional experience in hd , those with less time in practice ( up to 2 years ) tended to prescribe physical exercise as a treatment for these patients ( p<0.010 ) . the prevalence ratio for the prescribing of physical exercise was 10 times higher for those professionals with up to 2 years of experience , compared to those with 510 years ( 5.00 times ) , and more than 10 years ( 5.19 times ) of experience . among those who exercise regularly , 88.2% consider physical exercise to be important for hd patients ( p=0.009 ) . the nephrologists who exercise had a 1.32-fold likelihood of considering therapeutic exercise during hd to be important , compared to those who do not perform physical activity . no correlation was observed between prescribing physical exercise and the nephrologist s workplace ( public / private ) ( p=0.670 ) . the results of this study show that more than half of interviewees did not prescribe intradialytic therapeutic exercise for patients on hd , although they perceived it as an important component of the treatment process . most interviewees did not receive information on the use of physical exercise as a therapeutic tool for ckd patients during residency ; they recognize that the level of physical activity of their patients is poor and that most do not perform physical exercise . this study also found that nephrologists with less work experience are more likely to prescribe physical exercise for these patients . the lack of information about the use of physical exercise in patients undergoing hd that was provided during training in nephrology is similar to that reported by delgado and johansen16 , johansen et al.25 , painter et al.26 , and kontos et al27 . these authors identified a lack of knowledge and commitment to discuss the issue , and consider these to be one of the primary barriers to patient access to intradialytic therapeutic exercise programs . delgado and johansen28 and kontos et al.27 reported the lack of prescribing , despite the recognition of the importance of exercise . the reasons for this may lie in the curriculum of the medical residency16 . delgado and johansen28 emphasize that physicians need tools , training , and dialogue on exercise recommendations ; in their absence , they feel insecure about prescribing . painter et al . reported that nonmedical professionals at hd centers recognize that patients might benefit from an exercise program , and that these patients could have a better level of physical functioning26 . the incorporation of physical activity evaluation as a routine for these patients could alter this situation , adding to the recommendations and guidelines of the nkf kdoqi16 . most of the nephrologists did not usually evaluate or express concern about the level of physical activity of their patients , despite describing the activity level as poor . the lack of encouragement by nephrologists could be one of the major factors that inhibit the practice of physical activity among patients27 , which conflicts with the kdoqi suggestion that all dialysis patients should be advised and encouraged to exercise regularly by a nephrologist and other team members16 . this study found that nephrologists with up to 2 years of experience the area had a 10 times greater likelihood of prescribing exercise for these patients , compared to those with more professional experience . contrary to the present results , delgado and johansen observed that the most experienced nephrologists ( older than 55 years old ) queried their patients and prescribed physical activity with greater frequency16 . it is possible that younger nephrologists have had the opportunity to work in hd services where contact with the physiotherapist and the availability of physical therapy for chronic renal patients is a part of the treatment plan , and that this contributed to the difference between the results of the present study and those of delgado and johansen16 . the limitations of this study are the omission of questions about the contact of nephrologists with physiotherapists in or outside of the hd service , and knowledge among these professionals about the physiotherapist s role in caring for patients during the intradialytic period . the prescribing of therapeutic exercise for patients with ckd undergoing hd needs to be common among nephrologists , so that patients can benefit from an exercise program directed and conducted by a physiotherapist . this study revealed that although nephrologists recognize the importance of physical exercise , few prescribe it during the intradialytic period . considering the adverse consequences of hd on the physical condition of these patients , the physiotherapist must find ways to promote exercise in this area , since the nephrologists interviewed reported exercise as an adjuvant to hd was neglected during their training in nephrology . this study found that the nephrologists interviewed do not usually prescribe intradialytic therapeutic exercises for hd patients , while noting it has an important role in the treatment process . part of this problem may be related to the lack of education during training of these professionals during residency , but also is probably due to the lack of knowledge among physiotherapists regarding the possibility of interacting with these patients through a supervised program of therapeutic physical exercise . thus , it is necessary to continue to investigate other possible factors related to solutions for these patients in clinical practice .

[ purpose ] to assess knowledge among nephrologists at hemodialysis services about routine intradialytic therapeutic exercise , in the city of recife . [ subjects and methods ] a cross - sectional study , consisting of 49 nephrologists working in public and/or private hemodialysis services , who responded to a semi - structured questionnaire about their academic background , medical residency , and knowledge about exercise during the intradialytic period . [ results ] about 56.3% practiced for more than 10 years as nephrologists , 69.4% did not receive information about intradialytic physical exercise while in residency , 81.6% considered intradialytic exercise to be important , and 53.0% did not prescribe exercise during hemodialysis . about 61.2% consider the level of physical activity among their patients to be poor . nephrologists graduating within 2 years were 10 times more likely to prescribe exercise , compared to those with more than 2 years since graduation . [ conclusion ] our study found that the nephrologists interviewed do not usually prescribe intradialytic therapeutic exercise , despite understanding its importance as part of the treatment process . it is necessary to update doctors about the importance of exercise for patients during the intradialytic period , as well as to incorporate this knowledge at the undergraduate level .

the association between dietary fat and risk of cancer has been extensively investigated , and the composition of polyunsaturated fatty acids ( pufa ) in diet seems to be of particular importance . however , the effects of single dietary n-3 and n-6 pufa , and the ratio n-3/n-6 pufa are not completely clear . the majority of case - control studies seem to support a protective role of dietary n-3 pufa and n-3/n-6 pufa ratio [ 24 ] , but these associations have been confirmed by only one cohort study , and have been contradicted or not found by several cohort studies [ 68 ] . studies on serum and erythrocyte membrane fatty acid composition , regarded as biomarkers for fatty acid intake , mainly support a protective role for the very long - chained n-3 pufa docosahexaenoic acid ( dha ) [ 811 ] . abnormalities in plasma pufa composition may also be interpreted as metabolic changes in crc patients . dietary and biomarker studies have not established the role of n-6 pufa in crc . based on changes in n-3 and n-6 pufa expression in colorectal tumours , compared to normal mucosal tissue [ 13 , 14 ] , also found at early stages of adenomas , it seems obvious that patients with crc have an altered pufa metabolism . literature on crc tumour pufa pattern is sparse , and previous studies have not concluded on which particular pufas are present in diseased and normal mucosa [ 13 , 14 ] . however , pufa pattern in normal mucosal tissue seems to be similar in crc patients and healthy subjects . this indicates that the changes in mucosal fatty acid pattern are tumour and normal tissue specific in general crc patients . the younger crc patients may be more genetically predisposed ; their tumour might be expected to be more aggressive and environmental factors relatively less pronounced in the etiology of the disease , compared to older patients . genetic predisposition is known in patients with familial adenomatous polyposis ( fap ) , who may have characteristics more similar to young crc patients . our previous study in fap patients showed increased concentrations of arachidonic acid ( aa ) and dha , and decreased concentrations of linoleic acid ( la ) and -linolenic acid ( ala ) in plasma phospholipids ( pl ) , opposite to the majority of findings of protective effect of dha against development of colorectal adenomas [ 911 , 17 ] . we therefore hypothesize that pufa concentration in plasma phospholipid does not reflect dietary pufa in young crc patients . we further hypothesize that pufa composition differs between crc tumour tissue and normal colonic mucosal biopsies , thereby investigating if the fatty acid pattern characteristic for fap patients could be observed in the colorectum of crc patients . we also wanted to study whether dietary pufa intake or concentration of pufa in plasma pl , tumour , or normal mucosa were associated with cancer stage ( duke 's stage ) . eighty - two patients were recruited from seven hospitals in the south - eastern region of norway . exclusion criterion was known familial syndromes affecting crc risk including hereditary non - polyposis colorectal cancer ( hnpcc ) and fap . the frozen samples were sent on dry ice to vitas as , oslo , norway , where they were stored at 20c until analyses . fatty acid contribution in plasma phospholipid fraction ( pl ) was quantitatively determined as follows : plasma samples were thawed overnight at 4c and vortexed for 5 sec . dichloromethane in methanol was added to plasma and the internal standard ( 1,2 diheptadecaonyl - sn - glycero-3-phosphatidylcholine ) . after shaking and centrifugation , polar lipids were washed out with dichloromethane / isopropanol and methyl tert - butyl ether ( mtbe)/formic acid . after evaporation to dryness in a vacuum centrifuge , phospholipids were transmethylated by sodium methoxide , and fatty acid methyl esters ( fames ) were extracted to hexane before gas - chromatographic ( gc ) analysis . the gc analysis was performed on a 7683b gc with a split / splitless injector , a 7683b automatic liquid sampler , and flame ionization detection ( agilent technologies , palo alto , ca , usa ) . separations were performed on a 30 m sp-2380 column ( supelco , sigma - aldrich , st . tumour and normal mucosa tissue samples , at least 2 1 0.5 cm in size , were collected immediately after resection of the specimen , prepared with rna - later , transported , and stored dry in 80c until analysis . the normal mucosa sample was taken from a normal appearing part of the resected specimen , at least 20 cm from the cancer . the frozen samples were sent on dry ice to vitas as , oslo , norway , where tissue fatty acid contribution was determined as follows : approximately 10 mg tissue was directly methylated with 3n meoh hcl , first 30 minutes at 70c on an ultrasonic bath , and later at 80c on a thermoblock with 500 rpm . fames were extracted with hexane , and the samples were subsequently neutralized with koh in water . after mixing and centrifuging the patients were contacted postoperatively by telephone and invited to give dietary and lifestyle data . dietary intake was assessed by a validated self - filled food - frequency questionnaire ( ffq ) , designed to cover as much of the total diet as possible . questions were related to habitual frequency of consumption and the amount of foods eaten during the one - year period prior to crc diagnosis . dietary supplements , such as cod liver oil , fish oil capsules , and vitamin and mineral supplements , were included . the ffq was mailed to the participants and filled in at home . after returning of the questionnaire by mail , the participants were contacted by a dietitian by telephone , and filling of the ffq was checked . dietary intakes were calculated by using a database and a software system developed at the department of nutrition , university of oslo ( kostberegningssystem , version 3.2 ; university of oslo , oslo , norway ) . in the previously developed lifestyle questionnaire , participants were asked to describe their smoking status by detailed questions on current and lifetime smoking status . participants were asked to assess their last weight before crc diagnosis as well as nonvigorous and vigorous physical activity ( minimum 20 min at the time ) . the alternative frequencies of activity were : never , less than once / week , 1 - 2 times / week , 3 - 4 times / week , 57 times / week , and more than 7 times / week . the participants were also asked to state their weekly hours of occupational activity prior to crc diagnosis , and possible changes in physical activity and dietary habits after the diagnosis . the study protocol was approved by the norwegian health authorities and the regional ethics committee . we tested differences in fatty acid concentrations between tumour and normal mucosa by paired , non - parametric wilcoxon signed ranks test , and differences in categorical variables between subgroups by chi - square statistics . we also tested correlations between dietary and plasma pl fatty acids , and between pufa in plasma pl and normal mucosa and tumour tissue in analysis stratifying by bmi ( < 25 and 25 kg / m ) . we conducted the statistical analysis using software spss 15.0 for windows ( spss inc . , a total of 69 patients provided sufficient dietary data , whereas 79 patients had data on plasma pl . totally 65 crc patients with median age of 47 years ( range 2754 years ) , and median bmi of 24.8 kg / m ( range 18.443.2 kg / m ) , had data on fatty acid composition in diet and plasma pl . altogether 32 patients had additional data on fatty acid composition in tumour and normal colonic mucosal tissue . forty - eight percent of the subjects were overweight or obese ( 25 kg / m ) , whereas six percent of the patients had low bmi ( < 20 kg / m ) . thirteen percent of the subjects reported themselves as current daily smokers , whereas 37 percent reported never having smoked . first or second degree family history of crc in 14 percent of the patients was confirmed by the cancer registry of norway . the self - reported physical activity level was not associated with dietary intake of energy or fat , or bmi ( not shown ) . mean dietary intake of total and saturated fat was somewhat higher than in the nordic nutrition recommendations . intakes of total and long - chained marine n-3 pufa were in line with the recommendations ( table 2 ) . dietary dha intake and the sum of epa and dha intake correlated positively with the respective concentrations in plasma pl ( r = 0.42 , p = 0.001 and r = 0.36 , p = 0.003 , resp . ) . other n-3 or n-6 pufa did not show correlation between diet and plasma pl , but the ratio of n-3/n-6 pufa showed a correlation between diet and plasma pl ( r = 0.29 , p = 0.02 ) ( table 2 ) . the correlations did not noticeable differ between lean and overweight patients ( not shown ) . when separating the crc patients into low and high dietary epa + dha intake at a median of 0.82 g / day , the patients with intake above the median had higher plasma pl concentration of dha and n-3/n-6 pufa ratio ( p = 0.004 and p = 0.006 , resp . ) , and borderline significantly lower plasma pl concentration of la + aa and ratio of aa / epa ( p = 0.05 for both ) ( table 3 ) . median concentrations of all individual fatty acids of interest differed significantly between normal mucosa and tumour tissue ( table 4 ) . median concentrations of all long - chained n-3 and n-6 pufa ( c20 - 22 ) were higher in tumours than normal biopsies , whereas concentrations of the c18-chained pufa la and ala were lower in tumour tissue than normal mucosa . aa and dha were higher in tumours than normal biopsies ( 7.5 versus 3.5 g/100 g , p = 0.001 , and 2.2 versus 1.3 g/100 g , p < 0.001 , resp . ) . the ratio n-3/n-6 pufa , but also the ratio aa / epa were higher in the tumour than normal tissue ( 0.15 versus 0.13 , p < 0.001 and 12.5 versus 9.7 , p = 0.03 , resp . ) ( table 4 ) . there was a significant correlation between plasma pl concentrations of epa and dha ( weight% ) and the concentrations of epa and dha in normal mucosa and tumour tissues . these correlations existed both for epa and dha separately , and for the sum of these ( table 5 ) . also the ratio n-3/n-6 pufa in normal mucosa and tumour tissue significantly correlated with plasma pl concentration of epa + dha . the significant correlation between plasma pl concentration of epa + dha , and aa concentration in normal mucosa ( r = 0.43 , p = 0.02 ) remained significant only in overweight patients ( r = 0.69 , p = 0.005 ) when stratifying by bmi groups . plasma pl concentration of la + aa ( weight% ) correlated positively with la concentration in normal mucosa tissue . there were no other correlations between plasma pl concentration of la + aa , and tissue pufa concentrations , but the ratio of n-3/n-6 pufa in normal mucosa was significantly inversely correlated with plasma pl concentration of la + aa ( table 5 ) . median dietary intake of dha was higher in crc patients with severe duke 's stage ( c + d ) as compared to patients with less severe duke 's stage ( a + b ) ( 0.56 versus 0.45 g / d , p = 0.02 ) ( table 6 ) . further , dietary ratio of n-3/n-6 pufa was higher in patients with duke 's stage c + d compared to a + b ( 0.21 versus 0.18 , p = 0.007 ) . there were no significant differences in dietary aa content or plasma pl concentrations of any n-3 or n-6 pufa between the duke 's stage groups ( table 6 ) . there was no difference between the duke 's stage groups in any of pufa concentrations or ratios in tumour and normal mucosal tissue ( not shown ) . in the present study of young crc patients , we found that dietary intake of total pufa and marine n-3 fatty acids epa and dha were according to national dietary recommendations . dietary dha intake was in this patient population related to its concentration in plasma pl , tumour tissue , and normal mucosa . dietary intake of single n-6 pufa , la and aa did not correlate with its concentration in plasma pl , tumour tissue or normal mucosa . on the other hand , high dietary intake of epa and dha was reflected as high concentrations of these fatty acids and a high ratio of n-3/n-6 pufa in plasma and tissues . concentrations of n-3 and n-6 pufa with chain length c20 - 22 ( aa , epa and dha ) and the ratio of n-3/n-6 pufa were higher in tumour tissue than normal mucosa , whereas concentrations of pufa with chain length c18 ( la and ala ) were higher in normal mucosa . surprisingly , dietary dha intake was positively related to cancer severity measured by duke 's stage . the present results suggest that fatty acid metabolism is altered in crc patients , as indicated by earlier studies . first , we did not find correlation between dietary intakes of single n-6 pufa and plasma pl concentrations of these in crc patients . the absence of correlation for la between diet and plasma pl agrees with an earlier similar finding in fap patients , but disagrees with large studies in average , diverse adult population , which have demonstrated a significant positive correlation between la concentration in plasma pl ( weight% ) and habitual diet assessed by ffq [ 21 , 22 ] . another sign of abnormal la metabolism in crc in the present study was that concentration of la in plasma pl significantly correlated with la concentration in normal mucosal tissue , but not in tumour tissue . this indicates an altered incorporation of la from plasma pl into tumour tissue , or increased elongation of la into aa in tumours . as a contrast , there were high correlations between intake levels , plasma pl and tumour and normal mucosal tissue concentrations for epa and dha in the present crc patient population . such high correlations between intake and biomarkers of these external pufa are found in average population [ 21 , 22 ] and were expected also for la . n-3 pufa concentrations in plasma pl were reflected in their concentrations in both normal mucosa and tumour tissue in the present study . an unexplainable finding was that n-3 pufa concentration in plasma pl correlated also with aa concentration in normal mucosa , and only in overweight and not in lean patients . the second major finding on altered fat metabolism in crc patients was that pufa concentrations of crc tumour tissue differed from non - diseased mucosal tissue in the present patient group ; aa , epa , and dha were more abundant in tumour tissue than in normal colonic mucosa . this reflects the pufa pattern in plasma pl found earlier in fap patients as compared to control subjects . similar results on increased aa and dha concentrations in human crc tumours and a higher concentration of aa in rat tumour tissue have been found . in contrast , the opposite has also been found ; lower aa concentration in phospholipids extracted from human colon cancer cells compared to normal mucosa cells , non - different concentrations of aa and dha , and lower concentration of epa in human diseased versus normal mucosa . the ratio n-3/n-6 pufa in plasma pl and tumour tissue was in the present study correlated to dietary intake of epa and dha and was higher in tumour tissue than in normal mucosa . this suggests that epa and dha are incorporated from diet into plasma and tissues in crc patients , but that these are particularly accumulated in tumour tissue . the lower concentrations of ala in tumour than normal mucosal tissue may also suggest an increased elongation of ala into epa and dha in tumours . the markedly increased aa concentration and lower la concentration concentrations of all other measured fatty acids were also significantly different between tumour tissue and normal mucosa . interpretation of differences in sfa and mufa concentrations is difficult and may reflect the differences in pufa concentrations . the third and most unexplainable finding of altered pufa metabolism in the present study was that patients with the most advanced cancer stage reported highest dietary intake of dha , and ratio of dietary n-3/n-6 pufa . however , this difference in intake between duke 's stage groups a + b and c + d was not reflected in plasma pl . one reasonable explanation might be overreporting of dietary marine n-3 pufa intake among those with the most severe diagnosis . the ffq was filled by the patients up to four weeks after the crc diagnosis . dietary habits may have changed after the diagnosis , biasing reporting of average diet before the diagnosis . a possible true relationship between higher dietary intake of dha and crc severity would be unexpected in the light of the major evidence for a protecting effect of n-3 pufa in colorectal carcinogenesis [ 25 , 26 ] . animal and cell line experiments suggest that n-3 pufa on the contrary may reduce colorectal tumour formation and suppress aa - induced proliferation in colon carcinoma cells . there also is a plausible pathway for the anticarcinogenic effect of n-3 pufa through inhibiting pge2 synthesis and consequently reducing cox-2 expression [ 29 , 30 ] , an important step in colorectal carcinogenesis . further , there is evidence that dietary n-3 pufa intake protects against inflammation in cancer patients by an effect on cox-2 levels [ 31 , 32 ] . a possible carcinogenic effect of dietary epa and dha might relate to proinflammatory activity of these highly oxidizable fatty acids . high - dose n-3 pufa intervention ( 2.54.8 g epa + dha per day ) in other patient groups has resulted in increased concentrations of soluble inflammatory markers of endothelial function [ 34 , 35 ] . these , as well as other inflammatory markers have been related to crc development and increasing duke 's stage [ 3638 ] . the median dietary intake of epa and dha in the present patient group was , however , according to national recommendations , and only two patients reported intake levels above 2.0 g per day . in spite of an average intake of fatty acids as recorded by the questionnaires , overweight was prevalent ( 48% of the patients with bmi 25 kg / m ) . malignancy at younger age may indicate genetic involvement , but also a direct relationship between overweight and colorectal neoplasia could be possible . nevertheless , we did not observe higher prevalence of overweight or obesity in the group with higher duke 's stage . future long term studies are needed to confirm a possible association between intake of high - dose n-3 pufa intake and duke 's stage . the study would also have been strengthened by additionally analyzing fatty acid patterns in adenomas , since colorectal neoplasia develops over many years and the metabolic situation in cancerous tissue is different from that in adenomas . further , it is unclear to what extent fatty acid intake right before the diagnosis , as assessed in the present study , is the critical period of intake with regard to tumorigenesis . the most optimal study design might therefore include dietary intake , plasma and tissue samples from patients with colorectal adenomas , and crc patients and healthy controls . the present study showed that metabolism of n-6 and n-3 pufa was altered in young crc patients . dietary la did not correlate with its concentration in plasma pl and normal tissue , indicating an increased elongation of la into aa in tumour tissue . the higher concentrations of aa , epa and dha , and ratio of n-3/n-6 pufa in colorectal tumours than in normal mucosal tissue warrants further investigations . we suggest that future prospective studies address the possible relation between dietary intake of n-3 pufa and duke 's stage in crc in comparison to normal mucosa and adenomas , both among young and old patients .

fatty acid metabolism is altered in colorectal cancer ( crc ) . we aimed to investigate incorporation of dietary n-6 and n-3 polyunsaturated fatty acids ( pufas ) into plasma phospholipids ( pls ) , tumour tissue , and normal mucosa in young crc patients . we also aimed to study differences in pufa composition between tumour and normal mucosa , and pufa status associated with cancer stage . sixty - five crc patients younger than 55 years were included in a multicenter study . we assessed dietary fatty acid composition by food - frequency questionnaire . fatty acid composition in plasma pl ( n = 65 ) and tumour and normal colonic biopsies ( n = 32 ) were analysed by gas chromatography . we observed a significant correlation for docosahexaenoic acid ( dha ) between dietary intake and concentration in plasma pl ( weight% ) ( r = 0.42 ; p = 0.001 ) , but not for any n-6 pufa . tissue concentrations of arachidonic acid , eicosapentaenoic acid , and dha ( weight% ) were 1.72.5 times higher in tumour than normal mucosa ( p 0.001 ) . concentrations of n-3 and n-6 pufa in plasma pl and tissues were not related to duke 's stage , although patients with more severe cancer stage reported higher intake of n-3 pufa . in conclusion , we found accumulation of the long - chained n-3 and n-6 pufa in tumour tissue in young crc patients .

alzheimer s disease ( ad ) is the most common cause of dementia worldwide , affecting an increasing number of people each year due to population aging . recently published diagnostic algorithms for ad enable its diagnosis independently of its symptoms , based on biomarkers , for instance on cerebrospinal fluid ( csf ) amyloid- 1 - 42 ( a1 - 42 ) and tau proteins , which improve the diagnostic accuracy for ad . csf levels of total - tau ( t - tau ) are typically elevated in ad and are associated with neuronal and axonal damage.1 csf levels of a1 - 42 are reduced in ad and reflect the higher amyloid plaque burden in the brain.2,3 consistent with histopathological findings , csf chemistry studies have identified that reductions of a1 - 42 occurs several years before symptom onset . at 90% specificity , a1 - 42 discriminates ad from cognitively healthy persons with 85% sensitivity.4 furthermore , a1 - 42 has a high positive predictive value for the conversion from mild cognitive impairment to dementia in ad.5 the 4 allele of the apolipoprotein e ( apoe 4 ) gene is the strongest genetic risk factor for sporadic ad known to date.6 cell culture and animal models have identified potential pathogenic mechanisms of apoe which are related to amyloid- production and clearance , to tau hyperphosphorylation , and to synaptic function.7 several studies have reported that apoe 4 carrier have lower csf a1 - 42 levels than noncarriers , ie,8,9 some studies have described an association between sex and apoe with women having a higher 4-associated risk of developing ad than men10 but the effects of sex on csf biomarker levels are still under debate . although several studies indicate that not only familial but also sporadic early - onset ad ( eoad ) might slightly differ from late - onset ad ( load ) with regard to amyloid- production and clearance pathways,11,12 studies on the possible different aspects of apoe on csf biomarker levels in eoad and load are scarce . we hypothesized that the effect of apoe on csf biomarker levels might differ between eoad and load and that sex might have additional impact on the effect of apoe . therefore , the aim of the present study was to examine the associations of apoe 4 allele frequency , genotype , age , and sex with the csf levels of a1 - 42 and t - tau in a monocentric , memory - clinic based patient with ad sample that includes a high proportion of patients with eoad . the research project has been approved by the institutional review board of the medical faculty , technische universitt mnchen , munich , germany . the data of 117 german patients with mild to moderate probable ad according to the national institute of neurological and communication disorders and stroke / alzheimer s disease and related disorders association diagnostic criteria13 were identified in a pre - existing database that contains the data of patients which had been diagnosed with ad at an outpatient memory clinic between 2005 and 2011 . the patient data were included in the present study only , if a detailed , standardized neuropsychological assessment had been conducted , as well as structural and functional cerebral imaging , if a lumbar puncture had been performed with csf biomarker analysis , if the patient s apoe genotype was available , and if the patients had provided written informed consent for including their data in research projects . clinical diagnosis was based on information gathered from neurological and neuropsychiatric examination , and informant interviews . all patients underwent a thorough medical screening including laboratory tests ( serum chemistry , blood count , thyroid stimulating hormone , vitamin b12 , and folate levels ) , and a neuropsychological evaluation using the german version of the consortium to establish a registry in alzheimer s disease neuropsychological battery14 which incorporates the mini mental state examination ( mmse).15 severity of dementia was estimated using the clinical dementia rating scale.16 all patients had got either cranial computed tomography or magnetic resonance imaging . in 71 patients , f - fluoro-2-deoxy - d - glucose positron emission tomography ( fdg - pet ) scans had been performed the results of which were consistent with ad in any case . the csf samples were analyzed sample by sample , using commercially available enzyme - linked immunosorbent assays ( innotest , innogenetics , ghent , belgium ) to determine the levels of t - tau , and a1 - 42 . the -test was used to evaluate the differences of family history between patient groups ( male / female and eoad / load ) . fisher s exact test was used to evaluate the differences of apoe allele frequencies between groups . p - values are two sided and subject to a local significance level of < 0.05 . possible effects of severity of disease as measured by mmse , and sex were included as a covariate in ancova models . the research project has been approved by the institutional review board of the medical faculty , technische universitt mnchen , munich , germany . the data of 117 german patients with mild to moderate probable ad according to the national institute of neurological and communication disorders and stroke / alzheimer s disease and related disorders association diagnostic criteria13 were identified in a pre - existing database that contains the data of patients which had been diagnosed with ad at an outpatient memory clinic between 2005 and 2011 . the patient data were included in the present study only , if a detailed , standardized neuropsychological assessment had been conducted , as well as structural and functional cerebral imaging , if a lumbar puncture had been performed with csf biomarker analysis , if the patient s apoe genotype was available , and if the patients had provided written informed consent for including their data in research projects . clinical diagnosis was based on information gathered from neurological and neuropsychiatric examination , and informant interviews . all patients underwent a thorough medical screening including laboratory tests ( serum chemistry , blood count , thyroid stimulating hormone , vitamin b12 , and folate levels ) , and a neuropsychological evaluation using the german version of the consortium to establish a registry in alzheimer s disease neuropsychological battery14 which incorporates the mini mental state examination ( mmse).15 severity of dementia was estimated using the clinical dementia rating scale.16 all patients had got either cranial computed tomography or magnetic resonance imaging . in 71 patients , f - fluoro-2-deoxy - d - glucose positron emission tomography ( fdg - pet ) scans had been performed the results of which were consistent with ad in any case . the csf samples were analyzed sample by sample , using commercially available enzyme - linked immunosorbent assays ( innotest , innogenetics , ghent , belgium ) to determine the levels of t - tau , and a1 - 42 . the -test was used to evaluate the differences of family history between patient groups ( male / female and eoad / load ) . fisher s exact test was used to evaluate the differences of apoe allele frequencies between groups . p - values are two sided and subject to a local significance level of < 0.05 . possible effects of severity of disease as measured by mmse , and sex were included as a covariate in ancova models . patient characteristics are summarized in table 1 . in all , 49% of the patients were male , 53% were patients with eoad , defined as having an age of disease onset below 65 years . csf biomarker levels , family history for dementia , and apoe allele frequency are also provided in table 1 . female patients had got a significantly lower mmse score than male patients but did not differ from the male patients in any other variable . in particular , csf levels of a1 - 42 and t - tau did not significantly differ between male and female patients as well as patients with eoad and load . likewise , the distribution of the six apoe isoforms was similar in female and male patients as well as patients with eoad and load . in the total ad group , 60% of the patients carried at least one apoe 4 allele and 18% were homozygous 4-carriers . table 2 shows the mean levels of csf biomarkers according to apoe genotype in the whole group of patients and in the subgroups of patients with eoad and load , respectively . 4 homozygous subjects in the total group of patients and in the load group but not in the eoad group had significant lower csf a1 - 42 levels compared with 4 heterozygous subjects and 4 noncarriers ( whole group p=0.002 ; load p=0.003 ) . csf levels of t - tau did not demonstrate significant differences with regard to the presence of one or two 4 alleles neither among the whole group nor among the eoad and load subgroups . unlike in the whole group and in patients with load , in the eoad group the t - tau levels did not show an increase from 4 noncarriers over homozygous to heterozygous carriers . a univariate ancova ( table 3 ) showed that in the total group of patients only the apoe 4 dose was significantly associated with csf a1 - 42 levels . sex as well as mmse score and age group ( eoad vs load ) did not have a significant effect . table 2 shows the mean levels of csf biomarkers according to apoe genotype in the whole group of patients and in the subgroups of patients with eoad and load , respectively . 4 homozygous subjects in the total group of patients and in the load group but not in the eoad group had significant lower csf a1 - 42 levels compared with 4 heterozygous subjects and 4 noncarriers ( whole group p=0.002 ; load p=0.003 ) . csf levels of t - tau did not demonstrate significant differences with regard to the presence of one or two 4 alleles neither among the whole group nor among the eoad and load subgroups . unlike in the whole group and in patients with load , in the eoad group the t - tau levels did not show an increase from 4 noncarriers over homozygous to heterozygous carriers . a univariate ancova ( table 3 ) showed that in the total group of patients only the apoe 4 dose was significantly associated with csf a1 - 42 levels . sex as well as mmse score and age group ( eoad vs load ) did not have a significant effect . the main finding of this monocentric , memory - clinic based study that included 117 patients with ad ( 53% eoad , 47% load ) is the significant association between apoe 4 allele frequency and csf a1 - 42 levels . homozygous 4-subjects had significantly lower csf a1 - 42 levels compared to heterozygous 4-carriers and 4 noncarriers , suggesting a dose - dependent effect of apoe 4 on csf a1 - 42 levels . neither sex nor age or severity of dementia as measured by mmse was significantly associated with csf a1 - 42 levels . no significant association was detected between apoe 4 and csf t - tau - levels . as early as 1993 , genetic analyses identified apoe 4 as the major risk factor for ad.6 since then , apoe has been established as the most important susceptibility gene for late - onset sporadic ad.17 the apoe 4 isoform appears to enhance amyloid- production and cerebral plaque deposition and seems to alleviate the clearance of amyloid- ( for a detailed review , see yu et al18 ) . in line with these findings , several studies have found a dose effect of apoe 4 on csf a1 - 42 levels not only in ad patients but also in subjects with mild cognitive impairment and cognitively healthy controls : low csf levels of a1 - 42 , a marker of amyloid- plaque load , are linked to the presence of apoe 4 . for example , vemuri et al found a clear apoe 4 dose effect on csf a1 - 42 levels in 98 patients with ad from an alzheimer s disease neuroimaging initiative ( adni ) study.9 a recent multicenter study by lautner et al which included 309 patients with ad ( mean age 77 years ) showed that apoe 4 carriers had lower csf a1 - 42 levels compared to noncarriers in a dose - dependent manner.8 it is still under debate whether the association of low csf a1 - 42 levels with the presence of apoe 4 should be considered for the definition of cutoff levels of csf a1 - 42 when using them as a biomarker for ad . some studies suggest that the apoe genotype should be taken into account ; others , however , conclude that the cutoff level for csf a1 - 42 should be the same for all apoe genotypes.19 it is important to highlight , that thus far , large studies have overlooked the association between apoe polymorphism and csf biomarker levels focusing only on load . this is surprising because the pathophysiology , particularly with regard to amyloid- production and clearance , appears to differ between load and ( not only familial but also ) sporadic eoad.11,12,20 therefore , this study is the first to examine the association of apoe 4 with csf biomarker levels specifically in patients with eoad and find that in eoad in contrast to load and the whole patient group the apoe 4 allele frequency was not significantly associated with csf a1 - 42 levels . however , given the relatively small sample of patients with eoad in the present study , this finding needs to be investigated further in larger studies . it appears to affect tau neuropathological changes in ad brains and in animal models.18 human studies of the relationship between apoe 4 and cerebral tau , however , showed contradictory results.21 nonetheless , several studies found that apoe 4 carriers have higher csf t - tau levels than noncarriers.9 in the present study , significant effects of the apoe 4 dose on csf t - tau levels were not detected . our findings reveal that neither sex nor age alone appeared to influence csf levels of a1 - 42 and t - tau . while it is well established that , alike apoe 4 , female sex and older age are risk factors for ad , the study results are inconclusive with regard to the effect of female sex or higher age on csf biomarker levels . for example , an adni study of 144 ad patients showed that women and apoe 4 carriers experience higher rates of cognitive decline ; however , in this study no significant effects of sex on csf levels of a1 - 42 and t - tau were detected.22 on the contrary , a meta - analysis reported an interaction between sex and apoe with women having a higher 4-associated risk of ad than men.10 in a recent large study of 2,375 swedish ad subjects , logistic regression models revealed that female sex increased the risk of having pathologic csf biomarker levels ( decreased a1 - 42 , increased t - tau and phospho - tau ) . in this model , apoe genotypes were not taken into account in this study.23 in all 60% of the patients in our study carried at least one apoe 4 allele . the reasons for the high proportion of 4 carriers in our sample can only be speculated . the patients included in the study had extensive diagnosis including detailed neuropsychology , csf analysis , structural imaging in all and 61% of the patients had fdg - pet - imaging . therefore , the probability of misdiagnoses is extremely low and thus a pure alzheimer cohort might explain higher apoe 4 rates than usual . the present study has some limitations : first and most important , the patient sample is relatively small . statistical power is limited with respect to analyses of csf biomarkers in relation to a small sample of patients that homozygous apoe 4 carriers . the unicenter design with patients from one specialized memory clinic may limit the generalization of the results to the whole population of patients with ad . however , this study reflects the real - life practice in a specialized memory clinic . furthermore , a unicenter study allows the use of harmonized procedures for the measurement of csf biomarkers , avoiding assay - related preanalytical and analytical factors between laboratory variability . the strengths of the study lie in its detailed cognitive assessment protocol and careful diagnostic ascertainment . the diagnoses were performed by a trained team of psychiatrists and neuropsychologists highly specialized in cognitive disorders who used all available clinical and technical data , including fdg - pet scans in 61% of patients . last but not least , the study included a high proportion of patients with eoad . this result is consistent with the findings of several previous studies . in the subgroup of patients with eoad larger studies are necessary to further investigate associations between apoe 4 allele frequency and csf biomarker levels in patients with eoad .

introductioncerebrospinal fluid ( csf ) biomarkers improve the diagnostic accuracy for alzheimer s disease ( ad ) , even at the predementia stage of the disease . the 4-allele of apolipoprotein e ( apoe 4 ) , female sex , and older age are well - known risk factors for ad . it is unclear how these risk factors affect the csf biomarkers in patients with ad.aimthe objective of this study was to investigate the associations of apoe 4 , sex , and age with csf biomarker levels in a unicenter sample of patients with ad that includes a high proportion of patients with early - onset ad ( eoad).methodsthe csf levels of amyloid- 1 - 42 ( a1 - 42 ) and total - tau of 117 subjects with mild to moderate ad ( 55 late - onset ad and 62 eoad ) were assessed . all subjects underwent apoe genotyping , clinical evaluation , comprehensive neuropsychological assessments , and neuroimaging . associations between csf biomarker levels , apoe 4 allele frequency , age , and sex were evaluated.resultsin the whole patient sample and in the late - onset ad subgroup 4 homozygous subjects had significantly lower csf a1 - 42 levels compared with 4 heterozygous subjects and 4 noncarriers . this association was not detected in the eoad group . age group , sex , and severity of cognitive decline did not have a significant impact on csf a1 - 42 levels . no significant associations were found between apoe 4 allele frequency and csf total - tau levels.conclusionapoe 4 allele is associated with a reduction of csf a1 - 42 levels . this result is consistent with the findings of several previous studies . in the subgroup of patients with eoad this association was not replicated . larger studies are necessary to further investigate associations between apoe 4 allele frequency and csf biomarker levels in patients with eoad .

a recently published article on the specificity and sensitivity of disease identification utilizing the ivue iwellnessexam test revealed that the data provided were sufficient for a well - trained eye clinician to review and accurately detect disease in a very high percent of subjects with either retinal and/or optic nerve ( on ) disease and to accurately confirm health in an extremely high percent of healthy controls . this sd - oct scan obtains a substantial amount of data for the assessment of both central retina and optic nerve integrity simultaneously [ 27 ] . a follow - up pilot study was undertaken with the same set of data to determine whether novice review of the same sd - oct data is an effective way to identify retinal and/or optic nerve disease and to confirm health in normal subjects . the previous study was designed to measure the specificity and sensitivity of a well - trained optometric clinician , utilizing only data obtained on the iwellnessexam test , in the identification of retinal and optic nerve disease in a cohort of confirmed normal ( cn ) and confirmed disease ( cd ) subjects . specificity data were obtained by evaluating patients within the primary care clinic at the university eye center ( uec ) at suny state college of optometry who were determined to be both without retinal and without on disorder ( cn subjects ) . sensitivity data were obtained by evaluating patients within the ocular disease and special testing service at the uec with known central retinal and/or optic nerve disorders ( cd subjects ) . suny irb approval was obtained prior to the initiation of the study , and all subjects signed a suny irb approved informed consent document . two groups of patients were examined : a confirmed normal ( cn ) cohort for the specificity aspect of the study ( 126 subjects ) and a confirmed disease ( cd ) cohort for the sensitivity aspect of the study ( 101 subjects ) . of the cd patients , ( sixteen ( 16 ) fell into both categories , with both retinal and on pathology . ) no glaucoma suspects were included for evaluation , as their status as a normal or as an on pathology subject could not be clearly established . it scans at 26,000 a - scans / second , with an axial resolution of 5 microns . all analyses were made utilizing the iwellnessexam , a one - step sd - oct scan , which images a 7 mm 7 mm area of the posterior pole centered on the fovea . the iwellnessexam report provides eight high - resolution cross - sectional retinal images , along with its data analysis results : a full retinal thickness map , a ganglion cell complex ( gcc ) map , and a report on superior / inferior ( s / i ) symmetry within the eye , and symmetry between eyes . note that these scans were obtained and reviewed before the release of the normative database for the ivue system . four individuals who were novices at reviewing sd - oct images were enlisted to participate in the clinical review of this data set . the novices were each of a different level of clinical and educational experience in the ophthalmic field . he has no interest in pursuing a career in clinical optometry or ophthalmic research . this individual has been accepted into the professional program at the suny state college of optometry . she has had 4 years of experience working in optometric and ophthalmological practices , including 18 months in an ophthalmological practice with 6 months as a technician , operating retinal scanning devices . this individual was in the middle of his first year of the professional program at the suny state college of optometry . prior to entering optometry school , he spent one full year in an internship / research program , involved with the publication of unusual cases evaluated with cutting - edge ophthalmic technology . this individual was in the middle of her third year of the professional program at the suny state college of optometry . she had previous experience in detecting pil abnormalities on sd - oct , based upon an unrelated independent study project . these four novices were provided with a single , 1.5-hour lecture with author js on the nature of the data obtained on ivue iwellnessexam , and on both numerical and pictorial data interpretation . prior to this subject data sets were given a randomized code number , which served as the only identifier for each subject . reviewers did not have access to any supplementary patient history , demographic , or clinical data . the novice reviewers were instructed to classify each subject into one of four categories : ( 1 ) normal , ( 2 ) retinal disease , ( 3 ) on disease , and ( 4 ) retinal + on disease . they were also requested to record the amount of time spent in review sessions so that an estimate of the amount of time spent per image could be made . novice reviewers accurately identified disease ( sensitivity ) in 90.6 6.3% of cd subjects and accurately identified health ( specificity ) in 84.3 5.2% of cn subjects , utilizing only the iwellnessexam data . see table 1 and figures 1 and 2 for a detailed display of reviewer sensitivity and specificity data . data were also evaluated for predictive value . these are measures of the reliability of a positive or a negative result on a test . positive predictive value ( ppv ) is the percent of time that a positive test result will indicate disease . ppv is calculated as the number of true positives relative to the number of subjects who were identified as positive for the condition in question . negative predictive value ( npv ) is the percent of time that a negative test result will indicate health . npv is calculated as the number of true negatives relative to the number of subjects who were identified as negative for any condition . ( thus , a reviewer with high sensitivity for a disease , but who tends to over - refer , will identify more subjects as positive for a test than are truly positive . all novice reviewers demonstrated a greater ppv for the general category of disease than for either subcategory and a greater ppv for retinal disease than for on disease ( see figure 3 ) . this implies that overreferrals for disease primarily occurred in subjects who had only retinal disease ( category 2 ) but were classified as category 4 ( retinal + on disease ) . the novices on the whole perform well on the most important factor : appropriate referral of patients who have any disease ( 82.4 5.0% , with a range of 7889% ) . retinal disease overreferrals in patients with on disease appear to abate with optometric education ( 3rd year more successful than 1st year at correctly identifying retinal disease ) . on disease by contrast , all reviewers performed with a high npv , ranging from 85% to 98% ( see figure 4 ; table 2 ) . if the novices identified a patient as normal , there was a 92.0 4.8% chance that disease was not present . with a small sample of novice reviewers , and with variations in their educational backgrounds , it is not easy to rank their relative exposures to ophthalmic conditions and expected disease identification ability . this evaluates each subject 's false positive rate ( 1 specificity ) relative to their true positive rate ( sensitivity ) . best overall performance is defined by minimizing the false positives while maximizing sensitivity , with the most desirable performance being plotted at the top left corner of the roc space . roc plots were used to compare ( 1 ) expert performance for overall disease and for the two subcategories of retinal and optic nerve disease ( figure 5(a ) ) and ( 2 ) the novices with the expert and with each other ( figure 5(b ) , all disease ; figure 5(c ) , retinal disease ; figure 5(d ) , optic nerve disease ) . for ease of comparison , the two - dimensional roc findings are also presented as an accuracy rating . accuracy is calculated as the sum of the true positives and true negatives divided by the sum of the total number of positives and negatives . figure 6 compares the novices ' accuracy , arranged by relative amount of time spent in optometric education . figure 7 also compares their accuracy , rearranged to reflect their relative amount of clinical exposure time . the novices conducted image review over an average of 4 sittings ( ranging from 2 to 6 sittings ) and spent an average of 59 13 sec per image set ( range 49 to 77 sec per image set ) . . there does seem to be a correlation between the amount of time spent per image set and the accuracy of the subject categorization among novices ( see figure 8) . above all , a screening protocol needs to be capable of disease detection . the data obtained on iwellnessexam may complement the clinical data obtained in the course of a routine exam [ 1 , 912 ] . once disease is detected or suspected , appropriate referrals can be made for follow - up testing and clinical evaluation . the results here show that individuals who are novices at reviewing sd - oct images can be trained in a short amount of time to achieve an impressive rate of detection of the presence of posterior segment disease , while maintaining high specificity for the affirmation of health in control subjects , using only the data provided on iwellnessexam. another study evaluating the learning curve of a novice relative to an expert in imaging interpretation showed a similar learning effect with good accuracy when compared to the expert . a study evaluating the value of problem - based learning as compared with more conventional teaching methods concludes that problem - based learning produces better educational results . thus , in a clinical environment , an ongoing feedback process between the evaluating clinician and the detecting technician will help technicians learn to interpret scans with even greater levels of accuracy . differences in educational versus clinical exposure are made apparent in the present pilot study . from an educational standpoint , the novices may be ranked : a < b < c < d ( refer to methods ) . however , from a clinical exposure standpoint , the amount of contact time with patients and with review of typical clinical data may be ranked : a < c < d < b , as the pre-1st - year technician has had 4 years of exposure to an ophthalmic environment and has collected clinical data from a typical cross section of the population . assuming this technician is representative of the value of clinical learning , her performance edifies the findings of the value of problem - based learning in medical education [ 11 , 14 ] . in some regards , the 1st - year student ( c ) may have had a relative challenge in identifying normal , as he spent a year in ophthalmic research , exposed to challenging cases of ophthalmic disease with subtle findings . this may have predisposed him to identify disease , even in subtle cases , but not to identify health ( i.e. , reduced specificity ) . the roc plots enable a two - dimensional perspective on reviewer accuracy , at a glance . the pre-1st - year optometry student who has experience as an ophthalmic technician ( pink square , figure 5 ) consistently outperformed the other novices . this performance supports the need for clinical exposure to general practice in order to help students contextualize their clinical observations . the 1st - year and nonoptometric reviewer have similar levels of clinical exposure . while they make different errors in reviewing the data in figures 5(b ) and 5(c ) ( one has more false positives with lower sensitivity ; the other has fewer false positive with higher sensitivity ) , their performance is similar in terms of their accuracy ( see figure 7 ) . the novices were asked to report on the amount of time spent reviewing the data and the number of sittings . novices b and d took a longer amount of time reviewing each subject 's data set ( which consisted of 3 image files ) . there is an apparent correlation between the amount of time invested in image review and the accuracy of the overall categorization exercise . interestingly , this correlation appears strongest for retinal disease ( r = 0.98 ) , which requires a higher level of image scrutiny than the determination of optic nerve disease ( r = 0.78 ) . the reduced ppv and reduced sensitivity for patients with optic nerve disease , as compared to retinal disease , may be attributed to the challenges of assessing rnfl in the presence of an irregular outer retina , or even inner retinal disturbances , such as vitreoretinal adhesions . the interpretation of these challenging situations has been explored in detail , interpreting the ganglion cell complex in the presence of retinal pathology [ 1 , 15 ] . the iwellnessexam offers the health care provider a very reliable technology for the clinical identification of eyes at risk . novices can be trained in a short amount of time to effectively use the data from the iwellnessexam to screen for disease with a high rate of sensitivity , while maintaining high specificity . accuracy of the novice reviewers covaries with both clinical exposure and time spent on image review per subject . this study shows a small sample of novice reviewers with different levels of clinical and educational exposure . it would be insightful to undertake this review with a larger sample of students at various stages of optometric education . in the interest of public health , a similar study could be undertaken with training of nonophthalmic medical technicians , to explore the potential for the identification of eye disease in patients who do not seek routine eye care but do manage their health with primary medical providers . indeed , it is often an eye exam which results in medical referrals following the identification of retinal pathology .

introduction . four novices to spectral domain optical coherence tomography ( sd - oct ) image review were provided a brief lecture on the interpretation of ivue iwellnessexam findings ( available on ivue sd - oct , optovue , inc . , fremont , ca ) . for a cohort of 126 ( confirmed ) normal , 101 ( confirmed ) disease subjects , iwellnessexam od , os , and ou reports were provided . each novice independently reviewed and sorted the subjects into one of four categories : normal , retinal disease , optic nerve ( on ) disease , and retinal + on disease . their accuracy is compared between the novices and with an expert reviewer . results . posterior segment disease was properly detected by novices with sensitivities of 90.6% , any disease ; 84.3% , retinal disease ; 88.0% , on disease ; expert sensitivity : 96.0% , 95.5% , and 90.0% , respectively ; specificity : 84.3% , novices ; 99.2% , expert . novice accuracy correlates best with clinical exposure and amount of time spent reviewing each image set . the novices ' negative predictive value was 92.0% ( i.e. , very few false negatives ) . conclusions . novices can be trained to screen for posterior segment disease efficiently and effectively using iwellnessexam data , with high sensitivity , while maintaining high specificity . novice reviewer accuracy covaries with both clinical exposure and time spent per image set . these findings support exploration of training nonophthalmic technicians in a primary medical care setting .

the development of dual - energy ( de ) ct scanners paved the road for new clinical applications , taking advantage of the energy dependence of x - ray attenuation in matter , a fact that formerly had been considered a drawback . this technology allows for exposing patients to two distinct spectra , acquiring information about the material composition of the interior of the body . besides scanners with two x - ray sources and detectors there also exist approaches that , for instance , apply a fast kvp switching technique or dual - layer detectors [ 3 , 4 ] to acquire spectrally resolved scan data . all those approaches have in common that they are hardly extendable to produce more than two spectrally well separated data sets . additionally , spectrally resolved data from dual - source dual - energy and fast kvp switching devices do not match exactly due to a time shift between measured sinograms or projections , respectively , making a correct statistical treatment of the problem in image reconstruction difficult . the continuous improvement of photon - counting detectors ( pcds ) over the past few years promises a remedy for this limitation and holds new possibilities for multienergy imaging . recent prototype - detectors already provide two to four [ 5 , 6 ] discriminator thresholds . this allows the acquisition of two to four sinograms in a single scan , each sensitive to a different part of the applied tube spectrum . apart from energy - resolved scan data , photon - counting technology can provide further benefits , like reduced image noise and enhanced contrast [ 8 , 9 ] . however , there are also issues with the new detector type concerning its capability of high x - ray fluxes which may occur in clinical ct examinations . additionally , k - escape events and crosstalk between adjacent pixels lead to a degradation in spectral resolution . so the challenge is to find a compromise between spectral sensitivity and high flux - rate capability to ensure the clinical usability of photon - counting detectors . in established dual - energy applications , the spectral information is used , for instance , to classify different body materials , identify high contrast agent concentrations that indicate pathological tissue , or support an early diagnosis of gout by helping to distinguish urate from small calcifications [ 11 , 12 ] . other applications utilize the spectral information to remove osseous structures in the patient image , revealing previously covered structures . usually , a linear material decomposition is performed subsequently to image reconstruction , although raw data based approaches have already been investigated . as a consequence of the material decomposition process , image noise is increased significantly , especially if more than two materials are to be separated . statistical reconstruction methods offer the opportunity to achieve images with reduced noise compared to convenient filtered back - projection ( fbp ) reconstructions by including mature regularization techniques . statistical methods account not for the physical effects governed by the absorption characteristics of matter only . they also respect the poisson nature of the emission and absorption processes in x - ray tubes and detectors , respectively . impinging photons are distributed into energy bins according to the energy assigned to them in the detection process . therefore , an increase in energy resolution implies smaller energy bins with reduced photon statistics for a constant number of impinging photons and constant spatial sampling . statistical algorithms converge to the most likely image that best fits the measured sinogram data , according to the underlying statistical model . the canonical measure for how well image and measured data agree is given by the logarithmic likelihood function ( log - likelihood ) , which must be maximized to find the best suiting image . since no analytic solution exists for the maximum of the poisson log - likelihood function it must be approximated by numerical methods . some years ago , a new class of statistical , polychromatic algorithms has been introduced [ 16 , 17 ] that exploit the convexity of the negative log - likelihood by minimizing surrogate functions instead . surrogate functions are successive local approximations of the negative log - likelihood function . with surrogates it is possible to formulate the optimization problem in a parallel manner , which allows quick computation on graphic processing units ( gpus ) . under certain circumstances the algorithm 's polychromatic nature also implies a beam - hardening correction ( bhc ) for the chosen basis materials ( cf . the algorithm can be applied to spectrally not resolved data ( e.g. , to sinogram data taken with a convenient ct system with an energy integrating detector ) with good results [ 19 , 20 ] . in our approach we drop these assumptions and reconstruct the material fractions directly from energy - resolved sinogram data . compared to our algorithm allows a simultaneous and parallel update of all material images and considers the detector response function of a photon - counting detector . the algorithm is a priori not limited to two spectrally well separated data sets but scales with the number of energy - resolved data sets . as the number of thresholds in future pcds might increase an extension of the material separation to the respective number of basis materials will be possible , as long as these materials are distinguishable by means of energy - resolved ct ( cf . section 2.1.3 ) . our intention is to evaluate the accuracy of the reconstructed material fractions and determine the anticipated gain in image quality due to image noise reduction from a correct statistical treatment of the problem . in section 2 the creation of x - ray photons and their absorption and registration in a counting detector pixel are statistical processes that can be described by a poisson random variable inini . in computed tomography the joint probability for the measurement of a complete sinogram is thus the product of the individual random variables of all sinogram pixels(1)pn n=i=1mpini ni=i=1mninini!eni.here i indexes all m projection values that constitute the complete sinogram . in photon - counting ct n i is the number of pulses registered in a single detector pixel and ni the corresponding expectation value . the negative logarithm of nn yields the canonical negative log - likelihood function which can be used as a measure for how well taken data n agree with their expectation values n ( 2)ln=logpn n=i=1mnilogni+ni.we use bold letters to indicate vector quantities . terms that are constant with respect to ni have been omitted , since the goal of statistical reconstruction is to find the expectation values ni that best fit the measured data , that is , the minimum of -l(n ) , which is not altered by terms constant with respect to ni . in multienergy imaging one can formulate separate log - likelihood functions lb(nn ) for all sinograms , each associated with one of the b energy bins provided by a pcd . if all random variables are statistically independent , which we assume here , an appropriate objective function for minimization is the sum over all b negative log - likelihood functions(3)lnb=1blbn=b=1b i=1mniblognib+nibb=1b i=1mhibnib.the statistical independence holds true only in the case of an ideal photon - counting detector . in realistic pcds dependencies between energy bins are introduced by k - escape , pixel crosstalk , electronic noise , and pulse pileup . our proposed algorithm accounts for correlations introduced by k - escape , pixel crosstalk , and electronic noise by considering the detectors response function , whereas pulse pileup is corrected in a preprocessing step directly on the sinogram raw data . in general , a minimization of the log - likelihood function alone leads to very noisy images . to compensate for this , a regularization term ( prior ) r it incorporates a priori knowledge about the material images f j that are to be reconstructed . they usually take the form(4)rkfk=j=1p lnjkfjkflk , where p is the number of image pixels and l labels the j neighboring pixels of a considered image pixel j. the index k labels the k material images that are to be reconstructed . we require the penalty functions to be strictly convex and twice continuously differentiable with respect to f j . this requirement is necessary to allow parallelization while assuring convergence of the final algorithm . the computational effort for this prior is higher compared to frequently utilized quadratic priors , but since it is a differentiable approximation of the huber prior it can better preserve contrast edges . the addition of the prior term to the negative log - likelihood function yields(5)f=lf+rfas new objective function , with the scalar product(6)rf=k=1kkrkfk.the parameter { } governs the strength of regularization . moreover , we made use of the fact that the number of pulses n i registered in a sinogram pixel depends on the fractions f of materials constituting the scanned object ; that is , n i = n i(f j ) . the dependence is given by lambert - beer 's law in ( 7 ) and ( 9 ) as described below . the goal of computed tomography is to reconstruct an attenuation map j describing the interior of a scanned object . the attenuation map can not be measured directly , but only via projections n i of the object . the link between the attenuation map j and its projections n i is given by lambert - beer 's law . but in clinical practice x - rays are created by x - ray tubes which emit quanta distributed over a continuous spectrum (e ) of energies . image reconstructions based on lambert - beer 's law , like filtered back - projection ( fbp ) , neglect the polychrome nature of the x - rays and can therefore lead to serious beam - hardening artifacts that degrade image quality . the artifacts need to be corrected , which usually happens in a preprocessing step . to avoid their generation already in the reconstruction process , it is necessary to know the materials composing the object and integrate lambert - beer 's law for polyenergetic x - rays ( cf . ) ( 7)nibemin , bemax , bni,0sbeexpj=1paijjede=ni,0beexpj=1paijjede , into the reconstruction algorithm . in ( 7 ) n i,0 ( e ) is defined as n i,0 ( e ) n i,0(e) ( e ) and is the spectrum the bth energy bin is sensitive to . for realistic pcds ( e ) can be calculated from the tube spectrum (e ) via(8)sbe=eminbemaxbsee , ede,where (e , e ) is the normalized detector response function . it states the probability of the detector assigning energy e to a measured photon with true energy e. the system matrix a { a ij } governs the contribution of the jth image pixel to the ith projection , so it implicitly contains the scanner geometry . neglecting rayleigh scattering , there are two physical effects relevant for the attenuation in clinical ct examinations : compton scattering and photoelectric effect . this fact would limit the number of materials separable with energy - resolved ct to k = 2 . fortunately , due to the unique spectral lines of each chemical element more than two materials can be separated , if at least k 2 of the k materials exhibit one or more individual spectral lines in the range of the applied x - ray spectrum and all of them are mutually exclusive regarding their constituting chemical elements . under that assumption , we can decompose each material into a linear combination of k basis materials with their respective energy - depending attenuation coefficient ( e)(9)je=k=1kfjkke.the number of materials k forming the material basis may not exceed the number b of available spectrally different data sets to guarantee that the system of equations is not underdetermined . the choice of the material basis must depend on the composition of the scanned object and is crucial for the accuracy of the composition estimation . a suitable choice for clinical applications should include water , since it resembles well the attenuation behavior of the majority of human tissue [ 2426 ] . calcium or a suiting mixture of materials could be included if osseous body parts are to be identified . if an injected iodine - based contrast agent is to be identified , which is frequently used in clinical ct examinations to enhance the contrast of cancerous tissue , iodine should be included . a correct and reliable separation of materials requires a different absorption behavior of each basis material in the range of the applied tube spectrum . if attenuation coefficients of different materials have the same energy dependency in the energy range covered by the spectrum , those materials can not be distinguished by means of spectrally resolved ct . the proposed algorithm applies the optimization transfer principle introduced by de pierro [ 16 , 27 ] . according to this principle , the objective function is locally approximated by surrogate functions that are easier to minimize ( cf . in fact , we will approximate the objective function by parabolic surrogates whose minima are known analytically . the following conditions imposed to a surrogate q are sufficient to guarantee convergence of the algorithm:(10)qfn;fn=fn,qf;fnfjkf = fn=ffjkf = fn , qf;fnf.equations ( 10 ) ensure that each surrogate always lies completely above the objective function (f ) and is tangential to it at the current iteration step . our derivation of the separable surrogate objective function and the update equation follows the procedure presented in . in a first step , the energy integral in the physical model , ( 7 ) , is moved out of the log - likelihood term of the objective function ( 5 ) . therefore , we define(11)like , fkj=1paijfjkke , ( 12)tie , fk=1ktike , fk = expk=1klike , fkexplie , f , ( 13)ib , nenib , ne , fntie , fn.with these definitions , the polychromatic lambert - beer law ( 7 ) can be rewritten as(14)nibf=ni,0beib , ne , fntie , fib , ne , fnde.since nib(f ) is a convex function and it holds that(15)ni,0beib , ne , fnde=1,we can replace l(f ) by the surrogate q 1(f , f ) by applying jensen 's inequality for convex functions(16)lfb=1b i=1nni,0beib , ne , fnhibtie , fib , ne , fndeq1f , fn.it can be shown that q 1(f , f ) fulfills conditions ( 10 ) . next , we want to approximate q 1 by another parabolic surrogate whose minimum is known analytically . to this end , expand q 1 into a taylor series up to the second order about the current line integrals { l i } ( 17)hibe , liqib , ne , lik ( 18)=hibe , lin+k=1khibe , liliklik = lik , nliklik , n+12k=1k m=1kcibkm , nliklik , nlimlim , n.we end up with the new surrogate function(19)q2f , fn=b=1b i=1nni,0beib , nqib , ne , likde.the curvature c i in ( 17 ) must be chosen such that the optimization transfer principle ( 10 ) is satisfied . practically , we will use the hessian of h i ( e , l i ) instead . with this choice the monotonicity of the algorithm if we forced the line integrals l i to be positive , there would exist curvatures that provably ensures monotonic convergence . however , this constraint might affect the capability of the algorithm to model materials not incorporated in the material basis . materials not part of the basis are represented as a linear combination of the basis materials ( see ( 9 ) ) which also requires negative coefficients f j . a weaker form of the positivity constraint merely postulates that k=1 f j 0 would be compatible with the model . whether this constraint still guarantees monotonic convergence and the existence of an optimal ( in the sense of convergence rate , cf . ) finally , we replace q 2 by a surrogate that allows independent updates of all image pixels , enabling parallel computation . to this end , we rewrite the kth line integral as a convex combination by transforming(20)like , fk=j=1paijfjkke=j=1pijaijkeijfjkfjk , n+likne=j=1pijijke , fjk , with(21)ij = aijj=1paij,j=1pij=1 , ( 22)likne=j=1paijkefjk , n.applying once again jensen 's inequality , we pull the sum over the image pixels j out of q i ( e , j=1 ij ij ( e , f j ) ) which yields our final surrogate function q 3(f , f ) ( 23)q3f , fn=b=1b i=1n j=1pni,0beib , nijqib , ne,ijkfjkde.for the deduction of the separable surrogate s(f , f ) of the regularization term we followed de pierro and ended up with(24 ) to minimize the surrogate objective function q 3 + s we apply the newton - raphson method:(25)fn+1=fnq3f , fn+sf , fnhq3+hs1f = fn , hq3 and hs are the hessian matrices , that is , the second derivatives of q 3 and s , respectively . hence , the ( n + 1)st iteration step for the kth material image does only depend on the measured data of the bth energy bin and the material images previously calculated in the nth iteration step . evaluating the gradient of q 3 at the current iteration f yields(26)q3f , fnfjkf = fn=b=1b i=1n1nibnib , nnib , nfjk.for the elements of the second derivative of q 3 one gets(27)2q3fjkfjm=b=1b i=1nni,0beib , nijcibkm , nijkfjkijmfjm=b=1b i=1nni,0beib , nijaij2kemecibkm , nde.using the hessian h i ( e , l i)/l i l i of h i ( e , l i ) instead of a curvature c i that satisfies the conditions of the optimization transfer principle we get(28)2q3fjkfjmb=1b i=1nni,0beib , nijaij2kemenibde=b=1b i=1naijnibnibj=1paijni,0bekemeexplinde.in the last step ij and i were replaced by their respective definitions ( 21 ) and ( 13 ) . the derivatives of the surrogates s(f , f ) of the regularization function are(29)sfjk=klnjwjlktanh2fjk , nfjk , n1flk , n1k,2sfjkfjm=kmklnj2wjlk21tanh22fjk , nfjk , n1flk , n1k , with the kronecker symbol km . in the following we give a brief step by step overview over the algorithm:(1)first , one has to compute the line integrals l i ( e , f ) of the material images by forward - projecting them ; see ( 11 ) . subsequently , determine t i(e , f ) as given by ( 12 ) . for an adequate initialization of the algorithm provide the initial material images from material separated fbp images reconstructed from the raw data.(2)next , determine the n i by multiplying t i(e , f ) with the mean number of photons n i,0 emitted by the tube and the respective spectrum the bth energy bin is sensitive to . finally , discretize the integral in ( 7 ) an carry out the summation . the spectra can be calculated from the detector response function as stated by ( 8).(3)additionally , calculate(30)ni,0bekeexpj=1paijjede , which equates to 1/aijnib,(n)/fjk .(4)finally , one can determine q 3(f , f ) following ( 26).(5)the hesse - matrix q 3/f j f j can be achieved in a similar fashion , using ( 28 ) . it should be mentioned that ( j=1 a ij ) in ( 28 ) is a simple forward - projection of an image containing all ones , so it can be precomputed.(6)multiplying the inverted hesse - matrix with the previously calculated gradient and subtracting the result from the current material images f yield the updated material images f . first , one has to compute the line integrals l i ( e , f ) of the material images by forward - projecting them ; see ( 11 ) . subsequently , determine t i(e , f ) as given by ( 12 ) . for an adequate initialization of the algorithm provide the initial material images from material separated fbp images reconstructed from the raw data . next , determine the n i by multiplying t i(e , f ) with the mean number of photons n i,0 emitted by the tube and the respective spectrum the bth energy bin is sensitive to . finally , discretize the integral in ( 7 ) an carry out the summation . the spectra can be calculated from the detector response function as stated by ( 8) . finally , one can determine q 3(f , f ) following ( 26 ) . the hesse - matrix q 3/f j f j can be achieved in a similar fashion , using ( 28 ) . it should be mentioned that ( j=1 a ij ) in ( 28 ) is a simple forward - projection of an image containing all ones , so it can be precomputed . multiplying the inverted hesse - matrix with the previously calculated gradient and subtracting the result from the current material images f yield the updated material images f . we started evaluating the accuracy of the algorithm by reconstructing k = 2 material images from ideal simulation data of a water filled acrylic glass cylinder , generated with the drasim software . in this context , ideal means that the detector perfectly separates the irradiating tube spectrum into b disjoint energy bins . effects like k - escape , charge sharing , electronic noise , and pulse pileup do not occur . for the evaluation of the algorithm we choose the number of energy thresholds and by that the number of energy bins to b = 2 , matching the number k of material images to be reconstructed . the cylinder phantom has a diameter of 30 cm , containing five small cylinders with various concentrations of iodine . the iodine concentration increases from f = 0.00243 counterclockwise to f = 0.01215 in equidistant steps , with the upper contrast cylinder ( cf . the scans were simulated in fan - beam geometry with a field of view ( fov ) of 52 cm . the isocenter to detector distance was r cd = 49 cm and the focus to isocenter distance r fc = 60 cm . the virtual detector features 4 rows with 3680 quadratic subpixels , each with a pitch of 250 m . each fifth column of small pixels is excluded to account for the covering due to a collimator grating . over an angular range of 540 1664 projections the additional angular range of 180 in addition to a full cycle is required by the employed rebinning algorithm . rebinning converts the sinogram data from fan - beam to parallel - beam geometry prior to reconstruction . in doing so the emitted spectrum , irradiated onto the cylinder phantom , was prefiltered with 0.9 mm of titanium and 3.5 mm aluminum . we did not account for a bowtie filter in the simulations , albeit it could be respected in the detector response function ( 8) via an additional factor that depends on the sinogram pixel index i. the simulated detector has two counter thresholds , providing two spectrally separated data sets . we chose iodine and water as basis materials and initialized the algorithm with the ground truth , that is , the correct material images f j . since acrylic glass is not part of the material basis , the borders of the cylinder phantom will be represented as a linear combination of iodine and water . the bin - spectra ( e ) are provided with the same resolution . bin - spectra are the parts of the tube spectrum the energy bins of a pcd are sensitive to ( see figure 3(a ) ) . the mass attenuation coefficients m ( e ) and the material densities for the calculation of attenuation values ( e ) = m ( e) were taken from the epdl library and kuchling , respectively . to evaluate the mass attenuation coefficients at the sample points of the bin - spectra the interpolation was conducted piecewise if the considered material possesses one or more absorption edges within the energy range of the bin - spectra . real photon - counting detectors are not able to separate the registered photons into perfectly disjoint energy bins . together with pulse pileup , it leads to a considerable overlap of the sensitive ranges of individual energy bins . hence , the spectral resolution and consequently the amount of spectral information gathered is reduced compared to ideal pcds . since the material separation capability strongly depends on the basis materials difference in absorption behavior among the energy bins , we expect the convergence rate of our algorithm to depend on the choice of energy bins . the algorithm requires knowledge of the specific bin - spectra ( e ) , that is , the part of the tube spectrum an energy bin is sensitive to , to correctly reconstruct material fractions ; see ( 7 ) . the specific spectra for arbitrary , realistic bins can be calculated via the detector response function (e , e ) ; see ( 8) . we acquired the response function with a resolution of 1 kev by simulating monoenergetic scans without phantom , with x - ray energies between 20.5 and 139.5 kev , using the simsd simulation tool [ 30 , 31 ] . the tool accounts for all relevant physical processes occurring in realistic pcds . to keep the influence of pulse pileup on the detector response low ( < 1.5% ) , we chose a small x - ray flux of 4.8 10 ( 1/s mm ) and used a clocked readout to discriminate pulses . the calculation of the response function assumes a virtual cdte - detector with a bulk thickness of 1.6 mm , biased with a voltage of 1 kv , and the same ( pixel ) size as the detector described in section 2.3.1 . we sample the monoenergetic response functions with thresholds between 4 and 173 kev , again with a resolution of 1 kev . the detector response to each photon energy is approximated by averaging the response of 10k detector pixels . figures 3(b)3(d ) show the sensitive range of two energy bins calculated from the detector response function for the chosen prefiltered 140 kev tube spectrum ( cf . [ 30 , 31 ] we also created realistic data sets from the cylindrical phantom for a 2-bin pcd , where realistic means that we consider pulse pileup , k - escape , charge sharing , and electronic noise . h2o)/ h2o ) 1000 of the cylindrical phantom with image values centered at c = 0 with a window width of w = 100 . to investigate the dependence of convergence speed on the selected counter thresholds , we fixed the low energy counter at 20 kev and varied the high energy counter between 50 kev and 80 kev in steps of 15 kev . we created two data sets for each of the three energy bin configurations that only differ in their noise realization . this enables us to evaluate the image noise in difference images , avoiding systematic errors . in comparison to ideal pcd data ( cf . section 2.3.1 ) , a pileup correction is applied to the realistic raw data before reconstructing them with the proposed algorithm . the correction is based on a high - order polynomial that we fit to the mean true count rate plotted versus the respective mean count rate measured by the detector . first , the fbp images are reconstructed from the simulated raw data after a combined water - bhc and pulse pileup correction had been applied . next , the fbp images on hounsfield scale are converted to attenuation values ( 31)jb = hu10001jb , h2owith(32)jb , h2o=eminbemaxbjh2oesbedeeminbemaxbsbede.finally , the initial material fractions f are calculated from the attenuation maps j via(33)f0=m1,with f { f the matrix contains the attenuation values of the k pure basis materials for the respective bin - spectrum ( e ) . we compared the results of two different reconstructions of the same raw data sets . in the first case we reconstructed the material images from the ideal pcd data without any regularization ; that is , = 0 . for the second case we employed regularization with = 1 10 . parameters were selected depending on the considered material k by taking into account the image noise contained in the respective initial material images reconstructed from realistic data . after 1000 iterations the material fractions as well as their uncertainties were evaluated for both cases within the two rois that are depicted in figures 4(a ) and 4(b ) . roi1 is indicated by a dashed circle located within the highest iodine contrast cylinder and at the respective position in the water image . roi2 is indicated by a solid circle located at the lowest iodine contrast cylinder and at the respective position in the water image . the table also lists the mean deviations from the ground truth f = |f f | within the rois . figure 4 shows the true material images for iodine ( a ) and water ( b ) , as well as the difference between the respective images and the ground truth after 1000 iterations for both investigated cases ( ( c)(f ) ) . the initial material images as well as the final material images after 1000 iterations are exemplarily shown in figure 5 for the data set featuring the ( 2065 kev , 65140 kev ) bin configuration . compared to the ground truth images shown in figures 4(a ) and 4(b ) significant beam - hardening artifacts are visible between the iodine contrast probes . they are caused by the fbp reconstruction based on the monoenergetic version of lambert - beer 's law . due to these artifacts , the iodine contrast is considerably underestimated and images of the iodine contrast probes also show up in the initial water material image . similarly to the ideal material images in section 2.3.1 , all material images have been evaluated within identically positioned rois . the image noise , that is , the standard deviation of the roi data , is not estimated directly in the reconstructed images . instead , the images reconstructed from the two data sets that only differ in their noise realization are subtracted and the image noise is evaluated within the resulting difference image . eventually , the measured image noise is scaled by a factor of 1/2 to account for gaussian error propagation . the deviation of the measured material fraction f from the ground truth as well as the respective image noise is plotted against iteration number in figure 6 for both basis materials and the various bin configurations . considering the results of the reconstruction with deactivated regularization ( see figures 4(c ) and 4(d ) ) , the chosen internal energy resolution of 5 kev seems sufficient , since the deviations f from the ground truth are very small after 1000 iterations . the final images from nonregularized reconstruction mainly exhibit moir pattern residuals that are typical discretization artifacts from the forward- and back - projectors . those artifacts are much more prominent than the high - frequency , random noise residuals expected from a statistical reconstruction algorithm . so we conclude that the standard deviations f calculated from the roi data are a rough measure of discretization artifacts . the standard deviation is of equal magnitude as the deviations f , which are mainly caused by the limited internal energy resolution . this confirms that for the utilized projection operator an internal energy resolution of 5 kev is adequate . if regularization is employed , standard deviation in both material images is reduced , especially in the contrast probes within the iodine image . in return though , the precision of the material fraction estimation is reduced slightly for data within roi1 , that is , the probe with the highest iodine contrast . we suppose that this loss in precision comes from the way regularization is applied to the material images . although the material images are linked by measured spectral data sets l i(e , f ) = k=1 j=1 a ij f j ( e ) , regularization is applied to each material image individually . if the regularization is not perfectly edge - preserving this leads to a bias , affecting the precision of the material image estimate . suppose that by regularization of the iodine material image the edges of a contrast probe are smoothed out as illustrated in figure 8(a ) . the requirement ( 35)fjiodineiodinee+fjh2oh2oe = const.and by far stronger attenuation coefficient of iodine in the considered energy window inevitably cause the smoothed out edge to reappear in the water image with increased amplitude ; see figure 8(b ) . the regularization of the water image assures that the imprinted edges from the iodine image get smoothed and blurred and in turn also influence the iodine , albeit to a much lesser extent . thus , accumulated over the number of iterations this effect slightly influences the mean contrast measured within the rois in both images . the more prominent the contrast edge , the stronger the resulting deviation caused by regularization ( cf . the huber prior might reduce this issue but was not tested , since it does not fulfill the convergence criteria according to and a divergence of the algorithm seemed likely . generally , the choice of the second derivative of h i ( e , l i ) seems to be valid , since in all investigated cases the algorithm converged . as conjectured , it proves true that the convergence speed depends on the selected thresholds . while for thresholds located at 2065 kev and 2080 kev the algorithm shows a similar convergence rate ( cf . figure 6 ) , it converges noticeably slower for the threshold combination 2050 kev . on the one hand , this is due to a reduced separation between the two bin - spectra ( e ) ; see figure 3(b ) . on the other hand , it is a consequence of comparably worse initial images which are affected by the large overlap of the bin - spectra as well . therefore , not only is an increase of the number of thresholds in a pcd necessary to allow a separation of more than two basis materials , but the respective data sets also have to be spectrally well separated . for instance , a separation of water and fat will hardly converge with the proposed algorithm due to the similar absorption behavior of these materials within the energy range of clinical ct . addressing the dark shades between the contrast probes , caused by beam - hardening of the polychromatic x - ray tube spectrum , remarkably , the image noise measured in roi1 within the highest contrast rod of the iodine image is increasing beyond the respective image noise within the fbp starting image . this happens since within the first iterations mainly the mean iodine contrast is scaled to match the respective sinogram data . since the initial estimate of the highest iodine contrast is not very accurate and regularization primarily limits high - frequency noise from being added to the material images , it can not prevent an initial increase of image noise in this case . for the smallest contrast , the initial image represents a decent estimate of the actual iodine fraction and changes made by the algorithm within the first few iterations are less severe , so an increase of image noise can be prevented by regularization . summarizing the results of figure 6 , the algorithm performs well in correcting beam - hardening artifacts and quantitatively determining the material fractions for a two - bin pcd , if the material basis is chosen properly . with the utilized regularization function and parameters a significant reduction of image noise in both material images was achievable ; see figure 7 . an iterative statistical reconstruction algorithm has been introduced that successively approximates the negative log - likelihood function by paraboloidal surrogate functions . with the proposed algorithm a direct reconstruction of a set of material images is possible from energy - resolved sinogram data . since the algorithm considers the polychromatic nature of x - rays generated by typical clinical x - ray sources the algorithm includes an implicit beam - hardening correction for the selected basis materials . apart from that the algorithm has been tailored for reconstruction of material images from data measured with photon - counting detectors by taking into account correlations introduced between energy bin data sets in the detection process via the detector response function . it was shown that an internal energy resolution of 5 kev is sufficient to yield quantitative results if a proper material basis is selected . compared to reconstruction algorithms utilized on current commercial scanners the proposed algorithm converges rather slowly . this might make an immediate implementation in those scanners unlikely , despite the possibility for parallel computation .

this work proposes a dedicated statistical algorithm to perform a direct reconstruction of material - decomposed images from data acquired with photon - counting detectors ( pcds ) in computed tomography . it is based on local approximations ( surrogates ) of the negative logarithmic poisson probability function . exploiting the convexity of this function allows for parallel updates of all image pixels . parallel updates can compensate for the rather slow convergence that is intrinsic to statistical algorithms . we investigate the accuracy of the algorithm for ideal photon - counting detectors . complementarily , we apply the algorithm to simulation data of a realistic pcd with its spectral resolution limited by k - escape , charge sharing , and pulse - pileup . for data from both an ideal and realistic pcd , the proposed algorithm is able to correct beam - hardening artifacts and quantitatively determine the material fractions of the chosen basis materials . via regularization we were able to achieve a reduction of image noise for the realistic pcd that is up to 90% lower compared to material images form a linear , image - based material decomposition using fbp images . additionally , we find a dependence of the algorithms convergence speed on the threshold selection within the pcd .

the current study was performed as a nested case - control study in subjects participating in the prevend study . this prospective community - based cohort study on the natural course of urinary albumin excretion ( uae ) with serial follow - up measurements details of the study protocol have previously been published ( 9 ) . in short , all inhabitants of the city of groningen aged 2875 years were sent a questionnaire and a vial to collect a first - morning void urine sample . of these individuals , a cohort consisting of 8,592 subjects was selected ( the prevend cohort ) . in this ongoing study , participants are invited to visit an outpatient clinic for detailed medical examination at intervals of 3 years . at each screening round , participants fill out questionnaires on demographics , medical history , and drug use . information on drug use is completed with data from community pharmacies , including information on class of antihypertensive medication ( ace inhibitor [ acei]/angiotensin-2 receptor blocker [ arb ] ) . at the study visits , participants deliver two 24-h urine collections , blood pressure is measured , anthropometrical measurements are performed , and fasting blood samples are taken . the prevend study was approved by the institutional ethics review board and was conducted in accordance with the guidelines of the declaration of helsinki . normoalbuminuria was defined as uae < 30 mg/24 h , microalbuminuria as uae 30299 mg/24 h , and macroalbuminuria as uae 300 mg/24 h. albuminuria status was based on the average of two consecutive measurements in 24-h urine collections . transition in albuminuria was defined as a transition from normo- to micro- or from micro- to macroalbuminuria with at least 30% increase in uae from baseline between two consecutive study visits . t2 dm was defined as the use of oral antidiabetes treatment ( self - reported or by information retrieved from the regional pharmacy database ) , a fasting plasma glucose > 7.0 mmol / l ( 126 mg / dl ) , or nonfasting plasma glucose > 11.1 was defined as the use of antihypertensive treatment or a systolic / diastolic blood pressure > 140/90 mmhg . for the current study , we selected patients with t2 dm and transition from normo- to microalbuminuria or from micro- to macroalbuminuria . as control subjects , we selected t2 dm patients who had persistent normoalbuminuria or microalbuminuria throughout the same time interval as case subjects ( fig . , we selected similar case and control subjects out of all nondiabetic patients with ht ( fig . control subjects were matched to the case subjects 1:1 based on diabetes / nondiabetes , age , sex , baseline normo- or microalbuminuria status , and , if applicable , duration of diabetes . for each subject that showed transition in albuminuria , a matched control subject was selected that most optimally resembled the case subject on these combined parameters . plasma samples of these patients were used from the visit prior to the transition from normo- to microalbuminuria or from micro- to macroalbuminuria ( baseline ) . the use of agents intervening in the renin - angiotensin - aldosterone system ( acei / arb ) was allowed , but the type of drug and their dose had to remain stable during the study period . individual courses of uae in progressors and nonprogressors in albuminuria ( case and control subjects ) . individual course of uae during follow - up for case ( black ) and control ( gray ) subjects in patients with t2 dm ( a ) or ht ( b ) stratified for albuminuria status at baseline ( left : normoalbuminuria ; right : microalbuminuria ) . of the 8,592 study participants , 318 had t2 dm , and 33 of these subjects had unambiguous transition in albuminuria and available samples of sufficient quality . of these participants , 75 subjects had unambiguous transition in albuminuria and available samples of sufficient quality . plasma samples were stored at 80c , and all samples underwent one freeze - thaw cycle . gdf-15 was measured with a novel precommercial assay based on the eclia principle ( roche diagnostics ) with a lower limit of detection ( lld ) of 200 pg / ml and intraindividual coefficient of variation ( cv ) of 6.79.2% . blood pressure was measured twice , in the supine position , every min for 10 min with an automatic device ( dinamap xlmodel 9300 ; johnson & johnson medical , tampa , fl ) . egfr was estimated with the modification of diet in renal disease study equation , using sex , age , race , and serum creatinine ( 14 ) . serum creatinine was measured by dry chemistry ( eastman kodak , rochester , new york ) , with intra - assay cv of 0.9% and interassay cv of 2.9% . analyses were performed using stata version 11.2 ( statacorp , college station , texas ) . a study with 50 patients will provide at least 80% power to detect an odds ratio of 1.5 assuming a type 1 error of 5% and no residual confounding after matching case and control subjects ( http://www.bioconductor.org ) . variables with normal distribution are given as means sd and variables with skewed distribution as median ( interquartile range [ iqr ] ) . differences between the case and control subjects were tested with paired - sample t test for continuous variables and test on paired proportions for categorical variables . differences between nonpaired groups were tested with independent - sample t test for continuous variables and test for categorical variables . to investigate the association between the levels of the markers and transition in albuminuria , we used conditional logistic regression because of the paired study design . in multivariable analyses , we adjusted for differences in baseline uae and egfr between case and control subjects , as these two markers are important risk factors for transition in albuminuria stage . we added several sensitivity analyses , adjusting for other important risk markers for transition in albuminuria , and combining patients with t2 dm and patients with ht . we tested for interaction between patients who made a transition from normo- to microalbuminuria and those who made a transition from micro- to macroalbuminuria by adding an interaction term for baseline albuminuria status and gdf-15 in the model . to assess whether the markers improved risk prediction and discrimination , we determined the c statistic and integrated discrimination improvement ( idi ) . we calculated the c statistic ( discriminatory ability ) based on the most important established risk factors ( uae and egfr ) and compared those to c statistics after addition of gdf-15 to the established model . in addition to the c statistic , we calculated the idi , another measure of discrimination ( 15 ) . the idi is the difference in discrimination slopes between case and control subjects before and after the addition of the biomarker(s ) to the model . calculation of the idi is done by computing average predicted probabilities of the event in case and control subjects in models with and without the biomarker(s ) and subtracting the values from case and control subjects from each other . the increase in difference between case and control subjects after addition of the biomarker(s ) is the integrated reclassification improvement . the idi is often more sensitive than the rather conservative c statistic because when several highly predictive markers are in the model , enormous odds ratios are required to meaningfully increase the c statistic ( 17 ) . for all analyses , two - sided p values < 0.05 were considered statistically significant . normoalbuminuria was defined as uae < 30 mg/24 h , microalbuminuria as uae 30299 mg/24 h , and macroalbuminuria as uae 300 mg/24 h. albuminuria status was based on the average of two consecutive measurements in 24-h urine collections . transition in albuminuria was defined as a transition from normo- to micro- or from micro- to macroalbuminuria with at least 30% increase in uae from baseline between two consecutive study visits . t2 dm was defined as the use of oral antidiabetes treatment ( self - reported or by information retrieved from the regional pharmacy database ) , a fasting plasma glucose > 7.0 mmol / l ( 126 mg / dl ) , or nonfasting plasma glucose > 11.1 mmol / l ( > 200 mg / dl ) . hypertension ( ht ) was defined as the use of antihypertensive treatment or a systolic / diastolic blood pressure > 140/90 mmhg . for the current study , we selected patients with t2 dm and transition from normo- to microalbuminuria or from micro- to macroalbuminuria . as control subjects , we selected t2 dm patients who had persistent normoalbuminuria or microalbuminuria throughout the same time interval as case subjects ( fig . , we selected similar case and control subjects out of all nondiabetic patients with ht ( fig . control subjects were matched to the case subjects 1:1 based on diabetes / nondiabetes , age , sex , baseline normo- or microalbuminuria status , and , if applicable , duration of diabetes . for each subject that showed transition in albuminuria , a matched control subject was selected that most optimally resembled the case subject on these combined parameters . plasma samples of these patients were used from the visit prior to the transition from normo- to microalbuminuria or from micro- to macroalbuminuria ( baseline ) . the use of agents intervening in the renin - angiotensin - aldosterone system ( acei / arb ) was allowed , but the type of drug and their dose had to remain stable during the study period . individual courses of uae in progressors and nonprogressors in albuminuria ( case and control subjects ) . individual course of uae during follow - up for case ( black ) and control ( gray ) subjects in patients with t2 dm ( a ) or ht ( b ) stratified for albuminuria status at baseline ( left : normoalbuminuria ; right : microalbuminuria ) . of the 8,592 study participants , 318 had t2 dm , and 33 of these subjects had unambiguous transition in albuminuria and available samples of sufficient quality . of these participants , 75 subjects had unambiguous transition in albuminuria and available samples of sufficient quality . plasma samples were stored at 80c , and all samples underwent one freeze - thaw cycle . gdf-15 was measured with a novel precommercial assay based on the eclia principle ( roche diagnostics ) with a lower limit of detection ( lld ) of 200 pg / ml and intraindividual coefficient of variation ( cv ) of 6.79.2% . blood pressure was measured twice , in the supine position , every min for 10 min with an automatic device ( dinamap xlmodel 9300 ; johnson & johnson medical , tampa , fl ) . egfr was estimated with the modification of diet in renal disease study equation , using sex , age , race , and serum creatinine ( 14 ) . serum creatinine was measured by dry chemistry ( eastman kodak , rochester , new york ) , with intra - assay cv of 0.9% and interassay cv of 2.9% . analyses were performed using stata version 11.2 ( statacorp , college station , texas ) . a study with 50 patients will provide at least 80% power to detect an odds ratio of 1.5 assuming a type 1 error of 5% and no residual confounding after matching case and control subjects ( http://www.bioconductor.org ) . variables with normal distribution are given as means sd and variables with skewed distribution as median ( interquartile range [ iqr ] ) . differences between the case and control subjects were tested with paired - sample t test for continuous variables and test on paired proportions for categorical variables . differences between nonpaired groups were tested with independent - sample t test for continuous variables and test for categorical variables . to investigate the association between the levels of the markers and transition in albuminuria , we used conditional logistic regression because of the paired study design . in multivariable analyses , we adjusted for differences in baseline uae and egfr between case and control subjects , as these two markers are important risk factors for transition in albuminuria stage . we added several sensitivity analyses , adjusting for other important risk markers for transition in albuminuria , and combining patients with t2 dm and patients with ht . we tested for interaction between patients who made a transition from normo- to microalbuminuria and those who made a transition from micro- to macroalbuminuria by adding an interaction term for baseline albuminuria status and gdf-15 in the model . to assess whether the markers improved risk prediction and discrimination , we determined the c statistic and integrated discrimination improvement ( idi ) . we calculated the c statistic ( discriminatory ability ) based on the most important established risk factors ( uae and egfr ) and compared those to c statistics after addition of gdf-15 to the established model . in addition to the c statistic , we calculated the idi , another measure of discrimination ( 15 ) . the idi is the difference in discrimination slopes between case and control subjects before and after the addition of the biomarker(s ) to the model . calculation of the idi is done by computing average predicted probabilities of the event in case and control subjects in models with and without the biomarker(s ) and subtracting the values from case and control subjects from each other . the increase in difference between case and control subjects after addition of the biomarker(s ) is the integrated reclassification improvement . the idi is often more sensitive than the rather conservative c statistic because when several highly predictive markers are in the model , enormous odds ratios are required to meaningfully increase the c statistic ( 17 ) . for all analyses , two - sided p values < 0.05 were considered statistically significant . in total , 33 case subjects with t2 dm and transition from normo- to microalbuminuria or from micro- to macroalbuminuria were identified . these case subjects were pair matched to 33 control subjects with t2 dm and stable albuminuria . mean age of case subjects was 62.7 years , 25 ( 75.8% ) patients were male , and the median uae rate was 18 g/24 h ( iqr 944 ) . case subjects had a significantly higher baseline uae than control subjects , more frequently received lipid - lowering treatment , and more often received acei / arb treatment compared with matched control subjects . baseline characteristics for patients with t2 dm and nondiabetic patients with ht change in albuminuria concentrations during follow - up , according to the selection of case and control subjects , is shown in fig . patients with transition in albuminuria ( case subjects ) had a median increase in albuminuria of 31 mg/24 h ( 164% ) compared with 0 mg/24 h ( 0% ) in control subjects . the mean concentration of gdf-15 in case subjects and for control subjects , prior to transition in albuminuria , is presented in fig . 2b , these concentrations are presented separately for transition from normo- to micro- and from micro- to macroalbuminuria . gdf-15 concentrations were significantly higher in case vs. control subjects ( median 1,288 pg / ml [ iqr 8851,546 ] vs. 948 pg / ml [ 6601,016 ] , p < 0.001 ) . the concentrations were lower in normoalbuminuric case and control subjects than in microalbuminuric case and control subjects ( 910 pg / ml [ 7371,162 ] vs. 1,008 pg / ml [ 7631,470 ] , p = 0.03 ) . gdf-15 levels in progressors and nonprogressors in albuminuria ( case and control subjects ) for patients with t2 dm and nondiabetic patients with ht . a : gdf-15 levels ( pg / ml ) ( mean se ) in progressors and nonprogressors in albuminuria for t2 dm and ht . b : gdf-15 levels ( pg / ml ) ( mean se ) in progressors and nonprogressors in albuminuria for t2 dm and ht stratified for baseline albuminuria ( normoalbuminuria [ normo ] or microalbuminuria [ micro ] ) ; * p < 0.05 , * * p < 0.001 . the odd ratio for transition in albuminuria was 3.58 [ 95% ci 1.518.47 ] ( per sd increment in gdf-15 ) ( table 2 ) and 2.87 [ 1.107.53 ] after adjustment for baseline albuminuria and egfr . there was no statistical significant difference between the odds for transition from normo- to microalbuminuria and from micro- to macroalbuminuria with each sd increment in gdf-15 ( p for interaction = 0.55 ) . gdf-15 significantly improved the c statistic on top of a baseline model consisting of uae and egfr ( increase from 0.87 to 0.92 , p = 0.03 ) . gdf-15 also improved the idi ( 0.148 , p = 0.001 ) , indicating that gdf-15 improved discrimination between case and control subjects beyond baseline uae and egfr . odds ratios for gdf-15 and transition in albuminuria per log - unit increase and per sd increase in the level of the biomarker for patients with t2 dm and nondiabetic patients with ht the replication study was a second nested case - control study , which consisted of 75 ( nondiabetic ) case subjects with ht and transition to increasing stages of albuminuria who were pair matched to 75 control subjects with ht and stable albuminuria ( table 1 ) . characteristics of patients with ht were remarkably similar to those of patients with t2 dm , as was the median follow - up time ( 2.8 years [ iqr 2.33.9 ] in ht vs. 2.7 years [ 2.24.0 ] in t2 dm ) . case subjects with ht had a median increase in albuminuria of 70 mg/24 h ( 314% ) compared with 0.2 mg/24 h ( 5% ) in control subjects . gdf-15 concentration in patients with ht was similar to the concentration in patients with t2 dm ( median 910 [ iqr 7381,210 ] vs. 992 [ 7991,347 ] ; p = 0.63 ) . in case subjects with ht , gdf-15 concentration was borderline significantly higher than in control subjects ( 975 pg / ml [ 7391,222 ] vs. 872 [ 7261,172 ] ; p = 0.09 ) . the odds of transition to micro- or macroalbuminuria was significantly increased per sd increment in gdf-15 ( odds ratio 1.96 [ 95% ci 1.123.45 ] ) . also , in patients with ht no difference was observed in the odds for transition from normo- to microalbuminuria or micro- to macroalbuminuria with each increment in gdf-15 ( p for interaction = 0.90 ) . addition of gdf-15 to the prediction model of transition in albuminuria , consisting of baseline uae and egfr , did not result in significant improvement of the c statistic ( from 0.88 to 0.89 , p = 0.8 ) but significantly improved idi ( 0.059 , p = 0.04 ) . in a multivariate model with additional adjustment for risk markers known to be associated with progression in albuminuria and with baseline differences between case and control subjects ( baseline albuminuria , acei / arb use , use of lipid - lowering drugs , and history of coronary heart disease ) , gdf-15 remained statistically significantly associated with progression in albuminuria both in t2 dm ( odds ratio 4.84 [ 95% ci 1.2918.3 ] , p = 0.02 ] ) and in ht ( 1.73 [ 1.022.95 ] , p = 0.02 ] ) per sd increment in gdf-15 . because of the risk of overfitting the multivariate model , we were unable to include more risk markers for progression in our multivariate model . after combination of patients with t2 dm and ht , the odds ratio for transition in albuminuria was 2.63 ( 95% ci 1.464.75 ) , p = 0.001 , for each sd increase in gdf-15 , after adjustment for baseline albuminuria , egfr , systolic and diastolic blood pressure , cholesterol , fasting plasma glucose , bmi , smoking , acei / arb use , use of lipid - lowering drugs , and history of coronary heart disease . the mean concentration of gdf-15 in case subjects and for control subjects , prior to transition in albuminuria , is presented in fig . 2a . in fig . 2b , these concentrations are presented separately for transition from normo- to micro- and from micro- to macroalbuminuria . gdf-15 concentrations were significantly higher in case vs. control subjects ( median 1,288 pg / ml [ iqr 8851,546 ] vs. 948 pg / ml [ 6601,016 ] , p < 0.001 ) . the concentrations were lower in normoalbuminuric case and control subjects than in microalbuminuric case and control subjects ( 910 pg / ml [ 7371,162 ] vs. 1,008 pg / ml [ 7631,470 ] , p = 0.03 ) . gdf-15 levels in progressors and nonprogressors in albuminuria ( case and control subjects ) for patients with t2 dm and nondiabetic patients with ht . a : gdf-15 levels ( pg / ml ) ( mean se ) in progressors and nonprogressors in albuminuria for t2 dm and ht . b : gdf-15 levels ( pg / ml ) ( mean se ) in progressors and nonprogressors in albuminuria for t2 dm and ht stratified for baseline albuminuria ( normoalbuminuria [ normo ] or microalbuminuria [ micro ] ) ; * p < 0.05 , * * p < 0.001 . the odd ratio for transition in albuminuria was 3.58 [ 95% ci 1.518.47 ] ( per sd increment in gdf-15 ) ( table 2 ) and 2.87 [ 1.107.53 ] after adjustment for baseline albuminuria and egfr . there was no statistical significant difference between the odds for transition from normo- to microalbuminuria and from micro- to macroalbuminuria with each sd increment in gdf-15 ( p for interaction = 0.55 ) . gdf-15 significantly improved the c statistic on top of a baseline model consisting of uae and egfr ( increase from 0.87 to 0.92 , p = 0.03 ) . gdf-15 also improved the idi ( 0.148 , p = 0.001 ) , indicating that gdf-15 improved discrimination between case and control subjects beyond baseline uae and egfr . odds ratios for gdf-15 and transition in albuminuria per log - unit increase and per sd increase in the level of the biomarker for patients with t2 dm and nondiabetic patients with ht the replication study was a second nested case - control study , which consisted of 75 ( nondiabetic ) case subjects with ht and transition to increasing stages of albuminuria who were pair matched to 75 control subjects with ht and stable albuminuria ( table 1 ) . characteristics of patients with ht were remarkably similar to those of patients with t2 dm , as was the median follow - up time ( 2.8 years [ iqr 2.33.9 ] in ht vs. 2.7 years [ 2.24.0 ] in t2 dm ) . case subjects with ht had a median increase in albuminuria of 70 mg/24 h ( 314% ) compared with 0.2 mg/24 h ( 5% ) in control subjects . gdf-15 concentration in patients with ht was similar to the concentration in patients with t2 dm ( median 910 [ iqr 7381,210 ] vs. 992 [ 7991,347 ] ; p = 0.63 ) . in case subjects with ht , gdf-15 concentration was borderline significantly higher than in control subjects ( 975 pg / ml [ 7391,222 ] vs. 872 [ 7261,172 ] ; p = 0.09 ) . the odds of transition to micro- or macroalbuminuria was significantly increased per sd increment in gdf-15 ( odds ratio 1.96 [ 95% ci 1.123.45 ] ) . also , in patients with ht no difference was observed in the odds for transition from normo- to microalbuminuria or micro- to macroalbuminuria with each increment in gdf-15 ( p for interaction = 0.90 ) . addition of gdf-15 to the prediction model of transition in albuminuria , consisting of baseline uae and egfr , did not result in significant improvement of the c statistic ( from 0.88 to 0.89 , p = 0.8 ) but significantly improved idi ( 0.059 , p = 0.04 ) . in a multivariate model with additional adjustment for risk markers known to be associated with progression in albuminuria and with baseline differences between case and control subjects ( baseline albuminuria , acei / arb use , use of lipid - lowering drugs , and history of coronary heart disease ) , gdf-15 remained statistically significantly associated with progression in albuminuria both in t2 dm ( odds ratio 4.84 [ 95% ci 1.2918.3 ] , p = 0.02 ] ) and in ht ( 1.73 [ 1.022.95 ] , p = 0.02 ] ) per sd increment in gdf-15 . because of the risk of overfitting the multivariate model , we were unable to include more risk markers for progression in our multivariate model . after combination of patients with t2 dm and ht , the odds ratio for transition in albuminuria was 2.63 ( 95% ci 1.464.75 ) , p = 0.001 , for each sd increase in gdf-15 , after adjustment for baseline albuminuria , egfr , systolic and diastolic blood pressure , cholesterol , fasting plasma glucose , bmi , smoking , acei / arb use , use of lipid - lowering drugs , and history of coronary heart disease . to the best of our knowledge , this is the first study to indicate that gdf-15 precedes and predicts the development of micro- or macroalbuminuria in patients with t2 dm . gdf-15 was not only independently associated with transition in albuminuria during a follow - up of 3 years but also improved discrimination between patients who did and did not develop micro- or macroalbuminuria beyond conventional risk markers . previous studies mainly implicated gdf-15 as a predictor for cardiovascular events and all - cause mortality in patients with previous myocardial infarction ( 1012 ) . the only previous study focusing on gdf-15 and renal outcome was performed by lajer et al . ( 13 ) in patients with type 1 diabetes mellitus and macroalbuminuria . in this observational study , higher gdf-15 levels were associated with a faster decline of egfr and higher risk of development of end - stage renal disease . our study extends these latter findings to earlier stages of renal disease during which appropriate intervention is most beneficial . future validation studies in larger cohorts are needed to confirm the value of gdf-15 in prediction of renal disease progression and to define an optimal threshold for risk of progression in the different stages of renal disease . despite a growing body of literature demonstrating that gdf-15 is a valuable biomarker for cardiovascular disease , relatively little its expression is markedly increased in response to injury in various tissues , including heart and kidney , and is believed to be a protective factor by reducing apoptosis and influencing cellular proliferation ( 1820 ) . as endothelial cells appear to be a prominent source of gdf-15 , the circulating levels of gdf-15 most likely represent generalized endothelial and microvascular damage ( 21 ) . the relation of gdf-15 with microvasculature may explain the link with micro- or macroalbuminuria , as increased albuminuria supposedly reflects established microvascular damage in the renal and peripheral vasculature ( 22 ) . alternatively , as gdf-15 correlates with age and decreased telomere length in the general population , gdf-15 may be a marker of ( vascular ) aging . in diabetes and hypertension , stress factors such as inflammation , oxidative stress , and dyslipidemia could contribute to accelerated aging , indicating that circulating levels of gdf-15 may reflect the relative age of the vasculature and its ability to resist leakage of albumin ( 23 ) . regardless , since our case and control subjects were at the same stage of albuminuria ( either normo- or microalbuminuria ) at the time of gdf-15 measurement , gdf-15 seems to already be increased before microalbuminuria becomes detectable . our finding that gdf-15 also contributed in identifying subjects at risk for transition in albuminuria stage in a nondiabetic ht cohort implies that gdf-15 is not specifically related to diabetes . future research will be necessary to delineate the exact pathophysiological role of gdf-15 in diabetes or ht and to assess its relation with albuminuria . traditional risk factors associated with transition in albuminuria are age , sex , dyslipidemia , insulin sensitivity , hyperglycemia , duration of diabetes , increased blood pressure , duration of hypertension , bmi , and smoking ( 7,24 ) . despite identification of these important risk factors for transition in albuminuria stage , if confirmed , raise the possibility of identification of subjects at risk even when traditional risk factors do not indicate risk , facilitating early identification of those with an increased chance of developing micro- or macroalbuminuria . because early intervention is more effective than late intervention ( 4 ) , preventive treatment could thus reduce the risk of renal and cardiovascular events in patients with t2 dm ( 5,6 ) . our findings also applied to nondiabetic ht patients , in whom albuminuria is also strongly associated with renal and cardiovascular events ( 25,26 ) . we incorporated prediction analyses in the current study to determine whether gdf-15 could add to risk prediction in individual subjects beyond traditional risk markers . whereas the c statistic is most commonly used , the c statistic is not very sensitive in detecting small but meaningful contributions of biomarkers in correctly classifying individuals . hardly any improvement of the c statistic can be reached when the model already includes one or several important risk markers ( 17 ) . because of the insensitivity of the c statistic , other measures of risk classification such as the idi have recently been proposed ( 15,16 ) . in our analyses , we also found significant improvement in idi for gdf-15 , both in patients with t2 dm and in nondiabetic subjects with ht . the availability of patient samples from the large general population cohort prevend was a unique feature of this study . because this study followed the natural course of albuminuria by performing repeated measurements of albuminuria for > 10 years , we were able to measure gdf-15 in samples prior to the transition in albuminuria , whereas many studies have a cross - sectional design and test biomarkers in patients with established increased albuminuria . other strengths include the well - defined phenotype of the population , with albuminuria status based on two consecutive 24-h urine collections , the rigorous definitions for transition in albuminuria stage , and the availability of samples that had never been thawed . because of these strict criteria and the limited number of patients with diabetes in this general population cohort , we were only able to obtain 33 valid cases . a limitation of this study was that there was a modest difference in baseline albuminuria . this small baseline difference in albuminuria was inherent to the design of the study , as the level of albuminuria in itself is related to transition to increasing stages of albuminuria and we matched by albuminuria stage rather than by albuminuria level . adjustment for baseline albuminuria showed that the association of gdf-15 with albuminuria transition was independent of baseline albuminuria . a second limitation is the fact that the nested case - control design of the study may overestimate the true predictive capacity of the models because of the relative high event rate in case - control studies ( 27 ) . the improvement of the c statistic and the level of significance , however , are unaffected by case - control design of studies . lastly , whereas these results are promising and could be confirmed in nondiabetic ht patients , replication of the current results in other studies , preferably large prospective cohort studies , is warranted before final conclusions can be reached . in conclusion , we identified gdf-15 as a marker for prediction of transition in albuminuria in t2 dm subjects . gdf-15 , moreover , had significant additive value on top of conventional risk markers in the prediction of albuminuria transition . if these findings prove to be replicable in other studies , gdf-15 might be a valuable marker for individual risk stratification , facilitating start or intensification of treatment in high - risk patients to prevent or delay the progression of nephropathy .

objectivedevelopment of micro- or macroalbuminuria is associated with increased risk of cardiorenal complications , particularly in diabetes . for prevention of transition to micro- or macroalbuminuria , more accurate prediction markers on top of classical risk markers are needed . we studied a promising new marker , growth - differentiation factor ( gdf)-15 , to predict transition to increasing stage of albuminuria in type 2 diabetes mellitus ( t2 dm ) . in addition , we looked at the gdf-15 potential in nondiabetic subjects with hypertension ( ht).research design and methodscase and control subjects were selected from the prevend cohort , a large ( n = 8,592 ) , prospective general population study on the natural course of albuminuria , with > 10 years of follow - up and repeated albuminuria measurements . we found 24 t2 dm and 50 ht case subjects transitioning from normo- to macroalbuminuria and 9 t2 dm and 25 ht case subjects transitioning from micro- to macroalbuminuria ( average follow - up 2.8 years ) . control subjects with stable albuminuria were pair matched for age , sex , albuminuria status , and diabetes duration . gdf-15 was measured in samples prior to albuminuria transition.resultsprior to transition , gdf-15 was significantly higher in case subjects with t2 dm than in control subjects ( median [ iqr ] 1,288 pg / ml [ 8851,546 ] vs. 948 pg / ml [ 6601,016 ] , p < 0.001 ) . the odds ratio for transition in albuminuria increased significantly per sd of gdf-15 ( 2.9 [ 95% ci 1.17.5 ] , p = 0.03 ) . gdf-15 also improved prediction of albuminuria transition , with significant increases in c statistic ( from 0.87 to 0.92 , p = 0.03 ) and integrated discrimination improvement ( 0.148 , p = 0.001 ) . in ht , gdf-15 was also independently associated with transition in albuminuria stage ( 2.0 [ 1.13.5 ] , p = 0.02 ) and improved prediction significantly.conclusionswe identified gdf-15 as a clinically valuable marker for predicting transition in albuminuria stage in t2 dm beyond conventional risk markers . these findings were confirmed in nondiabetic ht subjects .

the monoclonal anti - c5 antibody eculizumab is an inhibitor of the terminal complement complex formation , successfully tested in atypical haemolytic - uraemic syndrome ( ahus ) , with or without mutations in the regulatory proteins of the alternative pathway of the complement , and less convincingly in enterohaemorrhagic escherichia coli - associated hus [ 13 ] . several drugs , including mitomycin - c and interferon- , may also trigger hus , but the therapeutic impact of eculizumab in this setting remains elusive . here , we show that eculizumab may prove efficient in drug - induced hus unresponsive to plasma exchanges . a female patient developed severe hus during mitomycin - c treatment for relapsing metastatic breast cancer . enterohaemorrhagic hus was ruled out , serum levels of complement components c3 and c4 were normal and screening for mutations in the genes encoding complement regulatory proteins cfh , cfi , mcp , and anti - adamts13 or anti - cfh autoantibodies were negative . before genetic testing and report , all patients gave a written informed consent as recommended by french law . despite termination of drug exposure and daily plasma exchanges , the lack of improvement of haematological parameters and the requirement for dialysis ( see figure 1 ) prompted us to consider eculizumab therapy . following the first infusion ( 900 mg ) , we observed an early and dramatic improvement of haematological parameters with normalization of platelet counts and lactate dehydrogenase levels within 7 days . seven additional infusions of eculizumab were performed , allowing partial renal recovery at 3 months . renal function remained stable ( egfr : 20 ml / min/1.73 m ) and no relapse of hus occurred during 18 months of follow - up . fig . 1.response to eculizumab therapy in a patient with drug - induced haemolytic uraemic syndrome . shown are the data indicating a rapid biological and clinical improvement of thrombotic microangiopathy after administration of the monoclonal anti - c5 antibody eculizumab . lactate dehydrogenase level ( left y - axis ) , platelet count and plasma creatinine level ( right y - axis ) are represented by white triangle , cross and black triangles , respectively . are the data indicating a rapid biological and clinical improvement of thrombotic microangiopathy after administration of the monoclonal anti - c5 antibody eculizumab . lactate dehydrogenase level ( left y - axis ) , platelet count and plasma creatinine level ( right y - axis ) are represented by white triangle , cross and black triangles , respectively . the striking coincidence of eculizumab infusion and improvement of hus parameters suggest a beneficial role of transient complement blockade in this patient . the pathophysiology of drug exposure - related hus remains poorly elucidated . in mitomycin - c - related hus , circulating immune complexes that cause platelet aggregation , complement activation and subsequent endothelial damage have been reported , supplying a molecular explanation for the striking effect of eculizumab . since no change in plasma levels of c3 and c4 was detected , the rapid normalization of haemolysis parameters and platelet counts in both of the patients despite suspension of plasma exchanges might be related to local blockade of complement activation . thus , our data plead for a role of complement blockade in the management of refractory , drug - related hus .

the monoclonal anti - c5 antibody eculizumab has been successfully tested in atypical haemolytic - uraemic syndrome ( ahus ) , with or without mutations in the regulatory proteins of the alternative pathway of the complement , and less convincingly in enterohaemorrhagic escherichia coli - associated hus . here , we report a patient with mitomycin - c - induced hus unresponsive to plasma exchanges . eculizumab infusion was followed by a dramatic improvement of haematological parameters and renal function , suggesting a role of complement blockade in the management of refractory , drug - related hus .

schizophrenia is a common disabling mental disorder , affecting approximately 1% of the general population , presenting with delusions , hallucinations , disorganized behaviors , and negative symptoms ( a deterioration in personal and social performance ) . some experts suggest that there is an association between this mental disorder and existence of subtle differences of the brain anatomy . for instance , patients with schizophrenia have larger lateral and third ventricles and smaller thalamic , hippocampal , and superior temporal volumes when compared to control healthy subjects . among these structural differences , midline and medial abnormalities , such as those related to corpus callosum , septum pellucidum , and cerebellar vermis have been suggested as a substrate of symptoms of schizophrenia , since midline circuits are responsible in mediating attention and information processing . in particular , the thalamus region has been identified with a critical role in pathophysiology of the illness , because of its unique location and connectivity . the adhesio interthalamica ( ai ) is a diencephalic midline structure formed by fusion of medial borders of both thalami , composed of neurons and five commissural fiber systems connecting thalamic nuclei , which develops in fetal period or after the birth . however , post - mortem and radiological studies have shown that in 15 - 30% of the cases , this structure never develops , as a normal variety . absence of ai has been found to be more frequent in males than in females , suggesting an important structural difference in connection of two hemispheres as an underlying factor for gender - related difference of cognitive function . some evidence also connected the presence and the size of ai structure with age , suggesting the atrophy and even disappearance of ai in elderly . nevertheless , studies have not found a relationship between development of ai and the size of the thalami or brain weight . recently , a few postmortem and mri studies have evaluated the presence or absence of ai in patients with schizophrenia . , reported that absence of ai is more common in patients with schizophrenia , in comparison with healthy controls , meisenzahl and agrawal refused any difference between them . therefore , this discrepancy of results may be partly due to the heterogenicity of schizophrenia subtypes . in order to address these issues , this study was designed to compare the rate of absent ai in patients suffering from three common clinical subtypes of schizophrenia ( paranoid , undifferentiated , and residual ) and a gender- and age - matched healthy group using mri method . in this case - control study , 29 patients with schizophrenia ( 19 males and 10 females ) referring to outpatient and inpatient departments of avicenna educational psychiatry hospital of mashhad , northeastern iran during 2010 were selected using purposive sampling method . the diagnosis of schizophrenia was confirmed by two psychiatrists using a semi - structured interview based on dsm - iv criteria . paranoid , undifferentiated , and residual subtypes of schizophrenia included 10 , 10 , and 9 patients , respectively . exclusion criteria were a history of head injury , neurological disorders , other mental co - morbidities , previous electroconvulsive therapy , and alcohol or drug abuse in the current 12 months . as a control group , 29 gender - and age- ( 5 years ) matched healthy volunteers participated and their mental health was confirmed by a psychiatrist using ghq-28 questionnaire . subjects with history of head injury , neurological disorders , family history of mental disorders , and alcohol or drug abuse in current 12 months were excluded from the study . all case and control participants signed an informed consent after being fully informed about the research process and approval by ethics committee of mashhad university of medical sciences . then , all participants underwent a 3-dimensional brain magnetic resonance imaging ( mri ) , using the brand named magnetom symphony maestro class with a 1.5-t scanner made by siemens corporation , germany . images were taken in complete coronal planes with a 1.5-mm slices without inter - slice gaps , tr=1.920 ms , te=3.93 ms , ti=1.100 ms , matrix size=256 179 , fov=240 240 mm , and scanning time=5 min and 40 s. presence or absence of the ai structure in mri was assessed by a neuroanatomist of our team , who was unaware of the participants diagnosis , gender , and other variables . the images were displayed on a computer screen using osiris v. 409 software interpolated to 4 magnification , allowing easy side by side comparisons or a stack flow . when the grey matter band connecting the thalami could be identified , at least on two coronal adjacent slices , the ai was considered as present [ figure 1 ] , otherwise it was considered as absent . two coronal adjacent slices showing adhesio interthalamica ( ai ) in a participant ; the ai in such cases was considered as present data were analyzed by spss software version 15.0 and the rate of absence of ai was compared between two groups using chi - square and fisher 's exact tests . the correlation between absence of ai with age and duration of schizophrenia the mean age in case ( n=29 ) and control ( n=29 ) groups was 36.412.7 and 36.512.6 years , respectively . the ai structure was absent in 8 patients ( 27.58% , including 7 males and 1 female ) and 4 healthy controls ( 13.79% , including 3 males and 1 female ) , but the difference was not statistically significant ( chi - square=0.84 , df=1 , p=0.11 ) . there were no significant difference between patients and healthy controls regarding the absence of ai , when the comparisons were restricted to either male ( chi - square=2.17 , df=1 , p=0.35 ) or female subjects ( p=0.53 , fisher 's exact test , one - tailed ) . also , there was not any difference in the absence of ai between females and males , neither in the case ( chi - square=2.36 , df=1 , p=0.12 ) or in the control group ( p=0.40 , fisher 's exact test , one - tailed ) . spearman test showed that absence of ai was not significantly correlated with age , in either patients with schizophrenia ( r=1.05 , df=27 , p=0.30 ) or healthy controls ( r=1.36 , df=27 , p=0.18 ) . also , there were no significant correlation between the absence of ai and duration of schizophrenia ( r=1.28 , df=27 , p=0.21 ) . patients with paranoid schizophrenia ( n=10 , m : f=7/3 ) and their matched control group ( n=10 , m : f=7/3 ) were also similar regarding age , body weight , and handedness . one paranoid patient ( 10% , including one male ) and 2 healthy controls ( 20% , including one male and one female ) did not have ai , which was not significantly different ( p=0.41 , fisher 's exact test , one - tailed ) . there were no difference between males and females ( p=0.7 , fisher 's exact test , one - tailed ) and also between male patients and male controls ( p=0.54 , fisher 's exact test , one - tailed ) and between female patients and female controls ( p=0.57 , fisher 's exact test , one - tailed ) . patients with undifferentiated schizophrenia ( n=10 , m : f=7/3 ) and their matched control group ( n=10 , m : f=7/3 ) were similar considering age , body weight , and handedness . three patients ( 30% , including 2 males and 1 female ) and 2 healthy controls ( 20% , including 2 males ) had absence of ai , which was not significantly different ( p=0.37 , fisher 's exact test , one - tailed ) . there were no difference between males and females ( p=0.52 , fisher 's exact test , one - tailed ) and also between male patients and male controls ( p=0.44 , fisher 's exact test , one - tailed ) and between female patients and female controls ( p=0.6 , fisher 's exact test , one - tailed ) . patients with residual schizophrenia ( n=9 , m : f=5/4 ) and their matched control group ( n=9 , m : f=5/4 ) were also similar considering age , body weight , and handedness . four patients ( 80% , including 4 males ) and none of healthy controls had absence of ai , which was significantly different ( p=0.041 , fisher 's exact test , one - tailed ) . the absence of ai was significantly more common in males than in females with residual schizophrenia ( p=0.039 , fisher 's exact test , one - tailed ) . also , there were a significant difference between patients with residual schizophrenia and controls when the comparisons were restricted to males only ( p=0.023 , fisher 's exact test , one - tailed ) , but it was insignificant when the comparison was restricted to female subjects ( p=0.50 , fisher 's exact test , one - tailed ) . as it was observed , patients with schizophrenia as well as paranoid and undifferentiated subtypes were not different from control group regarding the rate of ai absence . however , it was more common in residual subtype of schizophrenia as compared to healthy controls ( p=0.041 ) . the results of our study among all patients with schizophrenia are supported by post - mortem part of their study . they failed to detect any significant difference between post - mortem patient with schizophrenia and healthy controls . however , interestingly , their mri data of live patients showed that ai absence is higher in patients with schizophrenia compared to controls . since they used old mri device and relative thick slices ( 3.1 mm ) , it is possible that they missed some small ais in mri , which may be detected by post - mortem technique . therefore , post - mortem assessment seems to be more accurate than mri , but , if required , mri with thin slices is preferred . the results of the present study is also consistent with the studies conducted by meisenzahl , nopoulos , and de souza crippa , which could not find any significant differences in prevalence of ai absence between patients with schizophrenia and healthy controls . however , our results are not supported by erbagci 's study in which higher prevalence of ai absence has been reported in patients in comparison with healthy controls . some researchers stated that this difference with healthy people is limited to certain groups of patients with schizophrenia . snyder et al . , confined this structural difference only to patients with first episode of schizophrenia and excluded the chronic patients . in contrast , takahashi et al . , reported that the absence of the ai was more common in patients with chronic schizophrenia , when compared to first - episode psychosis and meisenzahl and takahashi stated that the absence of ai may be associated with more severe negative symptoms ( that are mostly observed in chronic patients ) . they claimed that patients with schizophrenia may manifest progressive brain changes , including atrophy of ai following the onset . however , this was inconsistent with our results , which showed that ai absence is not related to duration of illness . nopoulos et al . , found that female patients with schizophrenia had a significantly higher prevalence of absent ai compared to female controls , but this difference was not present in males . in the present study , the difference between patients and controls was absent , even when the comparison was limited to either females or males . but in the residual subgroup , male patients had a higher rate of absent ai as compared to healthy males ; this pattern was absent in females . the present results was also consistent with the prior studies conducted by erbagci and shimizu , that did not show a difference between male and female patients . however , de souza crippa et al . , found that absence of ai is more prevalent in male patients with schizophrenia than in female patients . this result was similar to our study on residual subtype of schizophrenia ; males had a higher rate of absent ai than females . in healthy controls , also , there was no differences between males and females in our study , as well as the studies performed by erbagci and de souza crippa . however , nopoulos and shimizu found that ai absence was more common in healthy males than in healthy females . this inconsistency may be due to differences in sample size , which was much larger in nopoulos and shimizu 's studies , in comparison with erbagci 's , de souza crippa 's , and ours . we believe that these controversies may be , also , partly due to differences in subtype distribution of the samples , which was not considered in prior studies . more precisely , if some subtypes of schizophrenia have a more constant relationship with ai absence than other subtypes , it can be concluded that studies including the more number of patients of these subtypes report an association between ai absence and schizophrenia , but studies with scant number of patients of these subtypes fail to show the relationship . the present study was the first one that assessed the rate of absent ai in different subtypes of schizophrenia and showed a higher rate of absent ai in patients with residual schizophrenia . however , it seems consistent with the result of studies performed by meisenzahl and takahashi , who perceived that patients without ai structure are more prone to have negative symptoms ( prominent symptoms of residual subtype of schizophrenia ) than patients with ai . on the other hand , ai has vast connections to the prefrontal cortex , and prefrontal abnormalities have been accused for development of negative symptoms ( most important symptoms of residual subtype ) in patients with schizophrenia . these series of relationships suggest that ai absence may be related to residual subtype of schizophrenia that again supports our results . although inconsistent with some prior studies , the rate of ai absence in patients with heterogenous subtypes of schizophrenia is not different with control group , even when patients and controls of each gender are compared separately . so , heterogenous subtypes of schizophrenia may have different neuroanatomical characteristics . in residual subtype of schizophrenia , patients showed a significant priority in ai absence in comparison with control group , which was not seen in paranoid and undifferentiated subtypes . small sample size and lack of a structured interview for a confident diagnosis of schizophrenia was the major limitations and should be corrected in subsequent studies . although we tried to evaluate all patients of all different subtypes of schizophrenia , we could not find an adequate number of patients in disorganized and catatonic subtypes . subsequent extensive investigations are required to assess this rare subtype and clarify the big deal of inconsistency in basic science about the anatomical differences in ai between patients with schizophrenia and control subjects . longitudinal studies are also suggested to determine the probable role of this structural difference in development of schizophrenia : is it a cause or an effect ? we expect that the future enhancements in the basic knowledge about the schizophrenia result in the better treatment approaches and policies and even open the horizons toward cure or prevention . small sample size and lack of a structured interview for a confident diagnosis of schizophrenia was the major limitations and should be corrected in subsequent studies . although we tried to evaluate all patients of all different subtypes of schizophrenia , we could not find an adequate number of patients in disorganized and catatonic subtypes . subsequent extensive investigations are required to assess this rare subtype and clarify the big deal of inconsistency in basic science about the anatomical differences in ai between patients with schizophrenia and control subjects . longitudinal studies are also suggested to determine the probable role of this structural difference in development of schizophrenia : is it a cause or an effect ? we expect that the future enhancements in the basic knowledge about the schizophrenia result in the better treatment approaches and policies and even open the horizons toward cure or prevention .

context : previous studies have suggested subtle anatomical brain differences between patients with schizophrenia and healthy control subjects . however , the results are inconsistent and there is no study investigating the various subtypes of this mental disorder separately.aim:this study was conducted to compare the rate of absence of adhesio interthalamica ( ai ) , a midline brain structure , between 3 subtypes of schizophrenia ( paranoid , undifferentiated , and residual ) and healthy control group , using magnetic resonance imaging ( mri).materials and methods : a total of 29 schizophrenia patients ( 21 men , 8 women ) of three subtypes ( paranoid , undifferentiated , and residual ) were compared with 29 age- and gender - matched healthy controls . all subjects underwent 3-d brain mri of full coronal series , 1.5-mm slices without interslice gaps . if the grey matter band connecting the thalami could not be identified on two or more coronal adjacent slices , the ai was considered as absent . the results were statistically analyzed.results:the incidence rate of ai absence in patients with heterogenous subtypes of schizophrenia was was similar to control group , even when patients and controls of each gender were compared separately ( p>0.05 ) . in residual subtype , patients showed a significant priority in ai absence in comparison with the control group ( p=0.041 ) , which was not seen in paranoid and undifferentiated subtypes ( p>0.05).conclusion : residual subtype of schizophrenia is associated with higher rate of ai absence in this study . subsequent studies are required to determine if the absence of ai is a cause of residual schizophrenia or an effect .

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tretter and walster 1980 ; song et al . 2012 ) , solving \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$r_{\nu } ( t ) = \rho $ $ \end{document } should conveniently be achievable by standard iterative root finding techniques , provided that good starting approximations are available ( which is particularly important in the right tail of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$r_\nu $ $ \end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$r_\nu $ $ \end{document } is rather flat ) . ( 2005 ) suggest the approximation1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } r_{\nu } ^{-1}(\rho ) \approx \frac{\rho } { 1 - \rho ^2 } ( 2(\nu + 1 ) - \rho ^2 ) = : q_\nu ( \rho ) \end{aligned}$$\end{document}obtained by truncating the gauss continued fraction representation of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$r_\nu $ $ \end{document } and adding a correction term determined empirically , pointing out that this initial approximation can subsequently be improved by newton raphson iterations . ( 2007 ) show that for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\nu \ge 0$$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$0 \le \rho < 1$$\end{document},\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } r_{\nu } ^{-1}(\rho ) = \frac{\rho } { 1 - \rho ^2 } ( 2(\nu + 1 ) - c ) \end{aligned}$$\end{document}for some suitable \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$0 \le c = c(\nu , \rho ) \le 2$$\end{document } , or equivalently,2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } \frac{2\nu \rho } { 1 - \rho ^2 } \le r_{\nu } ^{-1}(\rho ) \le \frac{2(\nu + 1)\rho } { 1 sra approximation assuming \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$c \approx \rho ^2$$\end{document}. the upper and lower bound differ by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$2\rho / ( 1 - \rho ^2)$$\end{document } which is independent of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\nu $ $ \end{document } but tends to infinity as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\rho \rightarrow 1-$$\end{document}. tanabe et al . approximation with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$c = 1$$\end{document } , i.e. , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$r_\nu ^{-1}(\rho ) \approx ( 2\nu + 1 ) \rho / ( 1 - \rho ^2)$$\end{document } as the starting value for iterative schemes for solving \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$r_\nu ( t ) = \rho $ $ \end{document } , such as the fixed - point iteration \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t_{n+1 } = t_n \rho / r_\nu ( t_n)$$\end{document}. in this paper , we use a family of bounds for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$r_\nu $ $ \end{document } first introduced in amos ( 1974 ) to provide substantially sharper bounds for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$r_\nu ^{-1}$$\end{document } , which have approximation error at most \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$3\rho /2$$\end{document } , and use these results to suggest a new approximation . we establish that these improved bounds also hold for the dhillon sra approximation , which thus has the same maximal approximation error . we also show that the error of the suggested new approximation tends to zero for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\rho \rightarrow 1-$$\end{document } , whereas the error tends to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$-1/2$$\end{document } for the dhillon sra approximation , which thus is too large for large \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\rho $ $ \end{document}. finally , we investigate whether the rational bounds for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$r_\nu $ $ \end{document } developed by nsell ( 1978 ) can be used to obtain improved explicit bounds for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$r_{\nu } ^{-1}$$\end{document } , and show that for the rational bounds which can be inverted by solving quadratic equations , no such improvement is possible . let\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } g_{\alpha , \beta } ( t ) = \frac{t}{\alpha + \sqrt{t^2 + \beta ^2 } } , \end{aligned}$$\end{document}where in what follows we take \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta \ge 0$$\end{document } without loss of generality . amos ( 1974 ) gives the bounds\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } g_{\nu + 1/2,\nu + 3/2}(\kappa ) \le r_\nu ( \kappa ) \le g_{\nu + 1/2,\nu + 1/2}(\kappa ) , \qquad \kappa , \nu \ge 0 \end{aligned}$$\end{document}(equation 16 ) and\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } g_{\nu + 1,\nu + 1}(\kappa ) \le r_\nu ( \kappa ) \le g_{\nu , \nu + 2}(\kappa ) \le g_{\nu , \nu } ( \kappa ) , \qquad \kappa , \nu \ge 0 \end{aligned}$$\end{document}(equations 9 and 11 ) . the bounds are actually valid for larger \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\nu $ $ \end{document } domains ( see for example nsell 1978 ; yuan and kalbfleisch 2000 ) . it is trivial that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g_{\nu + 1/2,\nu + 1/2}(t ) < g_{\nu , \nu } ( t)$$\end{document } for all \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t > 0$$\end{document}. for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta ( t ) = ( \nu + 1/2 ) + \sqrt{t^2 + ( \nu + 3/2)^2 } - ( ( \nu + 1 ) + \sqrt{t^2 + ( \nu + 1)^2})$$\end{document } we have \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta ( 0 ) = 0$$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta ' ( t ) = t / \sqrt{t^2 + ( \nu + 3/2)^2 } - t / \sqrt{t^2 + ( \nu + 1)^2}$$\end{document } , which is negative for all \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t > 0$$\end{document}. thus , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta ( t ) < 0$$\end{document } for all \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t > 0$$\end{document } and hence \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g_{\nu + 1/2,\nu + 3/2}(t ) > g_{\nu + 1,\nu + 1}(t)$$\end{document } for all \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t > 0$$\end{document}. let\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } \beta _ { ss}(\nu ) = \sqrt{(\nu + 1/2)(\nu + 3/2)}. \end{aligned}$$\end{document } simpson and spector ( 1984 ) show that with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$v_\nu ( t ) = t / r_{\nu } ( t)$$\end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$v_\nu ( t)^2 - ( 2\nu + 1 ) v_\nu ( t ) - ( t^2 + \nu + 1/2 ) > 0$$\end{document } for all \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\nu \ge 0$$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t > 0$$\end{document } , which is readily seen to imply \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$v_{\nu } ( t ) \ge \nu + 1/2 + \sqrt{t^2 + \beta _ { ss}(\nu ) ^2}$$\end{document } and hence \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$r_\nu ( t ) \le g_{\nu + 1/2,\beta _ { ss}(\nu ) } ( t)$$\end{document } , which is clearly smaller than \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g_{\nu + 1/2,\nu + 1/2}(t)$$\end{document } for all \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t > 0$$\end{document}. altogether , we thus have that for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\nu \ge 0$$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t \ge 0$$\end{document},3\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } g_{\nu + 1/2,\nu + 3/2}(t ) \le r_{\nu } ( t ) \le \min \left ( g_{\nu , \nu + 2}(t ) , g_{\nu + 1/2,\beta _ { ss}(\nu ) } ( t ) \right ) . \end{aligned}$$\end{document}what makes these amos - type bounds particularly attractive is that they can be inverted explicitly , as shown in the following lemma . let \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\alpha \ge 0$$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\alpha + \beta > 0$$\end{document}. then \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g_{\alpha , \beta } $ $ \end{document } is strictly increasing on \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[0 , \infty ) $ $ \end{document } , and for all \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$0 \le \rho < 1$$\end{document } the equation \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g_{\alpha , \beta } ( t ) = \rho $ $ \end{document } has a unique solution \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t = g_{\alpha , \beta } ^{-1}(\rho ) $ $ \end{document } given by4\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } g_{\alpha , \beta } ^{-1}(\rho ) = \frac{\rho } { 1 - \rho ^2 } \left ( \alpha + \sqrt{\rho ^2 \alpha ^2 + ( 1 - \rho ^2 ) \beta ^2 } \right ) . \end{aligned}$$\end{document } the derivative of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g_{\alpha , \beta } $ $ \end{document } is given by\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } g_{\alpha , \beta } ' ( t)&= \frac{1}{\alpha + \sqrt{t^2 + \beta ^2 } } - \frac{t}{(\alpha + \sqrt{t^2 + \beta ^2})^2 } \frac{2t}{2\sqrt{t^2 + \beta ^2 } } \\&= \frac{\alpha \sqrt{t^2 + \beta ^2 } + \beta ^2 } { ( \alpha + \sqrt{t^2 + \beta ^2})^2 \sqrt{t^2 + \beta ^2 } } , \end{aligned}$$\end{document}where the numerator has value \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta ( \alpha + \beta ) $ $ \end{document } at \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t = 0$$\end{document } and hence is positive for all \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t > 0$$\end{document } if and only if \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\alpha \ge 0$$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\alpha + \beta > 0$$\end{document } in which case \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g_{\alpha , \beta } $ $ \end{document } is strictly increasing , and as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g_{\alpha , \beta } ( 0 ) = 0$$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\lim _ { t \rightarrow \infty } g_{\alpha , \beta } ( t ) = 1$$\end{document } the equation \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g_{\alpha , \beta } ( t ) = \rho $ $ \end{document } has a unique solution for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$0 \le \rho < 1$$\end{document}. now \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g_{\alpha , \beta } ( t ) = \rho $ $ \end{document } iff \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t = \rho ( \alpha + \sqrt{t^2 + \beta ^2})$$\end{document } , giving \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t$$\end{document } as the larger root of the quadratic equation \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$(1 - \rho ^2 ) t^2 - 2 \rho \alpha t + \rho ^2 ( \alpha ^2 - \beta ^2 ) = 0$$\end{document } , so that\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } g_{\alpha , \beta } ^{-1}(\rho ) & = \frac{1}{2(1-\rho ^2 ) } \left ( 2 \rho \alpha + \sqrt { 4 \rho ^2 \alpha ^2 - 4 ( 1 - \rho ^2 ) \rho ^2 ( \alpha ^2 - \beta ^2 ) } \right ) \\&= \frac{\rho } { 1 - \rho ^2 } \left ( \alpha + \sqrt{\rho ^2 \alpha ^2 + ( 1 - \rho ^2 ) \beta ^2 } \right ) \end{aligned}$$\end{document}(the smaller root does not converge to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\infty $ $ \end{document } as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\rho \rightarrow 1-$$\end{document } ) . let \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\nu \ge 0$$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$0 \le \rho < 1$$\end{document}. then5\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } \max \left ( g_{\nu , \nu + 2}^{-1}(\rho ) , g_{\nu + 1/2 , \beta _ { ss}(\nu ) } ^{-1}(\rho ) \right ) \le r_\nu ^{-1}(\rho ) \le g_{\nu + 1/2,\nu + 3/2}^{-1}(\rho ) . \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ g_{\alpha , \alpha } ^{-1}(\rho ) = 2\alpha \rho / ( 1 - \rho ^2)$$\end{document } , so that the lower and upper bound in eq . 2 equal \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g_{\nu , \nu } ^{-1}(\rho ) $ $ \end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g_{\nu + 1,\nu + 1}^{-1}(\rho ) $ $ \end{document } , respectively , and hence correspond to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g_{\nu + 1,\nu + 1}(t ) \le r_\nu ( t ) \le g_{\nu , \nu } ( t)$$\end{document } , which was already shown to be strictly weaker than the bounds in eq . 3 . we also see that the mid - point approximation \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$r_\nu ^{-1}(\rho ) \approx ( 2\nu + 1 ) \rho / ( 1 - \rho ^2)$$\end{document } equals \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g_{\nu + 1/2,\nu + 1/2}^{-1}(\rho ) $ $ \end{document } , which for positive \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\rho $ $ \end{document } is strictly smaller than \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g_{\nu + 1/2,\beta _ { ss}(\nu ) } ^{-1}(\rho ) $ $ \end{document } , and hence strictly under - estimates \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$r_\nu ^{-1}(\rho ) $ $ \end{document}. let\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } g(\nu ) = \frac{(\nu + 3/2)}{2 \nu + 1}. \end{aligned}$$\end{document}then \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g$$\end{document } is monotonically decreasing on \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[0,\infty ) $ $ \end{document } with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g(0 ) = 3/2$$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\lim _ { \nu \rightarrow \infty } g(\nu ) = 1/2$$\end{document}. let \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\nu \ge 0$$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$0 \le \rho < 1$$\end{document}. then\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } 0 \le g_{\nu + 1/2,\nu + 3/2}^{-1}(\rho ) - g_{\nu + 1/2,\beta _ { ss}(\nu ) } ^{-1}(\rho ) \le \rho g(\nu ) \end{aligned}$$\end{document}and for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta _ { ss}(\nu ) \le \beta \le \nu + 3/2$$\end{document},\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } \big | r_{\nu } ^{-1}(\rho ) - g_{\nu + 1/2,\beta } ^{-1}(\rho ) \big | \le \rho g(\nu ) . \end{aligned}$$\end{document } using the mean value theorem , for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$u_1 \ge u_0 > 0$$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\alpha \ge 0$$\end{document } with a suitable \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\tilde{u } \in ( u_0 , u_1)$$\end{document},\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } 0 \le \left . \sqrt{\alpha + u } \right| _ { u = u_0}^{u = u_1 } = \frac{u_1 - u_0}{2 \sqrt{\alpha + \tilde{u } } } \le \frac{u_1 - u_0}{2 \sqrt{\alpha + u_0 } } \end{aligned}$$\end{document}and hence\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } 0&\le g_{\nu + 1/2,\nu + 3/2}^{-1}(\rho ) - g_{\nu + 1/2,\beta _ { ss}(\nu ) } ^{-1}(\rho ) \\&= \frac{\rho } { 1 - \rho ^2 } \left . \sqrt{(\nu + 1/2)^2 \rho ^2 + \beta ^2 ( 1 - \rho ^2 ) } \right| _ { \beta = \beta _ { ss}(\nu ) } ^{\beta = \nu + 3/2 } \\&\le \frac{\rho } { 1-\rho ^2 } \frac { ( 1 - \rho ^2 ) ( ( \nu + 3/2)^2 - ( \nu + 1/2)(\nu + 3/2 ) ) } { 2 \sqrt { ( \nu + 1/2)^2 \rho ^2 + \tilde{\beta } ^2 ( 1 - \rho ^2 ) } } \\&\le \frac{\rho ( \nu + 3/2)}{2 ( \nu + 1/2)}. \end{aligned}$$\end{document}for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta _ { ss}(\nu ) \le \beta \le \nu + 3/2$$\end{document } , both \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$r_{\nu } ^{-1}$$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g_{\nu + 1/2,\beta } ^{-1}$$\end{document } are bounded below by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g_{\nu + 1/2,\beta _ { ss}(\nu ) } ^{-1}$$\end{document } and above by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g_{\nu + 1/2,\nu + 3/2}^{-1}$$\end{document } , implying that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$| r_{\nu } ^{-1 } - g_{\nu + 1/2,\beta } ^{-1 } | \le g_{\nu + 1/2,\nu + 3/2}^{-1 } - g_{\nu + 1/2,\beta _ { ss}(\nu ) } ^{-1}$$\end{document } , whence the result from the bounds on this difference . let \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\nu \ge 0$$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$0 \le \rho < 1$$\end{document}. then\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } 0 \le r_{\nu } ^{-1}(\rho ) - \max \left ( g_{\nu , \nu + 2}^{-1}(\rho ) , g_{\nu + 1/2 , \beta _ { ss}(\nu ) } ^{-1}(\rho ) let \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\nu \ge 0$$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$0 \le \rho < 1$$\end{document}. then\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } \max \left ( g_{\nu , \nu + 2}^{-1}(\rho ) , g_{\nu + 1/2 , \beta _ { ss}(\nu ) } ^{-1}(\rho ) \right ) \le q_\nu ( \rho ) \le \end{aligned}$$\end{document } for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta _ \alpha ( \rho ) = \sqrt{(\alpha + 1)^2 - \rho ^2}$$\end{document},\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } \alpha ^2 \rho ^2 + \beta _ \alpha ( \rho ) ^2 ( 1 - \rho ^2)&= \alpha ^2 \rho ^2 + ( ( \alpha + 1)^2 - \rho ^2 ) ( 1 - \rho ^2 ) = ( \alpha + 1 - \rho ^2)^2 . \end{aligned}$$\end{document}hence , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\alpha + \sqrt{\alpha ^2 \rho ^2 + \beta _ \alpha ( \rho ) ^2 ( 1 - \rho ^2 ) } = 2 \alpha + 1 - \rho ^2$$\end{document } so that in particular,\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } q_{\nu } ( \rho ) = g_{\nu + 1/2,\beta _ { \nu + 1/2}(\rho ) } ^{-1}(\rho ) . \end{aligned}$$\end{document}as clearly \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta _ { ss}(\nu ) \le \beta _ { \nu + 1/2}(\rho ) \le \nu + 3/2$$\end{document } for all \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$0 \le \rho \le 1$$\end{document } , we thus obtain \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g_{\nu + 1/2,\beta _ { ss}(\nu ) } ^{-1}(\rho ) \le g_{\nu + 1/2,\beta _ { \nu + 1/2}(\rho ) } ^{-1}(\rho ) = q_\nu ( \rho ) \le g_{\nu + 1/2,\nu + 3/2}^{-1}(\rho ) $ $ \end{document}. writing \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta ( \sigma ) = \sigma + \sqrt{(\nu + 2)^2 - 4(\nu + 1)\sigma } - ( \nu + 2)$$\end{document } we have \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g_{\nu , \nu + 2}^{-1}(\rho ) - q_\nu ( \rho ) = \delta ( \rho ^2 ) \rho / ( 1 - \rho ^2)$$\end{document}. as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta ' ( \sigma ) = 1 - 2(\nu + 1 ) / \sqrt{(\nu + 2)^2 - 4(\nu + 1)\sigma } $ $ \end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta ' ' ( \sigma ) = - 4 ( \nu + 1)^2 ( ( \nu + 2)^2 - 4(\nu + 1)\sigma ) ^{-3/2}$$\end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta $ $ \end{document } is strictly concave with its unique maximum at the solution \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\sigma ^*$$\end{document } of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta ' ( \sigma ) = 0$$\end{document } , or equivalently \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$(\nu + 2)^2 - 4(\nu + 1)\sigma = 4(\nu + 1)^2$$\end{document } , from which\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } \sigma ^ * = \frac{(\nu + 2)^2 - 4(\nu + 1)^2}{4(\nu + 1 ) } = \frac{-3\nu ^2 - 4\nu } { 4(\nu + 1 ) } , \end{aligned}$$\end{document}which is non - positive for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\nu \ge 0$$\end{document}. thus , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta $ $ \end{document } is decreasing on \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[0 , 1]$$\end{document}. as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta ( 0 ) = 0$$\end{document } , we obtain that for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$0 < \rho < 1$$\end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta ( \rho ^2 ) < 0$$\end{document } and hence \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g_{\nu , \nu + 2}^{-1}(\rho ) < q_\nu ( \rho ) $ $ \end{document}. let \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\nu \ge 0$$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$0 \le \rho < 1$$\end{document}. then\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } \left| r_{\nu } ^{-1}(\rho ) - q_\nu ( \rho ) \right| sra approximation is not invalidated by the available inverse amos - type bounds ( in the sense of being outside the range provided by these bounds ) , and has the same maximal approximation error as these bounds ( indicating that it is indeed a good approximation ) . let \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\nu \ge 0$$\end{document}. then as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\rho \rightarrow 1-$$\end{document},\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } r_{\nu } ^{-1}(\rho ) - g_{\nu + 1/2,\beta _ { ss}(\nu ) } ^{-1}(\rho ) = o(\rho - 1 ) \end{aligned}$$\end{document}and\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } r_{\nu } ^{-1}(\rho ) - q_\nu ( \rho ) = - \frac{1}{2 } + o(\rho - 1 ) . \end{aligned}$$\end{document } using the asymptotic expansion of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$i_\nu $ $ \end{document } for large argument ( e.g.,watson 1995 , formula 7.23.2 ) , one can show that for arbitrary \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\nu $ $ \end{document},6\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } r_\nu ( t ) = 1 - \frac{\nu + 1/2}{t } + \frac{\nu ^2 - 1/4}{2t^2 } + o(1/t^3 ) , \qquad t \rightarrow \infty , \end{aligned}$$\end{document}see also ( schou ( 1978 ) , eq . 6 , assuming \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\nu \ge 0$$\end{document } ) . thus , we have \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$r_{\nu } ^{-1}(\rho ) = \omega _ { -1 } / ( \rho - 1 ) + \omega _ 0 + o(\rho - 1)$$\end{document } as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\rho \rightarrow 1-$$\end{document } with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\omega _ { -1 } \ne 0$$\end{document } , and the coefficients of this approximation can be determined by rewriting \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\rho = r_\nu ( t ) \approx 1 + \alpha _ 1 / t + \alpha _ 2 / t^2$$\end{document } as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$(\rho -1 ) t^2 - \alpha _ 1 t - \alpha _ 2 \approx 0$$\end{document } to obtain\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } t = r_\nu ^{-1}(\rho ) \approx \frac{\alpha _ 1 + \sqrt{\alpha _ 1 ^ 2 + 4 \alpha _ 2 ( \rho - 1)}}{2(\rho -1 ) } = \frac{\alpha _ 1}{\rho -1 } + \frac{\alpha _ 2}{\alpha _ 1 } + o(\rho -1 ) , \end{aligned}$$\end{document}giving ( with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\alpha _ 1 = - ( \nu + 1/2)$$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\alpha _ 2 = ( \nu -1/2)(\nu + 1/2 ) / 2$$\end{document})\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } r_\nu ^{-1}(\rho ) = - \frac{\nu + 1/2}{\rho - 1 } - \frac{\nu -1/2}{2 } + o(\rho - 1 ) , \qquad \rho \rightarrow { 1-}. \end{aligned}$$\end{document}for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\rho \rightarrow 1-$$\end{document},\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } \frac{\rho } { 1 - \rho ^2}&= - \frac{(\rho - 1 ) + 1}{(\rho - 1 ) ( 2 + ( \rho - 1 ) ) } \\&= - \frac{1}{2 } \left ( \frac{1}{\rho -1 } + 1 \right ) \left ( 1 - \frac{\rho - 1}{2 } + \frac{(\rho - 1)^2}{4 } + o((\rho -1)^3 ) \right ) \\&= - \frac{1}{2 } \left ( \frac{1}{\rho -1 } + \frac{1}{2 } - \frac{\rho - 1}{4 } + o((\rho -1)^2 ) \right ) . \end{aligned}$$\end{document}hence , if \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$f(\rho ) = \delta _ 0 + \delta _ 1 ( \rho - 1 ) + o((\rho - 1)^2)$$\end{document } as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\rho \rightarrow 1$$\end{document},\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } \frac{\rho } { 1 - \rho ^2 } f(\rho ) & = - \frac{\delta _ 0}{2 } \frac{1}{\rho - 1 } - \frac{\delta _ 0 + 2 \delta _ 1}{4 } + o(\rho - 1 ) \end{aligned}$$\end{document}as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\rho \rightarrow 1-$$\end{document}. in particular , as for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\alpha > 0$$\end{document}\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } \sqrt{\rho ^2 \alpha ^2 + ( 1 - \rho ^2 ) \beta ^2}&= \sqrt{\alpha ^2 + ( \beta ^2 - \alpha ^2 ) ( 1 - \rho ^2 ) } \\&= \alpha \left ( 1 + \frac{\beta ^2 - \alpha ^2}{2\alpha ^2 } ( 1 - \rho ^2 ) + o((\rho - 1)^2 ) \right ) \\&= \alpha - \frac{\beta ^2 - \alpha ^2}{\alpha } ( \rho - 1 ) + o((\rho - 1)^2 ) \end{aligned}$$\end{document}as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\rho \rightarrow 1$$\end{document},\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } g_{\alpha , \beta } ^{-1}(\rho ) & = \frac{\rho } { 1 - \rho ^2 } \left ( 2\alpha - \frac{\beta ^2 - \alpha ^2}{\alpha } ( \rho - 1 ) + o((\rho - 1)^2 ) \right ) \\&= - \frac{\alpha } { \rho - 1 } - \frac{1}{4}\left ( 2\alpha - 2\frac{\beta ^2 - \alpha ^2}{2\alpha } \right ) + o(\rho - 1 ) \\&= - \frac{\alpha } { \rho - 1 } - \alpha + \frac{\beta ^2}{2\alpha } + o(\rho - 1 ) , \qquad \rho \rightarrow { 1-}. \end{aligned}$$\end{document}for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\alpha = \nu + 1/2$$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta = \beta _ { ss}(\nu ) $ $ \end{document } we have \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$- \alpha + \beta ^2 / ( 2\alpha ) = -\nu /2 + 1/4$$\end{document } so that\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } g_{\nu + 1/2,\beta _ { ss}(\nu ) } ^{-1}(\rho ) = - \frac{\nu + 1/2}{\rho - 1 } - \frac{\nu - 1/2}{2 } + o(\rho - 1 ) \end{aligned}$$\end{document}and hence \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$r_\nu ^{-1}(\rho ) - g_{\nu + 1/2,\beta _ { ss}(\nu ) } ^{-1}(\rho ) = o(\rho - 1)$$\end{document } as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\rho \rightarrow 1-$$\end{document}. as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$2(\nu + 1 ) - \rho ^2 = ( 2\nu + 1 ) - 2(\rho - 1 ) - ( \rho - 1)^2$$\end{document},\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } q_\nu ( \rho ) = - \frac{\nu + 1/2}{\rho - 1 } - \frac{2\nu - 3}{4 } + o(\rho -1 ) , \qquad \rho \rightarrow { 1-}. \end{aligned}$$\end{document}as\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } - \frac{\nu - 1/2}{2 } - \left ( -\frac{2\nu - 3}{4}\right ) = -\frac{1}{2 } , \end{aligned}$$\end{document}we thus have \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$r_\nu ^{-1}(\rho ) - q_\nu ( \rho ) = -1/2 + o(\rho - 1)$$\end{document } as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\rho \rightarrow 1-$$\end{document } , and the proof is complete . nsell ( 1978 ) gives families \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$l_{\nu , k , m}$$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$u_{\nu , k , m}$$\end{document } of rational lower and upper bounds for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$r_\nu $ $ \end{document } , which converge to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$r_\nu $ $ \end{document } as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$m \rightarrow \infty $ $ \end{document } or \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$k \rightarrow \infty $ $ \end{document } ( nsell 1978 , theorems 2 and 3 ) . these bounds can be used for obtaining bounds for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$r_\nu ^{-1}$$\end{document } by applying numerical root finding techniques either directly to equations of the form \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p(t ) / q(t ) = \rho $ $ \end{document } with polynomials \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p$$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$q$$\end{document } , or by rewriting the equations of this form as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$r(t ) = p(t ) - \rho q(t ) = 0$$\end{document } and then determining a suitable root of the polynomial \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$r$$\end{document}. simple closed form expressions can be obtained when root finding amounts to solving a quadratic equation . as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$r_\nu ( t)$$\end{document } tends to 0 and 1 for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t \rightarrow 0$$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\infty $ $ \end{document } , respectively , we thus restrict ourselves to nsell bounds exhibiting the same limits , and having numerator and denominator degrees at most 2 . this leaves ( nsell 1978 , appendix ) the lower bounds \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$l_{\nu , 1,0 } < l_{\nu , 2,0}$$\end{document } and the upper bounds \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$u_{\nu , 1,1}$$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$u_{\nu , 3,0 } < u_{\nu , 2,0}$$\end{document } , where the inequalities again follow from theorems 2 and 3 in the reference . neither of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$u_{\nu , 1,1}$$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$u_{\nu , 3,0}$$\end{document } dominates the other , as they have different orders of approximation at 0 and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\infty $ $ \end{document}. in fact , writing\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } u_{\nu , 1,1}(t ) = t \frac{p_{\nu , 1,1}(t)}{q_{\nu , 1,1}(t ) } = t \frac{2(\nu + 2 ) + t}{4(\nu + 1)(\nu + 2 ) + 2(\nu + 1)t + t^2 } \end{aligned}$$\end{document}and\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } u_{\nu , 3,0}(t ) = t \frac{p_{\nu , 3,0}(t)}{q_{\nu , 3,0}(t ) } = t \frac{\frac{1}{2}(\nu + 1/2 ) + t } { ( \nu + 1/2)(\nu + 1 ) + \frac{3}{2}(\nu + 1/2 ) t + t^2 } , \end{aligned}$$\end{document}it is readily verified that\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } u_{\nu , 1,1}(t ) - u_{\nu , 3,0}(t ) = \frac{(\nu + 5/2 ) t^2 ( t - ( \nu + 2))}{q_{\nu , 1,1}(t ) q_{\nu , 3,0}(t)}. \end{aligned}$$\end{document}this implies that with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t_\nu = \nu + 2$$\end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$u_{\nu , 1,1}(t ) < u_{\nu , 3,0}(t)$$\end{document } for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$0 < t < t_\nu $ $ \end{document } , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$u_{\nu , 1,1}(t ) > u_{\nu , 3,0}(t)$$\end{document } for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t > t_\nu $ $ \end{document}. the best lower bound \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$l_{\nu , 2,0}$$\end{document } which only involves solving a quadratic equation is given by\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } l_{\nu , 2,0 } = t\frac{(\nu + 3/2 ) + t}{2(\nu + 1)(\nu + 3/2 ) + 2(\nu + 1)t + t^2}. \end{aligned}$$\end{document } let \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\nu \ge 0$$\end{document}. then for all \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t > 0$$\end{document},\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } l_{\nu , 2,0}(t ) < g_{\nu + 1/2 , \nu + 3/2}(t ) \end{aligned}$$\end{document}and\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } \min \left ( g_{\nu , \nu + 2}(t ) , g_{\nu + 1/2,\beta _ { ss}(\nu ) } ( t)\right ) < \min \left ( u_{\nu , 1,1}(t ) , u_{\nu , 3,0}(t)\right ) . \end{aligned}$$\end{document } all nsell bounds under consideration are of the form\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } t \frac{p(t)}{q(t ) } , \qquad p(t ) = t + \gamma , \quad q(t ) = t^2 + \delta _ 1 t + \delta _ 0 \end{aligned}$$\end{document}with non - negative coefficients \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\gamma $ $ \end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta _ 1$$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta _ 0$$\end{document}. all amos - type bounds \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g_{\alpha , \beta } $ $ \end{document } under consideration have \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\alpha \le \beta $ $ \end{document}. to show that the amos - type bounds dominate these nsell bounds we have to investigate when\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } \frac{p(t)}{q(t ) } - \frac{1}{\alpha + \sqrt{t^2 + \beta ^2 } } \end{aligned}$$\end{document}has no zeros on \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$(0 , \infty ) $ $ \end{document } , or equivalently , when\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } \delta ( t ) = \left ( q(t ) - \alpha p(t)\right ) ^2 - \left ( t^2 + \beta ^2\right ) p(t)^2 \end{aligned}$$\end{document}has no zeros on \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$(0 , \infty ) $ $ \end{document}. note that\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } \delta ( t ) = \left ( q(t ) - \left ( \alpha + \sqrt{t^2 + \beta ^2}\right ) p(t ) \right ) \left ( q(t ) - \left ( \alpha - \sqrt{t^2 + \beta ^2}\right ) p(t ) \right ) , \end{aligned}$$\end{document}where the second term is always positive for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t > 0$$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\alpha \le \beta $ $ \end{document}. hence , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta ( t ) > 0$$\end{document } for all \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t > 0$$\end{document } is equivalent to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$q(t ) - ( \alpha + \sqrt{t^2 + \beta ^2 } ) p(t ) > 0$$\end{document } , or \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p(t ) / q(t ) < 1 / ( \alpha + \sqrt{t^2 + \beta ^2})$$\end{document } for all \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t > 0$$\end{document } ; \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta ( t ) < 0$$\end{document } for all \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t > 0$$\end{document } is equivalent to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p(t ) / q(t ) > 1 / ( \alpha + \sqrt{t^2 + \beta ^2})$$\end{document } for all \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t > 0$$\end{document}. writing\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } q(t ) - \alpha p(t ) = t^2 + ( \delta _ 1 - \alpha ) t + ( \delta _ 0 - \alpha \gamma ) = t^2 + \omega _ 1 t + \omega _ 0 , \end{aligned}$$\end{document}we obtain\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } \delta ( t)&= ( t^2 + \omega _ 1 t + \omega _ 0)^2 - ( t^2 + \beta ^2 ) ( t + \gamma ) ^2 \\&= 2 ( \omega _ 1 - \gamma ) t^3 + ( \omega _ 1 ^ 2 + 2 \omega _ 0 - \beta ^2 - \gamma ^2 ) t^2 \!+\ ! 2 ( \omega _ 1 \omega _ 0 \!-\ ! \beta ^2 \gamma ) t + ( \omega _ 0 ^ 2 - \beta ^2 \gamma ^2 ) . \end{aligned}$$\end{document}comparing \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$l_{\nu , 2,0}$$\end{document } to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g_{\nu + 1/2,\nu + 3/2}$$\end{document } , we have \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\gamma = \nu + 3/2$$\end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta _ 0 = 2 ( \nu + 1 ) ( \nu + 3/2)$$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta _ 1 = 2 ( \nu + 1)$$\end{document } , from which \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\omega _ 0 = \delta _ 0 - \alpha \gamma = 2 ( \nu + 1 ) ( \nu + 3/2 ) - ( \nu + 1/2 ) ( \nu + 3/2 ) = ( \nu + 3/2)^2 = \beta ^2$$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\omega _ 1 = \delta _ 1 - \alpha = \nu + 3/2 = \beta $ $ \end{document } , giving\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } 2 ( \omega _ 1 - \gamma ) & = 0 , \\ \omega _ 1 ^ 2 + 2 \omega _ 0 - \beta ^2 - \gamma ^2&= \beta ^2 , \\ 2 ( \omega _ 1 \omega _ 0 - \beta ^2 \gamma ) & = 0 , \\ \omega _ 0 ^ 2 - \beta ^2 \gamma ^2&= 0 \end{aligned}$$\end{document}so that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta ( t ) = \beta ^2 t^2 > 0$$\end{document } for all \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t > 0$$\end{document}. hence , we have \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$l_{\nu , 1,0}(t ) < l_{\nu , 2,0}(t ) < g_{\nu + 1/2 , \nu + 3/2}(t)$$\end{document } for all \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t > 0$$\end{document}. comparing \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$u_{\nu , 1,1}$$\end{document } to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g_{\nu , \nu + 2}$$\end{document } , we have \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\gamma = 2(\nu + 2 ) = 2 \beta $ $ \end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta _ 0 = 4(\nu + 1)(\nu + 2)$$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta _ 1 = 2(\nu + 1)$$\end{document } , from which \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\omega _ 0 = \delta _ 0 - \alpha \gamma = 4 ( \nu + 1 ) ( \nu + 2 ) - 2 \nu ( \nu + 2 ) = 2 ( \nu + 2)^2 = 2 \beta ^2$$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\omega _ 1 = \delta _ 1 - \alpha = 2 ( \nu + 1 ) - \nu = \nu + 2 = \beta $ $ \end{document } , giving\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } 2 ( \omega _ 1 - \gamma ) & = -2 \beta , \\ \omega _ 1 ^ 2 + 2 \omega _ 0 - \beta ^2 - \gamma ^2&= 0 , \\ 2 ( \omega _ 1 \omega _ 0 - \beta ^2 \gamma ) & = 0 , \\ \omega _ 0 ^ 2 - \beta ^2 \gamma ^2&= 0 , \end{aligned}$$\end{document}so that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta ( t ) = - 2 \beta t^3 < 0$$\end{document } for all \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t > 0$$\end{document}. hence , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g_{\nu , \nu + 2}(t ) < u_{\nu , 1,1}(t)$$\end{document } for all \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t > 0$$\end{document}. finally , comparing \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$u_{\nu , 3,0}$$\end{document } to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g_{\nu + 1/2,\beta _ { ss}(\nu ) } $ $ \end{document } , we have \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\gamma = \frac{1}{2 } ( \nu + 1/2 ) = \alpha / 2$$\end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta _ 0 = ( \nu + 1/2 ) ( \nu + 1 ) = \alpha ( \alpha + 1/2)$$\end{document}. \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta _ 1 = \frac{3}{2 } ( \nu + 1/2 ) = 3\alpha /2$$\end{document } , from which \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\omega _ 0 = \delta _ 0 - \alpha \gamma = \alpha ( \alpha + 1/2 ) - \alpha ^2 / 2 = \alpha ( \alpha + 1 ) / 2 = \beta ^2 / 2$$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\omega _ 1 = \delta _ 1 - \alpha = 3\alpha /2 - \alpha = \alpha /2$$\end{document } , giving\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } 2 ( \omega _ 1 - \gamma ) & = 0 , \\ \omega _ 1 ^ 2 + 2 \omega _ 0 - \beta ^2 - \gamma ^2&= 0 , \\ 2 ( \omega _ 1 \omega _ 0 - \beta ^2 \gamma ) & = - \alpha \beta ^2 / 2 , \\ \omega _ 0 ^ 2 - \beta ^2 \gamma ^2&= \beta ^2 ( \beta ^2 - \alpha ^2 ) / 4 , \end{aligned}$$\end{document}so that with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta ^2 - \alpha ^2 = ( \nu + 1/2)(\nu + 3/2 ) - ( \nu + 1/2)^2 = \nu + 1/2 = \alpha $ $ \end{document } we get\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } \delta ( t ) = - \frac{\alpha \beta ^2}{2 } t + \frac{\beta ^2\alpha } { 4 } = \frac{\beta ^2\alpha } { 4 } ( 1 - 2 t ) , \end{aligned}$$\end{document}which is negative for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t > 1/2$$\end{document } , so that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g_{\nu + 1/2,\beta _ { ss}(\nu ) } ( t ) < u_{\nu , 3,0}(t)$$\end{document } for all \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t > 1/2$$\end{document}. as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$u_{\nu , 1,1}(t ) < u_{\nu , 3,0}(t)$$\end{document } for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$0 < t < t_\nu = \nu + 2$$\end{document } , we infer that\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } \min \left ( g_{\nu , \nu + 2}(t ) , g_{\nu + 1/2,\beta _ { ss}(\nu ) } ( t)\right ) < \min \left ( u_{\nu , 1,1}(t ) , u_{\nu , 3,0}(t)\right ) \end{aligned}$$\end{document}for all \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t > 0$$\end{document } , and the proof is complete . as the nsell bounds considered in the previous theorem are dominated by the amos - type bounds used for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$r_\nu $ $ \end{document } , the same must be true for the respective inverses . let \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\nu \ge 0$$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$0 \le \rho < 1$$\end{document}. then\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } r_\nu ^{-1}(\rho ) \le g_{\nu + 1/2,\nu + 3/2}^{-1}(\rho ) < l_{\nu , 2,0}^{-1}(\rho ) \end{aligned}$$\end{document}and\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } \max \left ( u_{\nu , 1,1}^{-1}(\rho ) , u_{\nu , 3,0}^{-1}(\rho ) \right ) \le \max \left ( g_{\nu , \nu + 2}^{-1}(\rho ) , g_{\nu + 1/2 , \beta _ { ss}(\nu ) } ^{-1}(\rho ) \right ) \le r_{\nu } ^{-1}(\rho ) . in the following we compare the number of iterations required to reach convergence by two different algorithms based on nested intervals for finding roots using ( 1 ) the tanabe et al . the two algorithms used are ( a ) a one - dimensional root finding algorithm as implemented in function uniroot ( ) available in r ( r core team 2013 ) and ( b ) the variant of the newton - fourier method for the case of strictly increasing concave functions ( see e.g. , atkinson 1989 , pp . concavity of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$r_{\nu } $ $ \end{document } can be established by using that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$r_{\nu } $ $ \end{document } is the pointwise minimum of a set of amos - type functions ( see hornik and grn 2013 , theorem 11 ) . it is straightforward to show that the second derivative of these amos - type functions is non - positive and hence \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$r_{\nu } $ $ \end{document } is concave , because it is the pointwise minimum of concave functions . the numbers of iterations required are determined for a range of different \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$d$$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\kappa $ $ \end{document } values which are selected similar to those used in sra ( 2012 ) , i.e. , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\kappa $ $ \end{document}\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$=$$\end{document } 100 , 500 , 1,000 , 5,000 , 10,000 , 20,000 , 50,000 , 10,000 and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$d$$\end{document}\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$=$$\end{document } 500 , 1,000 , 5,000 , 10,000 , 20,000 , 10,000 . each value of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$d$$\end{document } is combined with each value of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\kappa $ $ \end{document } and they are used to determine the corresponding \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\rho $ $ \end{document}. for the given \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$d$$\end{document } and implied \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\rho $ $ \end{document } the root finding algorithms are employed to determine \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\kappa $ $ \end{document } again . for uniroot ( ) convergence is assessed using argument tol and the newton - fourier algorithm is stopped if the relative distance of the midpoint to the endpoints of the interval is smaller than tol . this pre - specified precision value tol is varied and set to values \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$10^{e}$$\end{document } for exponents \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$e \in \{-6$$\end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$-8$$\end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$-10$$\end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$-$$\end{document}12}. the results are given in table 1 . for the newton - fourier algorithm the newly established bounds always require the same or a smaller number of iterations . in 31 % of the cases one iteration less is required , reducing the number of iterations required by 60 or 20 % . when using uniroot ( ) , there is an improvement achievement in 55 % of the cases , the same number of iterations are required in 42 % of the cases and only in 3 % of the cases a deterioration occurs and one iteration more is required when using the newly established bounds for initialization . table 1number of iterations required by uniroot ( ) ( top ) and by the newton - fourier algorithm ( bottom ) using the tanabe et al . ( 2007 ) bounds ( t07 ) and the newly established bounds ( new ) for initializationt07new0123451430000222232200305463630400514905004311 t07new12187025245308 number of iterations required by uniroot ( ) ( top ) and by the newton - fourier algorithm ( bottom ) using the tanabe et al . ( 2007 ) bounds ( t07 ) and the newly established bounds ( new ) for initialization overall it has to be noted that the number of iterations is rather small to reach convergence when either of the two sets of intervals is used for initialization . however , the general tendency to require even less iterations of the newly established bounds will nevertheless be of interest from a computational point of view if these roots are solved repeatedly , which is required when for example the expectation - maximization algorithm is used to estimate mixtures of von mises fisher distributions ( banerjee et al . 2005 ) .

maximum likelihood estimation of the concentration parameter of von mises fisher distributions involves inverting the ratio \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$r_\nu = i_{\nu + 1 } / i_\nu $ $ \end{document } of modified bessel functions and computational methods are required to invert these functions using approximative or iterative algorithms . in this paper we use amos - type bounds for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$r_\nu $ $ \end{document } to deduce sharper bounds for the inverse function , determine the approximation error of these bounds , and use these to propose a new approximation for which the error tends to zero when the inverse of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$r_\nu $ $ \end{document } is evaluated at values tending to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$1$$\end{document } ( from the left ) . we show that previously introduced rational bounds for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$r_\nu $ $ \end{document } which are invertible using quadratic equations can not be used to improve these bounds .

major depressive disorder ( mdd ) is a highly prevalent psychiatric disorder1 and a leading cause of disease burden.2 currently , a diagnosis of mdd is made on the basis of clinical symptoms that are exhibited by patients . there are no established practical laboratory tests that can be used to discriminate patients with mdd from those without mdd . gene expression profiling of peripheral blood has emerged as a useful method not only to investigate the pathogenesis of mdd , but also to identify relevant biomarkers.35 recently , we examined the diagnostic performance of a multi - assay leukocyte gene expression - based test comprising 40 candidate genes , and developed a panel of 5 gene expression markers ( arhgap24 , hdac5 , pdgfc , prnp , and slc6a4 ) , which are able to distinguish patients with mdd from nonpsychiatric control subjects with high accuracy in discriminant analysis.6 however , it is not clear whether this panel of gene expression markers is useful when subjects with other psychiatric disorders , such as those with schizophrenia and bipolar disorder , are included . in some cases , it may be difficult for clinicians to accurately diagnose mdd at the patient s first visit . li et al7 have reported that a diagnosis of mdd was changed to bipolar disorder in 7.6%12% of patients initially diagnosed with mdd , with a mean time to change of 1.892.98 years.7 ruggero et al8 have reported that 16.4% of participants classified as having mdd with psychosis at baseline were later rediagnosed with schizophrenia or schizoaffective disorder . in the present study , we measured five gene expression levels of subjects with schizophrenia and bipolar disorder , and we evaluated whether our gene expression - based markers for mdd are able to discriminate patients with mdd from those without mdd ( controls , schizophrenia , and bipolar disorder ) in the combined dataset of our previous study6 and the present study . seventeen medication - free patients with schizophrenia and 39 patients with bipolar disorder were recruited from the tokushima university hospital in japan . diagnoses of schizophrenia and bipolar disorder were made by at least two experienced psychiatrists according to dsm - iv criteria on the basis of extensive clinical interviews and a review of medical records . all of the patients with bipolar disorder were treated with lithium , anticonvulsants , atypical antipsychotics , antidepressants , or combinations of the above . the patients had not used nonsteroidal anti - inflammatory agents or steroids within at least 2 months before the study initiation . all subjects who participated in this study were of unrelated japanese origin and signed written informed consent forms , this study was also approved by the institutional ethics committee of the university of tokushima graduate school . pax gene blood ribonucleic acid ( rna ) tubes ( qiagen , tokyo , japan ) and pax gene blood rna kits ( qiagen ) were used according to the manufacturer s recommendations to extract total rna from peripheral leukocytes taken from whole blood samples . after assessing rna quality and quantity , individual total rna samples ( 2 m each ) , random ( n6 ) primers , and quantiscript reverse transcriptase ( qiagen , tokyo , japan ) quantitative real - time polymerase chain reaction ( rt - pcr ) analysis was performed using an abi 7500 fast real - time pcr system ( applied biosystems , foster city , ca , usa ) using the taqman gene expression probes ( applied biosystems ) hs00229928_ml ( arhgap24 ) , hs00608366_m1 ( hdac5 ) , hs00211916_m1 ( pdgfc ) , hs01920617_s1 ( prnp ) , hs00984356_m1 ( slc6a4 ) , and hs99999905_m1 ( gapdh ) . gapdh was used as the reference gene , as in our previous study.6 all measurements were performed in duplicate . the ct method was used to determine relative expression of each gene in the subjects . the obtained value for each gene ( ctx ) was used to calculate the relative level of gene expression ( ctx ) by normalization to the reference gene , gapdh . for validation of our expression data , we used 10 samples from the current study and measured gapdh expression according to the same protocol , and we observed a high correlation between the two datasets ( r=0.986 , p=1.11e08 ) . expression data for 25 patients with mdd and 25 controls were obtained from our previous study.6 statistical analyses were performed using r ver3.1.1 . one - way analysis of variance ( anova ) was used to test for differences in age among the groups , and fisher s exact test was used to examine differences in sex distribution among the groups . wallis one - way anova and the bonferroni test was used for post hoc comparisons . a linear discriminant function was developed for discrimination between patients with mdd and without mdd , as in our previous study.6 a discrimination score ( d - score ) was calculated for each subject by multiplying the coefficients of the linear discriminants , obtained using the lda ( ) function of the mass package in r , to the standardized values of expression after z - score transformation of the five selected genes . the d - score indicates the probability of membership to the group of patients with mdd or the group of subjects without mdd ( schizophrenia or bipolar disorder or controls ) . the highest membership probability for each subject determined their classification into the mdd group or the other groups . a d - score was calculated for each sample as follows : d - score=0.73980239pdgfc+0.84889084prnp0.42559503arhgap240.07806493slc6a4 + 1.48110014hdac5 + 0.005 . in this plot , as an aid in understanding the discriminant , to set a linear decision boundary at the zero level , the value of the constant term was adjusted . patients with mdd , and those with a d - score < 0 was identified as seventeen medication - free patients with schizophrenia and 39 patients with bipolar disorder were recruited from the tokushima university hospital in japan . diagnoses of schizophrenia and bipolar disorder were made by at least two experienced psychiatrists according to dsm - iv criteria on the basis of extensive clinical interviews and a review of medical records . all of the patients with bipolar disorder were treated with lithium , anticonvulsants , atypical antipsychotics , antidepressants , or combinations of the above . the patients had not used nonsteroidal anti - inflammatory agents or steroids within at least 2 months before the study initiation . all subjects who participated in this study were of unrelated japanese origin and signed written informed consent forms , this study was also approved by the institutional ethics committee of the university of tokushima graduate school . pax gene blood ribonucleic acid ( rna ) tubes ( qiagen , tokyo , japan ) and pax gene blood rna kits ( qiagen ) were used according to the manufacturer s recommendations to extract total rna from peripheral leukocytes taken from whole blood samples . after assessing rna quality and quantity , individual total rna samples ( 2 m each ) , random ( n6 ) primers , and quantiscript reverse transcriptase ( qiagen , tokyo , japan ) quantitative real - time polymerase chain reaction ( rt - pcr ) analysis was performed using an abi 7500 fast real - time pcr system ( applied biosystems , foster city , ca , usa ) using the taqman gene expression probes ( applied biosystems ) hs00229928_ml ( arhgap24 ) , hs00608366_m1 ( hdac5 ) , hs00211916_m1 ( pdgfc ) , hs01920617_s1 ( prnp ) , hs00984356_m1 ( slc6a4 ) , and hs99999905_m1 ( gapdh ) . gapdh was used as the reference gene , as in our previous study.6 all measurements were performed in duplicate . the ct method was used to determine relative expression of each gene in the subjects . the obtained value for each gene ( ctx ) was used to calculate the relative level of gene expression ( ctx ) by normalization to the reference gene , gapdh . for validation of our expression data , we used 10 samples from the current study and measured gapdh expression according to the same protocol , and we observed a high correlation between the two datasets ( r=0.986 , p=1.11e08 ) . expression data for 25 patients with mdd and 25 controls were obtained from our previous study.6 one - way analysis of variance ( anova ) was used to test for differences in age among the groups , and fisher s exact test was used to examine differences in sex distribution among the groups . wallis one - way anova and the bonferroni test was used for post hoc comparisons . a linear discriminant function was developed for discrimination between patients with mdd and without mdd , as in our previous study.6 a discrimination score ( d - score ) was calculated for each subject by multiplying the coefficients of the linear discriminants , obtained using the lda ( ) function of the mass package in r , to the standardized values of expression after z - score transformation of the five selected genes . the d - score indicates the probability of membership to the group of patients with mdd or the group of subjects without mdd ( schizophrenia or bipolar disorder or controls ) . the highest membership probability for each subject determined their classification into the mdd group or the other groups . a d - score was calculated for each sample as follows : d - score=0.73980239pdgfc+0.84889084prnp0.42559503arhgap240.07806493slc6a4 + 1.48110014hdac5 + 0.005 . in this plot , as an aid in understanding the discriminant , to set a linear decision boundary at the zero level , the value of the constant term was adjusted . any subject patients with mdd , and those with a d - score < 0 was identified as there were no significant differences among the groups in terms of sex distribution , while there were significant differences in age among the groups . we measured mrna expression levels of five genes ( arhgap24 , hdac5 , pdgfc , prnp , and slc6a4 ) using quantitative rt - pcr . the expression levels of these five genes were not associated with sex or age in multivariable analyses . all of the aforementioned five genes had similar changes in expression in mdd , schizophrenia , and bipolar disorder compared to controls and significantly changed among the groups . compared to controls , significantly increased mrna levels of the arhgap24 gene were observed in schizophrenia and bipolar disorder ( p=8.510 , 0.017 , respectively ) . compared to controls , significantly increased mrna levels of the hdac5 gene were observed in schizophrenia and bipolar disorder ( p=4.910 , 1.610 , respectively ) . significantly increased mrna levels of the pdgfc gene were observed in mdd , schizophrenia , and bipolar disorder compared to controls ( p=0.015 , 5.810 , 0.042 , respectively ) . although mrna levels of the prnp gene significantly changed among groups , the significant differences in the comparison between each patient group and control group were not observed . significantly increased mrna levels of the slc6a4 gene were seen in mdd , schizophrenia , and bipolar disorder compared to controls ( p=0.0238 , 4.910 , 0.003 , respectively ) . the index in figure 1 indicates the order of the samples sorted by their d - scores . of the 25 patients with mdd , 16 had an mdd - positive d - score test . of the other 81 subjects ( 17 with schizophrenia , 39 with bipolar disorder , and 25 controls ) , 52 had an mdd - negative d - score . we were thus able to segregate the patients with mdd from those without mdd with a sensitivity of 64% and specificity of 67.9% . the discriminating capacities of each single gene and a combination of five genes are shown in tables s1 and s2 . we recently developed a multiplex expression - based biological test for mdd , which demonstrated good sensitivity , and specificity exceeding 80% in a cohort comprising patients with mdd and nonpsychiatric controls.6 in the preset study , we examined the diagnostic performance of this panel in a cohort , including subjects with other psychiatric disorders , and demonstrated moderate diagnostic performance with a sensitivity and specificity of 64% and 67.9% , respectively , in differentiating patients with mdd from those without mdd . multiple biomarkers can serve better than a single marker in reducing the impact of the variation between populations and subgroups.9 several blood - based multiplex diagnostic biomarkers for mdd have been reported . spijker et al10 demonstrated that 7 lipopolysaccharide stimulation - based gene expression markers discriminated 16 patients with mdd from 13 controls with a sensitivity and specificity of 87.5% and 61.5% , respectively . this result was replicated in an independent set of samples with a sensitivity and specificity of 76.9% and 71.4% , respectively ( 13 patients with mdd and 14 controls).10 numata et al11 demonstrated that the top 18 dna methylation markers of peripheral leukocytes distinguished 20 patients with mdd from 19 controls . this result was replicated in an independent set of samples with high accuracy ( 12 patients with mdd and 12 controls).11 zheng et al12 demonstrated that 17 plasma nuclear magnetic resonance - based markers distinguished 58 patients with mdd from 42 control subjects . this result was replicated in an independent set of samples with a sensitivity and specificity of 92.8% and 83.3% , respectively ( 14 patients with mdd and 12 controls).12 the same group also demonstrated that 17 gas chromatography mass spectrometry - based markers of peripheral mononuclear cells discriminated 50 subjects with mdd from 50 controls , yielding an area under the curve ( auc ) of 0.93 . this result was replicated in an independent set of samples with an auc of 0.87 ( 58 patients with mdd and 56 controls).13 liu et al14 demonstrated that 4 plasma ultra - high - performance liquid chromatography equipped with quadrupole - time - of - flight mass spectrometry - based markers distinguished 60 patients with mdd from 59 control subjects with good sensitivity and specificity . this result was replicated in an independent set of samples with an auc exceeding 0.8 ( 75 patients with mdd and 52 controls).14 papakostas et al15 demonstrated that 9 serum protein - based markers , which were measured using elisa , distinguished 36 patients with mdd from 43 nondepressed subjects with high accuracy . this result was replicated in an independent set of samples with a sensitivity and specificity of 91.1% and 81% , respectively ( 34 patients with mdd and 43 controls).15 furthermore , the authors later conducted a test using an improved model and confirmed their results in a larger independent sample set with an auc of 0.96.9 these results suggest that multiple blood biomarkers can be useful tools for diagnosing mdd . however , previous biological tests for mdd have examined whether biomarkers can distinguish individuals with mdd from nonpsychiatric controls . to our knowledge , this is the first study to evaluate the utility of a biological diagnostic test for mdd in the condition where not only controls , but also subjects with other psychiatric disorders are included in the analysis . as expected , we found that the sensitivity of the test in discriminating patients with mdd from those without mdd , including those with schizophrenia and bipolar disorder , was lower in the present study compared to its sensitivity in discriminating between patients with mdd and controls in our previous study . on the other hand , the best aucs of mdd , schizophrenia , and bipolar disorder in the combined dataset were 0.87 , 0.97 , and 0.84 , suggesting that this expression panel was not specific to mdd . further research to identify mdd - specific markers is needed to improve the performance of this biological test.16 our expression panel consisted of five genes , arhgap24 , hdac5 , pdgfc , prnp , and slc6a4 . altered expressions of the arhgap24 , pdgfc , and prnp genes in mdd were originally discovered using cdna microarray in our previous studies.17,18 the arhgap24 gene encodes a gtpase - activating protein , and dysregulation of this gene inhibits axon and dendrite outgrowth and branching.19 pdgfc is a member of the platelet - derived growth factor ( pdgf ) family and is critical for neuronal survival in the central nervous system.20 pdgf is involved in disrupted - in - schizophrenia-1 expression,21 gamma aminobutyric acid dysfunction,22 and white matter integrity in bipolar disorder.23 the prnp gene encodes the prion protein , which has a protective role in several neurological conditions and affects n - methyl - d - aspartate receptor function in hippocampal neurons.24 mice with disruptions of this gene show depressive - like behavior.25 in addition , cellular prion protein - positive glial cells have been shown to be significantly reduced in the cingulate gyrus of patients with bipolar disorder or mdd.26 hdac5 is a member of the histone deacetylase family , which remove acetyl groups from histones to produce a less - accessible chromatin structure . chronic administration of imipramine reversed downregulation of brain - derived neurotrophic factor ( bdnf ) expression and increased histone acetylation at the bdnf gene promoters in social defeat stress mice , which was associated with a selective downregulation of hdac5 expression.27 previous reports have indicated increased hdac5 expression in leukocytes of patients with mdd.28,29 furthermore , hdac5 expression was also associated with the treatment efficacy or clinical improvement in mdd.30 the slc6a4 gene is one of the most investigated genes in relation to mdd . 5httlpr polymorphisms are candidate genetic polymorphisms for development and interactions between gene and environment of mdd.31,32 several studies have consistently demonstrated increased expressions of the slc6a4 gene in leukocytes of mdd3335 and schizophrenia.36 in pet imaging studies , slc6a4-binding potential has been shown to be elevated in subjects with mdd who have more severe symptoms.37 there are several limitations to the present study . first , our sample size was not large , and we did not confirm our results in an independent cohort . in addition , we did not evaluate the mood status of these patients with bipolar disorder as well as that of the patients with schizophrenia . third , we did not take physical conditions , such as body mass index and smoking status , into consideration , due to lack of information . fourth , we combined our previous expression data of subjects with mdd and controls with the present expression data of schizophrenia and bipolar disorder although we conducted experiments according to the same protocol . further studies will be needed in other ethnic cohorts . finally , we measured relative mrna expression levels . an absolute quantitation across laboratories will be needed to develop a common and reliable biological test.38 we were able to differentiate patients with mdd from subjects without mdd , including those with schizophrenia and bipolar disorder , with moderate sensitivity and specificity using gene expression changes in our panel . as we developed this panel based on mdd candidate genes , further research to identify mdd - specific markers will be needed to improve the performance of this biological test . the discriminating capacities of each single gene abbreviations : auc , area under the curve ; ci , confidence interval ; min , minimum ; max , maximum . the discriminating capacities of each combination of five genes

purposerecently , we could distinguished patients with major depressive disorder ( mdd ) from nonpsychiatric controls with high accuracy using a panel of five gene expression markers ( arhgap24 , hdac5 , pdgfc , prnp , and slc6a4 ) in leukocyte . in the present study , we examined whether this biological test is able to discriminate patients with mdd from those without mdd , including those with schizophrenia and bipolar disorder.patients and methodswe measured messenger ribonucleic acid expression levels of the aforementioned five genes in peripheral leukocytes in 17 patients with schizophrenia and 36 patients with bipolar disorder using quantitative real - time polymerase chain reaction ( pcr ) , and we combined these expression data with our previous expression data of 25 patients with mdd and 25 controls . subsequently , a linear discriminant function was developed for use in discriminating between patients with mdd and without mdd.resultsthis expression panel was able to segregate patients with mdd from those without mdd with a sensitivity and specificity of 64% and 67.9% , respectively.conclusionfurther research to identify mdd - specific markers is needed to improve the performance of this biological test .

reagents were obtained from sigma chemical ( st . louis , mo , usa ) unless otherwise specified . the protocol was approved by the ethics committee for experimentation with animals of the akita prefectural experiment station . oocytes were collected from follicles ( 25 mm in diameter ) of bovine ovaries collected at a meat processing plant by aspirating follicular fluid containing cumulus oocyte complexes ( cocs ) with a 5-ml syringe equipped with an 18 g needle . the ivm medium was 25 mm hepes - buffered tcm-199 ( tcm-199 ; gibco , thermo fisher scientific , waltham , ma , usa ) supplemented with 0.4% ( w / v ) bovine serum albumin ( bsa , fraction v ) , 100 m cysteamine , 50 ng / ml epidermal growth factor , 100 ng / ml transforming growth factor , 0.05 iu / ml human menopausal gonadotropin ( serono laboratories , tokyo , japan ) and 10 g / ml gentamicin ( gibco ) . the cocs with two or more layers of adhered cumulus cells were washed three times with ivm medium . they were then placed in ivm medium covered with paraffin oil at a ratio of 5 l / cocs ( 1520 cocs / culture drop ) using repro c-1 plates ( research institute for the functional peptides , yamagata , japan ) . they were incubated for 20 h under gaseous - phase conditions of 38.5c , 5% co2 , 95% air and saturated humidity . frozen semen from one japanese black bull was thawed in hot water heated at 38c and was washed twice with the brackett and oliphant ( bo ) solution containing 10 mm caffeine ( caff - bo solution ) by centrifugation ( 500 g for 5 min ) . the caff - bo solution was then added to achieve a final sperm concentration of 1 10/ml . then the same volume of bo solution supplemented with 20 mg / ml bsa ( fraction v ) ( bsa - bo solution ) and 10 iu / ml heparin ( novo - heparin ; mochida pharmaceutical , tokyo , japan ) was added to achieve a final sperm concentration of 5 10/ml . after ivm of cocs , oocytes with adhered cumulus cells were washed three times with the bsa - bo solution . they were then transferred into ivf medium covered with paraffin oil at a ratio of 5 l / oocyte using repro c-1 plates and were incubated under gaseous - phase conditions of 38.5c , 5% co2 , 95% air and saturated humidity . after 6 h of ivf , cumulus cells and spermatozoa were removed from the surface of the zona pellucida by gentle pipetting with a fine glass pipette . the putative zygotes were then washed three times in sof1 medium , which was sofaa medium supplemented with 3 mg / ml bsa ( fraction v ) , 10 l / ml insulin - transferrin - selenium ( gibco ) , 1 ng / ml transforming growth factor 1 and 10 ng / ml fibroblast growth factor . twenty zygotes per repro c-1 plate were placed in 100 l of sof1 medium covered with paraffin oil and cultured with or without 0.1 m dmg at 38.5c under gaseous conditions of 5% co2 , 5% o2 and 90% n2 or 5% co2 , 95% air and saturated humidity . after 96 h of culture , the embryos were then transferred to sof2 medium , which was sof1 medium supplemented with 1.5 mm glucose and 5% ( v / v ) fetal bovine serum in place of bsa , and cultured with or without 0.1 m dmg under the same gaseous conditions . we changed the ivc medium based on a report by matsumoto et al . and performed ivc using the culture media supplemented with glucose thereafter because it was shown that the addition of glucose to the ivc medium immediately after ivf until 96 h of ivc is detrimental to bovine embryo development , although it is necessary later for embryo hatching . embryos were treated with 0.2% triton x-100-pbs supplemented with 0.1 mg / ml propidium iodide for 1 min . they were then transferred into 99.5% ethanol supplemented with 25 g / ml bis - benzimide ( 33342 ; hoechst ) . they were then washed with vectashield ( vector laboratories , burlingame , ca , usa ) . the treatment solution containing embryos observations were conducted using a fluorescence microscope ( imt-2 , olympus , tokyo , japan ) . the inner cell mass ( blue ) and trophectoderm ( pink ) cells were counted . in one experiment , embryos at the 8-cell to 16-cell stage were used to examine the antioxidant effect of dmg . the test was conducted with three groups : a group cultured with h2o2 ( 0.5 mm ) , a group cultured with h2o2 ( 0.5 mm ) and dmg ( 0.1 m ) and a control group cultured without h2o2 and dmg . the embryos were cultured in sof1 medium and then in sof2 medium until day 8 ( day 0 = the day of ivf ) , and the development rate was examined . after all percentage data were subjected to arcsine transformation , significance was tested using a one - way analysis of variance ( anova ) and the tukey - kramer test . differences with p

the antioxidant effect of n , n - dimethylglycine ( dmg ) on in vitro - produced ( ivp ) bovine embryos was examined . after in vitro fertilization , presumptive zygotes were cultured with or without 0.1 m dmg under different oxygen tensions . the percentage of embryos developing to the blastocyst stage was lowest under a 20% oxygen concentration without dmg , and it was significantly increased ( p < 0.05 ) by applying a 5% oxygen concentration . under the 20% oxygen concentration , supplementation of the medium with dmg significantly improved blastocyst development , which was nearly equal to that achieved under 5% oxygen without dmg . furthermore , a tendentious increase ( p = 0.06 ) in blastocyst cell numbers was observed when dmg was applied . in the second experiment , addition of h2o2 ( 0.5 mm ) to the culture medium significantly ( p < 0.01 ) reduced the percentage of embryos developing to the blastocyst stage . however , dmg supplementation prevented this reduction . in conclusion , dmg enhanced the in vitro development of ivp bovine embryos by acting as an antioxidant .

an important task in molecular biology is the accurate classification of biological specimens on the base of their gene expression profiles . methods for accurate and reproducible classification are increasingly required for the potential biomedical applications of expression profiles , such as disease classification , diagnosis and monitoring . in this paper we describe scudo , a web server implementing an original signature identification method . given a set of expression profiles from control and affected subjects , scudo verifies the existence of a signature ( actually , a set of signatures , as explained below ) that can be used to classify the profiles according to a phenotype of interest , such as the control / affected status . the method is composed of a sequence of simple processing steps , none of which is original ; however their combination results in a robust and novel model of profile data analysis , that corresponds to a different way of exploiting similarities and differences among biological samples for classification purposes . specifically , the originality of the method is that it introduces a new concept of subject - specific signatures , as opposed to disease - specific signatures , that also includes the benefits of rank - based classification , data dimensionality reduction and the power of network analysis in its design . our method was tested against a selection of the best current approaches in the course of the sbv improver diagnostic signature challenge , an open competition designed to assess and verify computational approaches for classifying clinical samples based on gene expression ( 1 ) . in an effort to increase robustness of the outcome with respect to chance and disease specificity , the sbv challenge required a diagnosis for all of four diseases , namely psoriasis , multiple sclerosis ( ms ) , chronic obstructive pulmonary disease ( copd ) and lung cancer . for each disease , the organizers ( a team of researchers from ibm research and philip morris international ) suggested a list of publicly available mrna expression datasets to use as possible training sets and then solicited a healthy / affected diagnosis for a previously unpublished set of profiles ( test set ) . the submitted predictions were scored by the organizers according to a combination of three previously selected metrics : the area under the precision - recall curve ( aupr ) , the belief confusion metric ( bcm ) and the correct class enrichment metric ( ccem ) . the method implemented in scudo ranked second overall , and first in the ms sub - challenge , out of 54 submissions . interestingly , the ms diagnostic appeared to be the most difficult of the four , according to the plots of the distribution of random versus observed scores prepared by the organizers . the overall first placement was achieved by the winning team through a skillful application of linear discriminant analysis ( lda ) ; the third place was obtained by a team that applied lasso regression ( 2 ) . ( see ( 3 ) and ( 4 ) , respectively , for the details of the winning methods and of the scoring algorithm and score distribution plots . ) the method on which scudo is based represents a completely new way of addressing the expression profile classification problem . contrary to current practices , instead of evaluating a set of profiles by means of a common yardstick ( a single list of highly discriminating mrna / mirna species ) , our method first seeks to summarize the characteristics of each profile by means of a rank - based signature and then it performs a systematic , all - to - all signature comparison . the comparison consists in the measurement of a distance that quantifies the degree of similarity between any two signatures using a simplified version of the enrichment score ( es ) ( 5 ) . the result of the comparison is a distance matrix , which is then visualized in the form of a map of individual signatures . in such a map , the spontaneous emergence of groups ( i.e. clusters of closely connected nodes ) of transcriptionally similar subjects is typically observed . in a diagnostic application , a diagnostic classification for an unseen individual can subsequently be performed by determining the position of the individual 's transcriptional signature relative to the control / affected groups . the map is in the form of a graph where the nodes correspond to signatures / subjects and the length of a connecting edge encodes the level of similarity between the connected nodes ( short edge = high similarity ; no edge = negligible similarity ) . scudo accepts two separate datasets as input , respectively a testing and a training dataset , but it can also be used with a single ( training ) dataset . if a testing set is specified , scudo runs the same rank - based classification algorithm on it , but it skips the feature selection step , using instead the list of features ( genes or mirnas ) already selected for the training dataset . the optimization of the signature length is left to the user and is performed by trial and error ; in practice the user needs to try and select the n1 and n2 values that provide the clearest separation between the affected and the control subjects ( n1 = number of up - regulated genes to consider , n2 = number of down - regulated genes , n1 + n2 being the total signature length ) . crucially , we have shown that our method is robust to variations of its input parameter values , therefore the selection process is not critical and satisfactory values can be found with only a few iterations ( 6 ) . the expected result is the emergence of a partitioning of the set of samples in separate , clearly visible groups on the basis of signature similarity . while for the training set the sample classification is known in advance and thus the correctness of the partitioning is immediately verified by the appearance of homogeneously colored groups , for the test set in general the sample classification is the desired final output and needs to be deduced from the graph . the classification problem for the test set is then reduced to the simpler task of identifying the control / affected phenotype associated to each of the emerging groups in the test graph , in what we call the labeling step . this relatively simple labeling step can be carried out by means of empirical methods that rely on some form of previous knowledge . one form of previous knowledge consists of a small number of samples for which the classification is known ; by adding these samples to the test set , one can deduce the labeling of the groups based on the membership of the known samples . another form of previous knowledge is represented by a list of literature - derived disease genes ; by computing the average expression values of these genes across all the members of a group , one can deduce the labeling of the groups by comparing averages and taking a majority vote . we used both these methods for the labeling of samples in the sbv improver competition . our rank - based signature method is quite general and we have tested it successfully with a large assortment of data types : mrna and mirna profiles for various diseases / treatments / experiments , obtained from biopsies , blood and urine and even some non - biological multidimensional datasets ( i.e. engineering data ) . with scudo we are making available an online implementation of our new method to encourage experimentation and discovery of novel applications . scudo accepts a testing and a training dataset as input ( figure 1 ) , but it can also be used with a single ( training ) dataset , in case the user is not interested in the testing step . if a testing set is specified , scudo produces a map of the profiles using the same list of features ( genes or mirnas ) that were used in producing the training profiles map . in a sense , the testing set is used to validate the quality of the feature list , obtained examining the training set , as a starting point in producing discriminating signatures for a different set of profiles . the input data fields for the training set are on the left , the ones for the testing set on the right . a microarray expression dataset to be analyzed needs to have been previously normalized ; the choice of the algorithm is not critical ( i.e. either mas5 or rma can be used for affymetrix platforms ; either calibrated or uncalibrated data can be used for rt - pcr profiles ) , the data does not need to be log transformed . the data must be in a tab - separated text format , with one gene or probeset per row , and one sample ( i.e. a patient profile ) per column ; the testing and training datasets must have the same number of rows and the same gene / probeset on corresponding rows . the first column of the file is reserved for a gene / probeset identifier while the first row of the file is used for sample identifiers . both set of identifiers must be unique character strings without spaces ; the leftmost field of the first row , corresponding to the probeset i d column , needs to be filled with a placeholder character string . assuming that the user is using both a training and a testing dataset , the typical analysis workflow is as follows : upload the two data files , specify the algorithm parameters , launch the analysis , examine the two resulting maps ( one for the training profiles , the other for the testing profiles ) . after inspecting the maps , the user can repeat the same process with different parameter values , for example with a different signature size . the input parameters to be specified on the start page are : column ranges corresponding to the two classes of profiles ( i.e. control , affected ) in the training datasetn1 = number of genes from top ranked genes to include in signaturen2 = number of genes from bottom ranked genes to include in signaturepval = p - value to be used for the initial feature selectionn = percentage of all of the signature - to - signature distances that will be used for drawing the map of subjects . column ranges corresponding to the two classes of profiles ( i.e. control , affected ) in the training dataset n1 = number of genes from top ranked genes to include in signature n2 = number of genes from bottom ranked genes to include in signature pval = p - value to be used for the initial feature selection n = percentage of all of the signature - to - signature distances that will be used for drawing the map of subjects . the user has the option to use the same file for the training and testing dataset , presumably selecting different ranges of columns as the training and the testing sets . the column ranges specified by the user for the testing set are also used to assign different colors to the two groups of samples . only the specified columns for both sets are used for the analysis . in the current version of scudo , the optimization of input parameters such as the signature length and the p - value of the feature selection step is left to the user and is performed by trial and error . in a typical session , the user needs to try and select the n1 and n2 values that provide the clearest separation between the affected and the control subjects in the output maps , starting with the suggested value of p - value ( 0.01 for mrna , 0.1 for mirna profiles ) . based on our experience with a large number of representative datasets , good starting values for n1 and n2 are 250/250 for mrna , 25/25 for mirna and these values can be repeatedly doubled or halved until good separation is achieved ; if unsuccessful , a more stringent p - value can be tried . the p - value is not critical because it does not represent a threshold for inclusion of crucial features in a signature ; rather , the main purpose of the initial feature selection step is to prevent pollution of signature composition by non - informative features ( i.e. to remove the probesets / genes that are stuck high or low ) . the value of n is finally adjusted to improve clarity of the map by either reducing the clutter ( smaller n ) or increasing the number of connecting edges ( larger n ) as needed . the output of the tool is a map for each of the input dataset ( see figure 2 ) . the nodes in the map correspond to subjects and the length of a connecting edge encodes the level of similarity between the connected nodes ( short edge = high similarity ; no edge = negligible similarity ) . the map is in the form of a network drawn using cytoscape web , which allows manipulation and setting of display attributes . the desired outcome of the analysis is a map of the testing set in which the profiles are clearly divided in two groups composed of nodes of the same color ; such result would give high confidence in the fact that using the selected feature list the user will be able to satisfactorily classify a new set of profiles using signatures composed of the n1 + n2 most differentially expressed genes of each profile . snapshot of the scudo result page ; the map for the training data is on the left , the one for the testing data on the right . once the user is satisfied with the result , he / she can download the maps as graphical files , or as network files ( edge list ) ready to be imported in cytoscape ( 7 ) or similar tools . a cytoscape plugin such as clustermaker ( 8) can be useful in identifying clusters in those maps in which the boundaries between groups are not immediately apparent ( as in the right map of figure 2 ) ; see reference ( 6 ) for an example of use of such plugin . the user can also download a file containing the subject specific signatures ( one column per subject ) plus two consensus signatures the consensus signature for each group is computed as follows : the rank of each gene in a profile is computed separately for each subject by sorting expression values from the most expressed to the least expressed , then the columns of gene ranks corresponding only to the subjects included in the group are summed row - wise and the resulting rank sums are sorted to provide a new set of consensus ranks ; the signature for the group is then composed by the first n1 and the last n2 gene ids ordered according to the new consensus rank list . the consensus signature is reminiscent of a disease gene list although derived using our unconventional approach and can be used to study the underlying molecular mechanism of a disease ( for an instance of such an application see ( 9 ) ) . a sample set of expression profiles and corresponding suggested parameter values are available in the initial page . the two datasets comprise profiles of circulating mirna in patient with early breast cancer and matching controls published by two different research groups ( geo accession numbers gse22981 and gse41526 , including 40 and 70 profiles respectively ) . gse41256 was used as the training set and gse22981 as the testing set , because the larger size of the former improves the performance of the feature selection step . the two datasets were used as downloaded from geo ( gse series matrix format ) except for the required changes in the formatting ; also , in the first set we had to remove six rows corresponding to mirna species that were missing from the other set ( both set use the same platform , illumina human v2 microrna expression beadchip ) . the recommended signatures sizes are 20/20 for both sets ; the recommended p - value of 0.001 , empirically found to work best , is uncharacteristically small for scudo , probably reflecting the existence of high noise levels or confounding signals in the two datasets . in the two resulting maps ( figure 2 ) it can be observed that ( i ) in the training set map , scudo is able to group together control and affected patients with good accuracy and ( ii ) in the testing set , scudo is able to separate control and affected subjects reasonably well as long as the analysis is restricted to the subset of caucasian american individuals . these results are of particular interest in view of the fact that the authors of the first set were not able to identify a reproducible diagnostic signature ( 10 ) and that the authors of the second set reported differences in terms of differentially expressed circulating mirnas between caucasian american and african american subjects ( 11 ) . scudo implements a new expression profile classification based on the degree of similarity between profile - specific signatures . as a consequence of being rank - based , scudo is quite robust to differences in lab protocols , data processing and batch effects , because it relies only on the relative ordering of the gene expression values within each profile , and not on the values themselves ( for more details on this point see ( 6 ) ) . this robustness can be appreciated in the analysis of the provided sample data files , which are from the same affymetrix platform but have been produced by different research groups and have undergone different normalizations . additionally , we have shown that our method is robust to wide variations of its parameter values therefore the choice of the values is not overly critical . our rank - based signature method is quite general and we have tested it successfully with a large assortment of data types : mrna and mirna profiles for various diseases / treatments / experiments , obtained from biopsies , blood and urine , and even some non - biological multidimensional datasets ( i.e. engineering data ) . using publicly available data , we have obtained promising results on the stratification of patients with respect to treatment response ( 12 ) and early diagnosis of cancer based on blood mirna ( 13 ) ; we have preliminary results for sample classification in toxicity studies . the reasons for this generality are that being completely data driven , our method is agnostic about the details of the mechanisms producing the transcriptional response . by making available an online implementation of our new method we want to encourage experimentation with new data types and discovery of novel applications . scudo has been used as part of a class project by the students of a graduate course held at the university of trento during the spring 2014 semester , whose feedback we gratefully acknowledge . funding for open access charge : the microsoft research university of trento centre for computational and systems biology ( cosbi ) .

scudo ( signature - based clustering for diagnostic purposes ) is an online tool for the analysis of gene expression profiles for diagnostic and classification purposes . the tool is based on a new method for the clustering of profiles based on a subject - specific , as opposed to disease - specific , signature . our approach relies on construction of a reference map of transcriptional signatures , from both healthy and affected subjects , derived from their respective mrna or mirna profiles . a diagnosis for a new individual can then be performed by determining the position of the individual 's transcriptional signature on the map . the diagnostic power of our method has been convincingly demonstrated in an open scientific competition ( sbv improver diagnostic signature challenge ) , scoring second place overall and first place in one of the sub - challenges .

the authors declare that the work described has not been published previously , that it is not under consideration for publication elsewhere , that its publication is approved by all authors and tacitly or explicitly by the responsible authorities where the work was carried out , and that if accepted , it will not be published elsewhere including electronically in the same form , in english or in any other language , without the written consent of the copyright - holder . obinna obinwa , caitriona canning and martin odohonoe acquired the data for publication , drafted the article and revised it critically for important intellectual content .

highlightsa case of a 78-year - old female with bilateral prevascular groin herniae following an emergency aorto - uniiliac evar and femoro - femoral bypass for a ruptured abdominal aortic aneurysm is presented.primary repair of the herniae was achieved using a preperitoneal approach.the case emphasises a safe approach for dealing with this rare complication .

skin wound healing in adult mammals is a dynamic process , involving inflammation , proliferation , and maturation or remodeling phases . inflammation is initiated first , with neutrophil accumulation in the wound area , followed by resident macrophages and circulating monocytes infiltrating the wound . proliferation is characterized by reepithelialization , angiogenesis , granulation tissues formation , and matrix deposition that inevitably leads to fibrotic scar tissue formation . remodeling is the accumulation and remodeling of collagen , which is largely regulated by fibroblasts and forms scars finally . these scars may cause problematic results , particularly when wounds across joints , in which they can impair the mobility and flexibility or affect conspicuous locations , which can result in devastating psychological consequences . in contrast to adult wound healing , fetal skin wounds healing without scars , to some extent , attributing to them do not mount an inflammatory response after injury until late in gestation . in another study , the postinjured skin of nude mice is characterized by lack of scar , since nude mice are immunodeficient . so control inflammation in the process is quite important for the final scarless healing . in view of the scarless healing of the early fetus for example , sufficient content of type iii collagen may prevent scar tissue formation , and excessive secretion of type i collagen may result in a disorganized fiber structure and hypertrophic scar formation . there are three highly homologous transforming growth factor- ( tgf- ) genes in mammals , tgf-1 , tgf-2 , and tgf-3 . in general , tgf-1 and tgf-2 are known to promote fibroplasia and scar , while tgf-3 is believed to reduce scar [ 911 ] and downregulate tgf-1 and tgf-2 . matrix metalloproteinases ( mmps ) constitute a family of zinc endopeptidases that are capable of degrading most of the structural components of the extracellular matrix ( ecm ) and are regulated by their tissue inhibitors ( timps ) . scarless wounds have a higher ratio of mmp to timp , favoring remodeling and less accumulation of collagen . in particular , tgf- can be secreted by neutrophils , lymphocytes , macrophages , and so on , which are critical in inflammatory response , and tgf- can promote ecm synthesis and inhibit matrix turnover through the regulation of mmps and timps as well . thus , the regeneration without scar through regulating the collagen , tgf- , mmp , and timp by controlling the inflammation desperately needs to be studied . for the treatment of skin lesions , several strategies are currently available , such as , topical application of complement c5 or microbial cellulose . these methods speed the healing process of wounded skin , but they neglected the scar formation during this process . it has been reported that basic fibroblast growth factor ( bfgf ) has a possible antiscarring effect . however , the bioactivity and bioavailability of bfgf applied in the wounded area are unwarrantable . electrospinning is a widely used technique for the production of nanofibers from various natural or synthetic polymers [ 20 , 21 ] . the electrospun fibrous scaffolds are three - dimensional ( 3d ) constructs , designed like ecm , to support cell attachment , proliferation , migration , and differentiation , promote cell secretion of bioactive molecule , and accelerate functionality formation of tissues or organs . and the high porosity and specific surface area of electrospun fibers enable them as the useful drug carriers for inhibiting diseased cells and tissue engineering scaffolds for cells growth , leading to the complete regeneration . therefore , drug - loaded electrospun fibrous scaffolds have good potential as drug carriers and cell growth scaffolds . consideration of the bacterial infection can cause the surrounding microenvironment to become acidic ; we designed a feasible acid - responsive controlled release systems as repairing tissue scaffolds , which are used to intelligently regulate the anti - inflammatory agent release with the change of acid microenvironment in regions where the ph is reduced below 7.4 , leading to a good restrain of inflammation on the early stage and a scarless reparation on later stage . the results may cast some highlight on the relationship between inflammation and scars , developing the novel drug - loaded biomaterials for scarless healing . poly(l - lactide ) ( plla , mw = 100 kda , mw / mn = 1.6 ) was purchased from sigma company . dichloromethane ( dcm ) , sodium bicarbonate ( sb , nahco3 ) , and n , n - dimethylformamide ( dmf ) were of reagent grade and were purchased from sinopharm chemical reagent co. , ltd . all other chemicals and solvents were of reagent grade or better and were purchased from guoyao regents company ( shanghai , china ) , unless otherwise indicated . 1.0 g plla was completely dissolved in 4 g dcm and 2 g dmf for electrospun plla fibers ( plla - ef ) . to prepare ibuprofen - loaded plla electrospun fibrous membranes ( i - plla - ef ) , 1 g plla and 40 mg ibu were completely dissolved in a mix - solvent containing 4 g dcm and 2 g dmf for electrospinning . for acid - responsive ibu - loaded electrospun fibers ( ar - i - plla - ef ) , 1 g plla and 40 mg ibu were dissolved in 4 g dcm and 2 g dmf ; then 0.12 ml nahco3 saturated water ( 96 mg / ml ) solution was added into the ibu / plla solution slowly with vigorous stirring for 30 min with magnetic stirrers . the blended solvent was then put in a syringe pump that was attached to the high voltage device . a negative electrode blanketed with aluminum foil environmental scanning electron microscopy ( esem , fei , quanta250 , the netherlands ) was used to investigate the morphology of the electrospun fibers . the electrospun fibrous scaffolds ( weight , 100 mg ) were simultaneously immersed in centrifuge tubes containing 25 ml phosphate - buffered solution ( pbs , ph = 7.4 ) or sodium acetate - acetic acid buffer solution ( ph = 5.0 ) . the samples were incubated at 37c for 48 h with mild shaking ( 100 rpm ) . at predetermined time intervals , 1 ml buffer solution was collected and replaced with 1 ml fresh buffer solution . the amount of released drug in the collected medium was determined by uv - vis spectroscopy at 224 nm . a standard calibration plot of ibu in the concentration range of 00.05 mg / ml was used to determine the concentration of the drug released . the percentage of the released drug was then calculated based on the initial weight of drug incorporated in the electrospun scaffolds . all the procedures followed the policies of shanghai jiao tong university school of medicine and the national institutes of health . all animals were from the animal breeding center , shanghai sixth people 's hospital affiliated to shanghai jiao tong university , school of medicine , china . male 8-week sprague - dawley rats ( n = 40 ) weighing 200250 g were housed one per cage with free access to food and water , in a room with controlled humidity and temperature ( 22c ) , on a 12 h light / dark cycle . the animals were divided into two groups ( 20 rats in each group ) and anesthetized using ketamine ( 1 ml / kg body weight ) . a circular full - thickness 2 cm diameter skin wound extending through the panniculus carnosus was aseptically made on the dorsum on each side of midline ( figure 1(a ) ) . in group one , the right lesion was untreated with any scaffolds ( control group ) and the left lesion was treated with electrospun fibrous scaffolds ( plla group ) . in group two , the left lesion was treated with ibuprofen - loaded electrospun fibrous scaffolds ( i - plla - ef group ) and the right lesion was treated with stable acid - responsive ibuprofen - loaded electrospun fibrous scaffolds ( ar - i - plla - ef group ) . the animals in each group were daily evaluated but euthanized on the 3 , 7 , 14 , and 21 days ( n skin tissue samples from the wound site were excised in full depth and bisected for morphologic observation and molecular biological analysis . in order to determine the wound healing rate , the open wounds were drawn on graph acetate paper with a marker pen on the 3rd , 7th , 14th , and 21st days and photographed with a digital camera . the surface area of wound was measured with a planimetric program on computer by scanning the acetate sheets . the rate of wound closure was calculated using the following formula : ( 1)wound closure rate = [ ( original wound areaopen area on final day)original wound area ] 100% . specimens were fixed in 10% formalin phosphate - buffered saline , dehydrated in ethanol , cleared in xylene , and in order arrangement embedded in paraffin . the above - treated specimens were then sectioned in 5 mm and stained with hematoxylin and eosin ( he ) for histological observation . further , masson 's trichrome stain was performed for observation of the collagen framework . as part of the histological evaluation , all slides were examined by a pathologist under a light microscope , without knowledge of the previous treatment . the immunohistochemical staining of bfgf and -smooth muscle actin ( -sma ) were conducted by respective antibody . sections were dewaxed with xylene and hydrated in decreasing concentrations of etoh ; endogenous peroxidase was blocked with 3% hydrogen peroxide for 10 min ; nonspecific binding was blocked with 1% bovine serum albumin ( bsa ) for 30 min . tissue sections were processed in 10 mm citrate buffer ( ph 6.0 ) and heated to 100c for 10 min for antigen retrieval . primaries were applied for bfgf ( 1 : 100 ) and -sma ( 1 : 100 ) overnight at 4c . biotinylated secondary antibodies were applied at 1 : 200 for 30 min . color development was performed with dab for 3 to 5 min for all samples , followed by haematoxylin counterstaining . to extract the whole protein fraction , frozen tissues were homogenized in the lysis buffer [ 50 mm tris ph 7.4 , 150 mm nacl , 1% triton - x 100 , 0.5% deoxycholic acid , 0.1% sds , and protease - inhibitor - cocktail ( 1 : 100 v / v ; sigma ) ] . after incubation for 30 min on ice , the samples were centrifuged for 15 min at 12,000 r.p.m . , at 4c , and the supernatant was transferred to a new tube as whole protein fraction . protein concentrations were determined using the bio - rad protein assay ( bio - rad laboratories , hercules , ca ) with bca ( thermo scientific , waltham , ma ) as standard . then 20 g of proteins of each sample was electrophoresed using 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis ( sds - page ) and then transferred to a polyvinylidene fluoride membrane ( millipore , bedford , ma ) which was blocked with 5% skim milk in tris - buffered saline- ( tbs- ) t ( 10 mm tris , 150 mm nacl , and 0.1% tween-20 ) for 1 hour at room temperature . the membrane was incubated with the primary antibody for 2 hours and was washed several times with tbs - t , and then it was incubated with a horseradish peroxidase conjugated secondary antibody for 1 hour . finally , the membrane was washed and developed with an enhanced chemiluminescence detection system ( millipore , bedford , ma ) . the primary antibody used in this study was anti--sma ( 1 : 500 ; proteintech , chicago , usa ) . gapdh was analyzed as internal control with anti - gapdh antibody ( 1 : 1500 ; thermo scientific , waltham , ma ) . after the skin wound was treated with different scaffolds or negative control for 14 days , total rna from the regenerate tissues was prepared using trizol reagent ( thermo scientific , waltham , ma ) according to the manufacturer 's instructions . the integrity of rna was verified by optical density absorption ratio od260 nm / od280 nm between 1.8 and 2.0 . cdna synthesis was conducted according to the rna pcr kit protocol ( thermo scientific , waltham , ma ) . quantitative polymerase chain reaction ( qpcr ) amplification was performed in triplicate , using the sybr green pcr reagent system . real - time quantitative pcr was carried out as follows : initial denaturation for 10 min at 95c and 40 cycles of pcr consisting of 15 sec at 95c and 45 sec at 60c . statistical significance was determined with the student 's t - test when there were two experimental groups . multiple comparisons between the groups were performed with one - way analysis of variance ( anova ) and post hoc tests ( sas 9.1 for windows ) . the key component of these drug carriers is sodium bicarbonate ( nahco3 ) , which can be incorporated into electrospun fibers by using coelectrospinning . as described by ke et al . , nahco3 reacted with acidic solution and the co2 quickly released out of the fibers when fibrous scaffolds were incubated into acidic solution , which accelerated drug release from the inner sections of the fibers . morphology of all electrospun fibrous scaffolds showed no beads in the fibrous structure , the fibers were uniform in size and randomly interconnected ( figure 1(d ) ) , and all samples were further tested by sem after incubation for 48 h at ph 5.0 ( figure 1(e ) ) . compared with the original formation ( figure 1(d ) ) , the 3d structure of electrospun fibrous scaffolds after incubation for 48 h at ph 5.0 maintained a 3d fibrous structure , even though fibers were swollen slightly compared with the original formation shown in figure 1(d ) . the in vitro release profiles of ibu - loaded electrospun fibrous scaffolds in ph 5.0 solutions were shown in figure 1(f ) . the total amount of released drugs was around 30.6% and 78.2% of i - plla - ef and ar - i - plla - ef after incubation for 48 h in ph 5.0 buffer solution , respectively . the release rate of the loaded ibu was accelerated with nahco3 in fibers in the acidic solution . wound size in all groups was recorded on days 0 , 3 , 7 , 14 , and 21 after operation and expressed as percentage of the initial wound area ( figure 2 ) . the residual wound area on the 7th day in control group was 73.11% and they were 58.52% , 42.25% , and 25.00% in plla group , i - plla - ef group , and ar - i - plla - ef group , respectively ( p < 0.05 ) . in addition , major differences happened on the 14th day after skin excision with the residual wound area being 51.84% , 39.06% , 16.00% , and 6.25% , respectively , in the same sequence as above ; these differences were statistically significant ( p < 0.05 ) . histologic analysis was performed to determine the cellular effects on wounds treated with different fibrous scaffolds . we found that the scaffolds integrated to the new tissues and showed degradation with time . they were not degraded on the 3rd day after operation but on the 21st day , and all types of the scaffolds implanted into the subcutaneous tissues had degraded and disappeared . as shown in figure 3 , three days after operation , the epidermis and dermis showed deficiency on the area of the wound , left subcutaneous tissues only . the inflammation was observed in every group in the subcutaneous tissues and appeared in the intact tissues around the wound as well . inflammatory cells were observed in dermis of the surrounding areas , and proliferation of fibroblasts and mild hemorrhage were seen . during this inflammatory phase of wound healing , the ibu - loaded groups showed a lighter degree of infiltration of inflammatory cells ; especially in ar - i - plla - ef group , the control of inflammatory reaction was better than i - plla - ef group . especially on the 7th day , in ar - i - plla - ef group , the surface of epidermis became even . after 21 days in ar - i - plla - ef group the defect had almost disappeared and the wound was filled with fibroproliferative tissue . the surface of the wounds was covered with new epithelium . masson 's trichrome stained histological sections of the wounded skin samples showed more collagen content ( blue staining ) in control group and plla group than the ibu - loaded two groups with time ( figure 4 ) . and on the 21st day after operation , the collagen content of ar - i - plla - ef group was less than other three groups . to evaluate bfgf expressions in situ ar - i - plla - ef group immunohistochemistry revealed significant staining of bfgf ( figure 5 ) . at postwounded day 3 , bfgf expression was present in four groups with the weak staining but with a relatively higher expression in ar - i - plla - ef group . on the 7th day after operation , the expression of bfgf was increased in all groups and it was still higher in ar - i - plla - ef group than others . the significant difference on the 14th day was that the expression of bfgf showed strong positive staining with large area in ar - i - plla - ef group . this superiority of ar - i - plla - ef group lasted to the 21st day after operation with a slightly higher expression than the 14th day . sections from the wound showed -sma expression in skin wounds after 14 days of the operation ( figure 6 ) . the positive staining of -sma in control group was the strongest and decreased in proper order successively in plla - ef group , i - plla - ef group , and ar - i - plla - ef group . the expression of -sma in ar - i - plla - ef group was less than other three groups . western blot analysis showed that levels of -sma expression decreased in ibu - loaded groups relative to the non - ibu - loaded groups ( figure 7 ) . the expression level of -sma in control group was the highest and decreased in plla - ef group , i - plla - ef group , and ar - i - plla - ef group , respectively . the expression of -sma in ar - i - plla - ef group was the least among four groups . on the 14th day , the relative expression of collagen i ( col - i ) and collagen iii ( col - iii ) of ar - i - plla - ef group was significantly different from the control group ( p < 0.05 ) as shown in figure 8(a ) . the expression of col - i of ar - i - plla - ef group was the lowest in the four groups with more than four times lower than control group , and the expression of col - iii of ar - i - plla - ef group was the lowest in the four groups as well , about two times lower than the control group . though the relative expressions of col - i and col - iii of ar - i - plla - ef group were both the lowest among the four groups , with a relative higher expression of col - iii than that of col - i , the ratio of col - i / col - iii of ar - i - plla - ef group was still lower than control group , almost two times lower as shown in figure 8(b ) ( p < 0.05 ) . interestingly , the relative expressions of col - i and col - iii of plla group were somewhat higher than control group , though there were no significant statistical differences between them , maybe owing to the stimulation to collagen deposition of plla scaffold without ibu . as shown in figure 8(c ) , the relative expression of tgf-1 and tgf-3 was increased from control group , plla group , and i - plla - ef group to ar - i - plla - ef group in turns , respectively . and there were significant statistical differences between control group and ar - i - plla - ef group , with the expression of tgf-1 of ar - i - plla - ef group being almost four times higher than control group and the expression of tgf-3 of ar - i - plla - ef group being even more than eight times higher than control group ( p < 0.05 ) . because of the relative higher expression of tgf-3 than tgf-1 , the ratio of tgf-1/tgf-3 of ar - i - plla - ef group was lower than control group as shown in figure 8(d ) ( p < 0.05 ) . from the control group , plla group , and i - plla - ef group to ar - i - plla - ef group , the relative expression of mmp-1 was increased and the relative expression of timp-1 was decreased as shown in figure 8(e ) . so the ratio of mmp-1/timp-1 was increased in turn as displayed in figure 8(f ) . the expression of mmp-1 of ar - i - plla - ef group was almost three times higher than control group and the expression of timp-1 of ar - i - plla - ef group was more than two times lower than control group ( p < 0.05 ) . and the ratio of mmp-1/timp-1 of ar - i - plla - ef group was more than six times higher than control group ( p < 0.05 ) . ideal wound treatments should accelerate healing and reduce scar complications . however , scarless healing is difficult in adult mammalian tissues . accordingly , increasing attention has been directed to screening and developing products that are effective in both accelerating wound healing and preventing scars . in some pathological conditions such as malignant tumor , infection , or inflammation one research put forward evidence that during the early fermentation period the bacterial abundance increased quickly and the ph values concurrently decreased rapidly without any initial ph increase . another study also displayed that , after controlling the infection of bacteria , the ph of or around the lesion area increased . and the alkaline ph can increase both bacteriostatic and bactericidal activities of both planktonic and biofilm - associated bacteria . therefore , the microenvironment around bacteria is inclined to acid , which is in favor of the growth and proliferation of bacteria . because of excessive inflammation , which resulted in inappropriate deposition of extracellular matrix which can lead to overgrowth , hardening , and/or scarring of tissues , we structured the acid - responsive ibuprofen - loaded plla fibrous scaffolds to control the excessive inflammation , hoping to achieve complete regeneration without scar . in our histochemistry studies , ibuprofen effectively controlled the excessive inflammation in the early stage of the healing process , and the inflammation of ar - i - plla - ef group was even lighter than i - plla - ef group , which because of the accelerating release of ibuprofen resulted from the nahco3 . besides , ibuprofen can block the biosynthesis of inflammatory prostaglandins , especially prostaglandin e2 [ 32 , 33 ] . therefore , ibuprofen can alleviate postoperative pain and itch to some extent as well , which may benefit the peaceful healing process , owing to the reduction of the scratching frequency , especially for children . except that fetal skin wounds healing does not mount an inflammatory response after injury , fetal wounds differ from adult wounds also in extracellular matrix ( ecm ) components , growth factor expression and cellular differences , and so forth . our masson 's trichrome stained histological sections showed more collagen deposition in control group and plla group than the ibu - loaded two groups with time . further through qrt - pcr analysis , we found that the relative expression of col - i and col - iii of ar - i - plla - ef group was significantly different from the control group ( p < 0.05 ) as shown in figure 8(a ) ; the expressions of col - i and col - iii of ar - i - plla - ef group were both the lowest in the four groups , while , with the relative higher expression of col - iii than that of col - i , the ratio of col - i / col - iii of ar - i - plla - ef group was still lower than control group as shown in figure 8(b ) ( p < 0.05 ) . bfgf , which is a potent mitogen and chemoattractant for endothelial cells , fibroblasts , and keratinocyte can stimulate the metabolism , growth of the ecm , and the movement of mesodermally derived cells . in our immunohistochemical examination , the staining for bfgf of ar - i - plla - ef group was stronger and the area of staining was larger than other groups as shown in figure 5 . this is in line with the study of shi et al . where the bfgf had antiscar effects on wound repair in vitro and in vivo , though other studies thought that decreased fgf activity is associated with scarless wound healing . in addition , -sma is critical for wound contraction and plays a major role in fibrosis as well , as it sustained the myofibroblasts to form the granulation tissue , which inevitably leads to fibrotic scar tissue formation . from our immunohistochemical examination and western blot analysis , the expression level of -sma of ar - i - plla - ef group was the lowest among four groups ; the potential of forming scars of ar - i - plla - ef group , to some degree , was less than other groups . there were lots of studies focused on tgf- and the expression ratio between tgf-1/2 and tgf-3 during the scarless healing [ 8 , 37 ] . and further studies found that , when tgf-3(/ ) fetal mice wounded , the wounds healed with scarring , and , to the contrary , when tgf-3 was administered in adult wounds , the scars were reduced [ 38 , 39 ] . as fetal skin possesses the ability to regenerate completely , adults with scarless healing may need higher ratios of tgf-3 to tgf-1/2 , and our results are consistent with the above studies . other important molecules in complete healing were mmps and timps . like other studies which implied that there were high levels of mmps expression in dermal fibroblasts from nude mice and scarless wounds have a higher ratio of mmp to timp , which may be due to the decreased expression of tgf-1 , as it decreases mmp and increases timp expression , favoring collagen accumulation and scarring , our studies showed that , from the control group , plla group , and i - plla - ef group to ar - i - plla - ef group , the relative expression of mmp-1 was increased and the relative expression of timp-1 was decreased as shown in figure 8(e ) ; the ratio of mmp-1/timp-1 was increased in turn as displayed in figure 8(f ) . a novel acid - responsive ibuprofen - loaded electrospun fibrous scaffold was easily fabricated based on nahco3 for antiscar skin healing . in general , the results of this study demonstrate that the acid - responsive ibuprofen - loaded electrospun fibrous scaffolds can prevent excessive inflammation in the early stage of the wound healing as drug carriers and provide scaffolds for cells to regenerate the achievement of a complete and scarless healing in the later stage .

skin injury in adult mammals brings about a series of events and inflammation in the wounded area is initiated first and provides lots of inflammatory factors , which is critical for the final scar formation . while the postinjured skin of fetus and nude mice heals scarlessly owing to the absence of inflammation or immunodeficient , we designed a feasible acid - responsive ibuprofen - loaded poly(l - lactide ) ( plla ) fibrous scaffolds via doping sodium bicarbonate to prevent excessive inflammation and achieve scarless healing finally . the morphological results of in vivo experiments revealed that animals treated with acid - responsive ibuprofen - loaded plla fibrous scaffolds exhibited alleviative inflammation , accelerated healing process , and regulated collagen deposition via interference in the collagen distribution , the -smooth muscle actin ( -sma ) , and the basic fibroblast growth factor ( bfgf ) expression . the lower ratios of collagen i / collagen iii and tgf-1/tgf-3 and higher ratio of matrix metalloproteinase-1 ( mmp-1)/tissue inhibitor of metalloproteinase-1 ( timp-1 ) in acid - responsive ibuprofen - loaded plla fibrous scaffolds group were confirmed by real - time qpcr as well . these results suggest that inhibiting the excessive inflammation will result in regular collagen distribution and appropriate ratio between the factors , which promote or suppress the scar formation , then decrease the scar area , and finally achieve the scarless healing .

adult muscle tissue is highly exposed to the damage by both internal and external factors throughout the life span and therefore requires the ability to effectively regenerate . the cells which are primarily responsible for skeletal muscle tissue restoration capacity are called satellite cells . the idea to deliver myoblasts which are satellite cells progeny into the dysfunctional muscles as a method of treatment was presented for the first time in 1978 by partridge and colleagues . since then myoblasts transfer therapy ( mtt ) has been a subject of extensive studies . at the beginning , myoblasts have been considered as a very promising alternative for treatment of muscular dystrophies ( mds ) ; however it became clear that this population is not able to engraft skeletal muscle after systemic delivery . the poor migratory capabilities hinder the potential use of myoblast - based therapy in duchenne md and narrow the possible applications to the disorders with more focal character . nevertheless , myoblasts transfer is still perceived as possible future alternative treatment in numerous conditions . the list includes sphincters dysfunction ( urethral , anal , esophageal , and pyloric ) , atrophy of reinnervated muscle , rectovaginal fistulas , local muscle loss due to injury , and some types of muscular dystrophies ( like oculopharyngeal or facioscapulohumeral mds ) . unfortunately , myoblasts transfer procedure is associated with another crucial unsolved problem , the poor survival rate of donor cells . it was demonstrated that the vast majority of graft is lost within first 3 days after transplantation . the massive elimination of injected myoblasts was identified in 1996 by fan and colleagues and subsequently confirmed by many other studies . this phenomenon was observed regardless of type of animal model ( murine or porcine ) , type of target area ( skeletal muscle , myocardium , or urethral sphincter ) , status of host muscle ( intact or injured ) , or status of the host organism ( immunodeficient or immunocompetent ) [ 49 ] . they include inflammatory response in the injection site , ischemia [ 10 , 11 ] , hypoxia , , and anoikis . the immune reaction after myoblasts injection has been studied intensively in allogeneic models [ 5 , 1417 ] but not after autologous transplantation as this type of transfer is believed to be nonimmunogenic . however , our previous study demonstrated that oxidative stress might play a role in elimination of myogenic cells after autologous transplantation . therefore , we hypothesized that autologous transfer of myogenic cells also triggers early immune response associated with oxidative burst . as autologous transplantation is considered to be the safest option in all clinical applications listed above , it seems to be of prime importance to evaluate the local tissue response for this kind of grafting . therefore , the aim of this study was to examine the presence and dynamics of cytokines expression and cells associated with innate immune reaction within first week after autologous mtt . all experiments were performed on 3-month - old lewis rats ( in - bred strain ) . animals were housed with free access to food and water and were maintained at a constant temperature . animal housing and experimental procedures were approved by local ethics animal welfare commission of the medical university of warsaw . skeletal muscle samples ( about 0.05 g ) for cell isolation were obtained from musculus gracilis during general anesthesia induced by administration of xylazine ( 10 mg / kg ; leciva , prague , czech republic ) , ketamine ( 40 mg / kg ; spofa , prague , czech republic ) , and butorphanol ( 1 mg / kg ; fort dodge animal health , fort dodge , ia , usa ) . isolation of muscle - derived cells was performed as described by burdziska et al . . the cells were suspended in standard growth medium ( gm ) , dmem supplemented with 10% ( v / v ) fetal bovine serum and antibiotic , antimycotic mixture ( all components purchased from invitrogen , carlsbad , ca , usa ) . in order to reduce number of fibroblasts in culture , the medium containing nonadherent cells was removed to another dish 24 h after cell seeding ( preplating ) . when the culture reached 70% of confluence , cells were harvested by trypsinization ( 0.25% trypsin and 0.02% edta ; invitrogen - gibco carlsbad , usa ) and reseeded in new dishes in a density of 5 10/cm . majority of cells were cultured for transplantation whereas part of population were seeded separately to perform in vitro characterization , desmin expression and differentiation potential analysis . to identify isolated cells , cells after the first passage were cultured in a lab - tek 4-chamber slide w / cover ( permanox slide sterile , nalge nunc international , naperville , il , usa ) until they reached 80% confluence ; then they were fixed in 4% ( w / v ) paraformaldehyde for 15 min at room temperature and permeabilized with 70% cold methanol for 20 min in 20c . samples were treated with blocking solution ( 1% bovine serum albumin/5% normal donkey serum in phosphate - buffered saline ) for 30 min in rt and then probed with mouse anti - desmin ( sigma - aldrich , st . louis , mo , usa , 1 : 50 v / v , 90 min , rt ) . afterwards , cells were washed and probed with a secondary antibody [ alexa - fluor 594 donkey anti - mouse ( jackson immunoresearch europe , suffolk , uk ) , 1 : 100 v / v , 60 min , rt ] . the other subsets of isolated cells were induced to differentiate by cultivation in dmem supplemented with 2% of horse serum ( hs ) for 3 days . the fusion index was determined as the ratio of nuclei in myotubes to the total number of nuclei in the same field calculated from at least 10 fields of view per animal and was expressed as a percentage ( 0% to 100% ) . the presence of intracellular lipid droplets in mdc population was confirmed with oil red o staining ( sigma - aldrich , st . louis , mo , usa ) . for injection procedure rats were sedated with xylazine / ketamine mixture . the skin in the area of injection was shaved and disinfected . in the transplanted animals , mdcs suspended in 200 cell suspension was given through 22 g needle in a single bolus directly to the intact muscle without any skin incision . sham animals were treated in the same way but received 200 l dmem vehicle only . at the same time , additional 100 l of cell suspensions was directed for microbiological tests ( bacteria and yeast ) . the injected cells were either unlabeled ( animals designated for gene and protein expression analyses ) or labeled with fluorescent membrane linkers , pkh26 ( red dye , animals designated for immunohistochemical staining ) or did ( diic18(5)-ds [ 1,1-dioctadecyl-3,3,3,3-tetramethylindodicarbocyanine-5,5-disulfonic acid ] in animals for in vivo imaging ) . before preparing the final suspension , the cells were washed twice in dmem to remove serum completely . the tissue surrounding the area of either cells or dmem administration was harvested at day 1 ( 24 hours ) , day 3 , or day 7 after the transplantation . in the untreated group , the tissue samples were immediately snap - frozen in liquid nitrogen and stored in 80c until analysis . the animals designated for gene and protein expression analysis were transplanted with equal amount ( 1 10 ) of cells ( n = 18 , 6 in each time point ) . mdcs for these experiments were unlabeled to avoid additional manipulations which are always associated with increased risk of acquired immunogenicity . untreated ( n = 7 ) and sham operated groups ( n = 18 , 6 in each time point ) served as controls . tissue samples collected at days 1 , 3 , and 7 were homogenized in tissuelyser homogenizer ( qiagen , gmbh , hilden , germany ) at a frequency of 25 hz for 5 minutes . total rna was isolated using rneasy fibrous tissue mini kit ( qiagen , gmbh , hilden , germany ) . rna concentration was quantified by spectrophotometer at 260 nm using nanodrop ( nd-1000 spectrophotometer , nanodrop technologies , inc . ) . reverse transcription of total mrna into cdna was performed using the superscript iii ( invitrogen , gibco , carlsbad , usa ) according to the manufacturer 's instruction . real - time pcr was performed on abi prism 7500 sequence detector ( applied biosystems , foster city , usa ) . specific primers and probes set were purchased from applied biosystems : il-1 ( rn0055700_m1 ) , il-1 ( rn00580432_m1 ) , il-6 ( rn00561420_m1 ) , tgf-1 ( rn00572010_m1 ) , and tnf- ( rn01525859_m1 ) . gapdh gene ( 4352338e ) was used for normalization . the values are expressed relatively to a reference sample ( calibrator ) : not treated muscle . the ct ( threshold cycle ) for the target gene and the ct for the internal control were determined for each sample . the evaluation of il-1 and il-1 concentration in tissue homogenates was performed by elisa . the muscle samples were homogenized in a buffer with phosphates and proteases inhibitors ( sigma - aldrich , st . louis , usa ) . then probes were clarified by centrifugation at 10 000 rpm for 5 minutes and addition of pmsf . cytokines concentrations in tissue lysates were determined using commercial available elisa kits ( r&d system , minneapolis , mn , usa ) . the results were presented as an absolute ratio : interleukin concentration / protein concentration ( 10 ) . to visualize injected cells in the host tissue cells were labeled with red fluorescent membrane linker , 5 m pkh26 ( n = 6 , 2 in each time point ) . histological and immunohistochemical staining were performed on frozen sections ( 10 m thick ) prepared with the use of cryostat microm hm 525 ( microm , walldorf , germany ) . immunohistochemical staining was performed using primary antibodies against antigens : cd43 ( 1 : 20 v / v ) and cd68 ( 1 : 20 v / v ) ( abd serotec , kidlington , uk ) . afterwards , cells were washed and probed with a secondary antibody [ alexa - fluor 488 donkey anti - mouse ] ( jackson immunoresearch europe , suffolk , uk , 1 : 100 v / v ) for 1 h at rt . finally , the slides were washed and covered with vectashield mounting medium with dapi ( vector laboratories ltd . the samples were evaluated with eclipse ni - u microscope ( nikon , tokyo , japan ) . for optical imaging an average of 3.3 10 of mdcs the cells were labeled prior to the transplantation with 7.5 m did , a membrane linker with ex - max 650 nm / em - max 670 nm ( aat bioquest , sunnyvale , ca ) . the area of imaging was carefully shaved and the adjacent part of the body was covered with dark fabric to avoid hair - derived autofluorescence . in vivo imaging was carried out using the ivis spectrum system ( caliper life sciences , hopkinton , ma ) . automatic algorithm for spectral unmixing of did dye against the food and autofluorescence backgrounds was used for visualizing the transplant - specific fluorescent signal , with excitation wavelength of 640 nm and six emission wavelengths ( 680 , 700 , 720 , 740 , 760 , and 780 nm ) . results from rt - pcr were presented as a fold change of gene expression in relation to the calibrator , whereas data from elisa were expressed as means ( sd ) . results were analyzed in pairs ( untreated control versus veh group and veh versus mdc groups in certain time points ) using nonparametric u mann - whitney test . a value of p < 0.05 was considered as statistically significant . for rt - pcr assay the significance of differences between groups was measured in ct values . within first days after isolation procedure , the mononuclear , colony - forming spindle shaped cells could be observed ( figure 1(a ) ) . the mean percentage of desmin expressing cells in obtained mdcs population after the first passage amounted to 77% ( figure 1(b ) ) . isolated mdcs cultured in dmem/2% hs differentiated into myotubes ( figure 1(c ) ) , which confirms their myogenic potential . the mean fusion index was 35% . however , some cells in culture spontaneously accumulated lipids , which was visualized by oil red o staining ( figure 1(d ) ) . the analysis of gene expression revealed that needle insertion and administration of 200 l vehicle into the muscle caused significant upregulation of il-1 , il-6 , and tgf- ( 7-fold , 2.3-fold , and 2.7-fold , resp . ) at day 1 in the injection site comparing to the untreated control . the significant elevation of il-6 gene expression in veh group comparing to ctrl group was maintained at day 3 . one week after the injection there was no statistical differences between veh and ctrl groups ( figure 2 ) . as the injection of vehicle itself caused significant differences in cytokines gene expression , the results obtained from transplanted samples were statistically analyzed in comparison to the sham control ( veh group ) . the administration of autologous cells caused significant increase in gene expression of il-1 and il-1 ( figure 2 ) . the elevation was 5.5-fold and 5.2-fold , respectively , comparing to the veh group and 36-fold and 17-fold , respectively , comparing to the untreated group . the expression of il-6 , tnf- , and tgf- displayed similar pattern ( the elevation peak at day 1 ) ; however the differences in those cytokines were not statistically relevant . significant changes in gene expression ( both il-1 and il-1 ) were verified by evaluation of protein level in analogous samples . in the case of il-1 , the protein concentration did not confirm upregulation of gene expression ; the differences between groups were not statistically significant ( figure 3(a ) ) . mdcs administration induced significant 4-fold increase of proinflammatory il-1 mean concentration in the site of injection 24 h after transplantation in comparison to the sham control ( figure 3(b ) ) . at the same time , vehicle injection itself also caused significant local elevation of il-1 level in comparison to the untreated control ( figure 3(b ) ) . the dynamics of evaluated genes expression clearly demonstrate that upregulation of proinflammatory cytokines after autologous mdcs transfer is acute and transient . at day 3 only il-1 expression was still significantly elevated in samples from mdc group in comparison to the sham group . at day 7 no significant differences in the mrna level of examined cytokines were observed either after the transplantation or after the vehicle injection ( figure 2 ) . to identify injected cells at the injection site in some animals ( n = 6 ) mdcs were labeled with red fluorochrome pkh26 prior to transplantation . the analysis of sectioned tissue samples revealed big , distinct clusters of pkh26 stained cells between muscle fibers ( figures 4 and 5 ) . the transplanted cells formed dense clusters at day 1 and day 3 but not after one week after injection . pkh26 positive cells in tissue samples collected at day 7 were rather rare and diffused throughout the injection site . to evaluate the presence of inflammatory cells in the injection site this preparation allowed for recognition of transplanted cell clusters in samples collected 1 and 3 days after injection . numerous inflammatory cells , especially polymorphonuclear granulocytes , could be observed around and within the clusters at day 1 . in specimens obtained 7 days after injection , inflammatory cells , the tissue sections were immunostained for cd68 , macrophage marker , and cd43 , leukosialin , present on majority of leukocytes and not on macrophages . the substantial number of both macrophages and leukocytes could be recognized in the direct vicinity of transplanted mdcs clusters 24 h after injection . at this time point the majority of cd68 expressing cells were surrounding the clusters , and only few macrophages could be observed between the injected cells ( figure 4(a ) ) . at day 3 cells expressing cd68 were totally covering the area of pkh26 clusters ( figure 4(b ) ) . one week after injection the number of macrophages was substantially reduced . in many cases , cd68 colocalized with pkh26 red fluorescence ( figures 4(b ) and 4(c ) ) . the presence of cd43 positive cells was the most prominent at day 1 ( figure 5(a ) ) . in vivo fluorescence imaging revealed the distinct reduction of did - derived signal in subsequent time points in all analyzed animals ( n = 3 ) . vehicle injection did not induce fluorescence in analyzed wavelengths which indicate the specificity of signal detected in the transplanted limb . these results confirm the poor cell survival after intramuscular injection , but for the first time this phenomenon is demonstrated in the same animal in subsequent time points after autologous transplantation . labeling of cells ex vivo allowed for identification of their fate in vivo . at day 7 pkh26 derived fluorescence was observed both in mononuclear cells located between muscle fibers and in the muscle fibers ( figures 8(a ) and 8(b ) ) moreover , pkh26 positive cells could be recognized in the central position of muscle fibers sections ( figures 8(c ) and 8(d ) ) indicating that transplanted cells possessed ability to participate in the regeneration process . the poor survival of myoblasts after transplantation is well - known problem which limits the introduction of mtt into the clinic . 24 h after myogenic cells transplantation only 20% or less of initial cell number can be detected in the area of injection . this amount is further decreasing to 15% at days 35 after transfer [ 5 , 15 , 16 ] . detected 60% of injected population after 24 h , but the percentage of survived cells diminished to about 10% at day 3 . herein , we confirm the limited cell survival after autologous intramuscular transplantation by in vivo imaging of the same individuals in subsequent time points . our previously published results suggest the role of oxidative stress ( possibly generated by phagocytic immune cells ) in cellular graft elimination after autologous transfer . therefore the aim of presented herein experiments was to describe the dynamics of acute local inflammatory reaction in response to autologous mdc transplantation . in our experiments we isolated cells exploiting preplate ( pp ) technique described by qu et al . . to reduce the number of fibroblasts in culture we used a subset of cells which adhered to the plastic surface between 24 h and 72 h after seeding . according to preplating schedule presented by qu et al we used mixed population of pp3-pp4 cells which mainly consists of satellite cells progeny , myoblasts . indeed , our results confirm that majority of isolated cells expressed desmin . the average fusion index 3 days after induction of myogenic differentiation amounted to 35% . in comparison , the average fusion index of c2c12 cell line ( mice satellite cells ) was shown to be about 50% . therefore , as obtained population still contained some nonmyogenic cells like fibroblasts or fibroadipogenic progenitors , we call it muscle - derived cells rather than myoblasts . the analysis of samples collected 24 h after transplantation indicated that autologous mdcs induce significant upregulation of il-1 and il-1 genes expression in comparison to the group treated with vehicle only ( figure 2 ) . the significantly increased expression of il-1 , especially il-1 isoform , in response to the intramuscular cell transplantation , was previously reported in several different models : allogeneic transfer of myoblasts into the intact myocardium , allogeneic transfer of mesenchymal stem cells ( mscs ) into the infarcted myocardium , and syngeneic transfer of mscs into intact skeletal muscle . in the present study we demonstrate for the first time that similar response in regard to il-1 expression is observed also after autologous transfer of mdcs . the evaluation of il-1 expression dynamics during the first week after the procedure revealed that the boost of il-1 is acute and transient and the expression at day 3 is distinctly lower than at day 1 and it is not any more significantly higher comparing to the veh group in analogous time point . suzuki et al . also evaluated the dynamics of cytokines expression in two time points ( 24 h and 3 days after transfer ) after allogeneic myoblasts grafting into intact myocardium and reported the same pattern of changes , peak upregulation at day 1 and downregulation at day 3 . as il-1 is the key proinflammatory cytokine associated with the activity of phagocyting immune cells , those data suggest that the dynamic of early inflammatory reaction after mtt is similar in both auto- and allogeneic transplantations . it is worth noting that il-1 was showed to be important in respect to grafted cell survival in allogeneic model . it was demonstrated that blocking of il-1 action resulted in increased transplantation efficacy [ 14 , 20 ] . our data suggest that the same effect could be obtained in autologous transfer . in the present study we also characterized early inflammatory infiltration in reaction to mdcs transfer . for this part of experiment cells this method of labeling was chosen as it is described to be nonimmunogenic in both manufacturer 's specification and a published report . the disadvantage of this cell tracker is substantial diluting during cell divisions which was analyzed in our recently published report . however in short term experiment in which we did not expect intensive cell proliferation membrane linker met our needs . in the tissue samples collected from mdc group at days 1 and 3 in those two time points they formed dense , demarcated cell clusters ( figures 4(a ) and 4(b ) ) . very similar appearance was observed after allogeneic transplantation of either myoblasts or muscle - derived stem cells at day 2 after injection . in our experiment , the distribution of grafted cells changed in specimens collected at day 7 ; the cells were much more diffused throughout the site of injection ( figure 4(c ) ) . the amount of pkh26 positive cells at this time point was distinctly lower comparing to either day 1 or day 3 . at day 7 pkh26 derived fluorescence was observed both in mononuclear cells located between muscle fibers and in the muscle fibers ( figure 8(a ) ) . moreover , pkh26 positive cells could be recognized in the central position of muscle fiber section ( figure 8(b ) ) which indicated that transplanted cells which survived in the injection site were able to participate in the regeneration process . the results indicating limited persistence of injected cells at day 7 were confirmed by semiqualitative graft survival analysis using in vivo fluorescence imaging ( fli ) . although this method has serious limitations because of considerable autofluorescence of the animal tissues , it was previously demonstrated that fli analysis of myoblasts survival after intramuscular injection can be valid . in our study , we have utilized a spectral unmixing algorithm offered by the living image software to alleviate this issue . using membrane linkers for in vivo imaging creates an additional problem ; these dyes can be absorbed by phagocytes and therefore impede interpretation of results . however , in our experiment the radiant efficiency of did - derived signal decreased markedly in subsequent time points ; therefore we can conclude that cells were indeed eliminated from the injection site . to confirm the presence of immune response to autologous mdcs injection the tissue sections were analyzed in order to identify inflammatory cells . basic h&e staining revealed that clusters of transplanted cells were infiltrated with polymorphonuclear granulocytes at day 1 ( figure 6(a ) ) . at day 3 , the presence of neutrophil in h&e stained sections was not so obvious ; most numerous cells within transplanted clusters at this time point were large and mononuclear which could indicate them to be macrophages . to identify the infiltration more precisely the sections were immunostained for cd43an antigen which is present on majority of leukocytes and not on macrophages . the analysis revealed the typical sequence of events in early innate immune reaction , the rapid infiltration of target area with neutrophils ( within first 24 h ) and delayed appearance of macrophages with the maximal intensity at day 3 . at day 7 the inflammatory reaction was almost completely resolved which is consistent with results of cytokines gene expression analysis . thus , our results demonstrate for the first time that autologous transplantation of muscle - derived cells induces classical early immune reaction in the site of injection . it can be claimed that the presence of macrophages in the injection site is beneficial for the grafting efficacy . it was previously demonstrated that macrophages enhance or even are required for muscle regeneration [ 27 , 28 ] . furthermore , it was shown that coinjection of myoblasts and macrophages improves survival , proliferation , and migration of transplanted muscle precursor cells [ 9 , 29 ] . however , results regarding coinjection were obtained after either allogeneic or xenogeneic injection into immunodeficient host . thus , it is difficult to predict if host derived macrophages could also improve transplanted cell survival . first , the inflammatory cells are activated because the injected cells become immunogenic during extracorporeal manipulations . second , injected cells die due to other reasons like ischemia , hypoxia , or anoikis and phagocytes infiltrate the graft to clean up dead cells but not to eliminate viable ones . verification of the mechanism which has predominant importance is crucial for assessment of future strategies for autologous mtt efficacy improvement . our previously published data demonstrated that cells with increased resistance to oxidative stress survive better after autologous intramuscular transplantation . similar findings were presented before by urish et al . on allogeneic model of transplantation . as the production of reactive oxygen species is associated with phagocyting activity , thus the importance of resistance to oxidative stress supports the hypothesis that inflammation is the cause and not the consequence of grafted cell death . the role of inflammation in myoblasts elimination after autologous administration was also shown by ito et al . . the authors demonstrated that strong systemic immunosuppression improved the efficacy of transplantation in nondystrophic dogs . on the other hand some reports suggest that neutrophils and macrophages are not responsible for early death of donor myoblasts even in allogeneic model [ 15 , 16 ] and thus support the theory that cells die due to other reasons and inflammation is only the secondary consequence of this death . as the two potential mechanisms are not exceptive , it is possible that only complex protective approach will be able to effectively prevent myoblasts ' death after transplantation . to conclude , presented data demonstrate that autologous intracellular mdcs transplantation induces classical early immune reaction in the site of injection . these results , when considered with previously published reports , suggest that innate inflammatory response can contribute to limited survival of theoretically nonimmunogenic autologous cellular graft .

the vast majority of myoblasts transplanted into the skeletal muscle die within the first week after injection . inflammatory response to the intramuscular cell transfer was studied in allogeneic but not in autologous model . the aim of this study was to evaluate immune reaction to autotransplantation of myogenic cells and to assess its dynamics within the first week after injection . muscle - derived cells or medium alone was injected into the intact skeletal muscles in autologous model . tissue samples were collected 1 , 3 , and 7 days after the procedure . our analysis revealed the peak increase of the gene expression of all evaluated cytokines ( il-1 , il-1 , il-6 , tgf- , and tnf- ) at day 1 . the mrna level of analyzed cytokines normalized in subsequent time points . the increase of il- gene expression was further confirmed at the protein level . analysis of the tissue sections revealed rapid infiltration of injected cell clusters with neutrophils and macrophages . the inflammatory infiltration was almost completely resolved at day 7 . the survived cells were able to participate in the muscle regeneration process . presented results demonstrate that autotransplanted muscle - derived cells induce classical early immune reaction in the site of injection which may contribute to cellular graft elimination .

the cochlear implant ( ci ) is the treatment of choice for the ( re)habilitation of individuals with severe and profound sensorineural deafness.1 the ci partially replaces the function of the cochlea , transforming sound energy into electrical signals . its function depends on the integrity of the cochlear nerve to conduct electrical stimuli to the cerebral cortex.2 the ganglion cells of the auditory nerve effectively respond to electrical stimuli released by the ci . therefore , the number , distribution , and function of these neural cells represent determinant factors in relation to successful use of the ci.3 therefore , the possibility to obtain information related to the permeability of the cochlear nerve to electrical stimulation data and the way in which some parameters of electrical stimulation interact with the neural structures are very important for research involving the ci , cochlear nerve , and causes of hearing loss.2 neural response telemetry ( nrt ) captures the action potential of the distal portion of the auditory nerve ( ecap ) in patients with ci ( nucleus ci , cochlear corporation , australia ) , using the ci itself to generate stimuli and to record responses.3 the ecap wave is typically formed by a negative peak ( n1 ) , with 0.2- to 0.4-millisecond latency , followed by a positive peak ( p2 ) , with 0.5- to 0.7-millisecond latency . the response amplitude ( measured between n1 and p2 ) varies with increasing stimulus intensity and is measured in microvolts ( ranging between 40 and 2,000 v).1 one way to assess the temporal processing capacity of a ci user is to measure the refractory properties of the auditory nerve , that is , the recovery function of the auditory nerve ( rec ) . recs are extracted from the neural response amplitude as a function of the interval between the stimulus and the masker stimulus ( interpulse interval ) and can be measured with nrt using the subtraction technique.4 5 the routine clinical applications of nrt are : ( 1 ) to confirm correct implant function and electrode array insertion by obtaining the ecaps ; ( 2 ) to track implant function over time ; ( 3 ) to assist the fitting process by using the ecap thresholds as estimation of audible stimulus levels and loudness.6 other implications are estimating channel interaction , degree of neural refractoriness , and auditory system status . the telemetry method has several advantages over other methods , such as obtaining larger amplitudes and prevention of muscle artifacts , and it can be done quickly and effectively.2 studies have shown variation in the threshold and growth curve of the ecap wave amplitude between individuals and between different etiologies of deafness.1 7 it is not clear in the literature , though , whether the responses from adults are the same as or differ from children . it is assumed that one should find differences in assessments of children compared with adults due to several factors such as duration of deafness , the maturation of the auditory pathways , and others . it has been suggested that the human auditory system reaches maturity between 1 and 3 years of age . in this scenario , children with implants would undergo electrical stimulation on immature auditory pathways , and adults , in already mature pathways . children with implants would therefore have their maturation influenced by electrical stimulation of the ci . on the other hand , adults may have long periods of deafness in relation to children , being subject to varying degrees of nerve degeneration in the auditory pathways from lack of stimulation.8 therefore , the objective of this study is to compare the results of nrt and rec between adults and children undergoing ci surgery intraoperatively . this study was approved by the ethics committee on human research of our hospital under caae : 13157113.4.0000.5529 . this is a cross - sectional and descriptive study of the results of telemetry patients receiving cis in our hospital in the year 2012 . the sample included patients undergoing ci surgery who underwent intraoperative nrt , without limits of age , of both sexes . exclusion criteria were patients who underwent surgery but did not undergo intraoperative nrt or who failed ( had no response ) in assessment of impedance telemetry or in obtaining intraoperative ecap . measurements were obtained during ci surgery through computer software nrt custom sound ep 3.2 ( 3.2.3855 ) , connected to the portable programming unit and speech processor and the transmission antenna of the ci ( software developed by cochlear corporation ) . we used apical , medial , and basal electrodes ( 1 , 11 , and 22 ) for the telemetry response measures and the t0 ( absolute refractory period , in seconds ) , a ( saturation level , in microvolts ) , and tau ( curve of the model function ) measures for the calculation of the exponential function of neural recovery for groups of children ( 0 to 18 years ) and adults ( > 18 years ) . the current level used for the recordings of rec was 15 to 20 units of current above the current level at which the nrt was obtained in each stimulated electrode ( apical , medial , and basal ) , only 20 units being used if there was need for higher current to obtain the neural response and not cause saturation of the amplifier . the parameters of the amplifier gain and delay used for this study were the same as determined by the series of optimization of the electrode stimulation frequency of 80 hz according to lai.9 the probe rate was 80 hz between stimuli , and 20 mpi ( masker probe interval ) measurements were performed from 100 to 10,000 s . in some cases , it was necessary to perform manual corrections of the measurement of n1/p2 ; in others , the calculations given by the software were used . the custom sound ep software automatically converts measurement of the recovery function ( rec ) in an exponential function : the comparison between nrt and rec averages between the two groups ( children versus adults ) and statistical analysis was performed using student t test and mann - whitney test , with p < 0.05 considered significant . fifty - two patients were assessed ( 26 children and 26 adults ) for nrt and 24 patients ( 12 children and 12 adults ) for the rec . the mean age of children was 4.15 2.92 years and for adults , 37.12 16.22 years . causes of impairment in the nrt adults ( 26 patients ) included one case of genetic cause , three causes of congenital rubella , one case of presbycusis , one case of meningitis , and one case of late stage meniere syndrome , and all the other causes were idiopathic . on the other hand , the children ( 26 patients ) included six cases of genetic cause , one case of meningitis , and one case of encephalitis , and all the others were of idiopathic cause ( tables 1 and 2 ) . in the rec adults ( 12 patients ) , there was one case of genetic cause , two cases of late - stage otosclerosis , and one case of congenital rubella . the children ( 12 patients ) included four cases of genetic cause and the others were idiopathic causes ( tables 3 and 4 ) . no patient failed in obtaining impedance response during the surgery , showing that the electrodes were positioned correctly . the results of intraoperative nrt are shown in table 5 , for the apical , medial , and basal electrodes . the results of the rec were evaluated for t0 , a , and tau for the apical , medial , and basal electrodes and are show in tables 6 , 7 , and 8 . for the three parameters , there was no significant difference , except for the level of saturation of the basal electrode . a ci for severe to profound sensorineural hearing loss unresponsive to hearing aids may be indicated for both children and adults , according to the criteria proposed in the guidelines . there is a great variability among the candidates for ci in relation to age , type of deafness , hearing deprivation , speech production , and communication skills . speech perception and word recognition tests assess the subjective result of the implant , which is the ability to enable communication skills . however , there is no standardization of these tests in our country , making it difficult to compare results among research centers.10 in this scenario , an objective test , characteristic of the ci model such as nrt , has great value in the evaluation and comparison of data among individuals , and it is easy to standardize analysis and interpretation among research centers.11 because it does not require external electrodes , the nrt is less susceptible to myogenic interference and needs fewer stimuli to trigger the ecap . assessment during surgery with either general or local anesthesia with sedation allows better evaluation of the responses without causing discomfort to the patient by increasing the current units needed to unleash the potential.1 furthermore , the nrt through the ecap is a direct way to evaluate in vivo the functional characteristics of ganglion cells and other auditory neural structures . knowledge of the function of these structures is essential for the development of improved technologies and increasing knowledge of the different etiologies of deafness.3 yet there are not many studies on the cochlear nerve function , especially in comparison between adults and children . the action potential measured by nrt reflects the synchronized firing of many auditory nerve fibers and consists of the sum of the electrical activity of hundreds of neurons . as ecap occurs immediately after stimulus presentation , it is not affected by the maturation of the auditory system . gordon et al found higher amplitudes of the ecap and lower latency in children compared with postlingual adults.12 these differences were attributed to auditory deprivation time or number of stimulated neurons . in our study , we found a secluded difference between the amplitudes of the saturation level between children and adults only in the basal electrode . studies have shown that periods of slower recovery and lower amplitudes were found in basal electrodes . these changes were attributed to the fact that the electrodes stimulate a smaller population of neurons , due to the greater distance of the cells or to a smaller number of surviving cells in this portion because of the neurosensory deafness itself.7 12 on the other hand , a more recent study found that slower ecap recovery , at equal loudness , is associated with larger neural populations.5 the importance of determining thresholds , amplitude , and refractory properties of the responses of the action potential of the viii nerve may indicate differences in the neural population of individuals , and even on the auditory perception performance.1 but conflicts in results among studies show that there is still a need for more research regarding this subject . measures of both the nrt and the rec in combination may be important indicators of success with the ci.7 therefore , more studies about them and their correlation to clinical data are indispensable . no differences were found in measures of nrt and rec intraoperatively between adults and children , except for the level of saturation at the basal electrode .

introduction neural response telemetry ( nrt ) is a method of capturing the action potential of the distal portion of the auditory nerve in cochlear implant ( ci ) users , using the ci itself to elicit and record the answers . in addition , it can also measure the recovery function of the auditory nerve ( rec ) , that is , the refractory properties of the nerve . it is not clear in the literature whether the responses from adults are the same as those from children . objective to compare the results of nrt and rec between adults and children undergoing ci surgery . methods cross - sectional , descriptive , and retrospective study of the results of nrt and rec for patients undergoing ic at our service . the nrt is assessed by the level of amplitude ( microvolts ) and rec as a function of three parameters : a ( saturation level , in microvolts ) , t0 ( absolute refractory period , in seconds ) , and tau ( curve of the model function ) , measured in three electrodes ( apical , medial , and basal ) . results fifty - two patients were evaluated with intraoperative nrt ( 26 adults and 26 children ) , and 24 with rec ( 12 adults and 12 children ) . no statistically significant difference was found between intraoperative responses of adults and children for nrt or for rec 's three parameters , except for parameter a of the basal electrode . conclusion the results of intraoperative nrt and rec were not different between adults and children , except for parameter a of the basal electrode .

some hounds became lethargic and weak ; in some , these signs progressed to loss of consciousness . postmortem examination of the hound that died ( case 1 ) and 1 that was euthanized ( case 2 ) showed subacute broncho - interstitial pneumonia ; virus was suspected as the cause . when they were puppies ( 8 weeks of age ) , the hounds had been inoculated with commercially available vaccines against the major canine respiratory and enteric viruses . postmortem tissue samples submitted to a canine infectious diseases laboratory were negative for known canine viral pathogens ( e.g. , canine herpesvirus , adenovirus , parainfluenza virus ) . the diagnosis as to the cause of the pneumonia , returned in 2002 , was unknown , suspected viral etiology . in january and march 2005 , serum samples were obtained from the hounds affected by the respiratory disease outbreak in 2002 ( pack 1 ) . serum samples were obtained from another 3 packs of foxhounds in the same region of the united kingdom during december 2004 through february 2005 . samples were collected from 3133 hounds ( equivalent numbers of males and females ) in each pack , ranging in age from 9 months to 9 years . the serum was screened for antibodies by using the single radial hemolysis assay ( 2 ) . none of the samples contained antibodies to the strains that were included in the assay to control for nonspecific reactivity : equine h7n7 subtype strain a / equine / prague/56 and the human influenza virus strain a / puerto rico/8/34 ( h1n1 ) . antibodies to the h3n8 subtype strains a / equine / newmarket/1/93 and a / equine / newmarket/2/93 were , however , detected in 9 of the samples obtained during the first visit to pack 1 ( table ) . of these , 8 were from hounds that had survived the outbreak in 2002 ; however , 1 was from a hound ( no . 22 ) born after the outbreak in another part of the united kingdom , which suggests that the 2002 outbreak might not have been the only incident of equine influenza to have infected hounds in the united kingdom . another 3 positive serum samples were obtained during a second visit to pack 1 , and a repeat sample from hound no . the specificity of the antibodies for equine influenza a ( h3n8 ) strains was confirmed by hemagglutination inhibition assays that included human influenza ( h3n2 ) strain a / scotland/74 ( data not shown ) . * measured by single radial hemolysis ( mm ) in serum samples . nk , not known . an immunohistochemical test to detect influenza a virus that used equine influenza specific rabbit polyclonal antiserum was applied to formalin - fixed paraffin - embedded ( ffpe ) tissues from the 2 hounds that were examined postmortem in 2002 ( 3 ) . immunostaining of lung tissue showed positive staining in areas of pneumonic change ; infected cells had the morphology of epithelial cells and macrophages ( figure 1 ) . immunostaining of visceral tissues ( lung , liver , spleen , myocardium , intestine , pancreas , and oropharynx ) was negative . immunohistochemical staining for equine influenza a virus ( brown stain ) in sections of respiratory tissue from english foxhounds involved in 2002 respiratory disease outbreak , united kingdom . a ) case 1 , showing focal staining of an apparently necrotic bronchiole in an area of pneumonia ; magnification x100 . b ) case 2 , showing a large amount of staining throughout the epithelium and inflammatory cells present in the brush border ; magnification x200 ; hematoxylin counterstain . deparaffinization of the ffpe lung tissue from the 2 hounds was performed as described previously ( 4 ) with a few modifications . rna was extracted from the sample pellets obtained using the qiaamp viral rna mini kit ( qiagen , hilden , germany ) according to the manufacturer s instructions . ten different primer pairs designed to amplify short ( < 250-bp ) products from the matrix hemagluttinin and neuraminidase genes were used ( details available from the authors on request ) . reverse transcription pcr ( rt - pcr ) was carried out by using the qiagen onestep rt - pcr kit . only 1 primer pair ( forward : 5-aggcaggataagcatatact-3 and reverse : 5-gtgcatctgatctcattaca-3 , amplifying nucleotides 735871 of the hemagglutinin gene ) yielded an amplification product , which was purified by using pcr kleen spin columns ( bio - rad , hercules , ca , usa ) and sequenced by using the bigdye terminator v1.1 cycle sequencing kit ( applied biosystems , foster city , ca , usa ) . a blast search with the 74-bp nt sequence obtained from this amplicon confirmed that the virus shared 100% identity with the equine influenza ( h3n8 ) strains newmarket/1/93 ( 5 ) and newmarket/5/03 ( 6 ) . lysine at position 261 indicates that the virus belongs to the american lineage ( 5 ) ; this is supported by the presence of isoleucine at position 242 because all european lineage strains isolated since 1998 have valine at 242 . an important factor in interspecies transmission is the ability of the hemagglutinin protein of the virus to bind to certain receptors on the host cells before the virus is internalized . although all influenza a viruses recognize cell surface oligosaccharides with a terminal sialic acid , their receptor specificity varies ; it is thought that species - specific differences in the distribution of linkages on respiratory epithelial cells influences the ability of influenza a viruses to transmit between species . respiratory tract tissue samples were obtained within 24 hours of death from a horse and a greyhound , each euthanized for reasons other than this study , and rinsed extensively to remove surface mucous . the tissues were stained by immunofluorescence by using the lectins sambucus nigra ( sna , specific for sa2,6 galactose(gal)/n - acetylgalactosaminide ) and maackia amurensis ( maa , specific for sa2,3 ) as previously described ( 7 ) . the maa lectin bound strongly to the equine tracheal epithelium ( figure 2 , panel a ) , which confirms the finding that the neuac2,3gal linkage preferentially bound by equine influenza viruses is found on sialyloligosaccharides in the equine trachea ( 7 ) . the maa lectin also bound strongly to the canine respiratory epithelium ( figure 2 , panel b ) at all levels of the respiratory tract examined ( distal , medial and proximal trachea ; primary and secondary bronchi ) , which suggests that receptors with the required linkage for recognition by equine influenza virus are available on canine respiratory epithelial cells , although further subtle differences in receptor specificity may exist . the sna lectin , specific for sa2,6gal , which did not bind to the equine tracheal epithelium , showed some binding to the canine epithelium ( data not shown ) . lectin staining for 2,3 sialic acid linkages on a ) equine trachea and b ) canine trachea ; magnification x200 ; cell nuclei counterstained with hoechst 33342 solution . because the hounds infected in 2002 were housed near horses , it is possible that the virus was transmitted from infected horses by the usual ( aerosol ) route . however , during the week before onset of clinical signs , the hounds had been fed the meat of 2 recently euthanized horses from independent sources . that viral antigen expression was confined to the lungs indicates a respiratory rather than oral route of infection . it is possible that eating respiratory tissue from an infected horse led to inhalation of sufficient virus particles to initiate a respiratory infection . consumption of infected bird carcasses has been implicated in the transmission of highly pathogenic avian influenza virus of the h5n1 subtype to tigers and leopards ( 8) and a dog ( 9 ) and was demonstrated experimentally by feeding virus - infected chicks to domestic cats ( 10 ) . although the mechanism remains unclear , we have demonstrated transmission of equine influenza virus to dogs in the united kingdom , independent of that in the united states . we have also shown that canine respiratory tissue displays the relevant receptors for infection with equine influenza virus .

we retrospectively demonstrated that an outbreak of severe respiratory disease in a pack of english foxhounds in the united kingdom in september 2002 was caused by an equine influenza a virus ( h3n8 ) . we also demonstrated that canine respiratory tissue possesses the relevant receptors for infection with equine influenza virus .