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biological samples are an important part of epidemiological investigations of toxic exposures and their disease outcomes . blood , urine , saliva , or hair has often been utilized to assess risk factors for a specific disease by comparing the concentrations of some environmental toxic substances between affected and unaffected individuals . while this is a useful approach in some endeavors , these biosamples are only measures of recent exposure and can not inform about distant past exposures . alternatively , the use of deciduous teeth has served as a biomarker of early developmental exposure to heavy metals . the mineralization of primary teeth begins prenatally between 14-and 16-week gestation and concludes postnatally at 1.5 to 3 months for incisors , 9 months for canines , and 5.5 to 11 months for molars . it has been demonstrated that metals in circulation , which are present during the period of tooth formation , become incorporated into forming dental tissue and are stored in the mineral component of teeth [ 24 ] . deciduous teeth have been used as biomarkers of heavy metal exposure in disease outcome studies for some time . needleman et al . demonstrated that lead measured in deciduous teeth was associated with lowered cognitive performance in children . since that landmark study , the concentrations of lead in deciduous teeth have been widely used as a biomarker for lead exposure and body burden in a variety of other studies . along with lead , other heavy metals such as cadmium , zinc , and copper have also been measured in deciduous teeth . a major development in the assessment of heavy metals using deciduous teeth has been the use of laser ablation electrospray ionization ( laesi ) methods . these methods have been used to determine the timing of exposure during development by taking measurements along the developmental pathway of the tooth . in particular , identification of the perinatal line this has been demonstrated with metals such as chromium , iron , mercury , zinc , and antimony and validated with lead , manganese , and other heavy metals [ 1013 ] . environmental point sources of exposure have been shown to be related to tooth concentrations as a function of age and proximity to the source [ 1416 ] . further , physiological factors ( i.e. , gender , tooth type , and weight ) as well as behavioral factors ( i.e. , socioeconomic status , home antiquity , and nail biting habits ) have been shown to also explain levels of lead and cadmium in teeth [ 17 , 18 ] . these studies demonstrate the advantage of analysis of primary teeth for determining early exposures that may be related to specific disease outcomes . indeed , the conclusions from a recent nato workshop on the effects of heavy metal pollution on child development recommend that depositions found in teeth can serve as an important tool in relating heavy metal pollution to childhood development outcomes . for example , adams et al . used deciduous teeth to investigate differential mercury exposure between children with and without autism . their study demonstrated that there was a higher concentration of mercury in baby teeth of children with autism compared to children without autism . however , a recent study by abdullah et al . failed to confirm these findings . other applications of using deciduous teeth as biomarkers of exposure have been with radiation exposure and its relationship to cancer . the majority of studies using deciduous teeth have been on investigating heavy metals and much less has been done to assess organic toxic exposures such as pesticides , plastics , or medications . while laesi methods have been used to precisely determine the period of exposure when assessing metal concentrations , there is concern that heat produced by sectioning the tooth for analysis of semivolatile organic chemicals ( svoc ) may vaporize or transform the svoc on the surface so they will no longer be detectable by laesi . notwithstanding , researchers are currently working to overcome the obstacles with laesi and other imaging methods for use with svoc . gas chromatography / mass spectrometry ( gcms ) has been used to determine nicotine in deciduous teeth as a result of household exposure to cigarette smoking . recently , camann et al . identified additional semivolatile organic chemicals ( svoc ) in deciduous teeth and hypothesized that organic chemicals or their metabolites circulating in the bloodstream during development may absorb into forming dental tissues and remain stored in the tooth thereafter . chemicals detected by liquid chromatography / tandem mass spectrometry in molars of 21 typically developing children included the endocannabinoid anandamide ( 86% of children ) , acetaminophen ( 43% ) , and specific metabolites mono-2-ethylhexyl phthalate ( mehp , of plasticizer di-2-ethylhexyl phthalate , 29% ) , 3,5,6-trichloro-2-pyridinol ( tcpy , of organophosphate ( op ) insecticide chlorpyrifos , 10% ) , and 2-isopropyl-6-methyl-4-pyrimidinol ( impy , of op insecticide diazinon , 10% ) . the use of deciduous teeth with gcms methods to understand exposures to organic chemicals has great potential for use in epidemiological case - control studies for conditions of unknown etiology such as autism and other childhood disorders . however , unlike laesi , which can precisely determine the timing of perinatal exposure , gcms methods are limited to detection of cumulative exposures or at best approximate timing of exposure based on tooth type . notwithstanding , gcms methods currently offer a wider range of detectable svoc that may be hypothesized to be associated with specific health conditions . the purpose of this study was to replicate the findings of camann et al . using the deciduous teeth from two different samples of children with autism ( in the united states and mexico ) . specifically , we investigated the replication of acetaminophen , arachidonic acid ( ara ) , 3,5,6-trichloro-2-pyridinol ( tcpy , specific chlorpyrifos metabolite ) , 2-isopropyl-6-methyl-4-pyrimidinol ( impy , specific diazinon metabolite ) , diethyl - m - toluamide ( deet ) , and five monoester phthalate metabolites ( monoethyl phthalate ( mep ) , mono - n - butyl phthalate ( mnbp ) , monoisobutyl phthalate ( mibp ) , monobenzyl phthalate ( mbzp ) , and mono-2-ethylhexyl phthalate ( mehp ) . the aforementioned chemicals are pertinent to autism research because several studies indicate that exposure to pesticides [ 2527 ] , acetaminophen [ 28 , 29 ] , plastics ( phthalates ) [ 30 , 31 ] , and other chemicals is biologically relevant to autism etiology . however , no studies have used objective measures of early life exposure such as deciduous teeth to identify these compounds . before future epidemiologic investigations ensue , replication studies are needed to determine whether svoc concentrations can reliably be detected in diverse samples . post hoc analysis . while this study was not designed to assess validity , mothers who donated their children 's teeth also completed an exposure survey which included their reported exposures to acetaminophen , deet , phthalates , and pesticides . we investigated if the mother 's self - reported use or their child 's exposure to these compounds would correspond to the concentrations found in their children 's teeth . seventy - one ( 71 ) deciduous teeth , primarily molars and canines ( without cavities or fillings ) , from children with autism spectrum disorders ( asd ) were chosen from our tooth repository consisting of 928 children 's deciduous teeth . these teeth were obtained through ongoing recruitment efforts as part of our irb - approved pilot studies on autism through the university of texas health science center ( uthscsa ) . a component of our program involves an established biological tissue biorepository from families of children with and without autism . we have recently added the collection of deciduous teeth through collaborative efforts with the interactive autism network ( ian ) . to control potential bias from using a mixed sample , we chose children recruited from the interactive autism network ( ian ) whose diagnoses of autism have been verified [ 33 , 34 ] . as a cross - cultural comparison in a separate analysis , we also included 12 teeth from autistic children obtained through uthscsa collaborations with an autism parent group from matamoros , mexico . 83 tooth crowns were pulverized , extracted in batches of approximately 20 samples , and analyzed for acetaminophen , ara , deet , tcpy , impy , and mehp using modified methods from camann et al . . the pulp and any fillings , attached roots , and/or cavities were removed with a scalpel , engraving tool , and/or heatless wheel . the tooth was gently swirled in dichloromethane ( dcm ) , with the wash retained as a quality measure to evaluate external tooth contamination . each prepared tooth crown ( enamel + dentin ) was pulverized with a mortar and pestle to a fine powder and weighed . prior to extraction , a 50 mg pulverized tooth aliquot was spiked with acetaminophen - d4 and mehp - c4 and conditioned for 24 hours . the tooth aliquot was separately extracted under neutral and acidic conditions to enhance the extraction efficiency of the target analytes . the first extraction occurred through sonication with 0.5 ml acetonitrile . after removing the acetonitrile fraction , the tooth powder was acidified with glacial acetic acid and equilibrated overnight , with a second sonication extraction conducted using acetonitrile . electrospray ionization liquid chromatography tandem mass spectrometry ( lc / ms / ms ) in multiple - reaction monitoring mode was used to determine the concentrations of most targeted chemicals in the pulverized tooth samples . acetaminophen and deet were measured in positive mode , and tcpy , impy , and the phthalate metabolites in negative mode . a matrix blank with 50 mg of pulverized kiln - fired synthetic hydroxyapatite was extracted with each batch of tooth samples and analyzed to assess laboratory introduced contamination . a matrix spike of all target analytes , into a second 50 mg aliquot of two pulverized teeth , was extracted and analyzed to assess measurement accuracy ( i.e. , analyte extraction efficiency ) in these teeth . the organic wash portion was analyzed as a qc measure to assess external contamination for specific samples containing high levels of detected target analytes . to determine the concentration of ara and other fatty acids ( ian sample only ) , a second 50 mg aliquot of each pulverized tooth was spiked with triheneicosanoin , decalcified with edta , and extracted three times with chloroform . tridecanoic and tricosanoic acids were added as internal standards , and the extract was blown down to dryness , saponified , and methylated . the fatty acid methyl esters were extracted with saturated sodium chloride and isooctane , and the concentrated isooctane extract was analyzed for ara by gas chromatography mass spectrometry in selected ion monitoring mode . seven dichotomous items on the survey , indicating use ( = 1 ) or no use ( = 0 ) of acetaminophen , were summed to create a total exposure score . the items captured assessed ( 1 ) mother 's use during pregnancy , ( 2 ) child 's use from 0 to 6 months , ( 3 ) child 's use from 7 to 12 months , ( 4 ) child 's use from 13 to 18 months , and ( 5 ) around the time before or after measles mumps and rubella ( mmmr ) vaccination ( 1215 months ) . we also utilized information about the effectiveness of acetaminophen based on the mother 's report of whether it works for her or her child ( 1 = works well versus 0 = works a little or not at all ) , reasoning that if this analgesic worked , then it would be used . a dichotomous variable was then formed using a median split roughly corresponding to a 50% split of the distribution ( 0 = lower half , n = 28 versus 1 = upper half , n = 43 ) . this self - report exposure variable was submitted to a chi - square analysis , using fisher 's exact test , to determine if the two self - reported exposure groups had statistically different rates of acetaminophen detection in teeth . this was constructed from five items asking the number of times insect repellant was used on their child at less than 1 years old , 1 - 2 years old , 2 - 3 years old , 3 - 4 years old , and 4 - 5 years old . each response was scored 0 for no use during that time , 1 = 14 times , 2 = 59 times , and 3 = more than 10 times . since 83 percent said they had used repellants sometime during their child 's life , a sum score was used to reflect the frequency of repellant use summed over the five time frame items . these self - reported insect repellant exposure variables were submitted to a chi - square analysis using fisher 's exact test to determine if the highest self - reported exposure group category had statistically different rates of deet detection in teeth compared to the lowest reported exposure . we also used a tobit regression ( suitable for data that are left truncated due to laboratory detection limits ) to assess the linear association between the continuous self - reported score and the continuous deet tooth concentration , adjusted for parental age and gender . this was constructed from responses to three areas of usage . ( 1 ) did anyone use sprays , dusts , powders , mothballs , or foggers ( including a pest control service ) in your home or place of work to kill bugs ? ( 2 ) did anyone use a lawn service or apply bug or weed killers on your yard , plants , or trees ? ( 3 ) did anyone use sprays , dusts , powders , soaps / shampoos , or skin applications for fleas or ticks on your pets ? each of the three questions had response options of 0 = not at all , 1 = once or twice , 2 = three to five times , and 3 = six or more times covering three developmental time periods : before pregnancy , during pregnancy , and from birth to six years old . a dichotomous summary variable based on a median split was formed to reflect high and low self - reported exposure . chi - square analysis was performed with this dichotomous self - reported use and a dichotomous variable reflecting pesticide metabolites detection in teeth . cumulative self - reported exposure to phthalates was determined using questions asking whether or not the child was exposed to fumes / chemicals from new paint , new floors / cabinets ( stains and paint strippers ) , new carpet , new walls / drywall , or carpet cleaning during three developmental periods : three months before pregnancy , during pregnancy , and during the child 's life . a dichotomous variable was formed based on a median split and used as a predictor in a tobit regression model with phthalates as an outcome . the teeth from ian children with autism are of parents who are largely middle class non - hispanic white families . the matamoros samples of children with autism are hispanic families with comparably lower income and education . table 2 shows the distribution of chemicals we sought to measure . there was a 44% detection rate for acetaminophen ( 31/71 ) and a 75% detection rate for deet ( 53/71 ) . the detection rate for both of the organophosphate insecticide metabolites tcpy and impy was 13% . detection rates of the low molecular weight monoester phthalate metabolites were ( monoethyl phthalate ( mep ) ( 100% ) , mono - n - butyl phthalate ( mnbp ) ( 86% ) , and monoisobutyl phthalate ( mibp ) ( 70% ) . relatively smaller detection rates were found for monobenzyl phthalate ( mbzp ) ( 6% ) and mono-2-ethylhexyl phthalate ( mehp ) ( 36% ) . the detection rates of linoleic acid ( la ) and arachidonic acid ( ara ) were 100 and 85 percent , respectively . for alpha - linolenic acid ( ala ) and docosahexaenoic acid ( dha ) , detection rates were much lower at 21 and 20 percent , respectively . table 3 shows the distribution of chemicals from matamoros . there was a 42% detection rate for acetaminophen ( 5/12 ) and a 100% detection rate for deet . the detection rate for tcpy was zero and for impy was 17% ( 2/12 ) . detection rates of the low molecular weight monoester phthalate metabolites were ( monoethyl phthalate ( mep ) ( 100% ) , mono - n - butyl phthalate ( mnbp ) ( 66% ) , and monoisobutyl phthalate ( mibp ) ( 100% ) . detection rates were found for monobenzyl phthalate ( mbzp ) ( 0% ) and mono-2-ethylhexyl phthalate ( mehp ) ( 100% ) . among those self - reporting the highest cumulative exposures , 53.5 percent ( n = 23/43 ) had detectable amounts of acetaminophen in teeth compared to 28.6 percent ( n = 8/28 ) in the lower self - reported exposure group ( fisher exact test = p < .041 ) . in a logistic regression model adjusting for age , gender , and tooth type , those reporting higher exposures were 3.18 times more likely to have a detection of acetaminophen than those with lower self - reported exposures ( p < .03 ) . we also created a dichotomous variable where 1 = any detection and 0 = no detection of deet . chi - square analysis showed that 90.9 percent of those in the higher self - reported use category had detectable amounts of deet in teeth compared to 63.3 percent detection in the lower self - reported use group . deet tooth concentration was regressed on self - reported insect repellant use ( on the children only ) in three models : ( a ) self - reported use before 2 years old , ( b ) over 2 years old , and ( c ) across the entire developmental timespan . after adjustment for parental age , child age and gender , and tooth type , exposure under 2 years old yielded a .34 standardized regression coefficient ( p < .01 ) ( r - square = .18 ) . exposures after three years old yielded a .38 standardized regression coefficient ( p < .001 ) ( r - square = .21 ) . exposures during the entire developmental period yielded a .36 standardized regression coefficient ( p < .005 ) ( r - square = .20 ) . tobit regression reveals that the highest self - reported tertile level of exposure had 55.7 ng / g higher levels of deet than the lowest one third ( p = .01 ) . the distribution of the organophosphate insecticide metabolites tcpy and impy in 71 teeth is shown in table 2 . the detection rate of both metabolites was 13% in the sample ( 9/71 ) , with no detection in 62 of the teeth . both variables were dichotomized to form a binary variable 0 = no detection ( n = 62 ) and 1 = detection ( n = 9 ) . we also summed both insecticide metabolites to form another dichotomous variable , where 0 = no detection ( n = 55 ) and 1 = detection ( n = 16 ) . there was no significant association between any of the self - reported pesticide usage variables and any of the chlorpyrifos and diazinon metabolite tooth measures . there was no significant association between self - reported fumes / chemicals exposure from listed products and measured phthalate metabolites in teeth for mnbp or mep . the tobit regression results indicated that relative to the low self - reported group , the high self - reported group had 94 ng / g higher concentration of mibp in teeth . when combining all phthalates together , the tobit regression results indicated that relative to the low self - reported group the high self - reported group had 97 ng / g higher concentration of mibp in teeth ; however this was marginally significant ( p < .08 ) . , which used 21 deciduous teeth from typically developed children , we have demonstrated that specific svoc can be detected in deciduous teeth from children with autism in two different populations . despite demographic differences in the two samples , there were similar rates of detection for all chemicals with slightly higher detection rates of phthalates in the matamoros sample . this provides evidence that teeth are useful biomarkers of exposure to these svoc compounds and therefore can be used in properly matched case - control studies to investigate potential differences in exposure between cases ( e.g. , children with autism ) and controls ( e.g. , children without autism ) . this method of assessing early life environmental exposures is relevant to a wide range of diseases suspected to involve environmental triggers . prenatal exposures to polycyclic aromatic hydrocarbons , pesticides , secondhand smoke , diester phthalates , and polybrominated diphenyl ethers are linked to reduced fetal growth and developmental problems in young children [ 3539 ] . similarly , previous studies report a link between in utero organochlorine pesticide exposure and impaired neurodevelopment in childhood , and there is emerging evidence of neurobehavioral consequences for infants and children who have been exposed to even low levels pesticides [ 39 , 41 , 42 ] . taken together , these studies suggest that there are multiple chemicals that may be associated with increased risks for asd . chlorpyrifos has been shown to disrupt development of the serotonin neurotransmitter system ; this is similar to serotonin system dysfunction which has been implicated in autism etiology [ 4446 ] . chlorpyrifos is widely used in agriculture and remains an exposure of concern for children through diet even though the epa has restricted its indoor use . there are additional reports addressing established associations between ubiquitous environmental chemicals , including pesticides with effects on neurodevelopment and/or immune system regulation . for example , an elevated risk for asd has been attributed to the commonly used pesticide permethrin , based on self - reports of exposure . given the male predominance in asd , it is also relevant that phthalates are potential endocrine disruptors which can interfere with normal male development . diethylhexyl phthalate ( dehp ) , one of the most common phthalates present indoors , is often used as a plasticizer in polyvinylchloride ( pvc ) materials , such as pvc flooring and water pipes . two recent small - scale studies have linked dehp with autism [ 30 , 51 ] . prenatal acetaminophen exposure has been associated with asthma symptoms at age five , while acetaminophen use within the first 18 months after birth may be associated with increased susceptibility to autism [ 53 , 54 ] . acetaminophen is an indirect agonist of the endocannabinoid system , affecting levels of the endocannabinoids anandamide and 2-arachidonoylglycerol . anandamide , an endogenous activator of the endocannabinoid system , and 4-aminophenol , the active metabolite of acetaminophen , have similar toxic effects on developing cortical neurons . the mode of action , metabolic activation , and detoxification of many ubiquitous environmental pesticides are well known and have been linked to the disruption of endocrine activity and to disruption in the function of the primarily inhibitory neurotransmitter , gamma - aminobutyric acid ( gaba ) [ 56 , 57 ] . various studies demonstrate abnormalities of the glutamate and gaba systems in the brain and serum of subjects with autism [ 58 , 59 ] . for example , there is a significant decrease in gaba and various gaba subunit receptor - binding sites in brain tissues of subjects with autism when compared with controls . similarly , perinatal exposure to these environmental pesticide compounds appears to be associated with asd through the disruption of gaba function ; there are similar reports that pesticides interfere with development by disrupting thyroid function [ 25 , 31 ] . in sum , there is a substantial amount of converging evidence to suggest that environmental chemicals may play a role in asd risk and/or etiology by acting independently or through interactions with genetic vulnerabilities . the post hoc analysis results indicate that mother 's self - report of her child 's usage / exposure to acetaminophen and products containing deet and phthalates are consistent with detection and concentration levels in the child 's deciduous tooth . until additional studies are completed , we can make no claim about the developmental timing of exposure to the aforementioned chemicals found in deciduous teeth using the laboratory methods described in this paper . it would therefore be useful for future validation studies to be performed involving laboratory experimental models that manipulate in vivo timing of exposures . while laesi methods have been used to precisely determine the period of exposure when assessing metal concentrations , there is concern that heat produced by the necessary sectioning of the tooth may vaporize or transform the svoc on the surface so they will no longer be detectable . however , soft ionization imaging techniques such as matrix assisted laser desorption ionization time - of - flight ( maldi - tof ) researchers are currently working to overcome the obstacles with sectioning methods for use with svoc . there are thousands of potential environmental chemicals in circulation to which humans are exposed . even within certain chemical classes , . therefore , exploratory and targeted gc and lc / ms studies with pulverized teeth to identify candidate chemicals will be an important first step . chemicals identified in these initial studies can then be targeted in future imaging studies to fine - tune the time period of exposure . we feel these initial investigations are important for informing future epidemiologic research that can more precisely identify the timing of exposures between cases and controls . limitations . while we have demonstrated that chemicals relevant to asd can be detected in deciduous teeth and are associated with mothers ' self - reported exposures , our results are limited in generalizability largely due to the sample consisting entirely of children with asd . future studies that include more diverse participants and neurotypical children as controls will allow case / control comparisons . further , until additional studies are completed , we make no claim about the developmental timing of exposure ( pre- or postnatal ) to the aforementioned chemicals found in deciduous teeth using the laboratory methods described in this paper . studies incorporating both unbiased environmental exposure measures ( e.g. , deciduous teeth ) and measures of individual variation in the ability to biologically detoxify specific toxins ( e.g. , gene expression assessments ) would be the next step in understanding gene - environment interactions that could effectively inform various prevention efforts .

the long strand of genomic dna must be organized three dimensionally in cells to scan and read genome information ; this mediates various cellular functions . genomic dna is wrapped around core histones and forms a nucleosome fiber ( bead - on - a - string or 10-nm fiber ) . the nucleosome has long been assumed to be folded into a 30-nm chromatin fiber , and further hierarchical regular structures . although the higher - order chromatin structure in eukaryotic cells is not fully understood , several lines of evidence , including our recent cryomicroscopy and synchrotron x - ray scattering analyses , have demonstrated that chromatin in cells consists of irregularly folded nucleosome fibers without a 30-nm chromatin fiber ; i.e. , a polymer - melt - like structure . compared with the 30-nm chromatin fiber and further hierarchical regular structures proposed previously , irregular folding of nucleosome fibers , leading to a polymer melt - like structure , implies a physically constrained state that could be locally dynamic . nucleosome fibers may be constantly moving and rearranging at the local level , which might be essential for protein factors to scan genomic dna and locate their target sequences . however , many previous studies , including ours , examined only static structures in fixed cells . therefore , how can we know the chromatin environment in living mammalian cells , in which many protein molecules are freely mobile ? how can proteins access their destinations on chromatin in living cells ? to address these fundamental questions , we utilized a combined in vivo - in silico strategy of fluorescence correlation spectroscopy ( fcs ) , single - molecule imaging and monte carlo computer simulations . based on this strategy , we uncovered the unexpected local dynamics of individual nucleosomes in living mammalian cells ( fig . the results obtained here indicate that nucleosome fluctuation increases chromatin accessibility , which is advantageous for many target searching biological processes , such as transcription , dna repair , replication , and recombination ( fig . ( a ) graphical abstract . in the cells , nucleosome fibers ( red spheres and lines ) are irregularly folded . ( b ) schematic diagram of fcs detecting the in - out motion of egfp molecules ( green spheres ) in a ~0.1-femtoliter volume ( white - out cylinder region in the blue ) as fluctuations in fluorescence intensity ( shown as a graph ) . ( c ) cartoon showing that fluorescent proteins ( represented as runners ) move more slowly in a crowded chromatin environment due to the many obstacles ( represented as hurdles ) . to investigate the chromatin environment in living cells , we first employed fluorescence correlation spectroscopy ( fcs ) . fcs detects the in- and out - motions of fluorescent probe molecules , such as enhanced green fluorescent proteins ( egfps ) , in a small detection volume ( ~0.4 m in diameter by ~12 m in height ) , as up- and down - fluorescence intensity fluctuations ( fig . d shows how far the probe molecules can move in a certain range of time . in the crowded molecular environment , the d of probe molecules becomes much smaller due to the many obstacles that the probes encounter ( fig . 1c ) . since the fcs detection size is much larger than human chromosomes ( ~0.7 m ) , especially in height , we used indian muntjac dm cells . these cells have giant chromosomes whose size ( ~2 m in diameter ) is much larger than the detection volume . we obtained three stable dm cell lines coexpressing either the egfp - monomer , -trimer , or -pentamer , and h2b - mrfp1 . the three types of egfp are probe molecules for detecting mobility in the fcs detection volume . the egfp - trimer ( ~90 kda ) and -pentamer ( ~150 kda ) are used to examine the effect of molecular size on diffusion compared with the egfp - monomer ( ~30 kda ) . using these cell lines optimized for precise fcs measurement , we compared the d values of egfp probes in the cytoplasm , interphase chromatin , and mitotic chromosomes ( table 1 ) . surprisingly , while the chromosomes showed a highly condensed structure ( ~0.5 mm at nucleosome concentration ) , egfp molecules were able to move inside chromosomes , and the d of mitotic chromosomes was only 30% lower than that of interphase chromatin ( ~0.050.1 mm ) . even in the case of the egfp - trimer and -pentamer , their diffusion profiles appeared to be similar . this suggests that high protein mobility in dense chromosomes can be observed by fluorescent probes of larger size . our results in table 1 indicate that interphase chromatin and mitotic chromosomes have considerable chromatin accessibility , implying a novel underlying mechanism , especially in dense chromatin regions . * to characterize chromatin accessibility in living cells further , we reconstructed a chromatin environment in silico . to examine the mobility of egfp - pentamers ( ~150 kda ) under various chromatin conditions , we used metropolis monte carlo computer simulation with parameters obtained from our data ( ds and nucleosome concentrations ) . in this simulation , the nucleosome was represented as a sphere with a 10-nm hydrodynamic diameter ( 10-nm sphere in fig . 2 ) , and the egfp - pentamer molecule was represented as a sphere with a 13-nm hydrodynamic diameter ( 13-nm sphere in fig . 2 ) . first , the concentration of simulated nucleosomes was set to 0.1 or 0.5 mm ( fig . the 0.5 mm condition corresponded to highly condensed chromatin environment such as mitotic chromosomes . in these virtual chromatin environments , 2b ) and decided at random the order of the spheres to be moved ( step 2 ) . its displacement followed the three - dimensional normal distribution with the standard deviation ( sd = 6dt ) . we turned to the next sphere ( step 4 ) and repeated this process a certain number of times . finally , we traced the movement of the 13-nm spheres ( egfp - pentamer ) . at 0.1 mm of the nucleosomes , ( a ) the nucleosome is represented as a 10-nm red sphere and fixed in a restricted space at a concentration of 0.1 mm ( left ) or 0.5 mm ( right , corresponding to mitotic chromatin ) randomly but in a manner that avoids any overlap . ( b ) a simple scheme of the simulation procedure . for the details , see text . ( c ) tracing patterns of three 13-nm spheres ( egfp - pentamers ) under various conditions . at 0.1 mm of fixed 10-nm spheres ( nucleosomes ) , the 13-nm spheres ( egfp - pentamers ) move around freely ( left image ) . however , at 0.5 mm of fixed 10-nm spheres ( nucleosomes ) , the 13-nm spheres ( egfp - pentamers ) are unable to move far from their starting points ( middle image ) . in the environment with a fluctuation of 0.5 mm of the 10-nm spheres ( nucleosomes ) , the 13-nm spheres ( egfp - pentamers ) can move around freely ( right image ) , in contrast to the case of the fixed 10-nm spheres ( nucleosomes , middle image ) . each 10-nm red sphere ( nucleosome ) behaves like a dog on a leash . the three different temporal trajectories of the 13-nm spheres ( egfp - pentamers ) for 0.2 ms are indicated in blue , green , and red . ( d ) cartoons showing that a protein ( green ) is stacked in a confined space of fixed nucleosomes ( left ) , and the protein is able to move freely with fluctuation of the nucleosomes ( right ) . under the 0.5 mm condition , which corresponded to highly condensed chromatin environment , if the nucleosomes were fixed in space , the egfp - pentamer could not move far from the starting point ( fig . they were stacked in a confined space , thus causing a serious mobility problem ( fig . the nucleosomes were mobile , but their movements were restricted to a certain range , resembling a dog on a leash situation . in this dynamic environment , we observed apparent free diffusion of the egfp - pentamer , even in 0.5 mm nucleosomes ( fig . 2c strikingly , a 10- to 20-nm fluctuation of the nucleosomes was sufficient for the egfp - pentamers to diffuse freely . furthermore , even at the low concentration ( 0.10.4 mm ) of nucleosomes such as interphase chromatin environment , the fluctuation facilitated mobility of the egfp - pentamers . these simulation results suggest that nucleosome fluctuation facilitates chromatin accessibility for the diffusing protein ; see also references 32 and 33 by langowski group . since the nucleosomes in cells , which are linked by linker dna , fill more space with less freedom , nucleosome fluctuation may be critical for chromatin accessibility . the next obvious question was whether nucleosome fluctuation could be detected in living mammalian cells . for this purpose , we performed single particle imaging of nucleosomes in living cells . to visualize single nucleosomes , we fused photoactivatable ( pa)-gfp with histone h4 ( fig . 3a ) , a stable core histone component , and expressed the fusion protein in dm and hela cells at a very low level . for single nucleosome imaging , we used oblique illumination ( highly inclined and laminated optical sheet , hilo ) microscopy ( fig . 3b ) . normally , pa - gfp shows gfp fluorescence only after activation by a 405-nm laser ( fig . however , we unexpectedly found that a very low number of pa - gfp - h4 in the stable dm and hela cells were spontaneously activated without 405-nm laser activation ( fig . we observed single - step photobleaching of the pa - gfp - h4 dots , although photobleaching of multiple pa - gfp - h4 complexes should occur in multiple steps or gradually . the estimated number of pa - gfp - h4 was less than 5% of endogenous h4 . these observations indicate that each dot represents a single pa - gfp - h4 molecule in a single nucleosome . in addition , since free pa - gfp - h4 should have a much larger d value , we could trace only the pa - gfp - h4 fraction that was stably incorporated into nucleosomes . ( a ) generally , pa - gfp shows gfp fluorescence only after activation by a 405-nm laser ( right ) . however , a small fraction of pa - gfp - h4 was spontaneously activated without laser activation ( left ) and was used for our single - nucleosome imaging . ( b ) a schematic representation of the oblique illumination microscopy ( hilo system , ref . 37 ) . we used nikon laser tirf microscope system ti with sapphire 488 nm laser ( coherent ) . using a sheet light ( blue ) , a limited area in the cell ( c ) single - nucleosome image of a dm cell nucleus expressing pa - gfp - h4 . pa - gfp - h4 observed as a bright dot using the oblique illumination microscopy system . for single - nucleosome tracking , the free software , u - track , was used . the dots were fitted to an assumed gaussian point spread function to determine the precise center of signals with higher resolution . we recorded nucleosome signals in interphase chromatin and mitotic chromosomes in living dm cells at a video - rate ( ~30 ms / frame ) as a movie . for signal the dots were fitted to an assumed gaussian point spread function to determine the precise centers of the dots with higher resolution . we were able to analyze the behaviors of the nucleosomes over short periods of time , ranging from 0 to 0.18 s. notably , the displacement ( movement ) distributions of single nucleosomes of interphase and mitotic chromatin appeared similar ( ~50 nm per 30 ms ) . as a control , we measured the displacement of fluorescent beads ( 100 nm in diameter ) fixed on a glass surface . the average displacement values were much smaller than those observed in living cells , excluding major contribution of microscopy system drift . to exclude the possibility that the detected movement was derived from global motion of the nuclei or chromosomes , we measured centroid movements for many nucleosomes in the same time frame . these values were again much smaller than the movements of individual nucleosomes in living cells therefore , we concluded that most of the observed displacement was due to the local movement ( fluctuation ) of nucleosomes in living cells . in addition , we found that hela cells expressing a low level of pa - gfp - h4 showed considerable nucleosome mobility , suggesting that local nucleosome movement in mammalian cells is a general phenomenon . recently , the mcnally group also published single - nucleosome tracking data using h2b - egfp , which appear to be consistent with our single nucleosome tracking with pa - gfp - h4 . to strengthen our conclusion in reference 23 and for further characterization of nucleosome fluctuation the expression was driven by a different expression promoter ( psv40 ) from those in the previous study ( cdk1 and ef1- promoters ) . to show the displacement distribution , we measured approximately 20 000 signal points with single nucleosome tracking with h4-pa - gfp from seven dm cells ( figs . 3d , 4a and 4b ) , instead of the approximately 1 000 points previously reported with pa - gfp - h4 in reference 23 . some representative trajectories of the nucleosome movements and the displacement distributions in the x - y plain were also shown in figures 3d and 4b , respectively . the results also showed that nucleosome fluctuation was not affected by differences in the fusion position of pa - gfp and the expression promoter . ( a ) displacement ( movement ) histograms of single nucleosomes in interphase dm cells for 30 ms ( left ) , 60 ms ( center ) , and 90 ms ( right ; n = 20 000 in seven cells ) . the mean displacement standard error for the 30 ms time point was indicated . ( b ) distribution of nucleosome displacements in the x - y plane for 30 ms ( left ) , 60ms ( center ) , and 90 ms ( right ) . standard deviations ( sdx and sdy ) were shown . in panels ( a ) and ( b ) , the originally calculated displacement was in two dimensions . to obtain their 3d values , the two - dimensional values of displacement ( c ) displacement histograms of single egfp on a glass surface for 30 ms ( left ) , 60 ms ( center ) and 90 ms ( right ; n = 1 300 ) . the mean standard error for the 30 ms time point was indicated . ( d ) distribution of egfp displacements in the x - y plane for 30 ms ( left ) , 60ms ( center ) , and 90 ms ( right ; n = 1 300 ) . second , the displacement data of purified gfp fixed on a glass surface were obtained ( fig . the average displacement values were 12.8 0.2 nm per 30 ms , which was much smaller than those of h4-pa - gfp observed in living cells ( 59.0 0.2 nm per 30 ms in fig . third , to evaluate the contribution of whole cell or nuclear movements to the single nucleosome displacements , we again calculated the centroid movements for many nucleosomes in the same time flame ( illustrated in fig . 5a ) . the centroid movements ( 5.5 0.1 nm per 30 ms ) ( fig . 5b ) are much smaller than those in figure 4a and b , suggesting that the detected nucleosome movement is not derived from the global motion of cells or nuclei . ( a ) schematic representation for calculating centroid movement for many nucleosomes in the same time frame . the black arrows represent the movements of nucleosomes and the small arrow represents centroid movement in the frame . ( b ) displacement histogram ( left ) and distribution in the x - y plane ( right ) of centroid movement in the frame for 30 ms ( n = 350 in seven cells ) . ( c ) plots of the mean - square displacements ( msds ) with standard errors of single nucleosomes in interphase chromatin ( left ) and single egfps on a glass surface ( right ) from 0 to 0.18 s. the plots for single nucleosomes were fitted as an anomalous diffusion , suggesting that nucleosome movement supports a restricted diffusion model . error bars represent the standard errors of the mean value . in the panel of msd for single nucleosomes , the originally calculated msd were in two dimensions . to obtain their 3d values , the two - dimensional values of msd were multiplied by 1.5 ( 6dt/4dt ) . to analyze local nucleosome movement in dm cells further , the msd values ( m ) of nucleosomes in the dm cells and fixed egfp were plotted with their standard errors ( fig . 5c ) . the plots for the nucleosomes fitted well with the exponential equation , msd = 0.022 t ( fig . the msd values increased rapidly in a short time and the steepness decreased over time , consistent with previous data . again it was reported that movements of large chromatin domains tagged with laco arrays ( ~100 kb and above ) were on the 10 and 100 nm length scale for 30 ms ( e.g. , ref . we could not exclude the possibility that movements of larger domains might contribute to the local nucleosome fluctuation that we observed . monte carlo computer simulations suggested that nucleosome fluctuation facilitates the mobility of diffusing proteins in the environment ( fig . in addition , such nucleosome fluctuation can expose dna sequences to the surface of chromatin domains more often , while static regular folding structures , such as 30-nm chromatin fibers , can hide or mask most dna sequences . it should be emphasized that both facilitation of protein mobility and dna exposure lead to an increase in chromatin accessibility . this is in good agreement with our finding that tight cross - linking of nucleosomes blocked antibody accessibility and targeting in dense chromatin regions . recently , we suggested that interphase chromatin forms numerous compact chromatin domains resembling chromatin liquid drops in interphase cells . this chromatin liquid drops view is in line with predictions of the chromosome territory - interchromatin compartment ( ct - ic ) model . in the ct - ic model , each ct is built up from a series of interconnected , megabase - sized chromatin domains , which were originally identified using a pulse labeling as the dna replication foci . recent high - throughput 3c studies , such as hi - c and 5c , have also proposed physical packaging of genomic dna , termed topologically associating domains , topological domains or physical domains . local nucleosome fluctuation would play an especially important role in such compact chromatin domains because the effect of nucleosome fluctuation on the facilitation of chromatin accessibility is more significant in a compact chromatin environment . many biological processes , including transcription , dna repair , replication , and recombination , are based on scanning genomic dna and targeting . for example , in transcriptional regulation , the dynamic movement of nucleosome fibers assists with the targeting of transcription factors and complexes . dynamic irregular folding can easily form loops , facilitating interaction between promoters and enhancer sequences . since the flexible and dynamic nature of nucleosome fibers in living mammalian cells drives such biological processes , regulation of local nucleosome dynamics , possibly by histone modification and/or specific proteins

patients ' and clinicians ' esthetic demands have led to the development of high - technology processes such as glass infiltrated spinell and alumina , fused alumina or zirconia materials that are becoming increasingly advanced in their optical properties.1 - 3 these restorations allow diffuse transmission as well as specular reflectance of light , reproducing a depth of translucency and color mimicking that of natural teeth.4 among a variety of core materials , zirconia has been proved to possess better mechanical properties . however , it has poor translucency and therefore , it is difficult to satisfy the esthetic requirements . furthermore , the optical property of core materials plays an important role in matching the affected shade of the artificial restorations with the natural appearance of teeth . in addition , the optical properties of the material depend on the amount of crystals within the core matrix , their chemical nature , and the size of the particles.5,6 although the characteristics of coping is important for the clinical success , the variables that contribute to a durable and predictable restoration includes the luting material , its polymerization behavior and mechanism , the nature and thickness of the restoration itself and the light polymerizing unit.3 the rapid advancement in esthetic dental restorative techniques has dramatically increased the use of light curing units ( lcus ) to cure resin based materials . nowadays , boosted versions of high intensity quartz - tungsten - halogen ( qth ) , plasma arc ( pac ) and light emitting diode ( led ) lcus that possess higher light intensity than conventional lcus have been developed.7,8 however , doubts about the effectiveness of these lcus still in doubt . since , the zirconia restorations permit the use of both resin luting agents and conventional cements ( such as zinc phosphate or resin - modified glass ionomer cements ) for cementation , the polymerization of resin - based luting agents is important for long - term stability of the restoration . in most clinical cases , dualcuring resin cement is used when bonding ceramic to enamel and dentin.9 dual polymerization , the combination of light and chemical polymerization , provides a better conversion of monomers . this is important because inadequate polymerization is usually associated with poor mechanical and biological properties of the resin cements.10 moreover , adequate polymerization of resin cements depends not only on resin cement but also on the lcu intensity , wavelength of the visible light and polymerization time . therefore , it is important to evaluate the effect of different light curing units or different thicknesses of zirconia on light transmittance of the zirconia restoration . ( 2 ) the thickness of zirconia would not affect the light transmittance of zirconia . zirconia disc - shaped specimens ( 5 mm in diameter ) ( n = 21 ) ( in a1 shade ) were manufactured from pre - sintered y - tzp disc shaped blocks ( copran zr , whitepeaks dental gmbh & co. kg , essen , germany ) ; composition according to the manufacturer in wt% , zro2 , 88.0 - 96.0% ; y2o3 , 4.0 - 6.0% ; and al2o3 , less than 0.4% ) with yenadent d40 unit ( yena cnc milling systems , yena makina san . sti , istanbul , turkey ) and then sintered to full density in a high - temperature furnace ( protherm , b&d dental origin milling , ut , usa ) ; 1450 for 2 hours ) according to the manufacturer 's instructions . three different thicknesses ( 0.3 , 0.5 and 0.8 mm ) ( n = 7/group ) of zirconia were evaluated . before measurement of translucency , the zirconia discs were ultrasonically cleaned in distilled water for 10 minutes and then dried with compressed air . the thickness of the discs was measured using a digital micrometer ( mitutoyo manufacturing company ltd , kawasaki , japan ) . the power outputs from a qth lcu ( blue swan digital , dentanet , ankara , turkey ) , led lcu ( elipar freelight 2 , 3 m espe , st . paul , mn , usa ) and pac lcu ( plasmastar , sp-2000 , monitex , taiwan ) were approved by a hand - held radiometer ( demetron , kerr , orange , ca , usa ) . the irradiation was performed top through the zirconia specimens . then , the light transmission value of each thickness was measured by placing the disc on the aperture of the radiometer and recording the average of resultant light readings through the disc . two - way analysis of variance ( anova ) and tukey 's posthoc tests were performed to determine the effects of different zirconia thicknesses and lcus on light transmittance ( =.05 ) . 1 . statistical analysis demonstrated that different zirconia thickness and light curing unit had significant effects on light transmission percentage values ( p<.001 ) . furthermore , no significant two factor interaction between zirconia thickness and lcus was observed ( p<.177 ) . tukey 's post - hoc test revealed that there were significant differences between lcus ( p<.001 ) . additionally , the transmitted light with respect to lcu can be ranked as follows : led > pac > qth . according to the zirconia thickness , the percentage of light transmittance was ranked as follows : 0.3 mm > 0.5 mm > 0.8 mm ( p<.001 ) . as a result , the first null hypothesis was rejected as statistical analysis revealed differences in light transmittance percentage values after light curing with different lcus . it is known that inadequate polymerization of resin cements might be a problem especially under ceramic restorations.9 therefore , to reach maximum physical properties of resin cements ; the conversion rate should be as high as possible.11 in the present study , to simulate clinical conditions , the lcus were used at the top of the zirconia discs , where the end of the light guide was in contact with the discs . previous studies reported mechanical properties of led lcu polymerized resin composites are as well or better than some qth lcus.8,12 similarly , in the present study , light transmittance values obtained with led lcu were higher than that of qth lcus . furthermore , pac lcus are often discussed as an alternative to high - power led and qth lcus.13 a previous study by rasetto et al . indicated that , pac polymerization units had comparable light transmission values when compared with qth lcus.3 in accordance with these previous studies , the present study indicated comparable light transmittance values of pac lcus with led lcus which are higher than that of qth lcus . the special spectrum of the pac , which has very high light intensities ( 2250 50 mw / cm ) at certain wavelengths , might have caused this better outcome . a previous study by price et al . investigated the effects of different lcus on resin polymerization and the results were explained by a formula that indicated the total energy that reached the resin composite.14 with reference to that formula , when the pac lcu delivered 2200 mw / cm and was used for 10 s , the resin - based material under the restoration should have received 22 j / cm , after light transmission . moreover , when both the qth lcu which delivered 1000 mw / cm and the led lcu which delivered 1200 mw / cm were used for 20 s , the resinbased material under the restoration might have received 20 j / cm2 and 24 j / cm , respectively ( assuming that the same wavelengths are delivered ) . this could provide an explanation about the rank of lcus for the present study ( led > pac > qth ) . the second null hypothesis that the light transmittance of zirconia decreases when the thickness is increased should be rejected , as the different thicknesses of zirconia had a significant effect on light transmittance values of the tested zirconia specimens . this finding corroborates with the results of a previous pilot study by rasetto et al . , suggesting the decrease in light transmittance with the increase in thickness of the ceramic.3 in addition , a previous study by o'keefe et al . indicated that the thickness of the porcelain veneer was the primary factor affecting the light transmission of the restoration.15 traditional luting agents might provide adequate retention for cementation of zirconium oxide restorations . however , a previous study indicated that cementation with dual - cured luting agents is necessary for ensuring better retention and marginal adaptation.16 with the increase in zirconia thickness , the decreased light transmittance might have an effect on the mechanical properties of the cements . in the current study , uniform thicknesses of zirconia specimens however , in a clinical condition , the zirconia restoration thickness may vary at different regions of tooth . therefore , although the tip of the lcu seems to be in direct contact with the restoration surface , the distance between the tip and the surface of the luting material can vary markedly over the entire restoration . accordingly , the differences in restoration translucency might have an effect on the light transmission . consequently , the optical behavior of zirconia restorations with respect to thickness should also be taken into consideration when the resin - based material is selected for luting of these restorations . within the limitations of this in vitro study , the following conclusions could be drawn : percentage of transmitted light was dependent on lcu . moreover , light transmission through even thin zirconia copings appears to be achievable with led lcus when compared with pac and qth lcus.light transmission through the zirconia copings was reduced with increased coping thicknesses for all three lcus . percentage of transmitted light was dependent on lcu . moreover , light transmission through even thin zirconia copings appears to be achievable with led lcus when compared with pac and qth lcus . light transmission through the zirconia copings was reduced with increased coping thicknesses for all three lcus .

ureteropelvic anastomosis is the most important step in pyeloplasty and has a large bearing on its success rate . although continuous sutures are more watertight compared with interrupted sutures , the possibility of seeing the purse - string effect and tissue damage may be a concern . we present the results of our retrospective analysis of the effects and outcome of continuous and interrupted sutures during laparoscopic pyeloplasty . we retrospectively reviewed all patients who underwent laparoscopic pyeloplasty at our center from june 1998 to march 2012 . all procedures were performed by a single surgeon with > 3 years of prior laparoscopic suturing experience . twenty - two patients were excluded from the study : 8 patients who underwent transperitoneal but nondismembered pyeloplasty ( which included 3 redo nondismembered pyeloplasties ) , 9 patients who underwent retroperitoneoscopy , 2 patients who underwent redo dismembered pyeloplasty , and 3 infants who underwent laparoscopy - assisted pyeloplasty ( pelvis and ureter brought out through a 10-mm flank port for extracorporeal anastomosis ) . the initial 65 patients ( until december 2007 ) underwent anastomosis using interrupted sutures , and the latter 42 patients had continuous sutures . all patients had a preoperative ultrasonogram , intravenous urogram or computed tomography urogram , and diuretic renogram ; a sterile urine culture before surgery ; and an indwelling ureteric stent placed during surgery . patients were followed up with a urine culture and diuretic renogram at 6 months and then annually for 5 years . data on the operation time , suturing time , drain quantity , hospital stay , success rate ( based on renogram done at 6 months ) , complications , and the overall cost of the procedures were analyzed . the cost analysis was done using the methodology of converting historic cost into present values and applying average rates of inflation in india obtained from the consumer price index . the cost included the combined costs of hospital stay , operation charges , and drug charges . fvschedule , which is designed to calculate future values as they relate to current values . the statistical analysis using student t test to assess the level of significance was done with spss 20.0 software ( spss , inc./ibm , armonk , new york ) . while under general anesthesia , the patient was placed in the lithotomy position and a retrograde pyelogram was done to assess the length of the adynamic segment of the ureteropelvic junction ( upj ) and ureter . a stent was not placed at the same time because it would have been a hindrance for spatulation . four ports were used ( 10-mm port 3 cm above and lateral to the umbilicus for the camera ; two 5-mm ports in the midclavicular line in the subcostal region and iliac fossa , and the fourth port in the anterior axillary line for suction or irrigation ) ( figure 1 ) . for the transperitoneal approach , the proximal ureter was mobilized , with care taken to preserve the vascularity and crossing vessels if present . in patients with left upj obstruction , if the vascular pattern of the mesocolon was conducive and if the fat in the mesentery was minimal , the upj was dissected using the transmesocolic approach . the size of the suture used depended on the age of the patient : 4 - 0/5 - 0 polyglactin and 4 - 0/5 - 0 polydioxanone sutures were used for interrupted and continuous sutures , respectively . the posterior layer suturing was done initially ( figure 2 ) , followed by antegrade stent insertion ( figure 3 ) . sutures were equidistant in both the continuous and interrupted sutures . in both the continuous suture group and the interrupted suture group , postoperatively , the urethral catheter was removed on the second or third day once the urine cleared . the tube drain was removed if the drainage was < 20 ml per day and if there was no perirenal or intraperitoneal collection on ultrasonography . the procedure was defined as successful if there was an improvement or stabilization of the renal function and improved drainage ( based on the measurement of glomerular filtration rate [ gfr ] and t on isotope renogram postoperatively ) , along with relief of symptoms . while under general anesthesia , the patient was placed in the lithotomy position and a retrograde pyelogram was done to assess the length of the adynamic segment of the ureteropelvic junction ( upj ) and ureter . a stent was not placed at the same time because it would have been a hindrance for spatulation . four ports were used ( 10-mm port 3 cm above and lateral to the umbilicus for the camera ; two 5-mm ports in the midclavicular line in the subcostal region and iliac fossa , and the fourth port in the anterior axillary line for suction or irrigation ) ( figure 1 ) . for the transperitoneal approach , the proximal ureter was mobilized , with care taken to preserve the vascularity and crossing vessels if present . in patients with left upj obstruction , if the vascular pattern of the mesocolon was conducive and if the fat in the mesentery was minimal , the upj was dissected using the transmesocolic approach . the upj was dismembered and the ureter spatulated laterally using scissors without using diathermy . the size of the suture used depended on the age of the patient : 4 - 0/5 - 0 polyglactin and 4 - 0/5 - 0 polydioxanone sutures were used for interrupted and continuous sutures , respectively . the posterior layer suturing was done initially ( figure 2 ) , followed by antegrade stent insertion ( figure 3 ) . sutures were equidistant in both the continuous and interrupted sutures . in both the continuous suture group and the interrupted suture group , postoperatively , the urethral catheter was removed on the second or third day once the urine cleared . the tube drain was removed if the drainage was < 20 ml per day and if there was no perirenal or intraperitoneal collection on ultrasonography . the procedure was defined as successful if there was an improvement or stabilization of the renal function and improved drainage ( based on the measurement of glomerular filtration rate [ gfr ] and t on isotope renogram postoperatively ) , along with relief of symptoms . sixty - five patients had interrupted suturing and 42 patients had continuous suturing for pelviureteric anastomosis . twelve patients had crossing vessel ( 7 in the interrupted suturing group and 5 in the continuous suturing group ) . six patients had secondary renal pelvic calculi ( 4 in the interrupted suturing group and 2 in the continuous suturing group ) . patient demographic characters and preoperative gfr the mean operative time was 219 minutes ( range , 180240 ) for the interrupted suturing group patients and 186 minutes ( range , 135225 ) for the continuous suturing group patients . in the interrupted suturing group , anastomosis was done using 4 - 0 or 5 - 0 vicryl interrupted sutures for the pelviureteric anastomosis . in the continuous suturing group , continuous 4 - 0 or 5 - 0 polydioxanone sutures were used for the anastomosis . operative and postoperative details the mean drain output ( 108 ml vs 175 ml ) , drain tube retention ( 2.8 days vs 4.2 days ) , and duration of hospitalization ( 2.7 days vs 4.6 days ) were significantly less in the continuous suturing group compared with the interrupted suture group ( table 2 ) . the overall cost of the procedure was reduced among the continuous suturing group ( mean 59 530 indian rupees [ 1101 usd ] vs 55 760 indian rupees [ 1031 usd ] ; p < .001 ) . the complication rate was 15.38% in the interrupted suturing group and 7.14% in the continuous suturing group ( tables 3 and 4 ) . the end of the stent extruded through the suture line , and in the other patient the stent came out during removal of the urethral catheter . in the first patient , the stent was replaced , but the patient developed a stricture and underwent an open re - pyeloplasty . in the other patient , stent replacement was also done , and there was also an unfavorable outcome . fungal pyelonephritis developed in one patient in the continuous suturing group ; he was both obese and diabetic . he had extravasation and prolonged ileus , which resulted in partial anastomotic disruption and he required an open re - pyeloplasty . one child in the continuous suturing group had omental protrusion through the drain site after drain removal , which required reduction under anesthesia . complications grading ( clavien dindo classification ) the mean duration of follow - up was 78 months ( range , 156 months to 6 months ) . overall , 5 patients had failure of pyeloplasty . among them , 3 patients were in the interrupted suturing group and 2 were in the continuous suturing group . among the 3 failed pyeloplasties in the interrupted suturing group , one patient preferred repeated long - term ureteric stenting and 1 patient underwent open re - pyeloplasty . one 3-year - old child who had gross hydronephrosis with a gfr of 15 ml / min had deterioration of gfr ( 3 ml / min ) over 2 years after pyeloplasty . the 2 failed pyeloplasty in continuous suturing group , 1 had laparoscopic redo pyeloplasty and 1 patient had open redo pyeloplasty . pyeloplasty comprises most of the laparoscopic procedures done at our center ( 22% of all the laparoscopic urological procedures performed at our center ) . we use freehand suturing for laparoscopic pyeloplasty . for the initial 65 patients ( 60% ) , interrupted sutures with intracorporeal knotting were used ( interrupted suturing group ) , and for the subsequent 42 patients ( 40% ) , continuous sutures using polydioxanone ( nonbraided suture ) were used ( continuous suturing group ) . the effect of continuous and interrupted sutures with regards to tissue vascularity and collagen damage is controversial . most of the experimental studies were done in laboratory animals and mainly involved bowel anastomoses . sarin et al concluded that there was no significant increase in the incidence of leak or tissue damage with continuous sutures , although previous reports suggest that there can be vascular compromise with continuous suturing . margo et al compared continuous and interrupted suturing techniques for ureteroneocystostomy in cats and inferred that continuous suturing causes more tissue ischemia and necrosis . kass et al have suggested using interrupted sutures , especially for pyeloplasty in infants , to prevent the purse - string effect . another difficulty with using continuous sutures in laparoscopy is maintaining appropriate tension during the procedure . when braided polyglactin sutures are used for continuous suturing , tightening of the suture after 3 or 4 continuous sutures may become difficult . the memory of nonbraided sutures can sometimes be a hindrance , but the advantage is that the sutures could be appropriately tightened at any stage . haudart et al studied continuous and interrupted sutures in bowel anastomosis and concluded that there is no difference in the leak rate between the 2 types of anastomosis . bruch et al reported that single - layer continuous suturing could be accomplished in bowels in less time and at a lower cost . in a study on porcine models , leiber et al concluded that interrupted sutures were more often associated with extensive muscular fibrosis compared with continuous suturing . the improvement in gfr among patients in the interrupted suturing group was 4.6 ml / min and in the continuous suturing group was 4.7 ml / min . however , reports of large studies of laparoscopic pyeloplasty by rassweiler et al and teber et al , wherein continuous sutures have been used , show comparable outcomes and complication rates . indicate that the suturing time is not significantly different between standard suturing and self - retaining barbed sutures . self - retaining barbed sutures are more frequently used for vesicourethral anastomosis during laparoscopic radical prostatectomy . the mean suturing time was considerably reduced using continuous sutures , from 64 minutes ( range , 5580 ) to 55 minutes ( range , 5065 ) ( p < .001 ) . papalia et al and shao et al have also mentioned similar findings in a study comparing interrupted and continuous sutures . however , papalia et al mentioned that the reduction in operative time was related to the combined use of continuous suture and retrograde stenting . drain volume and its relation to the continuous or interrupted suture were not studied . in our study , all patients had antegrade stenting . if a large redundant pelvis needs to be sutured , the time taken would be longer . the presence of secondary calculi may also prolong the procedure . the time taken for stenting may also vary from patient to patient . recently , a few reports of robot - assisted pyeloplasty have been published and show no difference in all the parameters , even though suturing may be easier . the volume of drainage was significantly less in the continuous suturing group , and hence the drain tube was retained for a shorter period ( mean , 2.8 days for the continuous suturing group vs 4.2 days for the interrupted suturing group , p < .001 ) . this may be caused by less leakage from the suture line with the use of continuous sutures . reduced drain output also had an impact on the duration of hospitalization ( 2.7 days vs 4.6 days in favor of continuous sutures ; p < .001 ) , because many of the patients preferred to be discharged after the drain was removed . the overall cost of the procedure was less among the continuous suturing group patients ( mean , 59 530 indian rupees or 1101 usd vs 55 760 indian rupees or 1031 usd ; p < .001 ) . the reduced overall cost of the surgery for the continuous suturing group patients may be attributed to the reduced duration of surgery and hospitalization and fewer complications . shao et al mention that the incidence of urine leak was negligible in the continuous suturing group . however , the mean hospital stay was 6 days in their study vs a mean stay of 2.7 days in the continuous suturing group in our study . similarly , papalia et al mention that one patient had a significant leak in the continuous suturing group . however , no comparison was made between the volume of drainage output and its impact on the hospital stay , as in our study . the urinary leak and drain output are more relevant to the transperitoneal approach than the retroperitoneal approach . in the study by shao et al , however , all of the procedures were done by a single surgeon with 3 years of laparoscopic suturing experience . ureteropelvic anastomosis using the continuous suturing technique has a comparable success rate with that using interrupted suturing . the time taken for suturing , postoperative drain output , and duration of drainage were lower , hence the duration of hospitalization was significantly less with continuous suturing . continuous suturing may be preferred to interrupted suturing for ureteropelvic anastomosis in patients undergoing laparoscopic pyeloplasty .

romantic relationships plays an important role in the development of many critical tasks of adolescence and young adulthood . healthy dating experiences are associated positive adjustment and elevated self - esteem whereas aggressive dating experiences are associated with negative outcomes such as internalizing and externalizing behaviors . one quarter to over one - half of dating relationships and significant others continued in these relationships despite the abuse . aggression in romantic relationship is a continuing factor for breakups , physical assault , kidnapping , acid throwing , rape and even murder . relational aggression also includes flirting with others to make a romantic partner jealous , threatening to breakup with a partner if the partner will not comply , or giving a partner the silent treatment when angry predisposed at risk for adjustment difficulties including peer rejection , depression and maladaptive personality features . , college students who had relational aggression within their romantic relationships were found to be less trusting of their current or most recent partner , frustrated , jealous , and clingy in their romantic relationships . approximately 10% of american high - school students experienced physical aggression from a dating partner over a 12-month period they also experienced mood symptoms . the individuals who reported perpetrating relational aggression in their romantic relationships believed that aggression was most acceptable ( social - cognitive risk ) , whereas individuals who reported experiencing relational aggression ( as victims ) were more likely to indicate that their self - worth was contingent on relationships and that romantic relationships were very important to them . studies have also documented the gender differences in the experience and expression of relational aggression . gender was found to moderate the association between anxiety and reactive relational aggression , males with high anxiety showed higher levels of reactive relational aggression than males with low anxiety . socially based negative cognitive errors also mediated the association between reactive relational aggression and anxiety . in men , higher physical aggression was associated with lower agreeableness and lower extraversion . in women , higher physical aggression was associated with higher conscientiousness , more depression , lower bar - on eqi stress management and higher adaptability . relational aggression was associated with lower agreeableness and a lower bar - on eqi overall score for both men and women . in men , higher relational aggression was also associated with lower conscientiousness , and lower bar - on eqi interpersonal factor scores . highly avoidant individuals were more likely to engage in hostile relational conflict with their partners and feel significantly less satisfaction from their adult romantic relationships than their nonavoidant counterparts . avoidance and hostility have influence on relationship satisfaction levels rather than anxiety and validation media has highlighted many such cases in the capital of india and other metro cities . variety of adverse psychological consequences often accrue from experiencing relational aggression , with anxiety and trauma symptoms from experiencing physical relationship aggression . the present work explored the influence of personality and relationship satisfaction in aggression among romantic partners . to study the personality correlates of aggression in romantic relationships among youth . to explore the relationship of aggression with the followinga . neuroticism , b. extraversion , c. openness , d. agreeableness and e. conscientiousnessto study the gender differences in aggression in romantic relationshipsto find the relationship satisfaction with aggression and personality characteristics in romantic relationships . to explore the relationship of aggression with the following a. neuroticism , b. extraversion , c. openness , d. agreeableness and e. conscientiousness to study the gender differences in aggression in romantic relationships to find the relationship satisfaction with aggression and personality characteristics in romantic relationships . participants meeting the inclusion ( participant in a mutually consenting heterosexual relationship ) and exclusion criteria ( married person and presence of psychiatric distress ) were taken into the study . socio - demographic schedule ( sds ) ( developed for the study ) , 2 . checklist for psychological aggression ( cpa ) ( developed for the study ) , 3 . the revised conflict tactics scale ( cts2 ) ( straus , hamby , boney - mccoy , and sugarman , 1996 ) , 5 . relationship satisfaction scale ( ras ) ( hendrick , s.s , 1988 ) . the schedule was developed by the investigator to obtain information about sociodemographic characteristics of participants the tool had a section on information relevant to the current relationship of the participant which included current relationship status , duration of current relationship , sexual activeness in current relationship and disclosure of current relationship . the checklist was developed by the investigator to obtain information about different verbal and nonverbal aggressive behaviors reported in romantic relationships . the psychological aggression scale in cts2 had the least internal consistency of all scales as the items were selected to increase the diversity of content of the scale rather than to increase the internal consistency reliability . these two subsumed direct and indirect aggressive behaviors , commonly reported in the context of relationships . the tool has face validity as checked by experts in the are neo ffi : a concise measure of the five major dimensions of personality . it consists of five 12 item scales that measure each domain of a normal adult personality . a revised measure of psychological and physical attacks on a partner in a marital , cohabiting or dating relationship ; and also use of negotiation . the theoretical basis of cts is conflict theory which assumes that conflict is an inevitable part of all human association , whereas violence as a tactic to deal with conflict is not . cts has been used successfully in at least 20 countries , including india ( kumagai and straus , 1983 ) . it consists of seven items with a five point likert rating scale of low to high satisfaction , with items 4 and 7 reverse scored . majority of the participants were contacted in their respective undergraduate or postgraduate colleges or workplaces . seven colleges were contacted out of which four permitted data collection . in the two colleges that granted permission , the investigator announced the research content to the students , explaining how important romantic relationships are in the stage of young adulthood and how there existed multiple problems in relationships . they were also explained the importance of the study and how it will contribute to our scientific knowledge about romantic relationships . after taking written informed consent , individuals were administered the questionnaires ( socio demographic schedule , relationship assessment scale , neo five factor inventory , conflict tactics scale 2 and checklist for psychological aggression ) in person . the investigator clarified participants doubts regarding certain items and provided standard meanings in case of difficulty in comprehending the meaning of certain items . the responses were coded for computer analysis . using the statistical package for social sciences , parametric and nonparametric tests were used . correlation analysis was carried out to examine the association between personality characteristics , relationship satisfaction and aggression . neuroticism , b. extraversion , c. openness , d. agreeableness and e. conscientiousnessto study the gender differences in aggression in romantic relationshipsto find the relationship satisfaction with aggression and personality characteristics in romantic relationships . to explore the relationship of aggression with the following a. neuroticism , b. extraversion , c. openness , d. agreeableness and e. conscientiousness to study the gender differences in aggression in romantic relationships to find the relationship satisfaction with aggression and personality characteristics in romantic relationships . participants meeting the inclusion ( participant in a mutually consenting heterosexual relationship ) and exclusion criteria ( married person and presence of psychiatric distress ) were taken into the study . socio - demographic schedule ( sds ) ( developed for the study ) , 2 . checklist for psychological aggression ( cpa ) ( developed for the study ) , 3 . the revised conflict tactics scale ( cts2 ) ( straus , hamby , boney - mccoy , and sugarman , 1996 ) , 5 . the schedule was developed by the investigator to obtain information about sociodemographic characteristics of participants the tool had a section on information relevant to the current relationship of the participant which included current relationship status , duration of current relationship , sexual activeness in current relationship and disclosure of current relationship . the checklist was developed by the investigator to obtain information about different verbal and nonverbal aggressive behaviors reported in romantic relationships . the psychological aggression scale in cts2 had the least internal consistency of all scales as the items were selected to increase the diversity of content of the scale rather than to increase the internal consistency reliability . these two subsumed direct and indirect aggressive behaviors , commonly reported in the context of relationships . the tool has face validity as checked by experts in the are neo ffi : a concise measure of the five major dimensions of personality . it consists of five 12 item scales that measure each domain of a normal adult personality . internal consistency values range from 0.74 to 0.89 . a revised measure of psychological and physical attacks on a partner in a marital , cohabiting or dating relationship ; and also use of negotiation . the theoretical basis of cts is conflict theory which assumes that conflict is an inevitable part of all human association , whereas violence as a tactic to deal with conflict is not . cts has been used successfully in at least 20 countries , including india ( kumagai and straus , 1983 ) . it consists of seven items with a five point likert rating scale of low to high satisfaction , with items 4 and 7 reverse scored . the schedule was developed by the investigator to obtain information about sociodemographic characteristics of participants the tool had a section on information relevant to the current relationship of the participant which included current relationship status , duration of current relationship , sexual activeness in current relationship and disclosure of current relationship . the checklist was developed by the investigator to obtain information about different verbal and nonverbal aggressive behaviors reported in romantic relationships . the psychological aggression scale in cts2 had the least internal consistency of all scales as the items were selected to increase the diversity of content of the scale rather than to increase the internal consistency reliability . these two subsumed direct and indirect aggressive behaviors , commonly reported in the context of relationships . the tool has face validity as checked by experts in the are neo ffi : a concise measure of the five major dimensions of personality . it consists of five 12 item scales that measure each domain of a normal adult personality . a revised measure of psychological and physical attacks on a partner in a marital , cohabiting or dating relationship ; and also use of negotiation . the theoretical basis of cts is conflict theory which assumes that conflict is an inevitable part of all human association , whereas violence as a tactic to deal with conflict is not . cts has been used successfully in at least 20 countries , including india ( kumagai and straus , 1983 ) . it consists of seven items with a five point likert rating scale of low to high satisfaction , with items 4 and 7 reverse scored . majority of the participants were contacted in their respective undergraduate or postgraduate colleges or workplaces . seven colleges were contacted out of which four permitted data collection . in the two colleges that granted permission , the investigator announced the research content to the students , explaining how important romantic relationships are in the stage of young adulthood and how there existed multiple problems in relationships . they were also explained the importance of the study and how it will contribute to our scientific knowledge about romantic relationships . after taking written informed consent , individuals were administered the questionnaires ( socio demographic schedule , relationship assessment scale , neo five factor inventory , conflict tactics scale 2 and checklist for psychological aggression ) in person . the investigator clarified participants doubts regarding certain items and provided standard meanings in case of difficulty in comprehending the meaning of certain items . incomplete response sheets were excluded from analysis . the responses were coded for computer analysis . using the statistical package for social sciences , parametric and nonparametric tests were used . correlation analysis was carried out to examine the association between personality characteristics , relationship satisfaction and aggression . 25.5% ( 28 ) were in the age range of 18 - 22 years ; 14.5% ( 16 ) in the age range of 28 - 32 years ; the majority of 60% ( 66 ) in the age range of 23 - 27 years . there were 24 ( 21.8% ) undergraduates , 69 ( 62.7% ) post graduates and 17 ( 15.5% ) into professional courses . dating ( dating one or more than one person ) relationship and 58 ( 52.7% ) reported being in acommitted ( which included committed , engaged and cohabiting ) relationship . seventy - one ( 64.5% ) , reported being in the current relationship for more than 1 year ; 24 ( 21.8% ) were in the relationship from 6 - 12 months and 15 ( 13.6% ) reported being in a relationship for less than 6 months . in the entire sample , 86 ( 78.18% ) of these , 34 ( 31.1% ) engaged in penetrative sexual practices and 43 ( 39.1% ) were into nonpenetrative sexual activities like kissing , fondling , etc . disclosure of the current relationship also significantly varied in the group , with 48 ( 43.6% ) participants reportedly having disclosed their relationship status to their friends , 29 ( 26.4% ) had disclosed to their family ; and 22 ( 20% ) were open to everybody . eleven participants ( 10% ) had not disclosed their current relationship status to anybody . in the entire group , 31 ( 28.2% ) there was significant difference between male and female respondents with respect to sexual aggression ( f=7.11 , p<0.01 level ) , i.e. , male respondents reported more aggression ( m=2.53 , sd=3.62 ) than female respondents ( m=1.11 , sd=1.53 ) . significant difference was also found between genders for the scale on physical injury ( f=5.72 , p<0.01 level ) . males reported more physical injury ( m=1.32 , sd=2.93 ) than females ( m=0.33 , sd=0.77 ) . significant difference was found for the negotiation skill among those who were sexually active and those who were not sexually active in the current relationship ( f=5.40 , p<0.01 ) . those who did not engage in any sexual activities in their relationship reported better negotiation skills ( m=8.16 , sd=3.27 ) compared to those who engaged in sexual activities . among those who were sexually active in the relationship , difference was seen between those who engaged in penetrative ( m=10.30 , sd=2.54 ) and those who engaged in nonpenetrative sexual activities ( m=10.06 , sd=2.44 ) sexual aggression was found to be significantly different between those who engaged in penetrative and those who engaged in nonpenetrative sexual activities ( f=12.92 , p<0.01 ) . individuals who engaged in penetrative sexual activities reported more sexual aggression ( m=3.34 , sd=3.39 ) than those who engaged in nonpenetrative sexual activities ( m=1.20 , sd=2.36 ) . in the domain of psychological aggression , passive aggression ( procrastination , irritability , stubbornness and hostile attitude ) was found to be significantly different between those who were sexually active and those who were not ( f=5.68 , p<0.01 ) . those who engaged in penetrative sexual activities reported more passive aggression ( m=6.09 , sd=4.46 ) than those engaged in nonpenetrative sexual activities ( m=3.81 , sd=3.69 ) and those who were not sexually active in their current relationship ( m=3.04 , sd=3.51 ) at 0.05 and 0.01 level of significance . fifty - two ( 47.3% ) participants reported being in a dating ( dating one or more than one person ) relationship and 58 ( 52.7% ) reported being in acommitted ( which included committed , engaged and cohabiting ) relationship . seventy - one ( 64.5% ) , reported being in the current relationship for more than 1 year ; 24 ( 21.8% ) were in the relationship from 6 - 12 months and 15 ( 13.6% ) reported being in a relationship for less than 6 months . in the entire sample , 86 ( 78.18% ) of these , 34 ( 31.1% ) engaged in penetrative sexual practices and 43 ( 39.1% ) were into nonpenetrative sexual activities like kissing , fondling , etc . disclosure of the current relationship also significantly varied in the group , with 48 ( 43.6% ) participants reportedly having disclosed their relationship status to their friends , 29 ( 26.4% ) had disclosed to their family ; and 22 ( 20% ) were open to everybody . eleven participants ( 10% ) had not disclosed their current relationship status to anybody . in the entire group , 31 ( 28.2% ) reported using a psychotropic drug ( alcohol , cannabis , etc . , ) and 79 ( 71.8% ) did not use any psychotropic drug . there was significant difference between male and female respondents with respect to sexual aggression ( f=7.11 , p<0.01 level ) , i.e. , male respondents reported more aggression ( m=2.53 , sd=3.62 ) than female respondents ( m=1.11 , sd=1.53 ) . significant difference was also found between genders for the scale on physical injury ( f=5.72 , p<0.01 level ) . males reported more physical injury ( m=1.32 , sd=2.93 ) than females ( m=0.33 , sd=0.77 ) . significant difference was found for the negotiation skill among those who were sexually active and those who were not sexually active in the current relationship ( f=5.40 , p<0.01 ) . those who did not engage in any sexual activities in their relationship reported better negotiation skills ( m=8.16 , sd=3.27 ) compared to those who engaged in sexual activities . among those who were sexually active in the relationship , difference was seen between those who engaged in penetrative ( m=10.30 , sd=2.54 ) and those who engaged in nonpenetrative sexual activities ( m=10.06 , sd=2.44 ) sexual aggression was found to be significantly different between those who engaged in penetrative and those who engaged in nonpenetrative sexual activities ( f=12.92 , p<0.01 ) . individuals who engaged in penetrative sexual activities reported more sexual aggression ( m=3.34 , sd=3.39 ) than those who engaged in nonpenetrative sexual activities ( m=1.20 , sd=2.36 ) . in the domain of psychological aggression , passive aggression ( procrastination , irritability , stubbornness and hostile attitude ) was found to be significantly different between those who were sexually active and those who were not ( f=5.68 , p<0.01 ) . those who engaged in penetrative sexual activities reported more passive aggression ( m=6.09 , sd=4.46 ) than those engaged in nonpenetrative sexual activities ( m=3.81 , sd=3.69 ) and those who were not sexually active in their current relationship ( m=3.04 , sd=3.51 ) at 0.05 and 0.01 level of significance . personality is known to play an important role in determining the likelihood of engaging in an aggressive act in an interpersonal context . most of the participants were from nuclear families indicating the current pattern of indian families . 31.1% engaged in penetrative sexual practices and 43 ( 39.1% ) were into nonpenetrative sexual activities like kissing , fondling etc . those who did not engage in any sexual activities in their relationship reported better negotiation skills ( m=8.16 , sd=3.27 ) compared to those who engaged in sexual activities . thirty - four percent had sexual intercourse in the previous 3 months and 39% did not use any protection during the sexual act . youth are particularly vulnerable to becoming involved in relationships that include dating violence and risky sexual activity . violent victimization was more likely to occur in romantic relationships that included sexual intercourse : 37% of the respondents reporting sexual relationships experienced at least one form of verbal or physical violence victimization , compared with 19% of those reporting relationships with no sexual intercourse . trends in the current study also presence of physical aggression among men , like pushing , grabbing or kicking the partner . sexual aggression was found to be significantly different between those who engaged in penetrative and those who engaged in nonpenetrative sexual activities . individuals who engaged in penetrative sexual activities reported more sexual aggression than those who engaged in nonpenetrative sexual activities . those who engaged in penetrative sexual activities reported more passive aggression ( procrastination , irritability , stubbornness and hostile attitude ) than those engaged in nonpenetrative sexual activities and those who were not sexually active in their current relationship ( m=6.09 , sd=4.46 ) males reported more physical injury ( m=1.32 , sd=2.93 ) than females ( m=0.33 , sd=0.77 ) . india 's social system is patriarchal and females are socialized to be subordinate and believe that males have a right to control them . recent literature on urban , upward mobile women aka alpha females state that women also show same amounts of aggression in relational contexts . women reported that they attacked their partners while under the influence of emotional states of intense anger , men said they did so in response to aggression received . a high percentage of casual daters reported more difficulties in their relationships than involved daters . those who were open to their families about their relationship status reported less aggression in all its styles . individuals have unique support needs and preferences for the amount of support they receive from their networks . therefore those who were open about their relationship to everybody reported more aggression than those who were open to their families alone . agreeableness was also negative correlated with psychological aggression and mildly negatively correlated with physical aggression , sexual aggression , passive verbal aggression and physical injury . conscientiousness was found to be negatively correlated with psychological aggression and verbal passive aggression table 2 . . verbal passive and verbal active sexual form of aggression were found to be mildly negatively correlated with relationship satisfaction [ table 3 ] . neurotic partners rated their relationship as more costly and as diverging more from their idealistic standards . participants who had experienced psychological aggression or were physically assaulted reported a lower level of relationship satisfaction than those who did not experience psychological aggression or were physically assaulted respectively . relationship between relationship satisfaction and personality characteristics relationship between personality characteristics and aggression styles relationship between aggression styles and relationship satisfaction sample consisted only of urban , college educated or working men and women , therefore the representation of the sample is limited . information regarding any previous romantic relationship would have given additional information about aggression in current relationship . the present study highlighted the prevalence and different forms of aggression in romantic relationships in youth . personality characteristics like , openness , agreeableness and conscientiousness were positively correlated with relationship satisfaction and negatively correlated with aggression in romantic relationships . significant proportion are in sexual activities , there is a need to educated regarding safer sexual behavior , use of emergency contraceptive and anger management . a further exploration of these variables may be significant in designing interventions for youth at an early stage .

pe is universally defined as hypertension and significant proteinuria developed at or after 20 weeks of pregnancy in an otherwise normotensive woman [ 13 ] . pe is a multisystem disorder which complicates 314% of all pregnancies and about 58% of pregnancies in the united states . the disease is mild in 75% of cases in the united states , and severe in 25% of cases . this finding might be related to an increased prevalence of predisposing disorders , such as maternal age , chronic hypertension , diabetes , prepregnancy obesity , and multiple births [ 57 ] . overall , 10%15% of direct maternal deaths are associated with pe in low- and middle - income countries and the proportion is similar in high - income countries [ 8 , 9 ] . furthermore , severe pe is a major cause of maternal morbidity ( i.e. , stroke and liver rupture ) and negative long - term outcomes ( i.e. , cardiovascular disease and diabetes mellitus ) as well as adverse perinatal effects , such as prematurity and intrauterine growth restriction [ 5 , 10 ] . while much research has been devoted toward this topic , the cause of pe still remains elusive . two different theories have emerged : ( 1 ) vascular - ischemic origin of pe and ( 2 ) impaired immune response . a current hypothesis unifies these concepts where an altered immune response leads to disturbed placental function early in pregnancy with consequent syncytiotrophoblast ischemia and shedding of products that extensively damage endothelial integrity . this in turn results in an exponential production of multiple factors such as cytokines and growth factors leading to the clinical manifestations of pe . how the immune response can activate the cascade process is still unknown but it is proposed to act in synergy with additional exacerbating factors such as predisposing maternal and ambient factors . the most widely studied serum markers for pe , to date , are vascular endothelial growth factor ( vegf ) and placental growth factor ( plgf ) . antagonists include soluble fms - like tyrosine kinase 1 ( sflt-1 , also known as svegfr1 ) , and soluble endoglin ( seng ) . sflt-1 is a truncated splice variant of the membrane - bound flt1 ; it consists of the extracellular binding domain without the intracellular signaling domain . several studies demonstrated the association of increased sflt-1 with pe [ 14 , 15 ] . evidence for the involvement of sflt1 in the occurrence of pe was initially provided by an animal model in which gravid rats were infected with a recombinant adenovirus encoding sflt1 and compared to animals infected with the empty control adenovirus . the animals infected with sflt1 developed a syndrome highly reminiscent of human pe : hypertension and proteinuria due to a glomerular endotheliosis [ 14 , 16 ] . similarly , seng is a truncated form of receptor for two subtypes of transforming growth factor beta ( tgf ) specifically , tgf1 and tgf2 which are highly expressed by vascular endothelial cells and syncytiotrophoblasts . like sflt1 , soluble endoglin ( seng ) is an antiangiogenic factor capable of inhibiting capillary tube formation in vitro . soluble eng also increases vascular permeability ; overexpression of both sflt1 and seng in rodents results in capillary permeability in the lungs , kidneys , and liver . overexpression of both seng and sflt1 in pregnant rats develops nephrotic - range proteinuria , severe hypertension , biochemical evidence of hellp ( h for hemolysis , el for elevated liver enzymes , and lp for low platelet count ) , and intrauterine growth restriction of the pups [ 19 , 20 ] . plgf concentration during pregnancy increases during the first 30 weeks of gestation , and then decreases . longitudinal studies have shown that a relatively low plgf concentration ( which could be explained by a high sflt1 concentration ) is also a characteristic feature of pe . decreased levels of urinary plgf and pigf : sflt-1 ratio during mid - gestation have been proposed as a predictive model for development of clinical pe , and quantification of sflt-1 levels has correlated directly with severity of disease and inversely with time to onset of proteinuria and hypertension . the exact molecular basis for placental dysregulation of these factors remains unknown but hypoxia is likely an important regulator . other factors such as alterations in the renin - angiotensin - aldosterone axis , immune maladaption , excessive shedding of trophoblast debris , oxidative stress , and genetic factors likely contribute to the pathogenesis of the abnormal placentation . to date the most successful treatment for pe is delivery . cathecol - o - methyltrasferase ( comt ) catalyzes the o - methylation of various circulating hormones such as catecholamines and catecholestrogens [ 23 , 24 ] . in the placenta , comt metabolizes certain forms of circulating estradiols to the molecule 2-methyl estradiol ( 2-me ) . this estradiol metabolite has several effects , one being the destabilization of hypoxia - inducible factor- ( hif- ) 13 in the cytoplasm . hifs are heterodimeric proteins that mediate the effects of hypoxia on gene expression by upregulating transcription of target genes including sflt1 . this role of comt in maintaining oxygen balance suggests that comt might somehow be involved in the pathogenesis of pe . to better elucidate the role of comt in pe , a genetic comt knockout ( comt/ ) mouse model was recently developed . interestingly , pregnant comt/ mice developed a pe - like phenotype characterized by proteinuria , increased blood pressure , and histopathological changes in the placenta and kidney . this phenotype was accompanied by lower plasma concentrations of 2-me and higher placental protein levels of hif-1a . , these results suggest that comt and 2-me deficiency might play a significant role in the development of pe . although 2-me was found to suppress hif-1a and sflt1 , recent experiments suggest that there might be several other mechanisms through which 2-me promotes vascular health . lastly , recent evidence suggests that 2-me is necessary for cytotrophoblast invasion of the maternal decidua and therefore contributes to the prevention of pe by promoting normal placental vascular formation . further studies on this topic will be required to ascertain a more exact mechanism of action . the complement system , composed of over 30 proteins that act in concert to protect the host against invading organisms , initiates inflammation and tissue injury and normally has a protective role toward off - infection . complement activation promotes chemotaxis of inflammatory cells and generates proteolytic fragments that enhance phagocytosis by neutrophils and monocytes . there is scant information about complement activation in normal and abnormal human pregnancy . during normal gestation , serum levels of c3 , c4 , and total hemolytic complement ( ch50 ) studies have shown significant elevations in levels of bb , c3a , c4d , and soluble c5b-9 in preeclampsia , indicating excess activation of both the classical and alternative complement pathway . conducted a large prospective study in human pregnancy to investigate whether elevated levels of complement activation fragment bb ( reflecting alternative complement pathway activation ) at a single point in early pregnancy ( less than 20 weeks gestation ) were predictive of preeclampsia later in pregnancy . adjusted for other risk factors , women with higher levels of bb in early pregnancy were almost four times more likely to develop preeclampsia later in pregnancy compared with women with levels less than the top decile in early pregnancy . products of complement pathway have been found in deciduas , chorionic villi , and as subendothelial deposits in vessel walls in normal and preeclampsia [ 34 , 35 ] . a recent study reported that the presence in c5b-9 mac on trophoblasts was associated with fibrin deposits at sites of villous injury in vivo in normal placentas , but especially in placentas from pregnancies complicated by iugr or preeclampsia . it has been demonstrated that in normal pregnancies , complement regulatory proteins that are highly expressed on trophoblast membranes prevent excessive complement activation ( membrane cofactor protein [ mcp ( cd46 ) ] , decay accelerating factor [ daf ( cd55 ) ] , and cd59 ) ) as well as circulating complement regulatory proteins ( complement factor h [ cfh ] , c4b binding protein , and complement factor i [ cfi ] ) [ 37 , 38 ] . defective regulation of the complement system allows for the excessive complement activation that leads to placental damage , abnormal placental development , generalized endothelial activation , and the release of antiangiogenic factors toxic to the fenestrated endothelium of glomeruli , the choroid plexus , and liver sinusoids a sequence of events that culminates in clinical preeclampsia . the link between complement activation and pathogenic events in preeclampsia identifies potential biomarkers to predict patients at risk for preeclampsia and new targets to prevent its complications . as implied in the preceding section , insufficient and/or inadequate trophoblastic invasion of the maternal spiral arteries leads to reduced uteroplacental blood flow causing focal decidual hypoxia , and thus vegf [ 4042 ] . this in turn results in activation of the decidual endothelial cells and the aberrant expression of tissue factor ( tf ) [ 40 , 43 ] . in addition , under pathological conditions , other cells , such as macrophages present in the maternal fetal interface , can also generate tf . both vegf and tf induce aberrant angiogenesis and poor vessel maintenance reflected by endothelial cell fenestrations and induction of a prothrombotic surface and uteroplacental vascular insufficiency . under physiological conditions , tf is not expressed by endothelial cells . by contrast , endothelial tf expression is observed in pathologic conditions such as sepsis , atherosclerosis , and rapid but malformed vessel growth associated with malignancies and allograft rejection [ 4547 ] . indeed , expression of tf by the endothelium is a pathological consequence of cross - talk between coagulation and inflammatory cytokines . expression of tf transforms endothelial cell membrane from an anticoagulant to a procoagulant surface and promotes intravascular thrombosis [ 48 , 49 ] . interestingly , the uteroplacental vasculature of pregnancies complicated by pe and iugr display vascular features similar to those seen in allograft rejection . a recent study by di paolo et al . demonstrated high expression of tf mrna in the vascular endothelium of vessels in the decidua basalis of pregnancies complicated by pe and iugr but not in normal pregnancies . tissue factor , the critical initiator of the coagulation cascade , is induced during pregnancy in the maternal decidua [ 52 , 53 ] and is plentiful in the placenta and amniotic fluid [ 5457 ] . it has been well documented that patients with diseases leading to aberrantly increased circulating tf expression have a much higher rate of vascular thromboembolism ( vte ) [ 5861 ] . specifically , patients are at risk for cardiovascular diseases , sepsis , hematologic , and coagulation disorders such as disseminated intravascular coagulation [ 6267 ] . in these diseases , as well as in cancer and diabetes , plasma tf interestingly , plasma levels of thrombomodulin activity , tissue factor activity , and procoagulant phospholipids were significantly elevated in women with pe versus normal pregnant and nonpregnant women . moreover , these mps are small vesicles released from injured or activated cells , primarily leukocytes and endothelial cells [ 58 , 69 ] . tf - bearing mps in pregnancy also arise from syncytiotrophoblasts and excess syncytiotrophoblast microparticles have been found in the circulation of women with pe . unfortunately , vte is one of the leading causes of morbidity and mortality during pregnancy . a higher prevalence of risk factors for vte has been found in women with pe and fetal loss .the principal factors that result in venous thrombosis consist of the classic triad of virchow : venous stasis , vascular damage , and hypercoagulability , all of which occur during normal pregnancies . a shift of the hemostatic balance in the direction of hypercoagulability during pregnancy is believed to be evolutionarily advantageous in reducing hemorrhage , however , this could also contribute to the increased risk of venous thromboembolic processes . studies from our laboratory have demonstrated that tf is normally expressed by decidual cells . indeed , immunohistochemical staining for tf has been observed in the decidualized endometrial stromal cells , but not in endothelial cells [ 7779 ] . in contrast , we noted that basal plate uteroplacental vessel segments from cases of low birth weight with villous evidence of maternal uteroplacental malperfusion had increased percent of strong positive endothelial immunostaining for tf . vascular injury initiates clotting when plasma - derived factor vii binds to the extracellular domain of perivascular cell membrane - bound tf . the resulting tf / viia complex promotes hemostasis via a series of changes initiated by the extracellular cleavage of prothrombin to thrombin . the aberrant expression of tf in vessels from deciduas with activated endothelium led us to study this phenomenon in further detail . we proposed that aberrantly expressed tf in decidual vessels would in turn result in thrombin production and further endothelial activation . in vitro experiments treatment with 2.5 u of thrombin demonstrated the induction of tf protein and mrna expression . specifically thrombin significantly upregulated mrna expression for regulated upon activation , normal t - cell expressed , and secreted ( rantes ) chemokine , growth - related oncogene ( gro)- , gro- , granulocyte chemotactic protein ( gcp)-2 , granulocyte - macrophage colony stimulating factor ( gm - csf ) , monocyte chemotactic protein-1 ( mcp-1 ) , interleukin-8 ( il-8 ) , and macrophage inflammatory protein 3 ( mip3 ) . this increase in proinflammatory chemokines are expected to result in endothelial dysfunction or inappropriate endothelial cell activation which are the most common clinical manifestations in pe , including enhanced endothelial - cell permeability and platelet aggregation . moreover , the overexpression of these chemokines is consistent with observations made in our laboratory [ 81 , 82 ] as well as others [ 83 , 84 ] indicating that the preeclamptic decidua contains an excess of macrophages . to date , no accurate test exists for predicting pe . in recent years , it has become accepted that early - onset and late - onset pes are associated with different biochemical , histological , and clinical features . moreover , markers such as sflt , seng , products of fetal and placental origin , markers of renal or endothelial damage , or markers of oxidative stress are secondary to the pathophysiological changes that precede the clinical onset of pe . nonetheless , a combination of markers may increase the detection accuracy earlier in the pregnancy and hopefully allow for more effective prophylactic strategies .

hypertension is the most common chronic disease in the developed world affecting up to 25% of the adult population . it remains the most important modifiable risk factor for coronary heart disease , stroke , congestive heart failure , renal disease , and peripheral vascular disease . suboptimal blood pressure control is responsible for 62% of cerebrovascular disease , 49% of ischemic heart disease , and an estimated 7.1 million deaths a year . because of the associated morbidity , mortality and economic cost to society early diagnosis and treatment within the established guidelines is imperative . a sizeable percentage of the hypertensive population does not manage to achieve adequate control in spite of receiving 3 or more antihypertensive medications . resistant hypertension is defined by the joint national committee 7 as blood pressure that is above the patient 's goal despite the use of 3 or more antihypertensive agents from different classes at optimal doses , one of which should ideally be a diuretic . patients whose blood pressure is controlled but require 4 or more medications to do so should also be considered resistant to treatment . the latter includes both patients with inadequately treated blood pressure due to poor adherence or inadequate treatment , as well as those with true resistant hypertension [ 3 , 4 ] . the importance of resistant hypertension lies in the identification of patients who are at high risk of suffering complications from reversible causes of hypertension and patients who may benefit from a particular diagnostic or therapeutic approach . the exact prevalence of resistant hypertension is unknown , in part because of its arbitrary definition . however , small studies estimate prevalence from 5% in general medical practice up to 50% in nephrology clinics . in a prospective analysis of framingham study data , a higher baseline systolic blood pressure along with older age , the presence of lvh and obesity ( bmi > 30 kg / m ) were the strongest predictors of lack of blood pressure control [ 6 , 7 ] . results were similar in allhat where the older , obese patients with higher baseline systolic blood pressure and lvh required 2 or more antihypertensive agents . other patient characteristics associated with resistant hypertension include excessive salt ingestion , diabetes , black race , and female gender . up to 92% of patients achieved target diastolic blood pressure while only 60%67% achieved systolic blood pressure goals [ 6 , 8 ] . it is likely that this condition will become increasingly common because of the aging population and a progressive increase in obesity and comorbidities such as diabetes . a finnish study found that certain variants of the and subunits of the epithelial sodium channel gene enac were significantly more prevalent in patients with resistant hypertension . other studies associate the allele of the cyp3a5 * 1 enzyme with both higher blood pressure levels in normotensive people of black race , as well as with hypertension resistant to treatment [ 10 , 11 ] . these and other genes that may be identified in the future hold the potential for the development of novel therapeutic targets . the first step in evaluating a patient with uncontrolled blood pressure is to establish whether it is a case of true resistant hypertension or just pseudoresistance . the latter refers to a lack of blood pressure control despite receiving treatment without true resistance . this can be caused by easily reversible causes such as suboptimal blood pressure measurement technique , poor adherence to prescribed medication , suboptimal dosing of antihypertensive agents or inappropriate combinations , the white coat effect , and clinical inertia . it is important to exclude these causes before labeling a patient as having resistant hypertension . poor blood pressure measurement technique is quite common , usually the result of not letting the patient rest before measurement and using a small cuff . patients should always rest in a chair with their back supported for a minimum 5 minutes prior to measurement and the cuff 's air bladder must encircle at least 80% of the arm circumference . the average of two readings taken a minute apart represents the patient 's blood pressure . approximately 40% of newly diagnosed patients will discontinue their antihypertensive medication the first year of treatment [ 13 , 14 ] . eventually , less than 40% will continue taking their medication after 5 to 10 years [ 13 , 15 ] . the most common causes are poor patient - physician communication concerning blood pressure goals and the importance of achieving them , potential side effects , high cost of treatment , and complex regimens [ 16 , 17 ] . adherence can be improved by choosing affordable agents with minimal side effects that are given once daily alone or in fixed dose combinations . clinical inertia can be described as a physician 's ignorance of treatment guidelines or reluctance to adhere to them due to lack of training or inexperience in antihypertensive medication , underestimation of cardiovascular risk , and overestimation of the treatment provided [ 18 , 19 ] . a large part of this problem could be resolved if physicians familiarize themselves with one or two drugs in each class of antihypertensives . proper training is imperative so that physicians realize the importance of treating to reach a goal blood pressure level of less than 140/90 mmhg and knowing when to refer patients to a hypertension specialist . a white coat effect should be suspected in patients whose clinical blood pressure measurements are consistently and significantly higher than reliable out of office measurements . other signs include repetitive symptoms of overtreatment such as orthostatic hypotension and persistent fatigue as well as absence of target organ damage including left ventricular hypertrophy , retinopathy , and chronic kidney disease [ 20 , 21 ] . one study found that 20 to 30% of a patient population believed to have resistant hypertension was actually well controlled when measured by 24-hour ambulatory blood pressure monitoring . accurate home blood pressure values are the best guide for therapy . in elderly patients , especially diabetics , arterial stiffness obese patients have increased sympathetic activity , higher cardiac output , and a rise in peripheral vascular resistance due to reduced endothelium - dependent vasodilation . plasma aldosterone and endothelin are also increased , while excessive surrounding adipose tissue results in increased intrarenal pressures and changes in renal architecture . as the body mass index increases , progressively higher doses of antihypertensive drugs are required to control blood pressure . weight loss has been found to reduce both systolic and diastolic blood pressure [ 3 , 26 ] . insulin resistance increases sympathetic nervous activity , vascular smooth muscle cell proliferation , and sodium retention leading to elevated blood pressure resistant to treatment . the common comorbidities of obesity , hypertension , and diabetes induce renal dysfunction , further hindering blood pressure treatment . although small amounts of alcohol ( 2 drinks / day ) have vasodilating effects and may lower blood pressure , consumption of more than 30 ml daily raises blood pressure and may increase cardiovascular risk . older patients , patients of african origin , and patients with chronic kidney disease are particularly susceptible to salt intake . current guidelines recommend that dietary sodium for a hypertensive person should be under 100 mmol / day ( 2.4 g sodium or 6 g sodium chloride ) and even lower in salt sensitive patients . excessive salt intake can be assessed by measuring sodium excretion in a 24-hour urine collection . perhaps the most common are nonsteroidal anti - inflammatories including cox-2 inhibitors and aspirin , decongestants ( phenylephrine and pseudoephedrine ) , stimulant agents used for weight loss , narcolepsy or attention deficit disorder , contraceptives , cyclosporine , and erythropoietin . corticosteroids increase blood pressure through fluid retention , particularly but not limited to those with increased mineralocorticoid activity . secondary causes of hypertension are common in patients with resistant hypertension , particularly in the elderly . these include obstructive sleep apnea , renal parenchymal disease , renal artery stenosis , and primary aldosteronism [ 28 , 29 ] ( table 1 ) . a small study of 41 patients with resistant hypertension discovered that 83% suffered from sleep apnea . the severity of sleep apnea is positively associated with the likelihood of resistant hypertension [ 31 , 32 ] . intermittent hypoxemia and increased upper airway resistance induce a sustained increase in sympathetic nervous system activity [ 33 , 34 ] . also , there seems to be a significantly higher prevalence of primary aldosteronism in patients with sleep apnea [ 35 , 36 ] . alternatively , obesity may be the common factor that increases risk for both obstructive sleep apnea and excess aldosterone production . a sleep study is indicated in patients with resistant hypertension and other signs and symptoms of sleep apnea including obesity , large neck size , excessive loud snoring , interrupted sleep , daytime somnolence , polycythemia , and carbon dioxide retention [ 37 , 38 ] . treatment with a continuous positive airway pressure device ( cpap ) has been shown to reduce blood pressure and thus is beneficial in resistant hypertension patients with obstructive sleep apnea . the recent studies suggest that primary aldosteronism is a much more common cause of hypertension than originally believed . particularly in patients with resistant hypertension , the prevalence of primary aldosteronism has been found between 10% and 20% [ 3 , 39 , 40 ] . aldosterone exerts a number of effects leading to increased systemic vascular resistance , such as endothelial dysfunction , vascular remodeling through collagen deposition , vascular damage , impairment of the baroreflex leading to loss of compensation for elevated blood pressure , and hypovolemia [ 4143 ] . it has been suggested that obesity is involved , causing a generalized activation of the renin - angiotensin - aldosterone system , perhaps through excretion of cytokines from adipocytes [ 44 , 45 ] . primary aldosteronism may be suggested by hypokalemia ; however this is often a late manifestation preceded by the development of hypertension [ 4648 ] . screening should be done with plasma renin and serum aldosterone ratio measurement ( which has a high sensitivity but low specificity ) and confirmed with sodium loading or fludrocortisone suppression testing [ 39 , 49 ] . renal parenchymal disease is both a cause and a complication of poorly controlled hypertension [ 50 , 51 ] . as was previously mentioned , in allhat serum creatinine above 1.5 mg / dl was the strongest predictor of failure to achieve goal blood pressure . resistant hypertension in chronic kidney disease is mainly due to activation of the renin - angiotensin system , sodium retention , and the resulting intravascular volume expansion . other factors include activation of the sympathetic nervous system due to decreased blood flow to the kidney , alterations in vasoconstrictor and vasodilator excretion from the endothelium , and increased arterial stiffness . renal artery stenosis is a relatively common finding in hypertensive patients undergoing cardiac catheterization with approximately 20% of patients having unilateral or bilateral stenosis above 70% . however the causative role of these stenosis in hypertension remains unknown since only a few patients actually benefit from surgical or endovascular revascularization [ 54 , 55 ] . the majority of cases ( 90% ) are due to atherosclerotic lesions and are seen in older patients , smokers , patients with known atherosclerotic disease and unexplained renal insufficiency . the other 10% are fibromuscular lesions , commonly in women under 50 years of age , and these are the patients that will usually improve blood pressure control after revascularization . bilateral renal artery stenosis should be suspected in patients with a history of flash pulmonary edema with preserved systolic heart function . screening can be done using magnetic resonance angiography ( mra ) , computer tomographic angiography ( cta ) , doppler ultrasonography , or angiotensin converting enzyme ( ace ) inhibitor renography [ 49 , 57 ] . pheochromocytoma is a rare cause of resistant hypertension with a prevalence of 0.1%0.6% among hypertensives [ 58 , 59 ] . the average time between initial symptoms and diagnosis is 3 years , and many cases are missed altogether according to autopsy studies . the best screening test for pheochromocytoma is 24-hour urinary metanephrines or plasma free metanephrines ( normetanephrine and metanephrine ) which carries a 99% sensitivity and an 89% specificity . however , other factors such as obstructive sleep apnea and insulin resistance also contribute substantially [ 65 , 66 ] . target organ damage in cushing 's syndrome is more severe than in primary hypertension because it is associated with many other cardiovascular risk factors such as diabetes , obstructive sleep apnea , obesity , and dyslipidemia [ 67 , 68 ] . patients with hyperthyroidism usually present with systolic hypertension and those with hypothyroidism have diastolic hypertension . most patients with primary hyperparathyroidism are diagnosed because of routine findings of hypercalcemia [ 69 , table 1 - 2 ] . pharmacological treatment should be based on the most common causes of resistant hypertension and focused on blocking all the physiological pathways to blood pressure elevation [ 18 , 70 ] . antihypertensive agent doses should be titrated upward until blood pressure is controlled or the maximum recommended dosage is reached , unless the patient experiences dose related adverse effects . it is then appropriate to add a drug from another class that has additive or synergistic effects with the first drug . in general , a typical regimen should include a diuretic , an ace inhibitor or angiotensin receptor blocker ( arb ) , a calcium channel blocker ( ccb ) , and a -blocker . switching one of 3 or more medications from morning to bedtime administration can result normalization of blood pressure in 21.7%37% of patients [ 71 , 72 ] . since volume overload is the most frequent underlying pathophysiology and suboptimal dosing the most frequent cause of resistant hypertension , adding , increasing or changing diuretic therapy is the key to successful treatment and will help over 60% of patients achieve target blood pressure [ 18 , 7377 ] . patients with normal kidney function should receive 12.525 mg / day of hydrochlorothiazide although some will benefit from doses up to 50 mg / day . chlorthalidone at 25 mg / day is an alternative that offers greater 24-hour blood pressure reduction than 50 mg / day hydrochlorothiazide with the greatest difference occurring overnight and may be preferred in certain patients with resistant hypertension . when given at the same dose as hydrochlorothiazide , chlorthalidone will reduce blood pressure an additional 8 mmhg according to a small study . patients should be monitored for hyponatremia and hypokalemia , and caution is warranted in patients with a history of prediabetes and gout . a common pitfall is not realizing that the patient 's kidney function is deteriorating and not switching diuretic class when it does . therefore , they must be replaced with loop diuretics when the estimated glomerular filtration rate ( egfr ) falls below 40 ml / min/1.73 m [ 27 , 73 , 80 ] . in a study of 12 elderly patients with hypertension whose blood pressure was uncontrolled on multidrug regimens the use of furosemide significantly improved blood pressure control . furosemide and bumetanide have a relatively short half - life and should be dosed twice daily in order to avoid reactive sodium retention due to intermittent natriuresis and consequent activation of the renin - angiotensin system [ 73 , 80 , 82 ] . subclinical aldosteronism is a common occurrence in resistant hypertension , low doses of spironolactone ( 2550 mg / day ) or eplerenone can be particularly helpful . patients most expected to benefit from mineralocorticoid blockade include those with primary hyperaldosteronism , the obese , and those suffering from obstructive sleep apnea . according to one study , the addition of spironolactone 12.525 mg / day to 76 patients with uncontrolled hypertension taking an average of 4 antihypertensive agents resulted in an average 25/12 mmhg reduction after 6 months . the blood pressure lowering arm of the ascot study had similar results for patients who were unselected for aldosterone / plasma renin activity . when spironolactone was added as a fourth line agent , blood pressure dropped by 21.9/9.5 mmhg . at this dosage spironolactone is safe , well tolerated and provides significant additive blood pressure reduction [ 84 , 85 ] . eplerenone may be more suitable for patients requiring spironolactone doses above 25 mg / day because breast tenderness is a common adverse effect at higher doses . one small study found that the addition of 2.5 mg / day amiloride decreased blood pressure by 31/15 mmhg . however , it has been shown that 10 mg of amiloride has half the blood pressure reduction capability of 25 mg spironolactone and therefore should be considered only when spironolactone is not tolerated . blockade of the renin angiotensin system with ace inhibitors or arbs in patients that are intolerant of ace inhibitors is particularly recommended in patients with diabetes mellitus , heart failure , postmyocardial infarction , chronic kidney disease , high coronary disease risk , and recurrent stroke prevention [ 27 , 88 ] . dosage should be increased to the maximum recommended dosage as long serum creatinine does not increase more than 35% above baseline and hyperkalemia does not develop [ 111 ] . dual ace and arb therapy is no longer recommended in most cases because of the possibility of adverse renal outcomes [ 89 , 90 ] . aliskiren , the only available direct renin inhibitor , is at least as effective as arbs in reducing end target organ damage but has not been directly tested in resistant hypertension . the allay trial showed that aliskiren monotherapy was as effective as losartan in reducing lvmi , although the combination of both did not achieve a statistically significant further lvmi regression . the addition of aliskiren to losartan did however seem to have additional renoprotective effects in another study , reducing the mean urinary albumin creatinine ratio by 20% in patients with diabetic nephropathy . the additional blood pressure reduction was marginal and therefore the role of direct rennin inhibitors in resistant hypertension remains undetermined . polypharmacy is difficult to avoid because blood pressure can be controlled by using one drug in only about 50% of patients . fixed dose combinations offer the convenience of taking fewer pills , combining antihypertensive agents with additive or synergistic effect and reducing dose - dependant adverse effects of individual components . the latter is evident in ace inhibitor or arb combinations with ccbs , as the ace inhibitors / arbs reduce the peripheral edema that frequently develops with dihydropyridine calcium channel blocker therapy . another popular fixed dose combination with synergistic effects is ace inhibitors / arbs with diuretics , since the latter enhance the antihypertensive efficacy of all the other classes . the recent introduction of triple agent combinations containing dihydropyridine ccbs as well is expected to lessen the burden of polypharmacy and further improve adherence . the accomplish study suggested that combining ace inhibitors with ccbs was more effective at preventing major cardiovascular and renal events than ace inhibitors with diuretics despite achieving similar blood pressure control rates . both regimens are available as fixed dose combinations and are useful options in different circumstances . in cases of true resistant hypertension , there are also data to support adding a complementary non dihydropyridine ccb to a regimen including a ras blocker , diuretic , and dihydropyridine ccb . such a combination of complementary ccbs results in additive bp reduction with a low - side effect profile and makes pharmacological sense [ 94 , 95 ] . beta - blockers are indicated in the setting of coronary artery disease , congestive heart failure , and postmyocardial infarction . when adding on combinations already including a diuretic , an ace inhibitor or arb and a calcium channel blocker , vasodilating -blockers should be preferred [ 73 , 96 ] . beta - blockers as well as loop diuretics are also usually necessary when administering direct vasodilators to overcome the reflex tachycardia and fluid retention , respectively . if bp control is still not achieved with full doses of a 4-drug combination , use of other agents such as centrally acting alpha - agonists ( methyldopa and clonidine ) or vasodilators ( hydralazine or minoxidil ) is needed . these agents are very effective for lowering bp but have poor tolerability , require frequent dosing , and lack positive outcome data . at this stage , the intervention of a hypertension specialist is warranted . besides these established treatments , new antihypertensive agents are being developed . endothelin receptor antagonists are a new family of antihypertensive medications that are currently being evaluated . darusentan is a selective antagonist for type a endothelin receptors , activation of which causes vasoconstriction and proliferation of vascular smooth muscle . it has demonstrated significant dose - dependant reductions in both systolic and diastolic blood pressures and has been positively evaluated in resistant hypertension [ 98 , 99 ] . unfortunately , another unpublished phase 3 clinical trial failed to meet its coprimary end point and the drug 's future remains uncertain . atrasentan is another highly selective endothelin receptor antagonist that has shown positive results in blood pressure reduction for 72 patients . another promising category under development is medication that combines inhibitors of vasoconstrictive mediators with drugs that potentiate vasodilating mediators by inhibiting their breakdown by neutral endopeptidases ( neps ) . vaccines targeting angiotensin i and ii are also being developed and tested [ 103 , 104 ] . in any case , treatment should be tailored to the patient 's profile , lifestyle , and comorbidities . constructing a regimen that is acceptable to the patient , well tolerated and will maintain long - term compliance is important . yet in some patients , optimal blood pressure control will not be achieved even with the most carefully designed regimen . in these cases , new device - based approaches for blood pressure control are being evaluated ( tables 1 and 2 ) . one of these devices is the rheos device ( cvrx , maple grove , minn ) which stimulates the carotid baroreceptors for better blood pressure control by taking advantage of chronic electrical activation of the afferent limb of the carotid baroreflex . the device consists of a pulse generator and bilateral perivascular carotid sinus leads that are implanted under narcotic anesthesia . according to the findings from the device - based therapy of hypertension ( debut - ht ) study that were recently presented , after four years of treatment , rheos reduced systolic blood pressure by an average of 53 mmhg ( 193 mmhg versus 140 mmhg ) . blood pressure was reduced significantly each year , with the largest decrease occurring in year four . many of these patients were able to reach their blood pressure goal and reduce the number of medications that patients were taking to treat their hypertension from an average of 5 at baseline to 3.4 medications at 4 years . baroreflex activation therapy also improved functional capacity and reduced left ventricular mass without any evidence of carotid injury or stenosis . another target for the interventional treatment of resistant hypertension is catheter - based renal nerve ablation . renal sensory afferent nerve activity directly influences sympathetic outflow to the kidneys and other highly innervated organs involved in cardiovascular control , such as the heart and peripheral blood vessels , by modulating posterior hypothalamic activity [ 106 , 107 ] . renal sympathetic nerve ablation is achieved percutaneously via the lumen of the renal artery , using a catheter connected to a radiofrequency generator . treatment has been administered to 45 patients with resistant hypertension taking a median of 4.7 antihypertensive agents . followup at 1 and 2 years has shown a sustained blood pressure reduction of 27/11 mmhg [ 108 , 109 ] . the multicentre simplicity htn-1 study that was recently presented at the european society of hypertension 's 20th meeting included 108 patients with persistently elevated blood pressure despite treatment with an average of five medications . catheter - based renal denervation produced a mean blood pressure reduction of 33/15 mmhg at 24 months without evidence of vascular or renal abnormalities . results from the simplicity htn-2 study comparing renal denervation treatment to rigorous medical therapy are expected by the end of 2010 . causes of resistance should be considered when blood pressure does not respond satisfactorily to a rational triple antihypertensive regimen that includes a diuretic . the workup of patients with resistive hypertension should be a two - step approach ( table 3 ) : first , confirmation that it is indeed true resistant hypertension by ruling out or correcting factors associated with pseudoresistance , and second , identification of the true factors involved in treatment resistance . the cornerstone of therapy remains a rigorous evaluation followed by correction of contributing causes and appropriate pharmacological treatment . newer interventional therapies may become a viable option in the future for those patients with uncontrolled hypertension despite receiving an optimal multiple medication antihypertensive regimen and those who can not tolerate the medication .

carbon dioxide ( co2 ) is a very stable form of carbon , the central element of life on this planet and one of the simplest molecules that was probably highly abundant in the primeval earth atmosphere . this gas reacts with water , leading to h2co3 , which is an unstable compound that is spontaneously transformed into bicarbonate and protons . however , the reaction between co2 and water is particularly slow at ph values of 7.5 or lower , which is usually the physiologic ph value in many tissues and organisms . carbon dioxide hydration becomes , on the other hand , very effective at higher ph values , being instantaneous at ph > 12 . moreover co2 is an important molecule in all life processes , being generated in high amounts in most organisms . to catalyze its rapid transformation into bicarbonate , catalysts evolved in all life kingdoms , that is , the enzymes known as carbonic anhydrases ( cas , ec 4.2.1.1 ) . six genetically diverse such enzyme families are presently known the - , - , - , - , - , and -cas with the last class discovered quite recently . cas not only face the conversion of the high amounts of co2 formed in the metabolic processes , transforming it in bicarbonate and protons , but they also manage the acid base equilibria connected to this reaction . in fact , the products formed in the catalyzed reaction are either ions with strong buffering activity ( bicarbonate ) or hydrated protons ( h ions ) . the regulation of ph is a highly important process in all life forms , since many biochemical reactions are tightly regulated by it . this is probably the reason why so many genetic ca families are presently known so that in some organisms , a multitude of different ca families with many isoforms have been described , each with specialized functions . the necessity of a tight / precise ph regulation may thus explain why most organisms investigated so far contain multiple ca isoforms , although they differ significantly by their catalytic activity , susceptibility to various classes of inhibitors , subcellular localization , and many other such features . for example , in humans , 15 different ca isoforms , all belonging to the -class , have been described . most mammals ( including humans ) possess two blood isoforms , denominated ca i and caii , with a total concentration of these proteins as high as 0.2 mm . however , the catalytic activity of the human ( h ) isoform hca i is much lower compared with that of hca ii , and in addition , hca i is also inhibited by the chloride and bicarbonate present in the plasma , leaving a lot of questions regarding the physiologic function of this isoform . on the other hand , the high activity isoform hca ii ( also known as the rapid blood enzyme , to distinguish it from the slow one , hca i ) is involved in the secretion of electrolytes in a multitude of tissues , such as the bicarbonate - rich aqueous humor in the anterior chamber of the eyes , and the cerebrospinal fluid , but also in ph and co2 homeostasis all over the body , as mentioned above . other functions include urine formation and bicarbonate reabsorption in the kidney tubules ; biosynthetic reactions , such as gluconeogenesis , lipogenesis , and ureagenesis ; bone resorption and calcification ; and probably many other less well understood physiological / pathological processes . indeed , a dysregulation of the activity of these isoforms in one or more tissues has important pathologic consequences , such as glaucoma , when excessive aqueous humor is secreted within the eye , with the subsequent increase in the intraocular pressure ( iop ) and edema , when not enough fluids are secreted / eliminated in the urine , leading to fluid accumulation in the body , processes in which ca ii together with several other isoforms such as ca iv , xii , and xiv , are involved in the kidneys , epilepsy ( the involvement of ca ii and other brain ca isoforms in this disease is poorly understood and certainly not irrelevant ) , and some forms of cancer , in which ca ii was observed to be overexpressed , alone or together with other isoforms such as ca ix and xii . ca ii is also involved in other pathologies , such as acute mountain sickness and apparently , atherosclerosis and osteoporosis . primary sulfonamides constitute the main class of ca inhibitors ( cais ) , with a number of such derivatives in clinical use for decades , mainly as antiglaucoma agents , diuretics , antiepileptics , or antiobesity drugs . recently , some sulfonamides with ca inhibitory properties entered phase i clinical trials as antitumor / antimetastatic agents targeting hypoxic tumors in which two ca isoforms , ca ix and xii , are overexpressed . the search for sulfonamide cais with various potentials in therapeutics is a dynamic research field , with many new classes being reported constantly and investigated in detail for inhibitory effects against mammalian and nonmammalian cas . here , we report a class of 4-sulfamoylphenyl--aminoalkyl ethers , a poorly investigated chemotype in the cais landscape , with interesting properties as antiglaucoma agents as well as for the design of fluorescent enzyme inhibitors with potential use for imaging cas in various tissues . benzenesulfonamides constitute a highly investigated class of cais , with most such compounds reported so far being derivatives of sulfanilamide , homosulfanilamide , or 4-aminoethylbenzenesulfonamide . derivatization of the primary aliphatic / aromatic amino group from these compounds by its transformation into carboxamides , secondary sulfonamides , ureas , thioureas , or by reaction with pyrylium salts has led to a considerable number of new derivatives that showed excellent inhibitory properties against ca isoforms of medicinal interest , such as ca ii , iv , va / vb , ix , or xii . surprisingly , very few benzenesulfonamides incorporating ether or thioether moieties have been reported so far . in fact , only one paper , by vernier et al . , considered these chemotypes for the design of sulfonamide - based cais . in a very interesting study , these authors reported compounds of the type ar - x - ar-so2nh2 , where x was o or s , and ar , ar aromatic / heterocyclic six - membered rings , which showed highly effective inhibitory properties against ca isoforms involved in important physiologic processes , such as ca ii and iv . these derivatives showed improved water solubility compared with structurally similar sulfanilamide derivatives , possessed low nanomolar inhibitory action against ca ii , and were shown to penetrate eye tissues , arriving at the ciliary processes where the enzyme is present within the eye , and participating in aqueous humor secretion . unfortunately no in vivo antiglaucoma studies have been performed with those compounds that possessed physicochemical properties appropriate for an antiglaucoma drug candidate . considering these facts , we decided to explore the synthesis and properties of ethers incorporating the benzenesulfonamide head and aliphatic ether moieties of the type h2n(ch2)n o c6h4so2nh2 . as mentioned above , such ethers were not investigated as cais until now , and considering the aromatic derivatives reported by the pfizer group , the presence of aliphatic , amino moieties should also promote the water solubility of the compounds . in fact , a considerable pharmacologic problem of the first generation cais , such as acetazolamide , aaz ; methazolamide , mza ; or dichlorophenamide , dcp , was their poor water solubility . only the second generation drugs , such as dorzolamide , drz ; and brinzolamide , brz , have an improved water solubility because these two topically acting antiglaucoma drugs are administered as hydrochloride salts ( both weak amines ) . thus , we designed the following strategy for obtaining the 4-sulfamoylphenyl -aminoalkyl ethers reported in this paper ( scheme 1 ) . reaction of -amino - alcohols 1a e ( n = 26 ) with tert - butyloxycarbonyl anhydride afforded the boc - protected amines 2a e , which by mitsunobu reaction with 4-hydroxybenzenesulfonamide 3 led to the boc - protected derivatives 4a e . removal of the protecting group in the presence of trifluoroacetic acid ( tfa ) afforded the trifluoroacetate salts of the 4-sulfamoylphenyl -aminoalkyl ethers 5a e ( scheme 1 ) . the alkyl chain present in the new derivatives ranged between 2 and 6 carbon atoms to investigate the influence of the spacer length for the enzyme inhibitory properties of the new derivatives . another aspect in the design of cais is related to the use of such compounds as diagnostic tools , for example , for the imaging of tumors in which some ca isoforms are overexpressed . we have reported , for example , fluorescein - based sulfonamides ( obtained again from sulfanilamide , homosulfanilamide or 4-aminoethylbenzenesulfonamide , which were reacted with fluorescein isothiocyanate ) that were essential for demonstrating the role of ca ix / xii in the acidification of the extracellular tumor milieu and also in the proof - of - concept studies regarding the druggability of these novel antitumor targets . however , like most aromatic thioureas , the fluorescent sulfonamides reported earlier showed a rather low water solubility , which may be a limiting factor for some of their applications . thus , we report here novel derivatives that were prepared by reaction of the 4-sulfamoylphenyl -aminoalkyl ethers 5a e with fluorescein isothiocyanate 6 or [ 9-(2-carboxy-4-isothiocyanato - phenyl)-6-dimethylaminoxanthen-3-ylidene]-dimethylammonium chloride 8 , leading to the novel fluorescent compounds of types 7a c and 9a , b , respectively ( scheme 1 ) . the last compounds ( 9a , b ) incorporate a fluorophore that was not investigated earlier for its interaction with cas . mean from three different assays ; errors are in the range of 10% of the reported value . inhibition data against four physiologically significant ca isoforms , that is , h ( human ) hca i , ii , ix , and xii , are shown in table 1 . the following structure activity relationship ( sar ) can be drawn from the data of table 1 : ( i ) the slow human isoform hca i effectively inhibited by some of the sulfonamides investigated here , such as 4a4e and 7a , 7b , which showed ki values in the low nanomolar range ( 5.341.2 nm ) , whereas other derivatives ( e.g. , 5d , 5e , and 7c ) were medium potency inhibitors , with ki s of 52.590.9 nm . like acetazolamide aaz , some of the new compounds , among which 5a5c and 9a , 9b were less effective hca i inhibitors , with ki s of 151826 nm . thus , the best hca i inhibitors were the boc - protected derivatives 4 , which showed a rather compact behavior of very effective inhibitor , except for the compound with the 4-carbon - atoms linker ( 4c ) which was less effective compared with its congeners 4a , b , d , and e. the deprotected amines 5 were less effective as hca i inhibitors compared with the corresponding boc derivatives ( table 1 ) . among the fluorescent cais reported here , the fluorescein - containing compounds 7a and 7b were effective hca i inhibitors , whereas the tetramethylrhodamine derivatives 9a and 9b were much less effective as hca i inhibitors compared with the fluorescein derivatives mentioned above . ( ii ) the physiologically dominant hca ii very effectively inhibited by most sulfonamides reported here . indeed , just four compounds ( 5a and 5b ) as well as 9a , b showed medium potency activity , with ki s of 36.6215 nm . the remaining sulfonamides showed very effective hca ii inhibitory properties , with ki s ranging between 3.9 and 8.9 nm ( table 1 ) and are thus more effective than the clinically used drug acetazolamide aaz . the sar is rather clear - cut : the five boc - protected derivatives 4a4e showed a very compact behavior with basically no variation of the inhibitory power , with the length of the linker from 2 to 6 ch2 moieties . however , the situation is changed for the amines 5a5e , which on one hand were weaker hca ii inhibitors compared with the corresponding boc - protected derivatives and on the other hand showed an increase in the inhibitory power with an increase in the linker chain from 2 to 6 ch2 moieties . indeed , between compounds 5a and 5e , there is a 17-fold difference in the inhibitory activity against this isoform . as for hca i inhibition , again , the fluorescein - tailed sulfonamides 7a7c were much more inhibitory compared with the tetramethylrhodamine derivatives 9a and 9b . ( iii ) the tumor - associated , transmembrane isoform hca ix was very well inhibited by all derivatives reported here , with ki s of 5.832.6 nm . the sar is almost impossible to delineate because all these compounds show excellent inhibitory activity . for example , the boc - protected derivatives 4a e have a minimal variation of the inhibition constants , ranging between 5.8 and 8.3 nm . this variation is slightly higher for the amines 5 ( between 6.5 and 32.6 nm ) and is again almost absent for the fluorescent sulfonamides 7 and 9 . it is interesting to note that for this isoform , both the fluorescein and the tetramethylrhodamine derivatives were equally effective as cais . ( iv ) the other transmembrane isoform investigated here , hca xii , was also effectively inhibited by most of the new sulfonamides reported in this paper . two compounds , 5a and 9b , were medium potency inhibitors ( ki s of 36.788.5 nm ) , and one ( 9a ) was an ineffective inhibitor ( ki of 609 nm ) . the remaining sulfonamides investigated here showed excellent hca xii inhibitory activity , with inhibition constants ranging between 5.4 and 9.6 nm , again with no obvious sar to be discussed ( table 1 ) . ( v ) although most of these sulfonamides were effective cais against all four isoforms investigated here , several interesting selectivity cases were observed : for example , 9a is a hca ix - selective sulfonamide inhibitor , with a ki of 9.6 nm against the tumor - associated isoform and > 215 nm against hca i , ii , and xii ( table 1 ) . compound 5b effectively inhibits the two transmembrane isoforms ( ki s of 9.617.9 nm ) ; it is a much less effective inhibitor of the two cytosolic isoforms hca i and ii ( ki s of 36.6452 nm ) . rfree is calculated in the same way as rcryst except it is for data omitted from refinement ( 5% of reflections for all data sets ) . electron density of compound 4c bound within the hca ii active site . the zn(ii ) ion ( gray sphere ) and its three his ligands ( his94 , 96 , and 119 ) as well as other residues involved in the catalytic cycle or binding with inhibitors are shown . because of the less - ordered electron density for the tail region of 4c , its map was contoured at a lower sigma level compared with compound 5c . the zn(ii ) ion ( gray sphere ) and its three his ligands ( his94 , 96 , and 119 ) as well as other residues involved in the catalytic cycle or binding with inhibitors are shown . overlay of sulfonamides 4c ( orange ) and 5c ( teal ) bound within the active site of hca ii . hydrophobic region , red ; hydrophilic region , blue ; zinc ion , gray sphere at the bottom of the activity . to rationalize some of the inhibition data presented above , two of the novel sulfonamides reported here , 4c ( incorporating the boc - aminobutyl moiety ) and 5c ( incorporating the 4-aminobutyl fragment ) were cocrystallized with hca ii , and their crystal structures were resolved at a high resolution ( table 2 ) . both inhibitors were observed bound within the enzyme active site , coordinating to the zn(ii ) ion by means of the deprotonated nitrogen of the sulfonamide moiety ( figures 1 and 2 ) , like all other sulfonamide or sulfamates investigated so far by means of this technique . the phenyl ring and the rather long , hydrophobic alkyl tails of both inhibitors were observed to interact only with residues of the hydrophobic half of the hca ii active site ( as shown in figures 13 ) , such as val121 , phe131 , leu198 , pro201 , and pro202 . the tail of compound 5c extends farther out into the enzyme s hydrophobic cleft , allowing it to form more stabilizing interactions with amino acids , such as pro201 and leu204 ; however , the shorter tail of compound 4c is unable to perform such interactions ( figure 3 ) . it should be mentioned that for the boc - protected derivative 4c , the electron density of the tail region was not completely defined ( figure 1 ) , probably because of its high flexibility and disorder when complexed to the enzyme . in contrast , for the amine 5c , all atoms from the tail region had the electron density well - defined , proving that this region is less disordered compared with the boc - protected compound 4c ( figure 2 ) . to account for this disorder and to ensure the ligand was built into the density correctly , the map for compound 4c was contoured at a lower sigma level ( 0.6 ) than that of 5c ( 1.0 ) . the fact that the tails of these compounds lie only in the hydrophobic cleft of the hca ii active site is a noteworthy finding , because we showed in earlier papers that the active site region ( hydrophobic versus hydrophilic halves ) in which a sulfonamide binds is indicative of its isoform - selectivity properties . in fact , we observed earlier that compounds that have their tails lying only in the hydrophobic half generally do not possess isoform - selective inhibitory properties , and this is also confirmed by the present findings ( although neither 4c nor 5c is highly effective as hca i inhibitors , table 1 ) . in fact , these two compounds inhibit hca ii , ix , and xii in rather similar ranges and are effective inhibitors against these three isoforms . because our interest was to obtain compound with antiglaucoma activity , for which both hca ii and xii effective inhibitory properties are desired , we consider the present observations of real interest . drop of intraocular pressure ( iop , mmhg ) versus time ( min ) in hypertonic saline - induced ocular hypertension in rabbits , treated with 50 l of 2% solution of compounds 4c , 5c , and drz as the standard drug and vehicle . ( from three different measurements for each of the four animals in the study group ) and were statistically significant ( p = 0.045 by the student s t test ) . both the boc - protected and the amino derivative sulfonamides reported here showed excellent water solubility and could be formulated as 2% eye drops at the neutral ph value ( dorzolamide , drz , the clinically used drug is a hydrochloride salt with a ph of the eye drops of 5.5 which produces eye irritation and stinging as side effects ) . we have investigated the intraocular pressure ( iop ) lowering properties of some of these compounds , more precisely , 4c and 5c ( for which the x - ray in adduct with hca ii was reported ; see the discussion section , above ) , in an animal model of glaucoma . indeed , both compounds were low nanomolar inhibitors of isoforms hca ii ( responsible for aqueous humor secretion ) and hca xii ( isoform that is overexpressed in the eyes of glaucomatous patients ) . as seen from the data of figure 4 , the boc - protected derivative 4c showed a small decrease of iop ( of 12.5 mmhg ) when given topically to the eye of the animals , whereas the free amine 5c ( as a trifluoroacetate salt ) was more effective , with an iop decrease of 4.4 mmhg at 2 h postadministration , more effective than drz , the standard drug , which caused an iop drop of 4 mmhg at 60 min postadministration . another notable difference between 5c and drz was the fact that the new compound investigated here had a prolonged efficacy compared to drz , for which after 4 h no iop decrease was seen . in contrast , 5c showed efficacy even after 4 h postadministration , with an iop drop of 3 mmhg at that time point . it should be mentioned that the animal model employed here is of normotensive rabbits , and this is why the absolute iop drops are not very high , but the advantage of this model is that the measurements can be done rapidly and are highly reproducible . we report a series of new 4-sulfamoylphenyl--aminoalkyl ethers that have been prepared by mitsunobu reaction . interesting sar has been observed for the inhibition of four physiologically relevant ca isoforms : hca i , ii , ix , and xii . many of the new compounds were highly effective inhibitors of all these isoforms , in the low nanomolar range , with few isoform - selective compounds also identified . these findings have been rationalized by resolving the x - ray crystal structures of two of the new sulfonamides . the tails of these derivatives were observed bound only in the hydrophobic half of the enzyme active site , making van der waals contacts with amino acids such as val135 , leu198 , leu204 , trp209 , pro201 . and pro202 . one of the compounds incorporating a free amine moiety , which was highly water - soluble as a trifluoroacetate salt , also showed effective iop lowering properties in an animal model of glaucoma . several fluorescent sulfonamides have also been reported that incorporate either fluorescein thiourea or tetramethylrhodamine anhydrous solvents and all reagents were purchased from sigma - aldrich , alfa aesar , and tci . all reactions involving air- or moisture - sensitive compounds were performed under a nitrogen atmosphere using dried glassware and syringe techniques to transfer solutions . nuclear magnetic resonance ( h nmr , c nmr , dept-135 , dept-90 , hsqc , hmbc ) spectra were recorded using a bruker advance iii 400 mhz spectrometer in dmso - d6 . chemical shifts are reported in parts per million ( ppm ) , and the coupling constants ( j ) are expressed in hertz ( hz ) . splitting patterns are designated as follows : s , singlet ; d , doublet ; sept , septet ; t , triplet ; q , quadruplet ; m , multiplet ; brs , broad singlet ; dd , double of doubles , appt , apparent triplet , appq , apparent quartet . the assignment of exchangeable protons ( oh and nh ) was confirmed by the addition of d2o . analytical thin - layer chromatography ( tlc ) was carried out on merck silica gel f-254 plates . flash chromatography purifications were performed on merck silica gel 60 ( 230400 mesh astm ) as the stationary phase and ethyl acetate / n - hexane were used as eluents . melting points ( mp ) were measured in open capillary tubes with a gallenkamp mpd350.bm3.5 apparatus and are uncorrected . aminoalcohol 1a e ( 1.0 equiv ) was dissolved in dichloromethane ( dcm ) and treated with a 1 m naoh aqueous solution or diisopropyl ethylamine ( dipea ) ( 1.0 equiv ) , then di - tert - butyl dicarbonate ( 1.0 equiv ) was added , and the mixture was vigorously stirred o.n . until consumption of starting materials ( tlc monitoring ) . the reaction was quenched with a 1 m hydrochloric acid aqueous solution , neutralized with nahco3 aqueous solution , and extracted with ethyl acetate ( 3 15 ml ) . the combined organic layers were washed with h2o ( 3 20 ml ) , dried over na2so4 , filtered , and concentrated under vacuo to give the titled product . boc - aminoalchol 2a e ( 1.0 equiv ) was dissolved in dry thf and transferred to a two neck - flask via cannula , followed by addition of ph3p ( 1.0 equiv ) and 4-hydroxybenzenesulfonamide ( 1.0 equiv ) . then the solution was cooled to 0 c , and diisopropyl azodicarboxylate ( diad ) ( 1.1 equiv ) was added dropwise . the reaction was warmed to r.t . and stirred at the same temperature until the starting materials were consumed ( tlc monitoring ) , quenched with slush , and extracted with ethyl acetate ( 3 15 ml ) . the combined organic layers were washed with h2o ( 3 20 ml ) , dried over na2so4 , filtered , and concentrated under vacuo to give a residue that was purified by silica gel column chromatography followed by crystallization when necessary . compounds 4a e ( 1.0 equiv ) was dissolved in dcm or 1,4-dioxane and treated with tfa . the reaction was stirred at r.t . until the starting material was consumed ( tlc monitoring ) . the solvent was removed under vacuo , and the obtained residue was crystallized from ipa or triturated from diethyl ether to obtain the titled compound as a white solid . ethanolamine 1a ( 1.0 g , 1.0 equiv ) was dissolved in dcm ( 16.5 ml ) and treated with a 1 m aqueous solution of naoh ( 1.0 equiv ) , and then di - tert - butyl dicarbonate ( 1.0 equiv ) was added . the reaction mixture was treated according to the general procedure a , previously reported , to give the titled compound 2a as a colorless liquid , which was used as it is . 66% yield ; silica gel tlc rf 0.18 ( ethyl acetate / n - hexane 50% v / v ) ; h ( 400 mhz , dmso - d6 ) 1.41 ( 9h , s ) , 3.0 ( 2h , q , j 6.0 ) , 3.38 ( 2h , t , j , 6.0 ) , 4.6 ( 1h , t , j 6.0 , exchange with d2o , oh ) , 6.71 ( 1h , brt , exchange with d2o , nh ) ; c ( 100 mhz , dmso - d6 ) 29.2 , 43.6 , 61.0 , 78.4 , 156.6 . 3-amino-1-propanol 1b ( 0.98 g , 1.0 equiv ) was dissolved in dcm ( 13 ml ) and treated with a 1 m aqueous solution of naoh ( 1.0 equiv ) , and then di - tert - butyl dicarbonate ( 1.0 equiv ) was added . the reaction mixture was treated according to the general procedure a , previously reported , to give the titled compound 2b as a colorless liquid , which was used as it is . 80% yield ; silica gel tlc rf 0.18 ( ethyl acetate / n - hexane 50% v / v ) ; h ( 400 mhz , dmso - d6 ) 1.41 ( 9h , s ) , 1.55 ( 2h , pent , j 6.4 ) , 3.0 ( 2h , q , j 6.4 ) , 3.42 ( 2h , q , j , 6.0 ) , 4.4 ( 1h , t , j 6.0 , exchange with d2o , oh ) , 6.76 ( 1h , brt , exchange with d2o , nh ) ; c ( 100 mhz , dmso - d6 ) 29.2 , 33.7 , 38.1 , 60.7 , 78.3 , 156.5 . 4-amino-1-butanol 1c ( 1.45 g , 1.0 equiv ) was dissolved in dcm ( 16 ml ) and treated with dipea ( 1.0 equiv ) and then di - tert - butyl dicarbonate ( 1.0 equiv ) . the reaction mixture was treated according to the general procedure a , previously reported , to give the titled compound 2c as a yellow liquid , which was used as it is . 70% yield ; silica gel tlc rf 0.16 ( ethyl acetate / n - hexane 50% v / v ) ; h ( 400 mhz , dmso - d6 ) 1.41 ( 13h , s ) , 2.93 ( 2h , m ) , 3.40 ( 2h , m ) , 4.39 ( 1h , t , j , 5.0 , exchange with d2o , oh ) , 6.80 ( 1h , t , j 6.0 , exchange with d2o , nh ) ; c ( 100 mhz , dmso - d6 ) 27.2 , 29.2 , 30.8 , 40.7 , 61.4 , 78.2 , 156.5 . 5-amino-1-pentanol 1d ( 1.5 g , 1.0 equiv ) was dissolved in dcm ( 14.5 ml ) and treated with a 1 m aqueous solution of naoh ( 1.0 equiv ) and then di - tert - butyl dicarbonate ( 1.0 equiv ) . the reaction mixture was treated according to the general procedure a , previously reported , to give the titled compound 2d as a yellow liquid , which was used as it is . 77% yield ; silica gel tlc rf 0.3 ( ethyl acetate / n - hexane 60% v / v ) ; h ( 400 mhz , dmso - d6 ) 1.221.32 ( 2h , m ) , 1.341.48 ( 13h , m ) 2.92 ( 2h , q , j 6.4 ) , 3.39 ( 2h , m ) , 4.36 ( 1h , t , j 5.2 , exchange with d2o , oh ) , 6.79 ( 1h , brt , exchange with d2o , nh ) ; c ( 100 mhz , dmso - d6 ) 23.8 , 29.2 , 30.3 , 33.1 , 40.8 , 61.6 , 78.2 , 156.5 . 6-amino-1-hexanol 1e ( 0.1 g , 1 equiv ) was dissolved in dcm ( 8.5 ml ) and treated with a dipea ( 1.0 equiv ) and di - tert - butyl dicarbonate ( 1.0 equiv ) . the reaction mixture was treated according to the general procedure a , previously reported , to give the titled compound 2e as a colorless oil , which was used as it is . 81% yield ; silica gel tlc rf 0.2 ( ethyl acetate / n - hexane 50% v / v ) ; h ( 400 mhz , dmso - d6 ) 1.231.34 ( 4h , m ) , 1.341.48 ( 13h , m ) , 2.89 ( 2h , q , j 6.7 ) , 3.36 ( 2h , q , j 5.2 ) , 4.3 ( 1h , t , j 5.2 , exchange with d2o , oh ) , 6.76 ( 1h , brt , exchange with d2o , nh ) ; c ( 100 mhz , dmso - d6 ) 26.2 , 27.1 , 29.2 , 30.5 , 33.4 , 40.7 , 61.6 , 78.2 , 156.5 . tert - butyl 2-hydroxyethylcarbamate 2a ( 1.0 g , 1.0 equiv ) was dissolved in dry thf ( 9.5 ml ) and was treated with ph3p ( 1.0 equiv ) , 4-hydroxybenzenesulfonamide 3 ( 1.0 equiv ) , and diad ( 1.1 equiv ) according to the general procedure b , previously reported . the reaction was stirred at r.t . until starting materials were consumed ( tlc monitoring ) . the reaction was quenched with slush and extracted with ethyl acetate ( 3 15 ml ) . the combined organic layers were washed with h2o ( 3 20 ml ) , dried over na2so4 , filtered , and concentrated under vacuo , and the obtained residue was purified by silica gel column chromatography eluting with ethyl acetate / n - hexane 60% v / v , followed by crystallization in etoh / h2o mixture to afford the titled compound 4a as a white solid . 40% yield , silica gel tlc rf 0.4 ( ethyl acetate / n - hexane 60% v / v ) ; mp 148149 c ; h ( 400 mhz , dmso - d6 ) 1.42 ( 9h , s ) , 3.33 ( 2h , t , j 5.6 ) , 4.07 ( 2h , t , j , 5.6 ) , 7.07 ( 1h , brt , exchange with d2o , nh ) , 7.1 ( 2h , d , j 8.8 ) , 7.24 ( 2h , s , exchange with d2o , so2nh2 ) , 7.77 ( 2h , d , j 8.8 ) ; c ( 100 mhz , dmso - d6 ) 29.4 , 40.3 , 68.0 , 79.2 , 115.7 , 128.9 , 137.2 , 156.9 , 162.0 . elemental analysis : calcd c 49.35 , h 6.37 , n 8.85 , s 10.14 ; found c 49.43 , h 6.03 , n 8.68 , s 9.97 ; m / z ( esi negative ) 315.6 [ m h ] . tert - butyl 3-hydroxypropylcarbamate 2b ( 0.88 g , 1.0 equiv ) was dissolved in dry thf ( 4.7 ml ) and was treated with ph3p ( 1.0 equiv ) , 4-hydroxybenzenesulfonamide 3 ( 1.0 equiv ) , and diad ( 1.1 equiv ) according to the general procedure b , previously reported . the reaction was stirred at r.t . until starting materials were consumed ( tlc monitoring ) . the reaction was quenched with slush and extracted with ethyl acetate ( 3 15 ml ) . the combined organic layers were washed with h2o ( 3 20 ml ) , dried over na2so4 , filtered , and concentrated under vacuo , and the obtained residue was purified by silica gel column chromatography eluting with ethyl acetate / n - hexane 55% v / v , followed by crystallization in ipa to afford the titled compound 4b as a white solid . 21% yield , silica gel tlc rf 0.35 ( ethyl acetate / n - hexane 55% v / v ) ; mp 134135 c ; h ( 400 mhz , dmso - d6 ) 1.41 ( 9h , s ) , 1.88 ( 2h , pent , j 6.4 ) , 3.10 ( 2h , q , j 6.4 ) , 4.08 ( 2h , t , j , 6.4 ) , 6.95 ( 1h , t , j 6.4 , exchange with d2o , nh ) , 7.09 ( 2h , d , j 8.8 ) , 7.23 ( 2h , s , exchange with d2o , so2nh2 ) , 7.77 ( 2h , d , j 8.8 ) ; c ( 100 mhz , dmso - d6 ) 29.2 , 30.0 , 37.7 , 66.6 , 78.5 , 115.3 , 128.6 , 137.0 , 156.6 , 161.9 . elemental analysis : calcd c 50.89 , h 6.71 , n 8.48 , s 9.70 ; found c 51.09 , h 7.01 , n 8.64 , s 9.54 ; m / z ( esi negative ) 329.40 [ m h ] . tert - butyl 4-hydroxybutylcarbamate 2c ( 1.11 g , 1.0 equiv ) was dissolved in dry thf ( 10.0 ml ) and was treated with ph3p ( 1.0 equiv ) , 4-hydroxybenzenesulfonamide 3 ( 1.0 equiv ) , and diad ( 1.1 equiv ) according to the general procedure b , previously reported . the reaction was stirred at r.t . until starting materials were consumed ( tlc monitoring ) . the reaction was quenched with slush and extracted with ethyl acetate ( 3 15 ml ) . the combined organic layers were washed with h2o ( 3 20 ml ) , dried over na2so4 , filtered , and concentrated under vacuo , and the obtained residue was purified by silica gel column chromatography eluting with 60% v / v ethyl acetate / n - hexane to afford the titled compound 4c as a white solid . 22% yield , silica gel tlc rf 0.40 ( ethyl acetate / n - hexane 60% v / v ) ; mp 9394 c ; h ( 400 mhz , dmso - d6 ) 1.41 ( 9h , s ) , 1.56 ( 2h , m ) , 1.74 ( 2h , m ) , 3.10 ( 2h , q , j 6.5 ) , 4.08 ( 2h , t , j 6.5 ) , 6.95 ( 1h , t , j 5.2 , exchange with d2o , nh ) , 7.09 ( 2h , d , j 8.8 ) , 7.23 ( 2h , s , exchange with d2o , so2nh2 ) , 7.77 ( 2h , d , j 8.8 ) ; c ( 100 mhz , dmso - d6 ) 26.9 , 27.0 , 29.2 , 30.2 , 68.6 , 78.4 , 115.4 , 128.6 , 137.0 , 156.6 , 162.0 ; elemental analysis : calcd c 52.31 , h 7.02 , n 8.13 , s 9.31 ; found c 52.65 , h 6.76 , n 8.01 , s 8.91 ; m / z ( esi negative ) 343.17 [ m h ] . tert - butyl 5-hydroxypentylcarbamate 2d ( 1.2 g , 1.0 equiv ) was dissolved in dry thf ( 9.0 ml ) and was treated with ph3p ( 1.0 equiv ) , 4-hydroxybenzenesulfonamide 3 ( 1.0 equiv ) , and diad ( 1.1 equiv ) according to the general procedure b , previously reported . the reaction was stirred at r.t . until starting materials were consumed ( tlc monitoring ) . the reaction was quenched with slush and extracted with ethyl acetate ( 3 15 ml ) . the combined organic layers were washed with h2o ( 3 20 ml ) , dried over na2so4 , filtered , and concentrated under vacuo , and the obtained residue was purified by silica gel column chromatography , eluting with 60% v / v ethyl acetate / n - hexane to afford the titled compound 4d as a white solid . 27% yield , silica gel tlc rf 0.42(ethyl acetate / n - hexane 60% v / v ) ; mp 9596 c ; h ( 400 mhz , dmso - d6 ) 1.371.51 ( 13h , m ) , 1.73 ( 2h , pent , j 6.8 ) , 2.94 ( 2h , q , j 6.4 ) , 4.04 ( 2h , t , j 6.4 ) , 6.80 ( 1h , t , j 5.7 , exchange with d2o , nh ) , 7.08 ( 2h , d , j 8.8 ) , 7.20 ( 2h , s , exchange with d2o , so2nh2 ) , 7.75 ( 2h , d , j 8.8 ) ; c ( 100 mhz , dmso - d6 ) 23.6 , 29.1 , 29.2 , 30.1 , 40.6 , 68.7 , 78.2 , 115.3 , 128.6 , 136.9 , 156.5 , 162.0 ; elemental analysis : calcd c 53.61 , h 7.31 , n 7.82 , s 8.95 ; found c 53.50 , h 7.01 , n 7.87 , s 8.69 ; m / z ( esi negative ) 357.60 [ m h ] . tert - butyl 6-hydroxyhexylcarbamate 2e ( 1.37 g , 1.0 equiv ) was dissolved in dry thf ( 9.0 ml ) and was treated with ph3p ( 1.0 equiv ) , 4-hydroxybenzenesulfonamide 3 ( 1.0 equiv ) , and diad ( 1.1 equiv ) according to the general procedure b , previously reported . the reaction was stirred at r.t . until starting materials were consumed ( tlc monitoring ) . the reaction was quenched with slush and extracted with ethyl acetate ( 3 15 ml ) . the combined organic layers were washed with h2o ( 3 20 ml ) , dried over na2so4 , filtered , and concentrated under vacuo , and the obtained residue was purified by silica gel column chromatography , eluting with 50% v / v ethyl acetate / n - hexane to afford the titled compound 4e as a white solid . 42% yield ; silica gel tlc rf 0.35 ( ethyl acetate / n - hexane 50% v / v ) ; 101102 c ; h ( 400 mhz , dmso - d6 ) 1.41 ( 17h , m ) , 1.75 ( 2h , q , j 8.0 ) , 2.94 ( 2h , q , j 8.0 ) , 4.08 ( 1h , t , j 5.2 , exchange with d2o , oh ) , 6.80 ( 1h , brt , exchange with d2o , nh ) , 7.11 ( 2h , d , j 8) , 7.23 ( 2h , s , exchange with d2o , so2nh2 ) , 7.77 ( 2h , d , j 8) ; c ( 100 mhz , dmso - d6 ) 23.6 , 24.8 , 26.4 , 29.2 , 30.1 , 39.5 , 68.3 , 78.2 , 115.4 , 128.7 , 136.9 , 156.3 , 162.1 . elemental analysis : calcd c 54.82 , h 7.58 , n 7.52 , s 8.61 ; found c 54.66 , h 7.58 , n 7.36 , s 8.56 ; m / z ( esi negative ) 371.25 [ m h ] . tfa ( 7.0 equiv ) was added to a stirring mixture of tert - butyl 2-(4-sulfamoylphenoxy)-ethylcarbamate 4a ( 0.5 g , 1.0 equiv ) in 10 ml of dcm . the solvents were evaporated under vacuo , and the obtained residue was triturated with diethyl ether and dried under vacuo to afford the titled compound 5a as a white solid . 88% yield , mp 142143 c ; h ( 400 mhz , dmso - d6 ) 3.30 ( 2h , t , j 5.2 ) , 4.27 ( 2h , t , j 5.2 ) , 7.17 ( 2h , d , j , 8.8 ) , 7.29 ( 2h , s , exchange with d2o , so2nh2 ) , 7.81 ( 2h , d , j 8.8 ) , 8.02 ( 3h , brt , exchange with d2o , nh3 ) ; c ( 100 mhz , dmso - d6 ) 39.2 , 65.7 , 115.7 , 118.2 ( d , jc f 299 ) , 128.7 , 137.8 , 159.4 ( q , jc f 31 ) , 161.2 ; f ( 376 mhz , dmso - d6 ) 73.5 ( 3f , s ) . elemental analysis : calcd c 36.37 , h 3.97 , n 8.48 , s 9.71 ; found c 36.76 , h 3.72 , n 8.08 , s 10.02 ; m / z ( esi positive ) 217.08 [ m cf3coo ] . tert - butyl 3-(4-sulfamoylphenoxy)-propylcarbamate 4b ( 0.8 g , 1.0 equiv ) was dissolved in 1,4-dioxane ( 10.0 ml ) , followed by addition of tfa ( 50.0 equiv ) . the reaction was treated according to the general procedure c , previously reported ( tlc monitoring ) . the solvents were evaporated under vacuo , and the obtained residue was triturated with diethyl ether and dried under vacuo to afford the titled compound 5b as a white solid . 91% yield , mp 129130 c ; h ( 400 mhz , dmso - d6 ) 2.05 ( 2h , pent , j 6.4 ) , 3.00 ( 2h , m , ) , 4.17 ( 2h , t , j 6.0 ) , 7.12 ( 2h , d , j , 8.8 ) , 7.26 ( 2h , s , exchange with d2o , so2nh2 ) , 7.78 ( 3h , brt , exchange with d2o , nh3 ) , 7.79 ( 2h , d , j 8.8 ) ; c ( 100 mhz , dmso - d6 ) 27.6 , 37.1 , 66.0 , 115.43 , 118.2 ( d , jc f 297 ) , 128.6 , 137.3 , 159.4 ( q , jc f 31 ) 161.6 ; f ( 376 mhz , dmso - d6 ) 73.64 ( 3f , s ) , elemental analysis : calcd c 38.37 , h 4.39 , n 8.14 , s 9.31 ; found c 38.42 , h 4.60 , n 7.95 , s 9.66 ; m / z ( esi positive ) 231.30 [ m cf3coo ] . tert - butyl 4-(4-sulfamoylphenoxy)-butylcarbamate 4c ( 0.38 g , 1.0 equiv ) was dissolved in dcm ( 5.0 ml ) , followed by addition of tfa ( 12.0 equiv ) . the reaction was treated according to the general procedure c previously reported ( tlc monitoring ) . the solvents were evaporated under vacuo , and the obtained residue was crystallized from ipa to afford the titled compound 5c as a white solid . 50% yield ; mp 110111 c ; h ( 400 mhz , dmso - d6 ) 1.74 ( 2h , m ) , 1.83 ( 2h , m ) , 2.90 ( 2h , t , j 7.4 ) , 4.11 ( 2h , t , j 6.0 ) , 7.10 ( 2h , d , j 8.0 ) , 7.25 ( 2h , s , exchange with d2o , so2nh2 ) , 7.79 ( 5h , m , ar h , nh3 ) ; c ( 100 mhz , dmso - d6 ) 24.8 , 26.4 , 39.5 , 68.3 , 115.4 , 118.2 ( d , jc f 298 ) 128.6 , 137.1 , 159.15 ( q , jc f 31 ) , 161.8 ; f ( 376 mhz , dmso - d6 ) 73.4 ( 3f , s ) . elemental analysis : calcd c 40.22 , h 4.78 , n 7.82 , s 8.95 ; found c 40.30 , h 4.63 , n 7.72 , s 9.56 ; m / z ( esi positive ) 245.17 [ m cf3coo ] . tert - butyl 5-(4-sulfamoylphenoxy)-pentylcarbamate 4d ( 0.46 g , 1.0 equiv ) was dissolved in dcm ( 5.0 ml ) , followed by addition of tfa ( 7.0 equiv ) . the reaction was treated according to the general procedure c , previously reported ( tlc monitoring ) . the solvents were evaporated under vacuo , and the obtained residue was triturated from diethyl ether to afford the titled compound 5d as a white solid . 90% yield ; mp 121122 c ; h ( 400 mhz , dmso - d6 ) 1.50 ( 2h , m ) , 1.63 ( 2h , m ) , 1.79 ( 2h , m ) , 2.85 ( 2h , m ) , 4.09 ( 2h , t , j 6.0 ) , 7.10 ( 2h , d , j 9.0 ) , 7.24 ( 2h , s , exchange with d2o , so2nh2 ) , 7.71 ( 3h , brt , exchange with d2o , nh3 ) , 7.77 ( 2h , d , j 9.0 ) ; c ( 100 mhz , dmso - d6 ) 23.4 , 27.6 ; 28.9 , 39.6 , 68.6 , 115.3 , 118.1 ( d , jc f 298 ) , 128.6 , 137.0 , 159.1 ( q , jc f 31 ) , 161.9 ; f ( 376 mhz , dmso - d6 ) 73.5 ( 3f , s ) . elemental analysis : calcd c 41.93 , h 5.14 , n 7.52 , s 8.61 ; found c 41.73 , h 5.23 , n 7.25 , s 8.34 ; m / z ( esi positive ) 259.17 [ m - cf3coo ] . tert - butyl 6-(4-sulfamoylphenoxy)-hexylcarbamate 4e ( 0.10 g , 1.0 equiv ) was dissolved in dcm ( 1.8 ml ) , followed by addition of tfa ( 5.0 equiv ) . the reaction was treated according to the general procedure c , previously reported ( tlc monitoring ) . the solvents were evaporated under vacuo , and the obtained residue was crystallized from ipa to afford the titled compound 5e as a white solid . 30% yield ; mp 122123 c ; h ( 400 mhz , dmso - d6 ) 1.45 ( 4h , m ) , 1.59 ( 2h , pent , j 7.5 ) , 1.76 ( 2h , pent , j 7.5 ) , 2.82 ( 2h , t , j 7.5 ) , 4.08 ( 2h , t , j 6.5 ) , 7.11 ( 2h , d , j 9.0 ) , 7.25 ( 2h , brs , exchange with d2o , so2nh2 ) , 7.63 ( 3h , brt , exchange with d2o , nh3 ) , 7.77 ( 2h , d , j 9.0 ) ; c ( 100 mhz , dmso - d6 ) 25.9 , 26.4 , 27.9 , 30.7 , 39.7 , 68.7 , 115.4 , 118.1 ( d , jc f 298 ) , 128.6 , 137.0 , 159.3 ( q , jc f 31 ) , 162.0 ; f ( 376 mhz , dmso - d6 ) 73.5 ( 1f , s ) . elemental analysis : calcd c 43.52 , h 5.48 , n 7.25 , s 8.30 ; found c 43.19 , h 5.24 , n 6.95 , s 8.16 ; m / z ( esi positive ) 273.40 [ m cf3coo ] . the o - alkylbenzenesulfonamide salt 5 ( 1.0 equiv ) and 2-(6-hydroxy-3-oxo-3h - xanthen-9-yl)-5-isothiocyanatobenzoic acid 6 ( 1.0 equiv ) were poured into a two - neck flask , and dry dma ( 1.0 ml ) was added , followed by addition of tea ( 1.5 equiv ) . the reaction was stirred at r.t . until starting materials were consumed ( tlc monitoring ) . it was then quenched with slush and a 6 m aqueous hydrochloric acid solution and then extracted with ethyl acetate ( 3 15 ml ) . the combined organic layers were washed with h2o ( 3 20 ml ) , dried over na2so4 , filtered , and concentrated in vacuo to afford the titled compound 7 as an orange powder . 2-(4-sulfamoylphenoxy)-ethylammonium trifluoroacetate salt 5a ( 50 mg , 1.0 equiv ) and 2-(6-hydroxy-3-oxo-3h - xanthen-9-yl)-5-isothiocyanatobenzoic acid 6 ( 1.0 equiv ) were treated according to the general procedure previously reported afford the titled compound 7a as an orange powder . 76% yield ; silica gel tlc rf 0.10 ( meoh / dcm 10% v / v ) ; mp 172173 c ( dec . ) ; h ( 400 mhz , dmso - d6 ) 3.98 ( 2h , brt ) , 4.32 ( 2h , t , j 5.2 ) , 6.616.66 ( 4h , m ) , 6.71 ( 2h , d , j 2.0 ) , 7.23 ( 5h , m , 3h ar h , 2h exchange with d2o , so2nh2 ) , 7.76 ( 1h , exchange with d2o , nh ) , 7.80 ( 2h ar h , d , j 8.8 ) , 8.28 ( 1h , s ) , 8.35 ( 1h , exchange with d2o , nh ) , 10.11 ( 1h , exchange with d2o , nh ) , 10.15 ( 2h , exchange with d2o , oh ) ; c ( 100 mhz , dmso - d6 ) 44.0 , 67.1 , 84.1 , 103.2 , 110.7 , 113.6 , 115.5 , 117.6 , 125.1 , 127.6 , 128.7 , 130.0 , 130.6 , 137.4 , 142.1 , 148.3 , 152.9 , 160.5 , 161.7 , 169.5 , 181.8 ( c = s ) . elemental analysis : calcd c 57.51 , h 3.83 , n 6.94 , s 10.59 ; found c 57.56 , h 4.03 , n 6.68 , s 10.84 ; m / z ( esi positive ) 606.50 [ m + h ] . 3-(4-sulfamoylphenoxy)-propylammonium trifluoroacetate salt 5b ( 50 mg , 1.0 equiv ) and 2-(6-hydroxy-3-oxo-3h - xanthen-9-yl)-5-isothiocyanatobenzoic acid 6 ( 1.0 equiv ) were treated according to the general procedure e , previously reported , to afford the titled compound 7b as an orange powder . 83% yield ; silica gel tlc rf 0.40 ( meoh / dcm 20% v / v ) ; mp 185186 c ( dec ) ; h ( 400 mhz , dmso - d6 ) 2.11 ( 2h , t , j 6.4 ) , 3.72 ( 2h , brt ) , 4.18 ( 2h , t ; j 5.8 ) , 6.62 ( 4h , m ) , 6.71 ( 2h , s ) , 7.15 ( 2h , d , j 8.8 ) , 7.23 ( 2h , s , exchange with d2o , so2nh2 ) , 7.79 ( 2h , d , j 8.8 ) , 8.26 ( 1h , exchange with d2o , nh ) , 10.00 ( 1h , exchange with d2o , nh ) , 10.15 ( 2h , exchange with d2o , oh ) ; c ( 100 mhz , dmso - d6 ) 29.0 , 41.9 , 66.9 , 85.2 , 103.3 , 110.8 , 113.7 , 115.5 , 117.7 , 125.1 , 127.6 , 128.7 , 130.1 , 130.6 , 137.2 , 142.4 , 148.0 , 153.0 , 160.6 , 162.0 , 169.5 , 181.6 ( c = s ) ; m / z ( esi positive ) 621.0 [ m + 2h ] . 4-(4-sulfamoylphenoxy)-butylammonium trifluoroacetate salt 5c ( 50 mg , 1.0 equiv)and 2-(6-hydroxy-3-oxo-3h - xanthen-9-yl)-5-isothiocyanatobenzoic acid 6 ( 1.0 equiv ) were treated according to the general procedure e , previously reported , to afford the titled compound 7c as an orange powder . 79% yield ; silica gel tlc rf 0.70 ( meoh / dcm 20% v / v ) ; mp 200201 c ( dec ) ; h ( 400 mhz , dmso - d6 ) 1.771.87 ( 4h , m ) , 3.64 ( 2h , brs ) , 4.14 ( 2h , t , j 6.1 ) , 6.606.66 ( 4h , m ) , 6.71 ( 2h , d , j 2.2 ) , 7.13 ( 2h , d , j 8.8 ) , 7.22 ( 3h , d , j 8.3 , ar h , 2h exchange with d2o , so2nh2 ) , 7.77 ( 2h , d , j 8.8 ) , 8.30 ( 2h , s , 1h exchange with d2o , nh ) , 10.09 ( 3h , brs , exchange with d2o , 2xoh , nh ) ; c ( 100 mhz , dmso - d6 ) 26.4 , 26.9 , 45.4 , 68.5 , 85.3 , 103.2 , 110.0 , 113.7 , 115.4 , 117.1 , 126.0 , 128.1 , 128.7 , 129.8 , 130.0 , 137.1 , 141.9 , 148.9 , 153.1 , 160.8 , 161.9 , 168.6 , 182.6 ( c = s ) ; m / z ( esi positive ) 634.5 [ m + h ] . compound 5 ( 1.0 equiv ) and [ 9-(2-carboxy-4-isothiocyanatophenyl)-6-dimethylaminoxanthen-3-ylidene]dimethylammonium chloride 8 ( 1.0 equiv ) were poured into a two - neck flask , and dry dma ( 1.0 ml ) was added , followed by addition of tea ( 1.5 equiv ) . the reaction was stirred at r.t . until starting materials were consumed ( tlc monitoring ) , and then it was quenched with slush and a 6 m aqueous hydrochloric acid solution . the precipitate formed was centrifuged , collected , dried under vacuo , washed with diethyl ether ( 3 10 ml ) , and dried under vacuo to afford the titled compound 9 as a red powder . 2-(4-sulfamoyl - phenoxy)ethylammonium trifluoroacetate salt 5a ( 6.9 mg , 1.0 equiv ) and [ 9-(2-carboxy-4-isothiocyanatophenyl)-6-dimethylaminoxanthen-3-ylidene]-dimethylammonium chloride 8 ( 1.0 equiv ) were treated according to the previously reported procedure to afford the titled compound 9a as a red powder . 70% yield ; silica gel tlc rf 0.40 ( meoh / dcm 20% v / v ) ; mp 177178 c ( dec ) ; h ( 400 mhz , dmso - d6 ) 3.30 ( 12h , s ) , 3.99 ( 2h , brt ) , 4.33 ( 2h , t , j 5.4 ) , 7.00 ( 4h , m ) , 7.14 ( 3h , m ) , 7.21 ( 2h , d , j 8.8 ) , 7.26 ( 2h , s , exchange with d2o , so2nh2 ) , 7.41 ( 1h , m ) , 7.72 ( 1h , d , j 8.8 ) , 7.81 ( 2h , d , j 8.8 ) , 8.46 ( 2h , brs , 1h , exchange with d2o , nh ) , 10.38 ( 1h , exchange with d2o , nh ) ; c ( 100 mhz , dmso - d6 ) 35.1 , 41.5 , 43.9 , 67.1 , 97.2 , 114.1 , 115.3 , 122.6 , 124.5 , 125.9 , 128.7 , 131.7 , 137.5 , 142.6 , 157.6 , 160.2 , 161.4 , 161.6 , 166.8 , 167.1 ( c = o ) , 181.5 ( c = s ) ; m / z ( esi positive ) 660.50 [ m cl ] . 3-(4-sulfamoylphenoxy)-propylammonium trifluoroacetate salt 5b ( 7.2 mg , 1.0 equiv ) and [ 9-(2-carboxy-4-isothiocyanatophenyl)-6-dimethylamino - xanthen-3-ylidene]-dimethylammonium chloride 8 ( 1.0 equiv ) were treated according to the previously reported procedure to afford the titled compound 9b as a red powder . 74% yield ; silica gel tlc rf 0.30 ( meoh / dcm 20% v / v ) ; mp 192193 c ( dec ) ; h ( 400 mhz , dmso - d6 ) 3.30 ( 12h , s ) , 4.13 ( 2h , t , j 6.2 ) , 4.21 ( 2h , t , j 6.2 ) , 6.98 ( 3h , m ) , 7.087.25 ( 8h , m ) , 7.40 ( 1h , d , j 8.8 ) , 7.75 ( 1h , d , j 8.8 ) , 7.79 ( 2h , d , j 8.8 ) , 7.87 ( 1h , brs ) , 7.94 ( 1h , d , j 8.8 ) , 8.58 ( 1h , brt , exchange with d2o , nh ) , 10.51 ( 1h , brs , exchange with d2o , nh ) , 10.65 ( 1h , s ) ; c ( 100 mhz , dmso - d6 ) 28.8 , 35.0 , 41.4 , 41.5 , 66.8 , 97.2 , 114.2 , 115.5 , 122.6 , 124.2 , 127.0 , 128.6 , 131.7 , 137.1 , 142.6 , 157.7 , 160.8 , 161.9 , 161.6 , 166.8 , 167.1 ( c = o ) , 181.5 ( c = s ) ; m / z ( esi positive ) 674.42 [ m cl ] . two microliters of a 600 mm concentration of 4c was added to a 500 l 1.6 m sodium citrate , 50 mm tris one microliter of this reservoir solution was added to 5 l of ca ii at a final concentration of 10 mg / ml so that the final drug concentration was at 0.24 mm . diffraction data for ca ii-4c and ca ii-5c complexes were collected on an in - house r - axis iv image plate detector using a ru - h3r rotating cu anode ( k = 1.5418 ) operating at 50 kv and 22 ma . images were collected every 1 with an exposure time of 5 min at a detector distance of 100 mm . phasing was carried out in the phenix suite of programs using the auto molecular replacement procedure to obtain the initial phases using a previously solved hca ii structure with water molecules removed ( pdb code 3ks3 ) . the graphics program coot was used to view the electron density map , and the structure was adjusted on the basis of the calculated electron density . topology files of the inhibitors were generated using the prodrg server , and these files were used to model the drug into the density generated . the geometric restraints of the final model were analyzed using procheck . the data diffraction and final model refinement statistics are summarized in table 2 . male new zeland albino rabbits weighing 15002000 g were used in these studies . animals were anaesthetized using zoletil ( tiletamine chloride + zolazepam chloride , 3 mg / kg body weight , i.m . injection ) and injected with 0.05 ml hypertonic saline solution ( 5% in distilled water ) into the vitreous of both eyes . iop was measured by using a digital tonometer ( tomo - pen avia tonometer , reichert inc . depew , ny 14043 , usa ) prior to hypertonic saline injection ( basal ) at 1 , 2 , and 4 h after administration of the drug . vehicle ( phosphate buffer , ph 7.0 plus dmso 2% ) or drugs were instilled immediately after the injection of hypertonic saline into the conjunctive pocket . experiments with animals were conducted in agreement with current ethical guidelines and norms approved by the ethical committee of our university .

table 1 summarizes the criteria for the 7 dsm - iv somatoform disorders.18 the only validated entities , somatization disorder ( sd ) and conversion disorder , are infrequent.18,20,2533 the failure to validate the other dsm - iv entities stems from extensive overlap of criteria.34 an abridged sd ( asd ) construct requires fewer symptoms and is more comprehensive , but it also lacks validation.26,35 multi - somatoform disorder ( msd ) 3638 defines mus patients of similar severity,39 and its reliability and validity presently are under investigation.36,38,40,41table 1dsm - iv somatoform disorderssomatization disorder is of many years duration , begins before age 30 , is more common in women , and has ( over a lifetime ) at least four pain symptoms , two gastrointestinal symptoms , one sexual symptom , and one pseudoneurological symptom.undifferentiated somatoform disorder , the vast majority of persistent somatizers , is a residual category for patients who do not meet criteria for other somatoform disorders , is of at least 6 months duration , has no gender or age limit , and has at least one symptom.conversion disorder usually occurs acutely and lasts about 2 weeks but may be recurring or chronic , is most frequent in women before age 35 , and exhibits one or more motor , sensory , or seizure ( pseudoneurological ) symptoms.pain disorder occurs at any age , more often in women , usually is chronic and persistent , and has one or more pain symptoms that are the predominant focus of the presentation and that are not restricted to dyspareunia.hypochondriasis occurs at any age in males and females , may be more common in early adulthood , is at least 6 months duration and often chronic and persistent , and has one or more symptoms that provoke an unwarranted fear ( which is not delusional or restricted to concerns about appearance ) of organic disease even after reassurance and appropriate investigation.body dysmorphic disorder begins in adolescence , occurs in males and females equally , is chronic and persistent , and is suggested by preoccupation with an alleged defect in appearance that causes patients to feel ugly ( anorexia nervosa is classified elsewhere ) ; when of delusional intensity , an additional diagnosis of delusional disorder , somatic type is made.somatoform disorder not otherwise specified includes disorders with somatoform symptoms that do not meet the above criteria , such as pseudocyesis and symptoms of less than 6 months duration . dsm - iv somatoform disorders consistent with others , 4,26,42 smith , gardiner , and colleagues demonstrated in 206 distressed , high - utilizing mus patients that less than 25% had any dsm - iv somatoform disorders ( 4.4% ) or asd ( 18.9% ) . nonetheless , 60.2% had nonsomatoform ( psychiatric ) diagnoses , primarily anxiety and depression.23 this study s gold standard definition of mus came from a reliable , physician - conducted chart review.23,43 the dsm - negative patients were less psychologically and physically distressed than those with dsm - iv somatoform diagnoses or asd , but they were more distressed than the normal ones . because researchers have relied almost entirely on dsm as the gold standard for mus , these large numbers of distressed dsm - negative patients have been completely overlooked by the field.43 an earlier study identified some of these patients as minor acute illness ( mai ) to highlight minor , transient symptoms that differ from the 41 in dsm - iv.44 in low - utilizing mus patients , most symptoms in primary care resolve spontaneously and permanently . for example , a prospective study of 500 ambulatory clinic patients showed that 70% improved after 2 weeks and that this improvement was sustained after 3 months.45,46 in medical settings , chronic mus patients typically are understood as difficult47 or as one of several named mus conditions , e.g. , chronic fatigue syndrome , irritable bowel syndrome , fibromyalgia . table 1 summarizes the criteria for the 7 dsm - iv somatoform disorders.18 the only validated entities , somatization disorder ( sd ) and conversion disorder , are infrequent.18,20,2533 the failure to validate the other dsm - iv entities stems from extensive overlap of criteria.34 an abridged sd ( asd ) construct requires fewer symptoms and is more comprehensive , but it also lacks validation.26,35 multi - somatoform disorder ( msd ) 3638 defines mus patients of similar severity,39 and its reliability and validity presently are under investigation.36,38,40,41table 1dsm - iv somatoform disorderssomatization disorder is of many years duration , begins before age 30 , is more common in women , and has ( over a lifetime ) at least four pain symptoms , two gastrointestinal symptoms , one sexual symptom , and one pseudoneurological symptom.undifferentiated somatoform disorder , the vast majority of persistent somatizers , is a residual category for patients who do not meet criteria for other somatoform disorders , is of at least 6 months duration , has no gender or age limit , and has at least one symptom.conversion disorder usually occurs acutely and lasts about 2 weeks but may be recurring or chronic , is most frequent in women before age 35 , and exhibits one or more motor , sensory , or seizure ( pseudoneurological ) symptoms.pain disorder occurs at any age , more often in women , usually is chronic and persistent , and has one or more pain symptoms that are the predominant focus of the presentation and that are not restricted to dyspareunia.hypochondriasis occurs at any age in males and females , may be more common in early adulthood , is at least 6 months duration and often chronic and persistent , and has one or more symptoms that provoke an unwarranted fear ( which is not delusional or restricted to concerns about appearance ) of organic disease even after reassurance and appropriate investigation.body dysmorphic disorder begins in adolescence , occurs in males and females equally , is chronic and persistent , and is suggested by preoccupation with an alleged defect in appearance that causes patients to feel ugly ( anorexia nervosa is classified elsewhere ) ; when of delusional intensity , an additional diagnosis of delusional disorder , somatic type is made.somatoform disorder not otherwise specified includes disorders with somatoform symptoms that do not meet the above criteria , such as pseudocyesis and symptoms of less than 6 months duration . consistent with others , 4,26,42 smith , gardiner , and colleagues demonstrated in 206 distressed , high - utilizing mus patients that less than 25% had any dsm - iv somatoform disorders ( 4.4% ) or asd ( 18.9% ) . nonetheless , 60.2% had nonsomatoform ( psychiatric ) diagnoses , primarily anxiety and depression.23 this study s gold standard definition of mus came from a reliable , physician - conducted chart review.23,43 the dsm - negative patients were less psychologically and physically distressed than those with dsm - iv somatoform diagnoses or asd , but they were more distressed than the normal ones . because researchers have relied almost entirely on dsm as the gold standard for mus , these large numbers of distressed dsm - negative patients have been completely overlooked by the field.43 an earlier study identified some of these patients as minor acute illness ( mai ) to highlight minor , transient symptoms that differ from the 41 in dsm - iv.44 in low - utilizing mus patients , most symptoms in primary care resolve spontaneously and permanently . for example , a prospective study of 500 ambulatory clinic patients showed that 70% improved after 2 weeks and that this improvement was sustained after 3 months.45,46 in medical settings , chronic mus patients typically are understood as difficult47 or as one of several named mus conditions , e.g. , chronic fatigue syndrome , irritable bowel syndrome , fibromyalgia . patients into 1 category , and other names have been suggested , e.g. , mus spectrum disorder or physical symptom disorder . 4,1724 table 2 provides a template and supporting data for an evolving unitary or continuum model , and it identifies where the categorical disease entities fall on the spectrum of mus . table 2the clinical spectrum of mus * normal to mild 80% 9,46,52moderate 15%severe 5%very severe < 1%common nameworried welldsm - negative ; maiasd ; msdsdutilizationlow 9high 23,44high 3high 3,91,92age of onsetanyanyany<30 years 18specific physical symptomsanyanyfrom dsm symptom list of 41 ( asd ) or 15 ( msd ) 18,3941 specific symptoms in dsm - iv from 4 areas : pain , gi , sexual , neurological 18body systems involvedanyanymusculoskeletal , gi , nervous , or ill - defined systems 93,94musculoskeletal , gi , nervous , or ill - defined systems 93,94symptom duration 95acute days to weekssubacute > 6 mos 18number of symptomsfewany>3 ( men ) & > 5 ( women ) for asd 35,96>7 18symptoms occur and recur with external stress and clear when it abates 95yesyes , but recur frequentlyno , but worsen with stressno , but worsen with stressdepression , anxiety , dysthymia , and other psychiatric problems?20% 23,97,9867% 358899% 61,99personality structurenormal?personality disorder 98,1006172% personality disorder 101,102 ; rarely , psychoticprevalence , community100% 6,103?4.422% 20,2527,35,1040.030.7% 18,20,2527prevalence , all outpatients??33% 357% 3,92prevalence , inpatients???9% 91prevalence , outpatients with > 5 visits per year?51% had mai 4414% ( includes very severe ) 44mus = medically unexplained symptoms ; mai = minor acute illness ( derived from chart rating ) ; dsm = diagnostic and statistical manual of mental disorders ; asd = abridged somatization disorder ; msd = multi - somatoform disorder ; sd = somatization disorder ; gi = gastrointestinal.*comorbid medical disease is frequent throughout the spectrum ; psychiatric disease also is prevalent , but increases with increasing severity and utilization in mus.because there are many data on sd , a separate column ( very severe ) has been included , although sd is very rare.after organic disease is excluded , these areas particularly lend themselves to the quantification needed for explicit , concrete criteria for mus subtyping , e.g. , an average of 15 visits yearly over many years with 8 mus symptoms during the last year that are chronic in a patient with severe depression = severe ; an average of 8 visits / year for the last 24 months for 5 mus symptoms that occur intermittently but are becoming regularly persistent in a depressed patient = moderate ; an average of 2 visits yearly for many years for 2 or 3 mus symptoms that always occur in relationship to stress and abate with its resolution in a non - depressed patient = mild . these examples highlight the proposed need for research to provide specific criteria for each sub - category of mus , e.g. , cutoff points for number of symptoms , number of visits , and the degree of depression.this study did not separate severe and very severe.areas where data are unavailable and where research is particularly needed . the clinical spectrum of mus * mus = medically unexplained symptoms ; mai = minor acute illness ( derived from chart rating ) ; dsm = diagnostic and statistical manual of mental disorders ; asd = abridged somatization disorder ; msd = multi - somatoform disorder ; sd = somatization disorder ; gi = gastrointestinal . * comorbid medical disease is frequent throughout the spectrum ; psychiatric disease also is prevalent , but increases with increasing severity and utilization in mus . because there are many data on sd , a separate column ( very severe ) after organic disease is excluded , these areas particularly lend themselves to the quantification needed for explicit , concrete criteria for mus subtyping , e.g. , an average of 15 visits yearly over many years with 8 mus symptoms during the last year that are chronic in a patient with severe depression = severe ; an average of 8 visits / year for the last 24 months for 5 mus symptoms that occur intermittently but are becoming regularly persistent in a depressed patient = moderate ; an average of 2 visits yearly for many years for 2 or 3 mus symptoms that always occur in relationship to stress and abate with its resolution in a non - depressed patient = mild . these examples highlight the proposed need for research to provide specific criteria for each sub - category of mus , e.g. , cutoff points for number of symptoms , number of visits , and the degree of depression . areas where data are unavailable and where research is particularly needed . in summarizing this classification , we also have been guided by klinkman , coyne , and colleagues who identified 3 parameters for classifying depression , and we have applied them to mus : severity , duration , and comorbidity.4851 in this continuum or dimensional model , sd is at the very severe end , whereas asd and msd also are labeled severe . those who do not resemble dsm - iv entities ( dsm - negative ; minor acute illness ) are in the moderate range of the spectrum , merging into mild and normal mus patients when health care seeking and psychological distress decrease . extrapolating from many9,46,52 , we estimate in table 2 that 80% of all mus patients in a clinical setting are mild : acute symptoms , low utilization , respond to reassurance and resolution of stressors , and present little difficulty for providers . although often receiving much testing , they typically are not recognized as mus at all ; they are viewed , for example , as noise in the system . the remaining 20% of mus patients are high utilizers , which vary from subacute to chronic , and exist on the severity spectrum from moderate to severe . this group features the physical disability and severe psychological problems that command most of our clinical attention , the ones providers usually think of as somatization , mus , or the difficult patient . comorbid organic and psychiatric diseases are common across the entire severity spectrum , but psychological dysfunction and psychiatric diagnoses increase as the mus becomes more severe,53 as do functional disability and joblessness,26,54 a history of physical or sexual abuse,5557 and prescription and nonprescription substance misuse.5860 in primary care patients with sd , 10 psychiatric problems were more prevalent than in the general population , in the order of decreasing prevalence : depression , anxiety , phobia , panic , alcohol abuse , obsessive - compulsive , antisocial personality , schizophrenia , cognitive impairment , and mania.61 some posit that personality disorders in general are comorbid conditions.57,62 mus can be diagnosed only by excluding organic diseases.18,63 after that , clinicians also can make dsm - iv somatoform diagnoses or asd or one of the named syndromes such as ibs . our focus on excluding organic diseases does not preclude the possibility of underlying , explanatory psychophysiological changes,64 nicely summarized recently for ibs,65 nor does it preclude that improved understanding in the future could provide organic disease explanations for what we now call mus.28 nevertheless , with our present universally applied , disease - based classification system , the only useful , broadly applicable way to diagnose mus patients is to exclude organic disease . our long - range goal , however , continues to be integrating psychosocial and biomedical aspects to produce the biopsychosocial diagnoses articulated by engel over a quarter of a century ago.29,66,67 bespeaking our progress toward biopsychosocial medicine , the isolated - disease focus needed for diagnosis does not apply to treatment because psychosocial factors already are demonstrably key elements in successful medical treatment.11,68,69 we recommend the following clinical guidelines to exclude organic diseases . normal to mild reflects the infrequent , appropriate seeking of reassurance for worrisome symptoms , a normal illness behavior.70 symptoms may be of any type and intensity but usually are few and mild , and they seldom require much laboratory or other diagnostic investigation . rather , excluding organic diseases occurs primarily by history and physical examination and by follow - up observation over time.44 making mild mus explicit as a diagnosis can help resolve the problem of excessive laboratory testing , unnecessary treatments , and iatrogenic complications.1216 when symptoms do not follow the expected acute clinical course ( prompt resolution ) , an organic disease or moderate mus with incipient high utilization is considered . moderate mus moderate mus also can have symptoms of any type and intensity , but this newly recognized group exhibits much greater psychological and physical distress and utilization than those with normal to mild mus.23 each episode of symptoms tends to be self - limited over a few weeks to months , but these patients exhibit high utilization during this symptomatic period and with recurrences of the same or a different episode , the subsequent episode often clearing completely as well ; some , however , have chronic , low - grade symptoms and merge into the next category . initially , after a careful history and physical examination , observation over time suffices to exclude organic diseases . nevertheless , with frequent recurrences or chronicity and increased utilization , diagnostic work - up to exclude organic disease usually is needed . severe mus in contrast to moderate mus , severe mus is characterized by more bothersome and persistent physical symptoms ( more often of the type found in dsm - iv ) , still greater utilization , and more physical and psychological dysfunction . these patients require definitive laboratory or consultative investigation or both to exclude organic diseases in many instances but only if not already performed 6,20,35,71 and if not resolved by the initial history and physical examination ( h & p ) where a diagnosis sometimes can be established without further investigation , e.g. , a clinical diagnosis of angina . the h & p , of course , also provides the guidance that specifies which lab tests to order.72 because of the frequent presence of serious current or lifetime psychiatric disorder , one also makes sure that the diagnostic process itself does not frighten an already distressed patient . making a clear , definitive diagnosis of moderate / severe mus is essential : it leads the provider to the next - step treatment11rather than repetition of testing and consultation in a few months for persisting symptoms . moderate and severe mus patients require work - up , even with prominent psychological complaints and without classical textbook criteria for disease , because there is a high prevalence ( prior probability ) of underlying organic diseases.5 patients with chronic low back pain , even with no objective neurological signs , usually require mri , ct , or myelography to exclude impending neurological compromise , infection , or tumor.71,73 for example , 1 study showed clinically significant disease ( beyond the common uncomplicated disc protrusion and degenerative changes ) in 15%.74 the sensitivity and specificity of clinical findings , except for sciatica , are not sufficient to exclude significant organic diseases in chronic low back pain.75 similarly , 35% of patients with chronic abdominal pain or altered bowel habits or both , the symptoms alone suggesting ibs , had underlying organic disease explanations . 76 investigation ( e.g. , colonoscopy ) is indicated before one can diagnose these patients as mus 77 , especially those over 45 years of age.78 chronic pelvic pain is often thought to be caused by mus because of prominent psychological symptoms and a negative physical exam . but , from 41% to 75% of these women have organic disease explanations , such as endometriosis , adhesions , and chronic pelvic inflammatory disease,7981 and laparoscopy usually is recommended.7983 we note that recognizing some organic diseases may still not lead to success in difficult - to - treat conditions such as endometriosis , and that severe chronic organic diseases , especially those with pain , can lead to illness behaviors similar to those found in chronic mus patients.68,84,85 the following illustrate the pitfalls of relying on symptoms alone to make a diagnosis of moderate and severe mus . physical symptom criteria alone ( the rome criteria ) for the diagnosis of ibs show a sensitivity of 0.85 and a specificity of only 0.71 when gastrointestinal ( gi ) symptoms ( e.g. , bloating , diarrhea ) are used to distinguish ibs from organic diseases.77,78,86 in another study , 1 of 3 of all organic diseases and one - half of patients with active peptic ulcer were missed using clinical symptom criteria alone.87 involving 11,366 patients , a review of 15 studies of upper gi symptoms concluded that physical symptoms did not distinguish between nonorganic ( mus ) and organic diseases.88 psychological symptom criteria alone did not distinguish ibs from those with subsequently proven organic diseases in a prospective study of patients with abdominal pain and altered bowel habits.76 both groups had similarly elevated psychological symptom scores compared to healthy population normals . these data can be predicted by the biopsychosocial model66 , which tells us that psychosocial factors are indeed ubiquitous among organic and nonorganic ( mus ) patients.85 table 3 provides several examples demonstrating how the proposed diagnostic classification might look . table 3examples of muscase 1the most common : mild musa 32-year - old man with controlled hypertension presented with the new onset of fatigue and distracting headaches , and he mentioned the threat of being laid off work . physical examination was negative , and you empathized , supported , reassured , ordered no tests , and recommended ibuprofen . he reported 2 weeks later the symptoms had cleared , and that he was back to work.diagnosismus severity mild duration acute comorbidity essential hypertensioncase 2less common : moderate musa 44-year - old woman presented with yet another episode of low back pain without radicular symptoms . the pain interfered with work , and she had been in the clinic with recurrences 7 times in the preceding 12 months . she was not enjoying her life and said that she had difficulty sleeping , but did not feel depressed . you obtained an mri of the spine that provided no explanation for the pain ( small disc without neurologic compromise ) , and you implemented a program of treatment for her mus and depression,10,11 advised exercise and weight control , and increased her metformin dose.diagnosismus severity moderate duration subacute comorbidity depression and poorly controlled diabetes mellituscase 3least common : severe musa 50-year - old man related a long history of severe neck pain and headaches , virtually constant over the last 5 years . he wanted a new approach because he was not getting better , even though he went to 4 doctors and 2 pain clinics in the last year . he denied depression but did have anhedonia ( lack of enjoyment ) , insomnia , difficulty concentrating , and weight gain over the preceding year . you did not repeat the neck and brain mri his previous doctor had obtained 3 months earlier but reviewed it with the radiologist and learned that several minor abnormalities ( a few white matter changes and mild disc protrusion without neurologic compromise ) were unrelated to his symptoms . you initiated treatment for his mus and depression10,11 and advised a short trial of antibiotics for his copd.diagnosismus severity severe duration chronic comorbidity depression , copd rare organic diseases ( such as wilson s disease ) , or those with vague or unusual presentations ( such as multiple sclerosis , lyme disease , and porphyria ) , or those that may have prominent psychological symptoms ( such as some with carcinoma of the pancreas , subdural hematoma , or ulcerative colitis ) may be misdiagnosed as mus if the physician does not have an appropriate index of suspicion . mus also must be distinguished from 2 rare psychiatric disorders : factitious disorder ( fd ) and malingering . for the sole purpose of assuming the sick role ( lack external incentives ) , patients with fd intentionally produce organic disease , the munchausen syndrome being an extreme example , or they feign psychological symptoms . unlike mus , patients with fd usually have obvious organic diseases , although the self - induction itself may not be recognized initially , e.g. , bleeding secondary to surreptitious anticoagulant ingestion or fever caused by self - injection of feces . fd patients feigning psychiatric illness , however , are much more difficult to differentiate . malingering patients do not induce organic diseases , but they feign or grossly exaggerate physical or psychological symptoms for some external incentive such as financial compensation or obtaining drugs . mus patients do not intentionally produce or feign their symptoms and usually do not have obvious external incentives . a much more common primary care differential diagnosis occurs when a patient known to have significant organic disease develops mus around the same symptoms and thus poses a difficult diagnostic problem,63 e.g. , the patient with a recent myocardial infarction who now complains daily of chest pain . after investigation to ensure stability , equally troublesome , how does one determine whether a known mus patient develops an organic disease ? it has been proposed that when a symptom represents a new organic disease , the patient will present in a clearly different way .5,89,90 if the physician carefully listens to and briefly examines the patient for objective evidence of disease , a significant organic disease seldom is overlooked.5 rare organic diseases ( such as wilson s disease ) , or those with vague or unusual presentations ( such as multiple sclerosis , lyme disease , and porphyria ) , or those that may have prominent psychological symptoms ( such as some with carcinoma of the pancreas , subdural hematoma , or ulcerative colitis ) may be misdiagnosed as mus if the physician does not have an appropriate index of suspicion . mus also must be distinguished from 2 rare psychiatric disorders : factitious disorder ( fd ) and malingering . for the sole purpose of assuming the sick role ( lack external incentives ) , patients with fd intentionally produce organic disease , the munchausen syndrome being an extreme example , or they feign psychological symptoms . unlike mus , patients with fd usually have obvious organic diseases , although the self - induction itself may not be recognized initially , e.g. , bleeding secondary to surreptitious anticoagulant ingestion or fever caused by self - injection of feces . fd patients feigning psychiatric illness , however , are much more difficult to differentiate . malingering patients do not induce organic diseases , but they feign or grossly exaggerate physical or psychological symptoms for some external incentive such as financial compensation or obtaining drugs . mus patients do not intentionally produce or feign their symptoms and usually do not have obvious external incentives . a much more common primary care differential diagnosis occurs when a patient known to have significant organic disease develops mus around the same symptoms and thus poses a difficult diagnostic problem,63 e.g. , the patient with a recent myocardial infarction who now complains daily of chest pain . after investigation to ensure stability , equally troublesome , how does one determine whether a known mus patient develops an organic disease ? it has been proposed that when a symptom represents a new organic disease , the patient will present in a clearly different way .5,89,90 if the physician carefully listens to and briefly examines the patient for objective evidence of disease , a significant organic disease seldom is overlooked.5 to maximize care and understanding , an emerging perspective indicates that mus be classified according to : ( 1 ) severity , ranging from mild moderate severe ; ( 2 ) duration , where most mus patients will be acute ( and mild ) , but the most difficult ones will be subacute and chronic ( moderate and severe ) ; and ( 3 ) comorbidity , psychiatric or medical or both . history and physical examination and observation over time suffice to make the diagnosis , by excluding organic diseases , in 80% of mus patients . these mild patients have a few acute visits and little ongoing physical or psychological distress . severe , have increased utilization for subacute / chronic symptoms and are more physically and psychologically distressed . they typically require laboratory evaluation to exclude organic diseases and make a diagnosis of mus . mus is largely untreated , common and costly , and attended by considerable distress and morbidity some iatrogenic . because improved recognition / diagnosis can ameliorate these problems , we recommend convening a group of experts to develop research - based , consensus definitions for each subtype of the mus spectrum . whereas the symptom and utilization parameters can not themselves exclude organic diseases to diagnose mus , they can be used to subclassify its 3 dimensions . the mild , moderate , and severe categories , we propose , must be more concretely defined , analogous to dsm criteria , if we are to maximize their potential for better defining treatment and prognosis at all levels of mus . only with explicit , agreed - upon criteria , for all their shortcomings , can the field move ahead . finally , we suggest that the consensus group , at some point , include patients and work jointly with them to develop a nonpejorative name for mus .

solid pseudopapillary neoplasm ( spn ) is a rare pancreatic tumors , characterized by low malignant potential and strong female predilection . this tumor had several names including frantz s tumor , solid and papillary tumor , solid - cystic tumor , papillary cystic tumor , and solid and papillary epithelial neoplasm until 1996 when world health organization defined it as solid pseudopapillary tumor of the pancreas . however , due to the recent advantages in medical imaging , the incidence of spn has increased to 5 - 6% of all diagnosed pancreatic tumors . spn is diagnosed much more common in children than in adults consisting of 26% of pancreatic tumors . spn shows a strong female predilection , 90% of them arising in adolescent and young adult women . the overall mortality rate of spn is about 2% and recurrence occurred in almost 10 - 15% of patients after resection . the predominant occurrence of spn in young women at the beginning of the reproductive period along with the presence of progesterone receptors , indicate the role of female hormones in the growth of this tumor . this idea has been supported by the fact that genital ridges are closed to the pancreatic anlage during embryogenesis . chromosomal abnormalities are the other mechanism that may have an etiological role in the development of this neoplasm . some studies suggested that mutation in b - catenin , could play a major role in the tumorogenesis of spn . according to immunohistochemical and electron microscopic studies , it has been suggested that these tumors originate from undifferentiated pluripotent emberyonal cells . herein , we describe the clinical , radiological and cytopathological features of solid pseudopapillary tumor of the pancreas ( spnp ) diagnosed by endoscopic ultrasound - guided ( eus - guided ) fine - needle aspiration ( fna ) and review of relevant literature . patients with final diagnosis of pseudo papillary tumor who referred for eus to shariati hospital , affiliated to tehran university of medical sciences from november 2010 to march 2015 were enrolled . the demographic , clinical and pathological features of all patients were recorded in the digestive disease research institute . the database included age , sex , associated symptoms , laboratory data , tumor size and location , arterial and perineural invasion , distant metastasis , lymphadenopathy , operative data , and postoperative complications . the first diagnosis in all patients was made by eus - fna cytology samples and finally by surgical tissue . the endosonographer inserted a 22 gauge needle into the target lesion and aspirated samples by three passes , almost without applying suction . aspiration of lesions located in the head of the pancreas or uncinate process was done through the transduodenal approach and lesions in the body and tail of the pancreas through the transgastric route . all samples were enough for cytologic evaluation and there was no need to repeat fna in any patient . details of fine - needle aspiration and surgical pathology results were recorded . ihc was done on fna cell block samples using dako real en vision detection system , peroxidase / dab , rabbit / mouse . the hematoxylin and eosin ( h and e ) stained sections of all fna and surgical samples were reviewed by an expert pathologist to confirm the diagnosis . the ihc markers ( primary antibodies ) used in the analysis , included vimentin ( clone ; v9 ) , pan cytokeratin ( ck ) ( clone ; ae1 and ae3 ) , -catenin ( clone ; ncl- -catenin ) , cd10 ( clone ; 56c6 ) , progesterone receptor ( pr ) ( clone ; pr88 ) , synaptophysin ( clone ; 27g12 ) , and chromogranin a ( clone ; lk2h10 ) . all of the patients underwent a variety of radiological examinations for preoperative diagnosis including transabdominal ultrasonography , computed tomography ( ct ) and magnetic resonance imaging ( mri ) . the study was approved by the ethics committee and institutional review board of the digestive disease research institute of tehran university of medical sciences , tehran , iran . data were analyzed using spss software , version 20 ( ibm , new york , usa ) . seven patients underwent surgical treatment for spn of the pancreas during the study period . preoperative diagnosis was made by eus in all patients . of these patients , 6(85.7% ) were female . the level of serum amylase and tumor markers was within the normal range in all patients except in one . the tumor cells were mostly positive for vimentin , pr and cd10 , each in 6 cases . synaptophysin was positive in 3 patients , followed by nse and cytokeratin , each in 2 patients . nse : neuron specific enolase , pr : progesterone receptor , pan ck : pan cytokeratin all patients underwent surgical treatment . the most common location of the tumor was the head and neck of the pancreas , each in 2 ( 28.5% ) patients , followed by the body , tail and both the body and tail , each in one patient . the mean diameter of the tumor was 42.5 mm ( range : 12 - 60 mm ) . two patients had perineural invasion ; however , none of the patients showed distant metastasis . the other three patients with lesions in the body and/or in the tail underwent distal pancreatectomy . a mixed solid and cystic appearance was identified in three patients , and others had predominantly solid appearance . the tumor was the head and neck of the pancreas , each in 2 ( 28.5% ) patients , followed by the body , tail and both the body and tail , each in one patient . the mean diameter of the tumor was 42.5 mm ( range : 12 - 60 mm ) . two patients had perineural invasion ; however , none of the patients showed distant metastasis . the other three patients with lesions in the body and/or in the tail underwent distal pancreatectomy . a mixed solid and cystic appearance was identified in three patients , and others had predominantly solid appearance . the most common presenting symptoms of spns are abdominal pain followed by increased abdominal girth , poor appetite and nausea resulting from tumor compression on the adjacent organs and a palpable mass . although almost 30% of patients are completely asymptomatic and discovered incidentally during routine clinical examination or diagnostic imaging procedures , some patients ( 8% ) present with acute abdomen as a result of abdominal blunt trauma or spontaneous rupture of the capsule . clinical presentations of our patients included abdominal pain , nausea and vomiting , weight loss , bloating and feeling of fullness . it has a strong female preponderance with female- male ratio of 5:1.9 and up to 10:1 . male patients presented much later compared with women ( mean age : 31 years ) . the diagnosis of spn has increased during the recent years due to advances in diagnostic imaging techniques . however , clinical and radiological findings are insufficient for reaching a definite diagnosis and tissue sampling should always be considered . eus - fna needle aspiration and cytology is the best technique for preoperative diagnosis of spn and less invasiveness than surgical procedures . eus - fna is increasingly using for identifying the tumor over the recent years , however it still constitutes fewer than 5% of performed imaging . spn lesions have been shown as well - circumscribed , hypoecho , heterogenous solid and cystic masses by eus . eus - fna is significantly more effective than ct in differentiating neoplastic from non - neoplastic , along the identifying malignant pancreatic cysts . furthermore , immunohistochemical staining and mutation analysis of beta catenin gene are helpful in distinguishing spn from other pancreatic tumors . complete surgical resection is the mainstay of treatment in all patients with spn even in the presence of local invasion or distant metastasis . the most common surgical procedures performed for pancreatic spn are distal pancreatectomy and pancreaticoduodenectomy ( whipple surgery ) . local invasion and distant metastasis were reported in 5% and 6% of spn patients , respectively . the most frequent sites affected by metastases include the liver ( 28% ) , wall of vena cava ( 27% ) and the spleen ( 17% ) . duodenum , omentum , colon , and lung are the other preference location of metastases . invasion to regional lymph nodes has been rarely reported which is consistent with our series . the efficacy of non - surgical approaches including chemotherapy and radiotherapy has not been proven . the prognosis of spn is favorable , even in the presence of metastases or invasion to other organs . considering the fact that the 5-year survival rate of spn is 94 - 97% , it is very important to differentiate it from other pancreatic lesions . however , if metastatic lesions are impossible to be removed , long - term survival is undeterminable . according to the largest report of patients with spn , pancreatic tail and the head of pancreas are the most common localizations of the tumor followed by pancreatic body , body and tail , head and body , neck and the uncinate process of the pancreas . however , in this series , only one of our patients had tumor in the tail region and most of the tumors were located in the pancreatic head and neck . in our series , perineural invasion our small sample size did not allow us to compare different tumor characteristics with the sex of the patients . lymph node metastases were not found in none of the patients in this series which is consistent with other reports . the mean follow - up period of our patients was 14 months ( 7 - 30 months ) which is short for giving a comprehensive opinion on recurrence rate . in previous studies , spn have a mean diameter of 6.08 cm ( range 0.5 to 34.5 cm ) . metastasis or local recurrence rate of spnp in patients treated by radical surgical excision is 9%- 15% . as we presented in our series , there was no association between spnp and laboratory test results including serum tumor markers . factors that can be useful in predicting the malignant potential of the tumor are perineural and vascular invasion , lymph node involvement and deep invasion of surrounding tissues . the morphological appearance is not a good predictor of clinical behavior of these tumors ; spn with subtle histological malignant appearance can show local invasion to adjacent structures . several studies indicated that male sex , large tumor size ( > 5 cm ) , and tumor necrosis could predict malignant transformation . however , most of the studies do not report any association between age and tumor location with malignant potential of spn . the clinical features and outcome of our patients were compared with recent reports from other countries in table 2 . in our study , ihc analysis showed strong positivity for vimentin , cd10 , and pr . these findings are consistent with previous studies that reported positivity for vimentin in 100% and 70% of cases , respectively . tumor cells of spn showed strong positivity for cd10 in two previous studies , in the present series , pr was positive in 6 out of 7 patients . machado et al , reported pr positivity in 80% of cases and the percentage of pr positivity was 64.5% in a study by uppin et al . taken together , the frequent expression of pr and strong female predilection of spn indicate the importance of hormone dependency in the pathogenesis of this tumor . eus - guided fna with preparing cell block from the aspiration and using ihc with relevant markers are a very helpful techniques for preoperative diagnosis of spn .

the horse industry is increasing because of its diversity , and involves business , sport , gaming , entertainment , or recreation . the raising of horses plays a significant role , due to the high number of animals for leisure , which has generated several direct jobs , such as those needed for handling and the sanity of the animals . nowadays , horses are being used in wild pastures and countries for the forest management , utilizing unwanted vegetation for production . for this reason , this industry deserves the attention of the general public and the governments also . this new situation requires more information about the sanitary status of these animals , and the appropriate measures for ensuring the horses are in an adequate fitness condition . among the diseases affecting horses , an oceanic climate ( also called marine west coast climate and maritime climate ) is the climate typically found along the west coasts at the middle latitudes of all the world 's continents and in southeastern australia . oceanic climates are characterized by a narrower annual range of temperatures than are encountered in other places at a comparable latitude and do not have the extremely dry summers of mediterranean climates . under those conditions , survival of parasitology resistant forms several investigations have shown the effect of some factors on the infection by parasites in horses . observed the highest output of strongyle eggs in animals aged 2 - 3 years from sweden . bucknell et al . reported that in australia cyathostomes were more prevalent in young horses , and p. equorum was found exclusively in horses less than 2 years of age , and the highest mean intensity of infection for anoplocephala perfoliata was in those horses . obtained similar results in equids from kentucky ( usa ) . nevertheless , there is a lack of knowledge about the influence of breed or sex on the infection by helminth parasites in horses from oceanic climate areas . this study reports the effect of various intrinsic factors ( breed , age , sex ) on the equine infection by helminth parasites . fecal samples were individually collected form 418 horses in an oceanic climate area ( nw spain ) and analyzed by the coprological flotation , sedimentation , and migration techniques . between april and september 2007 , fecal samples from the rectum of 418 horses from nw spain were collected . according to the breed , four groups were established ( table 1 ) : spanish sport horse ( ssh ) , spanish pure breed ( spb ) , english pure breed ( epb ) and the autochthonous pura raza galega ( prg ) . by considering the age , horses were divided into three groups : g-1 ( <3 years old ) , g-2 ( 310 ) , and g-3 ( > 10 ) ( table 1 ) . although some interesting information about horses management as hygienic conditions , treatment history , and so forth would be helpful to explain the results , it was not possible to achieve in many cases , so we decided not to include it in the current paper . many samples were collected from horses under an extensive management , and ask those questions to the owners was not possible . for that reason , the impossibility for ensuring the collection of all data belonging to the total population limited us to the analysis of the factors possibly involved in the infection of equines by helminth parasites . the factors we analyzed corresponded to parameters which can be observed and measured for us in all cases . the influence of climatic conditions on the dynamics of egg expulsion has been reported . nevertheless , by considering that the main goal in the current work was to gain more information about the presence of helminth infection in horses and the difficulty in taking more than one fecal sample from each animal in several cases , the data presented here must be comprehended as a coprological survey . the observation of parasitic forms in the feces of the horses was evaluated by using the coprological flotation , sedimentation , and migration techniques . five grams of each fecal sample were processed ( in duplicate ) by the flotation , sedimentation , and migratory techniques , with a sensitivity of 10 eggs per gram of feces ( epg ) . the laboratory technician conducting the microscope analysis was also blinded to the study design of the selection of the stool specimens . since it is not possible to distinguish strongyle eggs of different species morphologically , fecal samples are cultured for 1014 days at 2025c to allow the development of l3s , which may be collected by means of the baermann procedure [ 6 , 9 ] . that the egg elimination is not normally distributed , statistical analysis was done by means of the nonparametric kruskal - wallis and mann - whitney u two - sided tests ( = 0.05 ) , and significant differences were considered when p < .05 . prevalences were expressed as the percentage and the 95% confidence interval ( ci ) . the existence of correlation among the different parameters was assessed by using nonparametric spearman 's rank correlation test . eggs from gastrointestinal nematoda and cestoda were observed in the feces of the horses . the analysis of the gastrointestinal eggs showed that the horses were parasitized by parascaris equorum , strongyles , and oxyuris equi . by means of the coprocultures , we identified cyathostominea and gyalocephalus spp ( small strongyles ) and strongylus and triodontophorus ( large strongyles ) . no differences in the genera of strongyles identified according to breed , age , or sex were recorded . eighty nine percent ( 95% ci 86 , 92 ) of the samples had gastrointestinal eggs and 1% ( 0 , 2 ) cestoda . the prevalence of horses passing parascaris eggs by feces was 11% ( 8 , 14 ) , 88% ( 85 , 91 ) strongyles , and 3% ( 1 , 5 ) oxyurids . as drawn in table 2 , the highest prevalence of parascariosis was obtained in the youngest animals , english pure blood horses and females . significant differences were obtained by considering the age , breed , and sex of the animals . the prevalence of strongyles was higher in g-1 and g-2 , horses belonging to the autochthonous prg breed and females . significant differences were observed by taking into account the age and the breed of the animals . table 2 reflects that the greatest percentage of horses with oxyurids corresponded to the oldest animals ( g-3 ) , english pure blood , and females . finally , the highest prevalence of horses passing cestode eggs by feces was obtained in the equids from the g-2 ( 310 years ) , autochthonous ( prg ) , and females , although these differences were not significant . spearman 's rank test showed a statistical negative correlation between the age of the horses and the prevalence of ascarids and strongyles ( r = 0.141 , p = 0.005 and r = 0.143 , p = 0.004 , resp . ) . likewise , a weak and positive correlation between age and prevalence of pinworms was matched ( r = 0.113 , 0.023 ) . the highest number of parascaris eggs was observed in the foals , autochthonous horses ( prg ) , and males . the kruskal - wallis test showed significant differences according to the age of the animals . as drawing in table 3 , there were reached differences in the egg excretion of small strongyles regarding the breed and sex of the horses . the greatest numbers were observed in the english pure blood animals and in females , whereas no elimination was observed in spanish sport horses . no differences were obtained in the egg elimination of oxyurids and cestoda by considering the age , breed , or sex of the horses . by means of spearman 's rank test , a statistical negative correlation between the age of the horses and the elimination of ascarids and strongyles eggs was proved ( r = 0.445 , p = 0.001 and r = 0.167 , p = 0.001 , resp . ) . worldwide , horses are exposed to helminth parasites from different groups resulting in significant morbidity and mortality . the most common helminths of horses include the small and large strongyles , along with tapeworms , ascarids , pinworms , and the lungworms . in the current study , we proved the existence of infection by ascarids , strongyles ( small and large ) and pinworms in horses from nw spain , which agrees with data reported by bucknell et al . in australia , romaniuk et al . in polonia , and pereira and vianna in brazil , in horses housed under differing management schemes . the main helminth parasites observed were strongyles , in agreement with previous investigations in areas with a similar weather [ 1 , 14 , 15 ] . likewise , a low prevalence was obtained for tapeworms , although the low sensitivity of the fecal examination techniques in detecting cestode eggs could be the explanation to our results . . pointed that the coprological methods have a lower likelihood of diagnosing cestode infection when horses have less than 100 tapeworms . the lack of flukes and coccidia in the current study provides evidence to back up the rarity of infection by this class of parasite [ 1 , 13 ] . to get more knowledge about the factors influencing infections by helminths in horses , the coprological data were examined by considering the age , breed , and sex of the animals . the values of prevalence and elimination of p. equi - eggs were significantly the highest in horses less than 3 years old , which agrees with mfitilodze and hutchinson and bucknell et al . . the percentage of infection by strongyles was significantly greater in the horses less than 10 years old . it has been proved that age does not influence the cyathostome burdens , and klei and chapman reported that the naturally acquired immunity against small strongyles slowly develops with age , but remains incomplete . this could be the explanation to the finding of a negative correlation between the age and the infection by ascarids and strongyles . this explains why the autochthonous pura raza galega horses , maintained under extensive forest conditions and receiving an inadequate keeping , attained the highest prevalence of infection by ascarids and strongyles . in addition , no helminth parasites were observed in the fecal samples from spanish sport horses , and the spanish pure blood ones were few parasitized . several studies did not match the influence of the horse breed on the helminthosis [ 18 , 21 ] . it has been shown that helminth infection is ubiquitous in horses with access to pasture . the absence of positive results in the sport spanish horses seems to be attributable to a reduced possibility for pasturing and thus to contact to the infective phases ( larval 3rd stages for the gastrointestinal nematoda ) or to intermediate hosts ( oribatids in the case of tapeworms ) . another possible explanation could be that these are economically valuable horses , maintained under appropriate hygienic conditions , and receiving periodical veterinary attention . moreover , the observation of elevated prevalences for gastrointestinal worms in the english pure blood and in the spanish pure blood equids was an unexpected result , because of their economic value and the conditions of housing and keeping , similar to the sport spanish horses . our results showed that sex had influence on the prevalence of ascarids and strongyles , being the females more infected than the males , in agreement with fikru et al . . this is the first study about the effect of several intrinsic factors on the helminth parasites affecting horses from nw spain , so the lack of coprological surveys makes more difficult the discussion of the results obtained in the current investigation . it must be taken into account that the presence of seasonal dynamics of egg expulsion influences the results for the intensity of parasitization . the development of coprological surveys provides a very interesting and indispensable tool for knowing the main helminth parasites infecting the horses . in the light of our results we consider that the infections caused by helminths , especially the strongyles , are significantly common in the region of study , so that greater importance should be given to this situation the elevated prevalence in the english pure blood and autochthonous horses underlines the need for the application of appropriate measure to the control of helminth parasites . more information is needed to know the applied ways for the control of parasites in equids , in particular that related to chemoprophylaxis .

a functioning permanent vascular access that can be regularly utilized to access the systemic circulation is crucial for the survival of patients on maintenance hemodialysis . yet , hemodialysis access failure was reported to be the most frequent cause of hospitalization among patients with chronic kidney disease stage 5 by the united states renal data system . qualities of an ideal access include the ability to deliver a blood flow rate adequate for the dialysis prescription , good long - term patency and a low rate of complications such as infection , stenosis , thrombosis , aneurysm and limb ischemia . studies have shown that arteriovenous fistula survival is superior to grafts when used as patients ' first access . hemodialysis catheters play an important role in the delivery of dialysis to patients with end - stage renal disease . however , anatomic , thrombotic and infectious complications associated with their continued use lead to significant morbidity in this population . clinical practice guidelines emphasizing the importance of placing fistulae in long - term hemodialysis patients for vascular access were first published by the nkf kdoqi ( national kidney foundation kidney disease outcomes quality initiative ) in 1997 . central venous stenosis related to cannulation of central veins remains common in dialysis patients and is fraught with several complications that may lead to loss of arteriovenous access . we report the case of a 58-year - old male african - american patient who became dialysis dependent in 1990 after he had developed end - stage renal disease due to focal segmental glomerulosclerosis . he had received hemodialysis via a left - radial - artery - to - brachial - vein gore - tex graft for several years before he was noted to have poor arteriovenous access blood flow . he subsequently underwent creation of a left brachiocephalic arteriovenous fistula that provided reliable vascular access for hemodialysis during several months . months later , he developed a remarkable enlargement of the arteriovenous fistula ( fig . 1 ) and dilated jugular veins ( fig . 2 ) . venography revealed 50% stenosis of the left brachiocephalic vein accompanied by dilatation of the ipsilateral cephalic and subclavian veins ( fig . retrograde flow was noted in the left internal jugular vein . after successful dilatation of the left brachiocephalic vein stenosis by balloon angioplasty , venography showed restoration of the blood flow in the central venous circulation and disappearance of the previously noted retrograde flow in the left internal jugular vein ( fig . however , as the access arm remained massively enlarged , venography was repeated a few weeks later , revealing moderate - to - severe stenosis of the left cephalic as well as subclavian veins . successful balloon angioplasties of both lesions were performed . despite several interventions , the striking enlargement of the venous system persisted , and surgical ligation of the fistula central venous stenosis is a major , frequently encountered problem in the dialysis population . in a study of 69 consecutive patients being subjected to percutaneous placement of tunneled right internal jugular vein catheters who underwent venography prior to insertion of a guide wire , 29 patients ( 42% ) were found to have unexpected stenosis or angulation of central veins severe enough to necessitate additional fluoroscopy or abandonment of the procedure . much of the early attention was focused on hemodialysis catheters as the etiologic agents responsible for this problem . however , insertion of a variety of indwelling devices including central venous catheters , peripherally inserted central venous catheters and indwelling intracardiac wires leads to the development of central venous stenosis . central venous stenosis has also been reported to occur in the absence of any of these antecedents . it has long been recognized that dialysis catheters are associated with late obstructive complications that may adversely affect the outcomes of permanent vascular access . early case reports in the 1980s linked subclavian dialysis catheters to central venous stenosis . in a prospective study of 50 patients with subclavian catheters and 50 patients with internal jugular vein catheters , venography revealed central venous stenosis in 42% of the patients in the subclavian group compared with 10% of the patients in the internal jugular group . however , more recent studies show that , despite embracing preferential cannulation of the internal jugular vein , central venous stenosis remains widespread . in a study of 133 hemodialysis patients who underwent venography for access - related concerns over a 14-month period , 55 patients ( 41% ) had evidence of significant central venous stenosis . longer time on hemodialysis ( 43 vs. 34 months ) and a history of a previous hemodialysis catheter insertion ( 52/55 vs. 59/78 patients ) were the factors found to be associated with stenosis . in addition to the site of insertion , the incidence of central venous stenosis is contingent upon the laterality of the insertion and the type of the indwelling object , i.e. a central venous catheter , peripherally inserted central venous catheter or intracardiac wire . a tendency toward an increased incidence of stenosis was observed in hemodialysis patients following cannulation of the left compared with the right internal jugular vein . this led to the conjecture that the angulations between the left internal jugular vein , brachiocephalic vein and superior vena cava that the catheter must negotiate , as well as the greater length of the path traversed by a left - sided when compared with a right - sided catheter , are responsible for this increased incidence . the complex three - dimensional anatomy of the left central veins has also been implicated in the increased incidence of stenosis related to left - sided central venous catheters . angulation of the left brachiocephalic vein as it winds around the aortic arch is not apparent on two - dimensional radiographs or venograms . contact between the catheter and vessel wall as it traverses this fulcrum is thought to lead to endothelial irritation and possible subsequent venous stenosis . therefore , the kdoqi guidelines opine that the right internal jugular vein is the preferred site for the insertion of tunneled cuffed venous dialysis catheters . a majority of the affected patients remain asymptomatic . in a study of 202 patients who underwent permanent pacemaker implantations with indwelling intracardiac wires inserted via a transvenous approach , 129 patients ( 64% ) developed varying degrees of central venous stenosis . central venous stenosis becomes clinically significant in the presence of an ipsilateral arteriovenous dialysis access that drains into the affected central veins . the high venous pressure and blood flow due to the fistula may overwhelm the collateral venous and lymphatic drainage , resulting in the development of dilated and tortuous collateral veins over the ipsilateral upper arm , neck and upper chest . in severe cases , venous hypertension may eventually lead to disabling arm edema with pain and discomfort . vascular access stenosis is a harbinger of thrombosis , recurrent infections , reduced access blood flow and compromised dialysis delivery due to access recirculation . myriad complications can render the arteriovenous access unusable despite valiant efforts to salvage the access by endovascular and surgical therapies , leaving no choice but to occlude the access . a great majority of patients who face this predicament may not be as fortunate as our patient , who received a kidney transplant . kdoqi guidelines steadfastly recommend that fistula placement should be considered first , followed by prosthetic grafts if fistula placement is not possible in prospective hemodialysis patients . catheter - last approach after having exhausted all avenues including fistula , graft , peritoneal dialysis and preemptive kidney transplantation .

osteoarthritis ( oa ) of the knee is one of the most common conditions in the elderly , mostly associated with knee pain1,2 ) . radiographic oa ( roa ) is defined as a kellgren and lawrence grade of 2 , whereas symptomatic oa ( soa ) refers to roa accompanied by symptoms . approximately 12% of the total us population was estimated to present with signs or symptoms of oa ; in particular , roa was diagnosed in 17% of the population aged 60 and over and in 70% of those 65 years of age3,4 ) . due to the growth of the senior population and the influence of the joint disease on the public heath , the currently available oa treatment options include medication using non - steroidal anti - inflammatory drugs ( nsaids ) or selective cyclooxygenase - type-2 ( cox-2 ) inhibitors , physical therapy , intra - articular steroid injection , viscosupplementation , joint irrigation , arthroscopic knee surgery , and knee replacement surgery5,6 ) . nsaids have been widely used as the primary pain reliever in soa7 ) , but treatment discontinuation may be required due to gastrointestinal side effects associated with cox-1 inhibition8,9 ) . on the other hand , aceclofenac has been established as a safe and effective medicine that demonstrates better safety and tolerance than other nsaids due to its superior selectivity for cox-2 inhibition for the past 20 years in south korea10 ) . however , the downside of aceclofenac lies in its inconvenient dosing regimen of twice daily . cockburn et al.11 ) demonstrated a relation between patients ' medication compliance and the number of daily doses : the compliance with the medication that was only 38% with three - times - a - day dosing increased to 69% with twice - a - day dosing and then to 90% with once - a - day dosing . thus , the study provides evidence that a reduced number of daily doses would result in higher compliance among patients , which would eventually improve treatment efficacy11 ) . compared to aceclofenac immediate release ( ir ) 100 mg that requires twice - daily dosing , aceclofenac controlled release ( cr ) 200 mg is formulated for once - daily dosing and composed of two layers , an ir layer and a cr layer . the purpose of the current study was to evaluate non - inferiority of aceclofenac cr 200 mg to aceclofenac ir 100 mg with regard to it efficacy and safety in clinical setting . of the patients between 40 and 70 years of age with chronic knee oa diagnosed with radiography and clinical examination , those presenting with knee oa symptoms lasting 3 months and > 40 mm on a 100-mm visual analogue scale ( vas ) were included in this study . the followings were used as exclusion criteria : patients with a history of gastric ulcer , duodenal ulcer , gastrointestinal hemorrhage , or other abnormal bleeding ; use of intra - articular or oral corticosteroids within 3 weeks prior to visit 1 ( randomization ) ; intra - articular injection of hyaluronic acid within 1 week prior to visit 1 or scheduled injection during clinical trial ; patients with increased risk of asthma or asthma patients at risk of developing hives or acute rhinitis due to aspirin or prostaglandin synthetase inhibitors ; hypersensitivity to nsaids or other drugs of the same class ( diclofenac ) as the study drug ; presence of severe cardiovascular , respiratory , renal , endocrine , hematological , gastrointestinal , or neuropsychiatric disorders or malignant tumors . of the total 127 patients who agreed to participate in the clinical trial , excluding 2 screening failures ( 1 due to hepatic dysfunction and 1 due to inclusion / exclusion criteria violation ) , 125 patients ( experimental group , n=62 ; control group , n=63 ) ) were included in the intention - to - treat ( itt ) analysis . of these , excluding 22 patients due to low compliance , use of prohibited medications , or inclusion / exclusion criteria violation , 103 patients ( experimental group , n=50 ; control group , n=53 ) were evaluated in the per - protocol ( pp ) analysis , . in the safety evaluation , 115 of the 125 patients who were eligible for the itt analysis were included ( experimental group , n=57 ; control group , n=58 ) , except for 8 patients who violated the inclusion / exclusion criteria and 2 patients who were not available for the safety assessment after drug administration ( fig . 1 , table 1 ) . the safety evaluation was based on the incidence of adverse events , clinical laboratory test results , physical examination findings , vital signs , and electrocardiographic findings . patients with missing data were excluded only from the respective analyses on the missing variables . the phase 4 multicenter , open - label , randomized , non - inferiority , comparative clinical trial was conducted at seoul national university hospital , samsung medical center , seoul national university borame medical center , seoul national university bundang hospital between january 24 , 2011 and march 30 , 2012 . pareek et al.12 ) reported that the mean vas change between the baseline and at 4 weeks after treatment was 6.9 mm greater in the aceclofenac group ( left knee , -15.7 mm ; right knee , -15.7 mm ) than in the diclofenac group ( left knee , -8.8 mm ; right knee , -8.8 mm12 ) . based on the study , the mean vas change at 4 weeks after treatment for knee oa was assumed to be -15.7 mm , the actual mean vas change in the aceclofenac group of the above - mentioned study , with a standard deviation ( sd ) of 15.9 mm , the greatest sd described in the study . when the mean intergroup difference in vas at 4 weeks after treatment as assumed to be -6.1 mm , the mean vas change in the cr group was calculated as -21.8 mm and the non - inferiority limit the sample size for each group required for a confidence interval of 95% and power of 80% was calculated using the following formula : taking into account potential dropouts from the trial , the number of patients required was determined as 120 . if the patient diagnosed with chronic oa agreed to participate in the clinical trial , after one week of wash - out period depending on the medication prior to trial , the eligibility of the patient for the study was determined based on the inclusion / exclusion criteria . then , the patient was assigned with 1:1 randomization into the cr group or the ir group . the patient allocation was done independently at each participating institution using a computer - generated randomization table . to ensure balanced randomization between groups , a randomized block design with block sizes 4 and 6 and a ratio of 1:1 was used . the randomly assigned subjects were treated with either aceclofenac cr 200 mg once daily or aceclofenac ir 100 mg twice daily for 4 weeks . the efficacy and safety of the treatment were tested at each clinic upon the end of the treatment . the experimental group received oral administration of an aceclofenac cr tablet 200 mg ( clanza cr ; korea united pharm inc . , seoul , korea ) once daily , whereas the control group , an aceclofenac ir tablet 100 mg ( airtal ; dae - woong pharm co ltd . , the primary variable used for the evaluation of efficacy was the mean change in 100-mm vas between baseline and at 4 weeks after treatment . the secondary variables were the knee injury and osteoarthritis outcome score ( koos ) function ( pain , daily living , sports and recreational activities , and quality of life ) and range of motion ( rom , flexion contracture - further flexion ) . for the evaluation of safety , development of adverse events during the clinical trial , abnormal findings in physical examination , and changes in clinical laboratory test results were assessed . the efficacy evaluation was primarily based on the itt analysis and secondarily on the pp analysis . changes in the 100-mm vas for pain , the primary variable for the efficacy evaluation , between the baseline and at 4 weeks after treatment were compared . the pre- and post - administration differences were compared in each group . regarding the intergroup comparison , non - inferiority of the experimental group was evaluated by investigating whether the lower limit of a one - sided 95% confidence interval was above the equivalence margin of -15 mm . the signed rank test was used to compare the pre- and post - administration differences in each group . as for the intergroup comparison , the wilcoxon 's rank sum test was performed and a covariance analysis was carried out with the institutions taken as the covariate . the secondary efficacy evaluation variables included koos - pain , koos - daily living , koos - sports and recreational activities , and koos - quality of life . the koos - pain , designed to assess knee joint symptoms , was calculated as the sum of scores on a 9-item questionnaire , and the pre- and post - administration differences were compared . the koos - daily living was assessed as the sum of scores on a 17-item questionnaire , and the pre- and post - administration differences were compared . a higher score was interpreted to represent less difficulty in performing daily living activities . regarding the assessment of koos - sports and recreational activities , the pre- and post - administration differences in the sum of scores on a 5-item questionnaire the koos - quality of life was measured as the sum of scores on a 5-item questionnaire , and the pre- and post - administration differences were compared . intergroup comparisons regarding the four subscales of the koos were performed using the wilcoxon 's rank sum test . for the assessment of rom of the knee , the pre- and post - administration differences in the active range of flexion measured with the patient in a sitting position were compared , and intergroup comparison was performed using either the wilcoxon 's rank sum test or an unpaired t - test . on the safety evaluation , intergroup differences in adverse events , clinical laboratory test results , physical examination findings , vital signs , and electrocardiographic results were analyzed using either chi - square test or fisher 's exact test . of the patients between 40 and 70 years of age with chronic knee oa diagnosed with radiography and clinical examination , those presenting with knee oa symptoms lasting 3 months and > 40 mm on a 100-mm visual analogue scale ( vas ) were included in this study . the followings were used as exclusion criteria : patients with a history of gastric ulcer , duodenal ulcer , gastrointestinal hemorrhage , or other abnormal bleeding ; use of intra - articular or oral corticosteroids within 3 weeks prior to visit 1 ( randomization ) ; intra - articular injection of hyaluronic acid within 1 week prior to visit 1 or scheduled injection during clinical trial ; patients with increased risk of asthma or asthma patients at risk of developing hives or acute rhinitis due to aspirin or prostaglandin synthetase inhibitors ; hypersensitivity to nsaids or other drugs of the same class ( diclofenac ) as the study drug ; presence of severe cardiovascular , respiratory , renal , endocrine , hematological , gastrointestinal , or neuropsychiatric disorders or malignant tumors . of the total 127 patients who agreed to participate in the clinical trial , excluding 2 screening failures ( 1 due to hepatic dysfunction and 1 due to inclusion / exclusion criteria violation ) , 125 patients ( experimental group , n=62 ; control group , n=63 ) ) were included in the intention - to - treat ( itt ) analysis . of these , excluding 22 patients due to low compliance , use of prohibited medications , or inclusion / exclusion criteria violation , 103 patients ( experimental group , n=50 ; control group , n=53 ) were evaluated in the per - protocol ( pp ) analysis , . in the safety evaluation , 115 of the 125 patients who were eligible for the itt analysis were included ( experimental group , n=57 ; control group , n=58 ) , except for 8 patients who violated the inclusion / exclusion criteria and 2 patients who were not available for the safety assessment after drug administration ( fig . 1 , table 1 ) . the safety evaluation was based on the incidence of adverse events , clinical laboratory test results , physical examination findings , vital signs , and electrocardiographic findings . patients with missing data were excluded only from the respective analyses on the missing variables . the phase 4 multicenter , open - label , randomized , non - inferiority , comparative clinical trial was conducted at seoul national university hospital , samsung medical center , seoul national university borame medical center , seoul national university bundang hospital between january 24 , 2011 and march 30 , 2012 . pareek et al.12 ) reported that the mean vas change between the baseline and at 4 weeks after treatment was 6.9 mm greater in the aceclofenac group ( left knee , -15.7 mm ; right knee , -15.7 mm ) than in the diclofenac group ( left knee , -8.8 mm ; right knee , -8.8 mm12 ) . based on the study , the mean vas change at 4 weeks after treatment for knee oa was assumed to be -15.7 mm , the actual mean vas change in the aceclofenac group of the above - mentioned study , with a standard deviation ( sd ) of 15.9 mm , the greatest sd described in the study . when the mean intergroup difference in vas at 4 weeks after treatment as assumed to be -6.1 mm , the mean vas change in the cr group was calculated as -21.8 mm and the non - inferiority limit the sample size for each group required for a confidence interval of 95% and power of 80% was calculated using the following formula : taking into account potential dropouts from the trial , the number of patients required was determined as 120 . if the patient diagnosed with chronic oa agreed to participate in the clinical trial , after one week of wash - out period depending on the medication prior to trial , the eligibility of the patient for the study was determined based on the inclusion / exclusion criteria . then , the patient was assigned with 1:1 randomization into the cr group or the ir group . the patient allocation was done independently at each participating institution using a computer - generated randomization table . to ensure balanced randomization between groups , a randomized block design with block sizes 4 and 6 and a ratio of 1:1 was used . the randomly assigned subjects were treated with either aceclofenac cr 200 mg once daily or aceclofenac ir 100 mg twice daily for 4 weeks . the efficacy and safety of the treatment were tested at each clinic upon the end of the treatment . the experimental group received oral administration of an aceclofenac cr tablet 200 mg ( clanza cr ; korea united pharm inc . , seoul , korea ) once daily , whereas the control group , an aceclofenac ir tablet 100 mg ( airtal ; dae - woong pharm co ltd . , the primary variable used for the evaluation of efficacy was the mean change in 100-mm vas between baseline and at 4 weeks after treatment . the secondary variables were the knee injury and osteoarthritis outcome score ( koos ) function ( pain , daily living , sports and recreational activities , and quality of life ) and range of motion ( rom , flexion contracture - further flexion ) . for the evaluation of safety , development of adverse events during the clinical trial , abnormal findings in physical examination , and changes in clinical laboratory test results were assessed . the efficacy evaluation was primarily based on the itt analysis and secondarily on the pp analysis . changes in the 100-mm vas for pain , the primary variable for the efficacy evaluation , between the baseline and at 4 weeks after treatment were compared . the pre- and post - administration differences were compared in each group . regarding the intergroup comparison , non - inferiority of the experimental group was evaluated by investigating whether the lower limit of a one - sided 95% confidence interval was above the equivalence margin of -15 mm . the signed rank test was used to compare the pre- and post - administration differences in each group . as for the intergroup comparison , the wilcoxon 's rank sum test was performed and a covariance analysis was carried out with the institutions taken as the covariate . the secondary efficacy evaluation variables included koos - pain , koos - daily living , koos - sports and recreational activities , and koos - quality of life . the koos - pain , designed to assess knee joint symptoms , was calculated as the sum of scores on a 9-item questionnaire , and the pre- and post - administration differences were compared . the koos - daily living was assessed as the sum of scores on a 17-item questionnaire , and the pre- and post - administration differences were compared . a higher score was interpreted to represent less difficulty in performing daily living activities . regarding the assessment of koos - sports and recreational activities , the pre- and post - administration differences in the sum of scores on a 5-item questionnaire the koos - quality of life was measured as the sum of scores on a 5-item questionnaire , and the pre- and post - administration differences were compared . intergroup comparisons regarding the four subscales of the koos were performed using the wilcoxon 's rank sum test . for the assessment of rom of the knee , the pre- and post - administration differences in the active range of flexion measured with the patient in a sitting position were compared , and intergroup comparison was performed using either the wilcoxon 's rank sum test or an unpaired t - test . on the safety evaluation , intergroup differences in adverse events , clinical laboratory test results , physical examination findings , vital signs , and electrocardiographic results were analyzed using either chi - square test or fisher 's exact test . in the total 125 patients included in the itt analysis , the 100-mm vas decreased after administration compared to pre - administration . the vas decreased from 66.516.4 mm before administration ( visit 2 ) to 43.220.7 mm at 4 weeks after administration ( visit 3 ) by 24.019.2 mm in the experimental group ( p<0.001 ) . in the control group , the value decreased form 62.513.9 mm before administration ( visit 2 ) to 43.320.1 mm at 4 weeks after administration ( visit 3 ) , by 19.318.7 mm ( p<0.001 ) . the intergroup difference in the 100-mm vas decrease between the pre- and post - administration was 4.818.9 mm . the lower limit of the one - sided 95% confidence interval ( -1.17 , ) was above the equivalence margin of -15 mm , thus the non - inferiority of the experimental group to the control group was confirmed . the wilcoxon 's rank sum test showed there was no significant difference in the pre- and post - administration vas score changes between the groups ( p=0.106 ) . in the covariance analysis , the intergroup was not statistically significant when institutional difference was adjusted as a covariate ( p=0.177 ) ( table 2 ) . among the total 103 patients involved in the pp analysis the vas decreased from 66.915.6 mm before administration ( visit 2 ) to 43.219.1 mm at 4 weeks after administration ( visit 3 ) by 23.719.5 mm in the experimental group ( p<0.001 ) . in the control group , the decrease was from 63.214.1 mm before administration ( visit 2 ) to 44.220.2 mm at 4 weeks after administration ( visit 3 ) by 19.018.0 mm ( p<0.001 ) . the intergroup difference in the 100-mm vas decrease between pre- and post - administration was 4.718.7 mm . the lower limit of the one - sided 95% confidence interval ( -1.43 , ) was above the equivalence margin of -15 mm , thus non - inferiority of the experimental group to the control group was confirmed . the wilcoxon 's rank sum test showed there was no significant different between the groups in the pre- and post - administration vas changes ( p=0.130 ) . in the covariate analysis , the intergroup difference in the vas change was not statistically significant when institutional difference was adjusted as a covariate ( p=0.169 ) . the knee joint condition was assessed using the four koos subscales , including koos - pain , koos - daily living , koos - sports and recreational activities , and koos - quality of life . the pre- and post - administration change in the koos - pain score was 3.94.8 points in the experimental group and 2.94.9 points in the control group . the koos - daily living score change between pre- and post - administration was 6.910.5 points in the experimental group and 4.68.7 points in the control group . the pre- and post - administration koos - sports and recreational activities scores were different by 2.33.5 points in the experimental group and 2.24.1 points in the control group . the pre- and post - administration difference in the koos - quality of life score was 1.32.5 points in the experimental group and 1.52.2 points in the control group . the experimental group exhibited greater improvement in the koos - pain , koos - daily living , and koos - sports and recreational activities , whereas the control group demonstrated higher increase in the koos - quality of life ; however , the intergroup differences were not statistically significant with regard to any of the four koos subscales . ( p=0.367 , p=0.138 , p=0.870 , and p=0.787 , respectively ) ( table 3 ) . the rom ( further flexion - flexion contracture ) was improved from 131.211.0 before administration ( visit 2 ) to 134.49.3 at 4 weeks after administration ( visit 3 ) by 2.68.0 ( p=0.025 ) in the experimental group . during the same period , the rom in the control group was improved from 132.210.6 to 135.410.0 by 2.87.3 ( p=0.002 ) . however , the intergroup difference in the rom ( -0.17.6 ; rom of the experimental group - rom of the control group ) was not statistically significant ( p=0.965 ) ( table 3 ) . a total of 26 adverse events developed in 20 of the 115 patients ( 17.4% ) during the clinical trial : 15 cases in 13 of the 57 experimental group patients ( 22.8% ) and 11 cases in 7 of the 58 control group patients ( 12.1% ) ( p=0.129 ) . fourteen adverse drug reactions were found in 12 patients ( 21.1% ) in the experimental group and 11 cases were observed in 7 patients ( 12.1% ) in the control group ( p=0.195 ) . no significant intergroup differences were found in the numbers of adverse events and adverse drug reactions ( table 4 ) . the 15 adverse events in the experimental group included indigestion ( 10 ) , abdominal pain ( 1 ) , facial swelling ( 1 ) , peripheral edema ( 1 ) , palpitation ( 1 ) , and inappetence ( 1 ) , and the peripheral edema case was not counted as an adverse drug reaction . the 11 adverse events in the control group were indigestion ( 4 ) , facial swelling ( 2 ) , chest discomfort ( 1 ) , swelling ( 1 ) , increase of gamma - glutamyltransferase ( 1 ) , increase of alanine aminotransferase ( 1 ) , and increase of aspartate aminotransferase ( 1 ) . all of these adverse drug reactions were predictable , not unexpected , based on the previous literature , and serious adverse drug reactions were not found during the clinical trial . on the vital sign ( blood pressure and pulse ) examination , the post - administration heart rate was increased in the experimental group and decreased in the control group , but the values were within normal range in both groups , showing no insignificant difference between groups . the physical examination revealed abnormal findings before administration in 4 patients in the experimental group ( eye in 1 , skin in 2 , and musculoskeletal system in 1 patient ) and in 3 patients in the control group ( musculoskeletal system in 3 patients ) . after administration , 1 in 4 of the former group became normal and the abnormal findings in the 3 remaining patients were not considered clinically significant . pre- and post - administration clinical laboratory tests , including blood tests , blood chemistry tests , and urine tests did not reveal notable changes in both groups . electrocardiography showed normal findings in 41 patients ( 73.2% ) and clinically insignificant level of abnormality in 15 patients ( 26.8% ) in the experimental group before administration . after administration , 35 patients ( 64.8% ) were normal and 19 patients ( 35.2% ) had clinically insignificant abnormality . in the control group , 32 patients ( 57.1% ) were normal and 24 patients ( 42.9% ) had clinically insignificant abnormality before administration . after administration , 32 patients ( 57.1% ) were normal and 24 patients ( 42.9% ) had clinically insignificant abnormality . in the total 125 patients included in the itt analysis , the 100-mm vas decreased after administration compared to pre - administration . the vas decreased from 66.516.4 mm before administration ( visit 2 ) to 43.220.7 mm at 4 weeks after administration ( visit 3 ) by 24.019.2 mm in the experimental group ( p<0.001 ) . in the control group , the value decreased form 62.513.9 mm before administration ( visit 2 ) to 43.320.1 mm at 4 weeks after administration ( visit 3 ) , by 19.318.7 mm ( p<0.001 ) . the intergroup difference in the 100-mm vas decrease between the pre- and post - administration was 4.818.9 mm . the lower limit of the one - sided 95% confidence interval ( -1.17 , ) was above the equivalence margin of -15 mm , thus the non - inferiority of the experimental group to the control group was confirmed . the wilcoxon 's rank sum test showed there was no significant difference in the pre- and post - administration vas score changes between the groups ( p=0.106 ) . in the covariance analysis , the intergroup was not statistically significant when institutional difference was adjusted as a covariate ( p=0.177 ) ( table 2 ) . among the total 103 patients involved in the pp analysis the vas decreased from 66.915.6 mm before administration ( visit 2 ) to 43.219.1 mm at 4 weeks after administration ( visit 3 ) by 23.719.5 mm in the experimental group ( p<0.001 ) . in the control group , the decrease was from 63.214.1 mm before administration ( visit 2 ) to 44.220.2 mm at 4 weeks after administration ( visit 3 ) by 19.018.0 mm ( p<0.001 ) . the intergroup difference in the 100-mm vas decrease between pre- and post - administration was 4.718.7 mm . the lower limit of the one - sided 95% confidence interval ( -1.43 , ) was above the equivalence margin of -15 mm , thus non - inferiority of the experimental group to the control group was confirmed . the wilcoxon 's rank sum test showed there was no significant different between the groups in the pre- and post - administration vas changes ( p=0.130 ) . in the covariate analysis , the intergroup difference in the vas change was not statistically significant when institutional difference was adjusted as a covariate ( p=0.169 ) . the knee joint condition was assessed using the four koos subscales , including koos - pain , koos - daily living , koos - sports and recreational activities , and koos - quality of life . the pre- and post - administration change in the koos - pain score was 3.94.8 points in the experimental group and 2.94.9 points in the control group . the koos - daily living score change between pre- and post - administration was 6.910.5 points in the experimental group and 4.68.7 points in the control group . the pre- and post - administration koos - sports and recreational activities scores were different by 2.33.5 points in the experimental group and 2.24.1 points in the control group . the pre- and post - administration difference in the koos - quality of life score was 1.32.5 points in the experimental group and 1.52.2 points in the control group . the experimental group exhibited greater improvement in the koos - pain , koos - daily living , and koos - sports and recreational activities , whereas the control group demonstrated higher increase in the koos - quality of life ; however , the intergroup differences were not statistically significant with regard to any of the four koos subscales . ( p=0.367 , p=0.138 , p=0.870 , and p=0.787 , respectively ) ( table 3 ) . the rom ( further flexion - flexion contracture ) was improved from 131.211.0 before administration ( visit 2 ) to 134.49.3 at 4 weeks after administration ( visit 3 ) by 2.68.0 ( p=0.025 ) in the experimental group . during the same period , the rom in the control group was improved from 132.210.6 to 135.410.0 by 2.87.3 ( p=0.002 ) . however , the intergroup difference in the rom ( -0.17.6 ; rom of the experimental group - rom of the control group ) was not statistically significant ( p=0.965 ) ( table 3 ) . a total of 26 adverse events developed in 20 of the 115 patients ( 17.4% ) during the clinical trial : 15 cases in 13 of the 57 experimental group patients ( 22.8% ) and 11 cases in 7 of the 58 control group patients ( 12.1% ) ( p=0.129 ) . fourteen adverse drug reactions were found in 12 patients ( 21.1% ) in the experimental group and 11 cases were observed in 7 patients ( 12.1% ) in the control group ( p=0.195 ) . no significant intergroup differences were found in the numbers of adverse events and adverse drug reactions ( table 4 ) . the 15 adverse events in the experimental group included indigestion ( 10 ) , abdominal pain ( 1 ) , facial swelling ( 1 ) , peripheral edema ( 1 ) , palpitation ( 1 ) , and inappetence ( 1 ) , and the peripheral edema case was not counted as an adverse drug reaction . the 11 adverse events in the control group were indigestion ( 4 ) , facial swelling ( 2 ) , chest discomfort ( 1 ) , swelling ( 1 ) , increase of gamma - glutamyltransferase ( 1 ) , increase of alanine aminotransferase ( 1 ) , and increase of aspartate aminotransferase ( 1 ) . all of these adverse drug reactions were predictable , not unexpected , based on the previous literature , and serious adverse drug reactions were not found during the clinical trial . on the vital sign ( blood pressure and pulse ) examination , the post - administration heart rate was increased in the experimental group and decreased in the control group , but the values were within normal range in both groups , showing no insignificant difference between groups . the physical examination revealed abnormal findings before administration in 4 patients in the experimental group ( eye in 1 , skin in 2 , and musculoskeletal system in 1 patient ) and in 3 patients in the control group ( musculoskeletal system in 3 patients ) . after administration , 1 in 4 of the former group became normal and the abnormal findings in the 3 remaining patients were not considered clinically significant . pre- and post - administration clinical laboratory tests , including blood tests , blood chemistry tests , and urine tests did not reveal notable changes in both groups . electrocardiography showed normal findings in 41 patients ( 73.2% ) and clinically insignificant level of abnormality in 15 patients ( 26.8% ) in the experimental group before administration . after administration , 35 patients ( 64.8% ) were normal and 19 patients ( 35.2% ) had clinically insignificant abnormality . in the control group , 32 patients ( 57.1% ) were normal and 24 patients ( 42.9% ) had clinically insignificant abnormality before administration . after administration , 32 patients ( 57.1% ) were normal and 24 patients ( 42.9% ) had clinically insignificant abnormality . aceclofenac is a phenylacetic acid - derived nsaid that is used not only for chronic joint conditions , such as oa , rheumatoid arthritis and ankylosing spondylitis but also for relief of acute pain , especially following surgery8,13 ) . compared to other anti - inflammatory drugs , such as diclofenac and naproxen , it exhibits excellent therapeutic effects by easily penetrating into inflammatory tissue , such as a joint , and accordingly effectively suppressing prostaglandin production . it has superior selectivity for cox-2 inhibition and thus does not disrupt generation of normal prostaglandin in the stomach mucosa , resulting in reduced gastrointestinal problems and adverse reactions and a high tolerance . in addition , it inhibits generation of interleukin-1 that destroys cartilage in a joint and promotes generation of glycosaminoglycan , a articular cartilage component , contributing to prevention of deterioration of rheumatoid arthritis or oa . in particular , the drug has been known to reduce pain and symptomatic severity and improve functional capacity of an injured joint , especially in cases of knee oa10 ) . in a clinical trial that compares the efficacy of aceclofenac with piroxicam among 240 patients with oa of the knee , perez et al.14 ) showed that although there were no significant differences between the two drugs with regard to the vas , osteoarthritis severity index , severity index of gonarthritis , and rom after two months of administration , aceclofenac facilitated more rapid improvement in rom . according a critical trial by ward et al.15 ) on comparison with diclofenac in 397 patients and another clinical trial by kornasoff et al.16 ) on comparison with naproxen in 374 patients , aceclofenac did not demonstrate significant difference with the other two drugs in terms of efficacy after 3 months of treatment ; however , tolerability of aceclofenac was found better than the other two drugs because fewer patients presented with gastrointestinal adverse events after treatment with aceclofenac . this can be attributed to the fact that aceclofenac is less effective in cox-1 inhibition and more potent in cox-2 inhibition than naproxen17 ) . aceclofenac is either metabolized to 4'-hydroxyaceclofenac by cyp2c9 and then converted into 4'-hydroxy - diclofenac by hydrolysis or hydrolyzed to diclofenac and then converted into 4'-hydroxy - diclofenac by cyp2c918,19 ) . henrotin et al.20 ) reported that aceclofenac and 4'-hydroxyaceclofenac inhibits cox-2 only , which may explain why aceclofenac demonstrates better tolerance than naproxen . aceclofenac is a pro - drug that is activated by metabolites and has a short biological half - life of 2 - 4 hours . thus , once or twice daily administration is necessary to maintain the efficacy of aceclofenac and major metabolites , 4'-hydroxy - aceclofenac , 4'-hydroxydiclofenac , and diclofenac21,22 ) . in contrast , celecoxib and meloxicam are nsaids that have a long elimination half - life of 11 hours23,24 ) and 20 hours25 ) , respectively . thus , once daily dosing is sufficient to maintain their plasma concentration and patient compliance with the drugs is high . aceclofenac cr was designed to be composed of two layers , an ir and a cr , to maintain its plasma concentration with once - daily administration . in addition , considering that it has a shorter maximum drug concentration time than celecoxib and meloxicam and a longer elimination half - life than aceclofenac ir , it would offer considerable advantages in the management of acute / chronic pain21 - 25 ) . in a clinical trial for pharmacokinetic comparison of cr formulation and aceclofenac ir formulation in healthy korean subjects , the elimination half - life of the cr formulation was significantly longer than that of the ir formulation ( 5 hours vs. 2 hours)21 ) . in our clinical trial , the non - inferiority of the experimental group was confirmed with 100-mm vas , the primary variable for efficacy evaluation . the vas was decreased by 4.818.9 mm more in the experimental group than in the control group , indicating the cr formulation has a more effective pain relieving effect , although the difference was not statistically significant ( p=0.106 ) . the koos was employed instead of western ontario and mcmaster universities ( womac ) oa index because it is basically comprised of the same subscales of the womac26 - 29 ) . there was no notable intergroup difference in the incidence of adverse events and adverse drug reactions ( p=0.195 ) . with regard to vital signs ( blood pressure and pulse ) , there were no significant changes in the systolic / diastolic pressures and heart rates in both groups . the physical examination and clinical laboratory tests ( blood tests , blood chemistry tests , and urine tests ) did not reveal any abnormality . these results of the 4 weeks of clinical trial involving patients with chronic knee oa confirm that the safety of once daily use of aceclofenac cr 200 mg formulation are comparable to that of twice daily use of aceclofenac ir 100 mg . thus , it is our understanding that the aceclofenac cr would be as safe as the aceclofenac ir among knee oa patients whose long - term use of a painkiller is generally unavoidable . however , we think that more than 4 weeks of long - term safety and efficacy evaluations should be conducted to generalize the results obtained from our clinical trial . it is necessary to confirm whether the equivalent level of safety and efficacy can be established after more than 4 weeks of administration period . one of the limitations of this study is that it was not conducted in a double - blinded manner due to the dosage difference between the two drugs . the safety and efficacy of the two aceclofenac fomulations were compared in patients with chronic knee oa only in our study . we believe comparisons between aceclofenac and other nsaids in patients with other oa , rheumatoid arthritis , and ankylosing spondylitis should be conducted in further studies . this study showed that aceclofenac cr 200 mg formulation was equivalent to aceclofenac ir 100 mg formulation with respect to pain relief and safety among patients with chronic oa of the knee . in addition , the once daily dosage of aceclofenac cr contributed to enhanced patient compliance . therefore , we think that aceclofenac cr 200 mg is a safe and effective medicine that would provide effective pain management and improved quality of life in patients with chronic oa of the knee .

this study was conducted in accordance with the principles outlined in the declaration of helsinki . the local ethics committee ( radboud university nijmegen medical centre , nijmegen , the netherlands ) approved the study , and all subjects gave written informed consent before participation . the study population consisted of 16 patients with type 2 diabetes , recruited through advertisements in a local newspaper and through the outpatient clinic of the radboud university nijmegen medical centre . included were subjects with type 2 diabetes who were treated with metformin with or without sulphonylurea or thiazolidinediones , aged 3575 years , and had an hba1c < 8.0% . exclusion criteria were use of acetylsalicylic acid or vitamin k antagonists , heart failure ( new york heart association class iii or iv ) , smoking , pregnancy or breastfeeding , a serum alanine aminotransferase or aspartate aminotransferase level of more than three times the upper limit of the normal range , a serum creatinine level higher than 130 mol / l , and inability to give informed consent . after inclusion in the study , subjects received either vildagliptin 50 mg b.i.d . for 28 days or acarbose 50 mg t.i.d . for 7 days followed by acarbose 100 mg t.i.d . for the remaining 21 days participants were randomized into treatment with vildagliptin in the first treatment period and acarbose in the second treatment period or vice versa . investigational medication was given on top of their other medication in a randomized , double - blind , cross - over design . between the two treatment periods there was a wash - out period of 1 week . participants were contacted by telephone halfway each treatment period to check for compliance and possible side effects . at the end of each treatment period ( days 2528 ) endothelial function was measured , body weight and potential side effects were determined , and arterial blood was collected for serum hematological and chemical safety profiles . a schedule of the study protocol is shown in fig . compliance was monitored by pill counts . the study protocol ( a ) in which 4 weeks treatment with vildagliptin and acarbose are administered in a cross - over design . the protocol of venous occlusion plethysmography ( b ) started with insertion of arterial catheter at t = 0 , after which a 30-min equilibration period followed . baseline fbf measurements and subsequently fbf measurements during the infusion of acetylcholine ( dark gray ; 0.5 , 2.0 , and 8.0 g dl min ) are performed . again , after a 30-min equilibration period , baseline fbf and fbf during infusion of sodium nitroprusside ( light gray ; 0.06 , 0.20 , and 0.60 g dl min ) are assessed . the experiments started at 8.30 a.m. after an overnight fast in a quiet , temperature - controlled room ( 2324c ) . the brachial artery of the nondominant arm ( experimental arm ) was cannulated ( angiocath , 20-gauge ; becton dickinson , sandy , ut ) under local anesthesia ( xylocaine 2% ) for infusion of vasodilators and collection of blood samples . the brachial artery catheter was connected with an arterial pressure - monitoring line for continuous blood pressure monitoring ( hewlett packard , bblingen , germany ) . forearm blood flow ( fbf ) was measured using mercury - in - silastic strain gauge , venous occlusion plethysmography as previously described ( 16 ) . forearm volume was measured with the water displacement method , and all drugs were dosed per 100 ml forearm tissue with an infusion rate of 100 ml dl min . after complete instrumentation , a 30 min equilibration period was included , after which baseline measurements were performed with infusion of saline . subsequently , three increasing doses of acetylcholine ( 0.5 , 2.0 , and 8.0 g dl min , 10 mg / ml dry powder , dissolved to its final concentration with saline , novartis , greece ) were infused into the brachial artery . each dose was infused for 5 min and fbf was measured during the last 3 min of the 5 min period . after the infusion of all three doses of acetylcholine a 30 min equilibration period followed . subsequently , three increasing doses of sodium nitroprusside ( 0.06 , 0.20 , and 0.60 g dl min , 25 mg / ml , dissolved to its final concentration with 5% glucose solution ; clinical pharmacy , radboud university medical centre ) , endothelium - independent vasodilators ( 18 ) were infused in the brachial artery . again , each dose was infused for 5 min , and fbf was measured during the last 3 min of the 5-min period . fbf registrations of the last 2 min of each dosage of vasodilator were averaged to a single value for data analysis . we considered a 30% change in primary end point between treatment groups , acetylcholine - induced vasodilatation , as clinically relevant . assuming a test - to - test correlation coefficient of 0.5 and a mean fbf in response to acetylcholine of 19 ml dl min would require a total of 15 subjects to detect a 30% change in fbf with a power of 80% at a significance level of 0.05 ( z = 1.96 ) . before each set of drug infusion repeated - measures anova was used to assess differences in increments in fbf between groups . we used the factors treatment ( vildagliptin vs. acarbose ) and time ( three increasing dosages of vasodilator ) , and subjects were included as matched variables . differences in means of laboratory results and hemodynamic parameters were tested by paired student t test or wilcoxon signed rank test for nonnormally distributed data . results in tables and figures are expressed as mean sem , unless otherwise indicated . this study was conducted in accordance with the principles outlined in the declaration of helsinki . the local ethics committee ( radboud university nijmegen medical centre , nijmegen , the netherlands ) approved the study , and all subjects gave written informed consent before participation . the study population consisted of 16 patients with type 2 diabetes , recruited through advertisements in a local newspaper and through the outpatient clinic of the radboud university nijmegen medical centre . included were subjects with type 2 diabetes who were treated with metformin with or without sulphonylurea or thiazolidinediones , aged 3575 years , and had an hba1c < 8.0% . exclusion criteria were use of acetylsalicylic acid or vitamin k antagonists , heart failure ( new york heart association class iii or iv ) , smoking , pregnancy or breastfeeding , a serum alanine aminotransferase or aspartate aminotransferase level of more than three times the upper limit of the normal range , a serum creatinine level higher than 130 mol / l , and inability to give informed consent . after inclusion in the study , subjects received either vildagliptin 50 mg b.i.d . for 28 days or acarbose 50 mg t.i.d . for 7 days followed by acarbose 100 mg t.i.d . for the remaining 21 days participants were randomized into treatment with vildagliptin in the first treatment period and acarbose in the second treatment period or vice versa . investigational medication was given on top of their other medication in a randomized , double - blind , cross - over design . blinding of the study medication was performed by a double dummy technique . between the two treatment periods participants were contacted by telephone halfway each treatment period to check for compliance and possible side effects . at the end of each treatment period ( days 2528 ) endothelial function was measured , body weight and potential side effects were determined , and arterial blood was collected for serum hematological and chemical safety profiles . a schedule of the study protocol is shown in fig . compliance was monitored by pill counts . the study protocol ( a ) in which 4 weeks treatment with vildagliptin and acarbose are administered in a cross - over design . the protocol of venous occlusion plethysmography ( b ) started with insertion of arterial catheter at t = 0 , after which a 30-min equilibration period followed . baseline fbf measurements and subsequently fbf measurements during the infusion of acetylcholine ( dark gray ; 0.5 , 2.0 , and 8.0 g dl min ) are performed . again , after a 30-min equilibration period , baseline fbf and fbf during infusion of sodium nitroprusside ( light gray ; 0.06 , 0.20 , and 0.60 g dl min ) are assessed . the experiments started at 8.30 a.m. after an overnight fast in a quiet , temperature - controlled room ( 2324c ) . the brachial artery of the nondominant arm ( experimental arm ) was cannulated ( angiocath , 20-gauge ; becton dickinson , sandy , ut ) under local anesthesia ( xylocaine 2% ) for infusion of vasodilators and collection of blood samples . the brachial artery catheter was connected with an arterial pressure - monitoring line for continuous blood pressure monitoring ( hewlett packard , bblingen , germany ) . forearm blood flow ( fbf ) was measured using mercury - in - silastic strain gauge , venous occlusion plethysmography as previously described ( 16 ) . forearm volume was measured with the water displacement method , and all drugs were dosed per 100 ml forearm tissue with an infusion rate of 100 ml dl min . after complete instrumentation , a 30 min equilibration period was included , after which baseline measurements were performed with infusion of saline . subsequently , three increasing doses of acetylcholine ( 0.5 , 2.0 , and 8.0 g dl min , 10 mg / ml dry powder , dissolved to its final concentration with saline , novartis , greece ) were infused into the brachial artery . each dose was infused for 5 min and fbf was measured during the last 3 min of the 5 min period . after the infusion of all three doses of acetylcholine a 30 min equilibration period followed . subsequently , three increasing doses of sodium nitroprusside ( 0.06 , 0.20 , and 0.60 g dl min , 25 mg / ml , dissolved to its final concentration with 5% glucose solution ; clinical pharmacy , radboud university medical centre ) , endothelium - independent vasodilators ( 18 ) were infused in the brachial artery . again , each dose was infused for 5 min , and fbf was measured during the last 3 min of the 5-min period . fbf registrations of the last 2 min of each dosage of vasodilator were averaged to a single value for data analysis . the experiments started at 8.30 a.m. after an overnight fast in a quiet , temperature - controlled room ( 2324c ) . the brachial artery of the nondominant arm ( experimental arm ) was cannulated ( angiocath , 20-gauge ; becton dickinson , sandy , ut ) under local anesthesia ( xylocaine 2% ) for infusion of vasodilators and collection of blood samples . the brachial artery catheter was connected with an arterial pressure - monitoring line for continuous blood pressure monitoring ( hewlett packard , bblingen , germany ) . forearm blood flow ( fbf ) was measured using mercury - in - silastic strain gauge , venous occlusion plethysmography as previously described ( 16 ) . forearm volume was measured with the water displacement method , and all drugs were dosed per 100 ml forearm tissue with an infusion rate of 100 ml dl min . after complete instrumentation , a 30 min equilibration period was included , after which baseline measurements were performed with infusion of saline . subsequently , three increasing doses of acetylcholine ( 0.5 , 2.0 , and 8.0 g dl min , 10 mg / ml dry powder , dissolved to its final concentration with saline , novartis , greece ) were infused into the brachial artery . each dose was infused for 5 min and fbf was measured during the last 3 min of the 5 min period . after the infusion of all three doses of acetylcholine a 30 min equilibration period followed . subsequently , three increasing doses of sodium nitroprusside ( 0.06 , 0.20 , and 0.60 g dl min , 25 mg / ml , dissolved to its final concentration with 5% glucose solution ; clinical pharmacy , radboud university medical centre ) , endothelium - independent vasodilators ( 18 ) were infused in the brachial artery . again , each dose was infused for 5 min , and fbf was measured during the last 3 min of the 5-min period . fbf registrations of the last 2 min of each dosage of vasodilator were averaged to a single value for data analysis . we considered a 30% change in primary end point between treatment groups , acetylcholine - induced vasodilatation , as clinically relevant . assuming a test - to - test correlation coefficient of 0.5 and a mean fbf in response to acetylcholine of 19 ml dl min would require a total of 15 subjects to detect a 30% change in fbf with a power of 80% at a significance level of 0.05 ( z = 1.96 ) . before each set of drug infusion , baseline fbf measurements were performed . repeated - measures anova was used to assess differences in increments in fbf between groups . we used the factors treatment ( vildagliptin vs. acarbose ) and time ( three increasing dosages of vasodilator ) , and subjects were included as matched variables . differences in means of laboratory results and hemodynamic parameters were tested by paired student t test or wilcoxon signed rank test for nonnormally distributed data . results in tables and figures are expressed as mean sem , unless otherwise indicated . we considered a 30% change in primary end point between treatment groups , acetylcholine - induced vasodilatation , as clinically relevant . assuming a test - to - test correlation coefficient of 0.5 and a mean fbf in response to acetylcholine of 19 ml dl min would require a total of 15 subjects to detect a 30% change in fbf with a power of 80% at a significance level of 0.05 ( z = 1.96 ) . before each set of drug infusion , baseline fbf measurements were performed . repeated - measures anova was used to assess differences in increments in fbf between groups . we used the factors treatment ( vildagliptin vs. acarbose ) and time ( three increasing dosages of vasodilator ) , and subjects were included as matched variables . differences in means of laboratory results and hemodynamic parameters were tested by paired student t test or wilcoxon signed rank test for nonnormally distributed data . results in tables and figures are expressed as mean sem , unless otherwise indicated . seventeen of the 20 initially screened subjects underwent randomization and were enrolled in the study . one participant withdrew because of nausea and upper abdominal discomfort during the first week of vildagliptin treatment . the mean duration of diabetes was 7.9 5.3 years with an hba1c of 6.97 0.61% . ten participants ( 62% ) were treated with metformin and six were treated ( 38% ) with metformin in combination with a sulfonylurea . of these six participants , three used glimepiride , two used tolbutamide , and one used glibenclamide . baseline characteristics after completion of the trial but before deblinding one subject was excluded from statistical analyses because of unreliable measurement results , probably the result of a technical problem with one of the strain gauges . baseline fbf in the experimental arm in the vildagliptin group was higher than in the acarbose group . baseline values for fbf in the experimental arm were 3.3 0.3 ml dl min in the vildagliptin group and 2.5 0.2 ml dl min in the acarbose group ( p = 0.02 ) . corresponding values in the nonexperimental arm were 2.7 0.3 and 2.2 0.3 ml dl min ( p = 0.07 ) . to compensate for differences in baseline fbf , responses to drugs were expressed as absolute change in fbf ( fbf ) above baseline . infusion of acetylcholine induced a dose - dependent increase in fbf in the experimental arm , whereas fbf did not change in the nonexperimental arm . vildagliptin treatment augmented the absolute increase in fbf in response to acetylcholine from baseline compared with acarbose ( p = 0.01 by two - way anova ) . changes in fbf from baseline with vildagliptin were 3.1 0.7 , 7.9 1.1 , and 12.6 1.4 ml dl min in response to acetylcholine 0.5 , 2.0 , and 8.0 g dl min , respectively , compared with 2.0 0.7 , 5.0 1.2 , and 11.7 1.6 ml dl min with acarbose . results of the repeated - measures two - way anova for acetylcholine were as follows : p < 0.0001 for changes over time , p = 0.002 for differences between treatment , and p = 0.01 for their interaction . figure 2 shows the change in fbf from baseline in response to the different vasodilators . change in fbf in response to acetylcholine ( ach ) in the experimental arm in response to acetylcholine ( top panel ; dosage 0.5 , 2.0 , and 8.0 g dl min ) and sodium nitroprusside ( lower panel ; dosage 0.06 , 0.20 , and 0.60 g dl min ) , during vildagliptin ( filled circles , solid lines ) or acarbose ( open squares , dashed lines ) . comparable results were obtained when data were expressed as relative changes in fbf from baseline . when data were expressed as mean fbf ( without correction for baseline differences ) the repeated - measures two - way anova was still borderline significant . infusion of sodium nitroprusside induced a dose - dependent increase in fbf in the experimental arm . changes in fbf from baseline when treated with vildagliptin were 3.6 0.5 , 7.4 0.9 , and 11.9 1.0 ml dl min in response to sodium nitroprusside 0.06 , 0.20 , and 0.60 g dl min , respectively , compared with 3.5 0.4 , 6.4 0.8 , and 10.7 1.0 ml dl min with acarbose . results of the repeated - measures two - way anova were as follows : p < 0.0001 for changes over time , p = 0.06 for differences between treatment , and p = 0.36 for their interaction . both systolic and diastolic blood pressure ( measured intra - arterially during the venous occlusion plethysmography ) tended to be lower when treated with vildagliptin compared with acarbose ( 143.1 4.7 vs. 145.2 3.6 mmhg [ p = 0.37 ] and 73.3 2.4 vs. 74.9 2.3 mmhg [ p = 0.22 ] ) . mean arterial pressure was 100.7 3.2 during vildagliptin compared with 102.4 2.8 mmhg during acarbose treatment ( p = 0.27 ) . heart rate was 67.9 2.1 bpm when treated with vildagliptin and 66.3 2.5 bpm when treated acarbose ( p = 0.41 ) . fasting plasma glucose ( measured intra - arterially ) was lower during vildagliptin than during acarbose treatment ( 6.62 0.27 vs. 7.59 0.28 mmol / l ; p < 0.0001 ) . fasting insulin levels were 14.5 2.0 me / l after vildagliptin and 15.6 2.4 me / l after acarbose treatment ( p = not significant [ ns ] ) . hba1c levels tended to be lower during with vildagliptin compared with acarbose treatment ( 6.73 0.12 vs. 6.84 0.12% , respectively ; p = 0.07 ) . there was no change in body weight and lipid profile ( supplementary table 1 ) . most reported side effects during vildagliptin treatment were flatulence ( n = 4 ) and nausea ( n = 3 ) . one subject withdrew during the first week of vildagliptin treatment because of upper abdominal pain and nausea . flatulence ( n = 15 ) and diarrhea ( n = 5 ) were the most frequent side effects during acarbose treatment . no changes in blood count , renal function , and liver function were observed during vildagliptin treatment . baseline fbf in the experimental arm in the vildagliptin group was higher than in the acarbose group . baseline values for fbf in the experimental arm were 3.3 0.3 ml dl min in the vildagliptin group and 2.5 0.2 ml dl min in the acarbose group ( p = 0.02 ) . corresponding values in the nonexperimental arm were 2.7 0.3 and 2.2 0.3 ml dl min ( p = 0.07 ) . to compensate for differences in baseline fbf , responses to drugs were expressed as absolute change in fbf ( fbf ) above baseline . infusion of acetylcholine induced a dose - dependent increase in fbf in the experimental arm , whereas fbf did not change in the nonexperimental arm . vildagliptin treatment augmented the absolute increase in fbf in response to acetylcholine from baseline compared with acarbose ( p = 0.01 by two - way anova ) . changes in fbf from baseline with vildagliptin were 3.1 0.7 , 7.9 1.1 , and 12.6 1.4 ml dl min in response to acetylcholine 0.5 , 2.0 , and 8.0 g dl min , respectively , compared with 2.0 0.7 , 5.0 1.2 , and 11.7 1.6 ml dl min with acarbose . results of the repeated - measures two - way anova for acetylcholine were as follows : p < 0.0001 for changes over time , p = 0.002 for differences between treatment , and p = 0.01 for their interaction . figure 2 shows the change in fbf from baseline in response to the different vasodilators . change in fbf in response to acetylcholine ( ach ) in the experimental arm in response to acetylcholine ( top panel ; dosage 0.5 , 2.0 , and 8.0 g dl min ) and sodium nitroprusside ( lower panel ; dosage 0.06 , 0.20 , and 0.60 g dl min ) , during vildagliptin ( filled circles , solid lines ) or acarbose ( open squares , dashed lines ) . comparable results were obtained when data were expressed as relative changes in fbf from baseline . when data were expressed as mean fbf ( without correction for baseline differences ) the repeated - measures two - way anova was still borderline significant . infusion of sodium nitroprusside induced a dose - dependent increase in fbf in the experimental arm . changes in fbf from baseline when treated with vildagliptin were 3.6 0.5 , 7.4 0.9 , and 11.9 1.0 ml dl min in response to sodium nitroprusside 0.06 , 0.20 , and 0.60 g dl min , respectively , compared with 3.5 0.4 , 6.4 0.8 , and 10.7 1.0 ml dl min with acarbose . p < 0.0001 for changes over time , p = 0.06 for differences between treatment , and p = 0.36 for their interaction . both systolic and diastolic blood pressure ( measured intra - arterially during the venous occlusion plethysmography ) tended to be lower when treated with vildagliptin compared with acarbose ( 143.1 4.7 vs. 145.2 3.6 mmhg [ p = 0.37 ] and 73.3 2.4 vs. 74.9 2.3 mmhg [ p = 0.22 ] ) . mean arterial pressure was 100.7 3.2 during vildagliptin compared with 102.4 2.8 mmhg during acarbose treatment ( p = 0.27 ) . heart rate was 67.9 2.1 bpm when treated with vildagliptin and 66.3 2.5 bpm when treated acarbose ( p = 0.41 ) . fasting plasma glucose ( measured intra - arterially ) was lower during vildagliptin than during acarbose treatment ( 6.62 0.27 vs. 7.59 0.28 mmol / l ; p < 0.0001 ) . fasting insulin levels were 14.5 2.0 me / l after vildagliptin and 15.6 2.4 me / l after acarbose treatment ( p = not significant [ ns ] ) . hba1c levels tended to be lower during with vildagliptin compared with acarbose treatment ( 6.73 0.12 vs. 6.84 0.12% , respectively ; p = 0.07 ) . there was no change in body weight and lipid profile ( supplementary table 1 ) . most reported side effects during vildagliptin treatment were flatulence ( n = 4 ) and nausea ( n = 3 ) . one subject withdrew during the first week of vildagliptin treatment because of upper abdominal pain and nausea . flatulence ( n = 15 ) and diarrhea ( n = 5 ) were the most frequent side effects during acarbose treatment . no changes in blood count , renal function , and liver function were observed during vildagliptin treatment . the main finding of the current study is that 4-week treatment with vildagliptin improves endothelial function in patients with type 2 diabetes . this conclusion is based on the observation that vildagliptin augments the vasodilator response in the forearm vascular bed to acetylcholine , an endothelium - dependent vasodilator , whereas the vascular response to sodium nitroprusside , an endothelium - independent vasodilator , remains unaffected . baseline blood flow tended to be higher during vildagliptin , accompanied by slightly lower blood pressure values , but these changes did not attain statistical significance . that glp-1 is a physiological substrate of dpp-4 , dpp-4 inhibition by vildagliptin may be expected to increase circulating levels of glp-1 ( 19 ) . several studies have reported beneficial effects of glp-1 on the cardiovascular system . in humans , 20 ) have shown that 72-h infusion of glp-1 improved left ventricular function in patients with acute myocardial infarction and systolic dysfunction after successful reperfusion therapy , an effect that was observed in both diabetic and nondiabetic patients . they discussed that this observation might be explained by the insulinotropic and insulinomimetic properties of glp-1 , but alternatively glp-1 might also improve endothelial function . studies have shown that glp-1 improves endothelium - dependent vascular responses in the brachial artery while leaving endothelium - independent responses unaffected in healthy humans and patients with type 2 diabetes ( 11,21 ) . the cardiovascular actions of glp-1 may occur either directly through the glp-1 receptor or alternatively by a glp-1 receptor - independent effect of the degradation product of glp-1 , glp-1(936 ) ( 12 ) . apart from glp-1 , dpp-4 also degrades gip , and perhaps cytokines and certain chemokines ( including stromal - derived factor 1- ) , thus other substrates of dpp-4 may be responsible for the improvement in endothelial function . however , we did not find changes in fasting insulin levels when treated with vildagliptin . this is in correspondence with literature , in which dpp-4 inhibitors do not influence fasting insulin levels ( 19 ) . although we aimed for similar glucose levels during both treatments , this was not completely accomplished since the fasting glucose levels were slightly higher in the acarbose group ( 6.62 0.27 for vildagliptin vs. 7.59 0.28 mmol / l for acarbose ) . it seems unlikely that this small difference of 1.0 mmol / l causes the observed difference in vascular response . this is supported by the absence of any correlation between change in glucose and change in increase in fbf from baseline in response to acetylcholine ( r = 0.05 , data not shown ) . finally , vildagliptin might have a direct pharmacological , yet unidentified , effect on the endothelium . the improvement in endothelium - dependent vasodilatation in the forearm observed after 4-week treatment with vildagliptin might translate into a decrease in cardiovascular events . endothelial dysfunction is a marker of cardiovascular complications , and a close relation exists between endothelial function in the human coronary circulation and peripheral circulation ( 23,24 ) . this possibility is supported by a recent meta - analysis showing that treatment with vildagliptin 50 mg b.i.d . may decrease the relative risk ( rr ) of cardio - cerebrovascular events ( rr 0.84 , ci 0.621.14 ) ( 14 ) . of course these findings need to be confirmed by a long - term cardiovascular outcome study . as secondary findings , we found slightly lower diastolic and systolic blood pressure with a slightly higher heart rate after vildagliptin treatment . furthermore , baseline measurement of fbf was also higher after vildagliptin than after acarbose treatment ( 3.3 0.3 vs. 2.5 0.2 ml dl min in the experimental arm and 2.7 0.3 vs. 2.2 0.3 ml dl min in the nonexperimental arm ) . basal vascular tone can be affected by the no pathway as well as by the sympathetic nervous system ( 24 ) . the lower blood pressure and increased basal fbf might implicate that vildagliptin is a systemic vasodilator . this observation can be a result of an enhanced availability of no as a result of improved endothelial function or alteration in the activity of the sympathetic nervous system . however , 26-week treatment with dpp-4 inhibitor alogliptin did not cause clinical meaningful changes in either systolic or diastolic blood pressure ( 25 ) . this 4-week period was enough to show improved endothelial function but larger outcome trials are needed to evaluate a potential beneficial effect on cardiovascular outcome . second , although this study was double - blinded , more subjects experienced flatulence during acarbose ( 15 vs. 4 ) , which might compromise this . however , the investigator analyzed the fbf results of plethysmography of each treatment period separately and the flow measurements are objective . third , the forearm vasodilator response to acetylcholine is only one of the methods to study endothelial function . nevertheless , these functional measurements are one of the most representative indexes of endothelial function . the reason for using an active comparator drug in our study was to aim for equal glucose levels . our study design does not allow us to conclude that the effect of vildagliptin on endothelial function is glp-1 dependent . but if this were true , the observed difference in fbf would have been diminished by acarbose and larger differences would have been found using placebo . however , when using placebo our results might have been confounded by a larger change in fasting glucose . furthermore , acarbose is the optimal active comparator drug in this trial , since it is not absorbed into the circulation and thus can not influence endothelial function directly . the study has also strengths ; the effects of vildagliptin were studied in a relevant population metformin - treated patients with type 2 diabetes and is thus applicable to clinical practice . in addition , an appropriate control treatment arm was used to avoid major differences in glycemic control . in conclusion , 4 weeks treatment with vildagliptin these findings may be relevant in the context of future cardiovascular complications and provide a basis for further cardiovascular outcome studies .

continuous renal replacement therapy ( crrt ) has become the preferred modality for the management of critically ill - children with acute kidney injury ( aki ) and fluid overload ( f.o ) , especially in developed countries because of the advances in technology allowing more accurate treatment delivery and better control of blood flow and fluid removal over extended period of time , especially in hemodynamically unstable patients . crrt eliminates the compulsion for fluid restriction and allows the provision of medications , blood products , and nutrition in critically ill - children with multiorgan failure . it has the capacity to provide more efficient clearance of solutes than standard peritoneal dialysis . another potential use of crrt is the removal of inflammatory cytokines and endotoxins in septic patients . a descriptive study from india showed that , sepsis lead to 39.7% total multiorgan dysfunction syndrome ( mods ) in a tertiary care pediatric intensive care unit ( picu ) . data regarding the usage of crrt is very limited from developing countries as compared to developed countries . the authors report their experience with crrt in sepsis with mods from a tertiary level picu of a developing country . in this retrospective descriptive study , data were collected from the patient case records who received crrt for sepsis with mods in a tertiary care picu from september 2010 to february 2015 [ figure 1 ] . children who received crrt but excluded from the study were postoperative cardiac patients , children with inborn errors of metabolism , nonseptic mods . information was obtained by a predesigned data collection sheet which included the following : age , gender , primary disease leading to initiation of crrt , relevant co - morbid illnesses , reason for crrt initiation , time duration from icu admission to crrt initiation , fluid input from icu admission to crrt initiation ( l / day ) , fluid output from icu admission to crrt initiation ( l / day ) . f.o at the time of crrt initiation was calculated using the formula : study flow chart . sepsis with multiorgan dysfunction syndrome f.o ( % ) = ( fluid in fluid out)/picu admission weight 100% serum creatinine at the initiation of crrt , prism score at 12 and 24 h , inotropic score ( is ) at initiation and termination of crrt were also noted . in this study , ( [ is ] = dopamine dose [ mic / kg / min ] + dobutamine [ mic / kg / min ] + 100 epinephrine dose [ mic / kg / min ] ) . vasoactive is = is + 10 milrinone dose ( mic / kg / min ) + 10,000 vasopressin dose ( units / kg / min ) + 100 nor epinephrine dose ( mic / kg / min ) . plateau pressure required for mechanical ventilation at initiation and termination of crrt , pao2/fio2 ratio at initiation and termination of crrt , diuretic usage prior to crrt initiation ( yes / no ) , route of venous access , anticoagulation used , priming solution , crrt modality used continuous venovenous hemodiafiltration ( cvvhdf ) , continuous venovenous hemofiltration , continuous venovenous hemodialysis , complications during crrt were also analyzed . secondary outcome measures were ( a ) change in fluid balance ( b ) percentage of change in is , pao2/fio2 ratio and plateau pressure at the termination of crrt , and ( c ) effect of timing of initiation of crrt on survival . data were analyzed using spss ( version 19 , ibm - spss , spss inc . new york , usa ) software and significance was examined using t - test and mann whitney test . data were analyzed using spss ( version 19 , ibm - spss , spss inc . new york , usa ) software and significance was examined using t - test and mann whitney test . data were analyzed using spss ( version 19 , ibm - spss , spss inc . new york , usa ) software and significance was examined using t - test and mann whitney test . median age and weight of subjects were 11 years ( range 1.116 ) and 39 kg ( range 7.565 ) , respectively all children had shock requiring 2 inotropes and were on mechanical ventilation support at the time of initiation of crrt . oligo - anuria and f.o ( > 10% ) were presented in 90% and 48.1% , respectively . chief indications for initiation of crrt were aki ( 88% ) , f.o ( 50% ) , as a part of mods and anticipation of f.o . total 34 crrt sessions were given amounting to 1304.4 h with mean lifespan of each filter was 47.62 h. femoral venous access , internal jugular venous access , and subclavian venous access were used in 23 ( 85% ) , 3 ( 11.1% ) , and 1 ( 3.7% ) patients , respectively . blood and 5% albumin were used for circuit priming in 15 ( 55.5% ) and 12 ( 45.5% ) patients , respectively . heparin was used as an anticoagulant in only 10 ( 37% ) patients in view of coagulopathy . manageable dyselectrolytemias namely hypokalemia , hypophosphatemia , hypomagnesemia , and hypocalcemia were observed in 21 ( 77.8% ) , 16 ( 59.2% ) , 13 ( 41.8% ) , and 7 ( 25.8% ) , respectively . duration of icu stay , time from admission to crrt initiation , f.o , crrt duration , prism score at 24 h , creatinine at crrt initiation were not different between survivors and nonsurvivors . in contrast , percentage of decrease in inotrope score , plateau pressure , and percentage of increase in pao2/fio2 ratio were higher in survivor group compared to nonsurvivor group . the same clinical variables were compared between the children who received crrt within 48 h of admission and those received after 48 h of admission and are depicted in table 3 . there was no difference in duration of icu stay , time from admission to crrt initiation , f.o , crrt duration , prism score at 12 h , percentage of decrease in inotrope score , plateau pressure , and percentage of increase in pao2/fio2 ratios between the two groups . creatinine at initiation of crrt in children who received crrt within 48 h of admission is significantly lower than those who received after 48 h. fourteen patients of 27 patients ( 51.8% ) were survived . survival rate was 61.1% ( 11/18 ) in those patients who received crrt within 48 h of admission and was 33.3% in those who received crrt after 48 h of admission . clinical profile of the patients underwent crrt comparison of the parameters between survivors and non survivors comparison of the parameters between patients received crrt within 48 hours of admission and after 48 hours of admission to picu sepsis is a major contributor for mods in picu constituting 39.2% of mods etiology as reported by khilnani et al . from india . most pediatric centers from developed nations use crrt as an initial mode of renal replacement therapy ( rrt ) for critically ill - children with mods . recently , khandelwal et al . from india , reported their experience of pediatric crrt in which 15 out of 17 patients ( 88.2% ) had sepsis with mods at the time of initiation of crrt . in this study , the same parameters were also compared in those patients who were given crrt within 48 h of icu admission to after 48 h of admission . f.o was one of the major indication for initiation of crrt in our study population as it is an independent risk factor for poor outcome in critically ill - patients . one study found that survivors of acute respiratory distress syndrome had a net negative fluid balance as compared with nonsurvivors in an icu . we took the advantage of crrt to provide adequate nutrition , to transfuse blood and plasma products transfusion without much hesitancy . assessment of f.o in critically ill - patients is multidimensional such as clinical examination ( edema , hepatomegaly , bilateral basal lung crepitations ) , serial weight measurement , cumulative fluid balance ( ml fluid in - ml fluid out from picu admission)/picu admission weight in kg 100% ) , chest x - ray , oxygenation indices , lung ultrasound , echocardiography , intra - abdominal pressure , and bio - impedance analysis of body composition . these additional findings provide indications that prevention of excessive f.o may be a hallmark of optimal care for the pediatric patient with mods . hence , we assessed plateau pressure , pao2/fio2 ratio at the time of crrt initiation and crrt termination between the two groups . percentage of decrease in plateau pressure ( p = 0.00 ) and increase in pao2/fio2 ratio ( p = 0.03 ) in survivors is significant compared to nonsurvivors . the severity of illness as measured by prism score at 12 and 24 h of admission to icu is also similar between the two groups . in our cohort , survivor group had low serum creatinine at initiation of crrt as compared to nonsurvivor group ( 2.8 mg / dl vs. 3.7 mg / dl ) which may suggest that initiation of crrt at early stage of aki may give survival benefit . crrt registry subgroup analysis on children with mods receiving crrt showed survival rate of 52% which is similar to our cohort , that is , 51.8% . a study by proulx et al . showed that severe and life - threatening mods occur very early in pediatric patients during their icu course . in their study , 87% of children developed the maximum number of organ failures within 72 h of picu admission and 88.4% of deaths occurred within 7 days of mods evolution . hence , early and appropriate aggressive supportive measures , including rrt to treat or prevent aki sequelae , could conceivably improve patient outcome . hence , we compared clinical parameters , laboratory parameters as well as mortality in those who were given crrt within 48 ( early initiation ) hours of admission to those who were given crrt after 48 h ( late initiation ) of admission to icu . the survival rate in early initiation group ( 61.1% ) is statistically significant compared to late initiation group ( 33% ) ( p = 0.0001 ) . a recent retrospective study by modem et al . also showed that early initiation of crrt was associated with lower mortality in critically ill children requiring renal support for f.o or aki . a study in 2005 , had shown that plasma endotoxin and cytokines ( tumor necrosis factor - alpha , interleukin ( il)-1 beta , il-6 , and il-8 ) can be removed effectively with crrt in severely burned patients with sepsis . another animal study in 2014 showed that crrt reduces the systemic and pulmonary inflammation induced by veno - venous extracorporeal membrane oxygenation in a porcine model . even though , there is no statistically significant difference in the f.o between the patients who received early crrt and late crrt in our cohort , the early removal of inflammatory cytokines may be the key factor for mortality benefit . disadvantages of crrt are vascular access , requirement of technical expertise , vigilance over hemodynamic and coagulation parameters . . however , this is the first study done in children with severe sepsis and mods from a developing country . our study emphasizes the role of crrt in improving the oxygenation status and probably also the survival in children with severe sepsis with mods who receive crrt early in the course of illness .

post - thoracotomy pain can adversely affect coughing and deep breathing , resulting in respiratory complications such as hypoxia , atelectasis , chest infection and respiratory failure that may delay recovery and if severe , could be life - threatening . surgical approach to patent ductus arteriosus ( pda ) ligation surgery is carried through left posterolateral thoracotomy . this incision has often been reported to be associated with severe post - operative pain that may have deleterious effects on pulmonary function . a thoracic paravertebral block ( tpvb ) by local anaesthetic provides effective pain relief with few side effects . this technique also reduces the occurrence of chronic post - thoracotomy neuralgia , stress responses and preserves pulmonary function . efficacy of tpvb depends on properneedle placement , potency , concentration and volume of the local anaesthetic . ultrasonographic visualisation of in - plane needle advancement reduces the risk of pleural puncture as well as entry of the needle into the intervertebral foramen . it is a potent drug ; however toxicity is reported when higher dosages are injected . ropivacaine is a newer local anaesthetic that could be a useful alternative to bupivacaine for tpvb . there is paucity of literature comparing the efficacy of the two local anaesthetics in pda ligation surgeries , and hence this study was undertaken to compare the efficacy of bupivacaine with that of ropivacaine . after obtaining institutional ethics committee and scientific committee approval , we studied hundred consecutive patients aged between 2 and 10 years , of american society of anaesthesiologists ( asa ) physical status i and ii , undergoing pda ligation surgery . the duration of study was 1-year . patients with cutaneous infection at the site of needle puncture , pathology in the paravertebral space , allergy to local anaesthetic drugs , coagulopathy , kyphoscoliosis and previous thoracotomy were excluded from the study . written informed consent from the parents of the patients was taken . fifty patients received a pre - incisional single thoracic paravertebral injection of 0.2% ropivacaine ( group r ) , and another 50 patients received pre - incisional single thoracic paravertebral injection of 0.25% bupivacaine ( group b ) . all patients in this study were pre - medicated with syrup promethazine 0.3 mg / kg per orally , 90 min before induction of anaesthesia . patients were monitored using electrocardiogram ( ecg ) , invasive blood pressure , heart rate and pulse oximeter in the perioperative period . intravenous ( iv ) cannula was inserted with inhalational induction using oxygen , nitrous oxide and halothane and iv fluid ( ringer lactate or normal saline ) was administered . tracheal intubation was carried out with fentanyl 2 g / kg , midazolam 0.1 mg / kg , muscle relaxation with pancuronium 0.1 mg / kg body weight . after tracheal intubation , patients were ventilated with intermittent positive pressure ventilation with o2 and n 2 o in 50:50 ratio and later , anaesthesia maintained along with isoflurane ( minimum alveolar concentration 1 ) . tidal volume of 810 ml / kg and ventilatory frequency of 1624 breaths / min were adjusted to maintain normal end tidal co2 levels . paravertebral block was administered under ultrasound guidance by in - plane approach with patients in right lateral decubitus position to expose their upper thoracic region . after skin disinfection , phased array transducer of s8 frequency ( philips ultrasound machine hd 11 xe ) kept in sterile sleeve , was placed in an axial ( transverse ) plane on the rib at the selected thoracic level , just lateral to the spinous process . machine was optimised for imaging capability by selecting the appropriate depth of field ( within 23 cm ) , focus range and gain . the transverse process and rib were visualised as a hyperechoic line with acoustic shadowing below it . thoracic paravertebral space ( tpvs ) localisation was done by moving the transducer caudally into the intercostal space between adjacent ribs . the transverse process was visualised on the medial side as a hyperechoic convex line with acoustic shadowing beneath . the tpvs and the adjoining intercostal space could be visualised as a wedge - shaped hypoechoic layer demarcated by the hyperechoic lines of the pleura below and the internal intercostal membrane above . after ultrasound - guided identification of paravertebral space ( in plane approach ) , 0.25% bupivacaine 0.4 ml / kg was injected in group b patients and 0.2% ropivacaine 0.4 ml / kg was injected in group r patient using 21-gauge hypodermic needle , which was attached to 20 cm pressure monitoring line for convenience of administration of medication . local anaesthetic deposition translated as an anterior displacement of the parietal pleura on the ultrasound image . the haemodynamic parameters - heart rate and blood pressure ( systolic , diastolic and mean ) were measured . bradycardia was defined as < 80 bpm ( below 1-year ) , < 70 bpm ( 13 years ) and < 65 bpm ( 36 years ) and < 60 ( 612 years ) . however , intra - operatively , hypotension was induced momentarily during ligation of pda using infusion and bolus of injection nitroglycerine . after surgery , the patients were transferred to the post - operative intensive care unit ( icu ) with endotracheal tube in situ . patients were extubated within 30 min , after stabilisation of acid - base status and surgical drainage and confirmation of normal chest x - ray . monitoring of pulse rate , blood pressure , spo2 , ecg , respiratory rate and modified objective pain score ( mops ) was carried out in icu for 12 h after surgery . assessment of post - operative pain based on mops [ table 1 ] score ( 0 = no pain and 10 = worst pain imaginable ) was assessed hourly for the first 4 h , 2 hourly till 12 h after the surgery . post - operative analgesic ( injection ketorolac 0.5 mg / kg iv ) was given as a rescue analgesic only if the pain score on mops was more than 5 . modified objective pain score ( mops ) descriptive statistical analysis was carried out in the present study . results on continuous measurements are presented on mean , sd ( minimum - maximum ) and results on categorical measurements are presented in number ( % ) . chi - square / fisher exact test was used to find the significance of study parameters on a categorical scale between two groups . paired samples t - test was used to find the significance of study parameters on the continuous scale within the group ( intra - group analysis ) on metric parameters . student 's t - test ( two - tailed , independent samples ) was used to find the significance of study parameters on continuous scale between two groups ( inter - group analysis ) on metric parameters . the sample size of 50 patients in each group was based on previous study , with pain score and time of first rescue analgesic as parameters , with 90% statistical power , 5% level of significance and 95% confidence interval . chicago , il , usa ) was used for the analysis of the data and microsoft word and excel used to generate graphs and tables . demographic data was comparable between the groups with respect to age ( 5.68 2.59 vs. 5.78 2.66 years ) , weight ( 16.16 4.88 vs. 16.06 4.88 kg ) , height ( 107.36 15.54 vs. 107.88 16.36 cm ) and gender ( 28 male , 22 female vs. 26 male 24 female patients ) . the mean duration of anaesthesia in the ropivacaine group was 51.42 4.89 min as compared to 51.48 5.32 min in the bupivacaine group , and the difference was statistically not significant ( p = 0.372 ) . both the groups had a reduction in heart rate at 5 min and 10 min . reduction in heart rate from baseline was comparable in both the groups at 5 min and 10 min , and the difference was not statistically significant . during post - extubation period both the groups had a reduction in heart rate , which was more in ropivacaine group , but the difference was not statistically significant . there was no statistically significant difference in heart rate between the two groups throughout the study [ figure 1 ] . comparison of heart rate in two groups studied there was a reduction in blood pressure at 5 min , 10 min and 15 min from baseline in both the groups . fall in systolic blood pressure in group b was statistically significant at 10 min and 15 min . post - extubation , systolic blood pressure started rising after 6 h in both the groups . difference in diastolic blood pressure was statistically significant ( p < 0.001 ) between the two groups at 10 min . mean arterial pressure ( map ) was lower in bupivacaine group at 10 min and 15 min , which was statistically significant with p < 0.001 and 0.009 , respectively . map values were comparable in both the groups intra - operatively and post - extubation [ figure 2 ] . comparison of mean arterial pressures related to analgesia , other parameters assessed after extubation were the respiratory rate , spo2 , mops and time of rescue analgesic administered . there was no statistically significant difference in respiratory rate and spo2 of the two groups . mops increased in both the groups as time interval increased from the administration of block . difference between mops score was not statistically significant throughout the study [ figure 3 ] . comparison of mean objective pain scores in both the groups , 10% patients received rescue analgesic at 6 h. at 8 h , 44% patients received rescue analgesic in group r and 42% patients received rescue analgesic in group b. at 10 h , in both the groups 38% patients received rescue analgesic . at 12 h , time of first rescue analgesic ( h ) in group r , hypotension was observed in four patients , while in group b , 10 patients had hypotension . bradycardia was seen in three patients in group r and nine patients in group b , which was found to be statistically significant . two patients had vascular puncture in group r and one patient in group b. pneumothorax and arrhythmias were not seen in any of the groups . pre - emptive analgesia is a treatment that is initiated before the surgical procedure in order to prevent central sensitisation to painful stimulus , thereby reducing hyperalgesia . the primary goals of preemptive analgesia are to decrease acute pain following tissue injury , to prevent pathological modulation of the central nervous system due to this pain and to prevent the development of chronic pain . the various pharmacological options for preemptive analgesia include opioids , non - steroidal anti - inflammatory drugs , local anaesthetics , alpha agonists . the various routes of administration include local infiltration , iv route , neuraxial and peripheral blocks . iv opioids are not potent enough to control neurogenic pain without detrimental effects on respiratory outcome . thoracic epidural analgesia carries the risk of dural puncture , epidural haematoma , epidural abscess and side effects such as hypotension , bradycardia and urinary retention . compared to epidural block , the paravertebral block is associated with less haemodynamic changes and is a suitable alternative in patients with severe comorbidities and contraindications to neuraxial blocks . in cardiac surgery , where maintenance of stable haemodynamics is a prerequisite , tpvb has a vital role in pain management . in our study , the changes in heart rate , map and respiratory rate were comparable in both the groups except for a significant fall in map in bupivacaine group at 10 and 15 min which responded promptly to drug therapy . we did not find statistically significant difference in mean respiratory rate and spo2 between the two groups after extubation . similarly , bariskaner et al . found that neither drug had a significant effect on respiratory rate or blood gas values ( p > 0.05 ) in their study . ropivacaine has emerged as a suitable alternative with comparable quality and duration of analgesia , but with lesser degrees of motor block . in our study , mean values of mopss were comparable in both the groups throughout the study , and the difference in them was not statistically significant . the time observed for the demand of rescue analgesic was 8 to 10 h in over 80% of patients in both the groups indicating an adequate duration of analgesia post - operatively . but , lesser duration of analgesia was noted by ivani et al . who conducted a double - blind , multicentre trial in 245 children , aged 110 years undergoing elective minor surgery receiving a single caudal extradural injection of 1 ml / kg of either 0.25% bupivacaine or 0.2% ropivacaine after induction of general anaesthesia . the mean time duration to administer first analgesia was 233 min ( 3.8 h ) in the bupivacaine group and 271 min ( 4.5 h ) in ropivacaine group . bariskaner et al . , described that ropivacaine was less cardio depressant and arrhythmogenic than bupivacaine . bupivacaine has more negative inotropic effect and increases av conduction time more than ropivacaine . in our series , significantly more patients had hypotension and bradycardia in bupivacaine group compared to ropivacaine group . they reported that out of 43 patients , hypotension and bradycardia requiring treatment were experienced by seven and three patients , respectively , in the bupivacaine group , and by two and one patient , respectively , in the ropivacaine group . we found that overall , both the study drugs , bupivacaine and ropivacaine produced satisfactory analgesia , without any technical failure and with minimal side effects , but ropivacaine group was associated with lesser adverse effects . paravertebral block has been established to be a superior analgesic technique offering superior pain relief compared to ga alone . it has a better safety profile compared to iv and thoracic epidural analgesia and better preserves post - operative pulmonary function . ultrasound has been used to enhance efficacy and safety of the block by determining the location and depth of transverse process and parietal pleura . in our study , also reported block success rate of 100% using in - plane ultrasound - guided tpvb . likelihood of vascular puncture and pneumothorax had been reported to be higher in bilateral compared to unilateral block by nerve stimulator technique . safety profile of the chosen analgesic technique is of paramount importance in cardiac surgery as heparin may be administered in elective or emergent setting , exposing the patient to the risk of dreadful complications such as epidural haematoma . ultrasound - guided block minimises the risk of such complications , and is thus , a safe and effective modality . nevertheless , the present study had limitations . first , we chose only asa physical status i and ii patients , so efficacy and side effects of the two drugs in high - risk patients could not be assessed . also , in our study surgical procedure performed for pda cases was posterolateral thoracotomy , so the efficacy of paravertebral block in anterolateral thoracotomy and sternotomy could not be assessed . single pre - incisional ultrasound - guided paravertebral injection of 0.4 ml / kg of either 0.2% ropivacaine or 0.25% bupivacaine provided equipotent analgesia . ultrasound - guided paravertebral block appears to be a safe and effective mode of analgesia in paediatric patients undergoing pda ligation surgery .

in turkey , gastric cancer is the second most common cause of cancer - related mortality in men , and the fourth most common cause in women.1 it is very important to assess the presence or absence of metastases to establish appropriate treatment plans for gastric cancer . gastric cancer generally metastasizes to the peritoneal surfaces , liver , and distant lymph nodes ; metastasis to the spleen , adrenal glands , ovaries , lung , brain , or even the skin has also been noted frequently . there is limited data on the frequency and clinical significance of bone marrow metastasis in gastric cancer . bone marrow metastases are frequently observed with solid tumors and these may negatively affect patient survival . malignancies of the prostate , breast , lung , kidney , thyroid , and stomach are the most common neoplasms that may metastasize and involve the bone marrow.2,3,4,5,6,7,8,9,10 there are limited published data from the far east about gastric cancer with bone marrow involvement . in the last 4 years , 420 patients with gastric cancer were admitted at our clinics , of which 245 had metastatic cancer . of these , only 5 patients had bone marrow metastasis . we evaluated 5 ( 2% ) patients with bone marrow metastasis out of 245 patients with advanced gastric cancer . the median age was 45 years ( range , 22~55 ) , which was lower than that of the overall population of gastric cancer patients . all patients were male ; 3 patients had signet - ring cell type , 1 had undifferentiated type , and 1 had tubular type tumor , according to the world health organization classification . four patients had primary tumors located in the corpus and 1 patient had neoplasm of the cardia . the most common sites of synchronous metastasis in gastric cancer patients with bone marrow involvement were the bone ( 4/5 patients ) , followed by the liver ( 3/5 patients ) . all patients with bone marrow metastasis were diagnosed on the basis of bone marrow biopsy reports , and all patients had cytopenia at the time of admission . one patient had peritoneal metastasis at the time of diagnosis , and bone marrow metastasis developed 5 months later . only 1 patient had recurrence with bone and bone marrow metastasis after curative surgery and adjuvant chemoradiotherapy at the 32nd month of diagnosis . the median duration of survival after the diagnosis of bone marrow involvement was 47 days . the range of survival duration after diagnosis of bone marrow metastasis was 20~53 days among the 4 patients who died ; 1 patient was alive at 61 days after diagnosis . unfortunately , 1 patient died after the first cycle , and the other patient died after the second cycle of treatment . all patients had elevated levels of alkaline phosphatase ( alp ) with a median value of 615 u / l ( normal range , 40~150 u / l ) , and lactate dehydrogenase ( ldh ) with a median of 628 u / l ( normal range , 125~220 u / l ) . however , no patient developed hypercalcemia , although 4 patients had bone metastases . bone marrow infiltration with malignant cells is frequently observed in patients with solid organ malignancies . carcinomatosis of the bone marrow can originate from the primary prostate , lung , or breast carcinoma,2,3,4,5,6,7,8,9,10,11 and rarely from gastric carcinoma.3 there is limited data regarding the incidence and clinical significance of bone marrow metastasis in gastric carcinoma . this case series is one of the largest in the literature compared to other case series reported from the far east . kim et al.12 reported the prevalence of bone morrow metastasis to be 0.024 , which was confirmed by bone marrow biopsy reports . kusumoto et al.13 reported 9 gastric cancer patients with bone marrow metastases , and concluded that all patients had more aggressive histopathology ( signet ring cell or poor differentiated ) and were younger than patients without bone marrow metastasis . they also reported an elevation of alp and/or ldh levels in gastric cancer patients with bone marrow involvement , and 6 of 9 patients had disseminated intravascular coagulopathy . in gastric cancer cases , bone metastasis is usually rare compared to liver and peritoneal metastasis . in the 5 cases of gastric cancer with bone marrow involvement reported here , 4 patients had bone metastasis , which was consistent with reports from published literature . in the study by kwon et al.14 that included gastric cancer patients with bone marrow involvement , the incidences of metastases to the bone , lung , and liver were 57% ( 15/26 ) , 11% ( 3/269 ) , and 3.8% ( 1/26 ) , respectively . of the 2,150 patients with advanced cancer in the study by ahn et al.,15 there were 19 patients ( 0.9% ) with bone metastases ; however , another study revealed a higher incidence ( 13.4% , 33/246 patients ) among autopsy cases.16 in both series , bone metastasis was associated with poorly differentiated histology . all these findings suggest that there may be a relationship between bone and bone marrow metastasis , and possibly between poor differentiation and unusual metastatic sites , including the bone and bone marrow . kong et al.17 reported that there were 193 ( 0.96% ) patients with pulmonary metastases among 20,197 patients with advanced gastric cancer ; further , 34 ( 17% ) of the patients with lung metastasis also had bone metastasis and 11 ( 5.7% ) patients had bone marrow metastasis as well as lung metastasis . chemotherapy is known to prolong survival as compared to best supportive care ( bsc ) , for the management of advanced gastric carcinoma.18 however , these reports in published literature were limited to patients with adequate bone marrow function ; in other words , patients with bone marrow involvement were excluded . the incidence of bone marrow metastases in gastric cancer patients is low ; therefore , the clinical features and optimal treatment options for such patients has not yet been established . in the present study , 4 patients died approximately within 2 months after the diagnosis of bone marrow metastasis . in the study by kusumoto et al.,13 the median survival was only 3 months in the chemotherapy group and 2 months in the bsc group , with borderline significance in gastric cancer patients with bone marrow metastasis . kwon et al.14 published the largest case series in 2011 . in that report , 15 patients received palliative chemotherapy and 10 patients received bsc . the median survival was 11 days in the bsc group compared to 121 days in the palliative chemotherapy group ( p<0.001 ) . because of the lack of prospective studies of gastric cancer patients with bone marrow metastases , an optimal chemotherapy regimen is still unknown . bone marrow metastases in gastric cancer patients are characterized by aggressive histology and a younger age of onset . they are diagnosed on the basis of bone marrow biopsy findings and treated like cases of metastasis to other sites .

patients are provided with functional recovery and esthetical satisfaction at a lower cost.1 however , severe alveolar bone loss can be observed in patients who have worn complete dentures for a long time and inferior alveolar nerve may be near the alveolar ridge due to that process . this may lead to denture instability and soreness at sharpened alveolar ridge , especially in the mandible . to solve this problem , two to four implants can be placed into the mandibular anterior region to support overdenture . ball attachments , magnetic attachments , bar attachment systems , and telescopic crowns have been used to anchor the overdenture . among these systems , bar attachment system has the greatest retention.2,3 on the other hand , increased chair time and high cost of fabrication are problems in that system . in addition , for appropriate adaptation of the bar , soldering or laser welding procedure is often necessary to compensate the dimensional change due to the errors arising from some procedures such as impression making and laboratory process . the recently developed sfi - bar ( cendres + mtaux , biel / bienne , switzerland ) can overcome these weak points . according to manufacturer , sfi - bar aims to reduce chair time to practitioner and mechanical stress to implant . the sfi - bar system consists of two or four implant adapters which are mounted on the implants . two or four large ball joints are secured on the adapters by fixation screws and tube bar then connects the two ball joints . the length of tube bar can be adjustable according to the inter - implant distance , between 10 and 26 mm . the inter - implant distance is measured with a tube bar gauge , and subsequently the tube bar is shortened to adjust the bar to the ball joints . in addition , the adaptor is allowed to have 30 degree of implant angulation in order to compensate a range of the angular difference between the implants . this system is a " chairside " system , as all preparations can be inserted into the patient 's mouth after the implants have been put into place . it is also possible to make this system on the master model at the dental laboratory during fabrication of new denture . customization of the bar components may be done directly at the dental chair with existing denture or indirectly at the dental laboratory with new denture . this clinical report describes two - implant - supported overdenture using the sfi - bar system in a mandibular edentulous patient . a 67-year - old woman was presented at the department of prosthodontics , ajou university hospital , complaining of mobility in mandibular anterior teeth . she had been wearing removable partial denture ( rpd ) on the mandible and complete denture ( cd ) on the maxilla . the results of radiographs and clinical evaluation showed that the mobility and periapical pathosis of the mandibular anterior teeth were so severe that extraction was decided . she had adverse drug reaction from non - steroidal anti - inflammatory drug ( nsaid ) and was referred to the department of allergy . extractions were performed in the department of oral surgery after drug allergy tests to find drugs caused no allergy . after extraction , alginate impressions ( aroma fine df ii , gc , tokyo , japan ) were made for provisionalization . , occlusal plane of the wax rim was set with reference to the interpupillary line and camper plane . after vertical dimension ( vd ) was determined by using physiological rest position and willis method,4 facial appearance were assessed . centric relation ( cr ) bite was taken by bilateral manipulation guidance with vinyl polysiloxane bite registration paste ( blumousse , parkell , ny , usa ) , then transferred to semi - adjustable articulator ( hanau , waterpik , ny , usa ) with face - bow transfer . wax denture try - in for the maxilla and mandible were done and the patient was satisfied with her appearance . provisional cds were delivered and relined with coe - soft ( gc america , alsip , il , usa ) . six months after extraction , radiographic stent was made using existing provisional denture and was switched to surgical stent after taking of computerized tomography ( ct ) scan . following the analysis of ct , 13 mm - length implants were determined in the area of mandibular left canine and mandibular right canine . 4.013 mm us ii implants ( osstem , busan , korea ) were installed at both mandibular canine regions5 and cover screw was connected to each implant . three months later , secondary surgery was performed and 4 mm - height healing abutments were connected ( osstem , busan , korea ) . after 1 month of soft tissue healing , alginate impressions ( aroma fine df ii , gc , tokyo , japan ) were made for an individual tray . functional impressions were taken for cd in the maxilla and implant - supported overdenture in the mandible at the fixture level by pick - up impression technique using regular type vinyl polysiloxane impression materials ( express , 3 m espe , mn , usa ) . on master cast , sfi - bar adapters ( cendres + mtaux , biel / bienne , switzerland ) ( fig . 2a ) were connected to each fixture analog and the inter - implant distance was measured . the height of the adapters were 3 mm and 1 mm , respectively parallel with occlusal plane . after connecting ball joint to one side of tube bar ( cendres + mtaux , biel / bienne , switzerland ) ( fig . 2b ) , tube bar gauge ( cendres + mtaux , biel / bienne , switzerland ) was connected to the other side of the tube bar . then the ball joint was connected with one adapter and a curved surface of tube bar gauge was seated on the other adapter ( fig . tube bar was then cut off at the gap of tube bar gauge with premium disc ( cendres + mtaux , biel / bienne , switzerland ) ( fig . the sfi - bar female ( cendres + mtaux , biel / bienne , switzerland ) made up of gold alloy was cut and prepared according to the length of the bar . during laboratory procedure , metal portion above the bar was opened so that the gold female part can be attached to overdenture in clinical office . wax rim and wax denture try - in for the maxilla and mandible were performed in the same manner with provisional denture . vd was determined as the value of existing provisional denture . after curing of dentures in common way , adapters were then connected to the implant fixtures in the mouth with 35 ncm ( fig . the ball joints were secured to the adapters by fixation screws with 20 ncm , and prepared tube bar was also connected to the ball joints . marginal fits of the sfi - bar were evaluated with periapical radiograph . after checking occlusion , retention , and facial appearance ( fig . 5 ) , the final dentures were delivered without connecting the female part and daily maintenance care was instructed using interdental cleansing aids . at the next recall appointment , 6 ) and the gold female part was attached to the denture with self - curing processing material ( vertex - dentimex , zeist , netherlands ) in the mouth under habitual bite force ( fig . 7 ) . management of tooth brushing was performed through periodic recall check and peri - implant bone was assessed with radiographs ( fig . compared with complete denture , implant - supported overdenture improves the stability of the prosthesis . increased number of implants may guarantee more retention . however , there is a problem of increased cost and anatomical limitations in severely resorbed residual ridges , especially in the mandible . several studies demonstrated that the two - implants - supported overdenture therapy can be considered as a very reliable treatment for patients with an edentulous mandible.5 - 7 therefore , two - implants - supported overdenture in the mandible should be the standard treatment modality for full edentulous patients suffering from discomfort with their conventional denture.5 - 7 immediate or early loading protocols for the edentulous mandible offer the patient with many advantages in terms of decreased number of visits , early functional recovery , and reduction of surgical exposure.8 short - term outcomes of immediate or early loading protocols for mandibular implant - supported overdenture showed success , comparable to delayed loading protocols in curtained cases . however , this treatment option is not universally appropriate for all patients . although no evidence was found in long - term studies to support delayed loading protocols for mandibular implant - supported overdenture , delayed loading of prosthesis is expected to show better results than immediate or early loading.9 further long - term studies concerning loading protocol of mandibular implant - supported overdenture are required . implant can be placed either by 1-stage procedure or 2-stage procedure to set load - free for a few months . if the optimal implant stability is not achieved or if there is a risk that the provisional denture transmits excessive forces to the implants , the 2-stage procedure could be preferable.10 we conducted 2-stage submerged implants in this case with delayed loading . there is no difference in implant survival rates , peri - implant outcome and patient satisfaction regardless of splinting.11 however , several articles showed that the non - splinted design requires more prosthetic maintenance and the bar attachment system has been shown to be a more successful prosthesis.11 while bar system has the best maintainability,2 laboratory procedures such as soldering and welding give rise to errors and increase in cost . besides , several bar framework material such as gold alloy , silver - palladium alloy , commercially pure titanium , and cobalt - chromium alloy presented a lower effect on the stress distribution of an overdenture.12 sfi - bar was recently developed and made up for the weakness of casting bar attachment system . according to manufacturer , due to the telescopic design of the bar joints , no lateral stress is applied to the implants . however , further long - term studies concerning lateral stress on implants with this system are required . compared to other bar forms such as ovoid - shaped or parallel - wall shaped bar , round bar allows more rotational movements of the overdenture . described that there may be little denture - fracture probably due to the possible movement over the round bar.13 metal framework reinforcement is not required for a sfi - bar on two - implants . as some parts of the system are not fixed to bar by soldering or welding , there is room for stress release that avoids implant preload . moreover , the absence of soldering joints , which sometimes reduces the length of the retentively usable bar surfaces , also can be advantageous . since soldering is not required and titanium grade 5 is used as bar material , there is no risk of corrosion . one is the milled gold female part with asymmetrical design of the retention in the resin for more room in the tongue area and a better esthetic . the other is the titanium female part with replaceable plastic retention inserts for adjusting the retention force . the titanium matrix with plastic inserts as a female part for sfi - bar on two implants is not recommended due to excessive wear at the titanium male parts . in this case , the patient is satisfied with good function and favorable esthetics by using implant - supported overdenture . however , periodic recall check is necessary and long - term clinical results are required . fabrication of overdenture with sfi - bar is an advantageous treatment option because clinical results showed save of cost and time , which benefits both the patient and the clinician .

the coronary angiogram ( cag ) remains the gold - standard method for assessing coronary artery disease ( cad ) . however , the cag has inherent pitfalls , such as only showing the vessel lumen as an x - ray shadow image , created by the injection of contrast medium , and of often visualizing a " side - view . " thus , the apparent degree of coronary stenosis can be affected by the projection angle due to lesion eccentricity . additionally , diffuse coronary disease , lesion foreshortening , angulations , calcification , and vessel overlap can be challenges in the angiographic assessment of lesion severity . in some cases , an angiographically normal - looking coronary artery actually shows various degrees of atherosclerotic plaque by intravascular ultrasound ( ivus ) . ivus is an invasive imaging technique used to visualize coronary cross - sectional anatomy and is superior to cag in assessing vessel size , calcium content , and lesion severity . it also provides complementary procedural information in lesions requiring percutaneous coronary intervention ( pci ) when determining adequate stent sizing , and confirming optimal stent deployment and apposition without edge tearing in real time . thus , a growing number of interventional cardiologists attain optimal procedural results with reduced complications when using ivus in pci . although the routine use of ivus in daily pci remains controversial , stent optimization by ivus during stenting procedures , especially in the era of drug - eluting stents ( dess ) , may have an important role in improving long - term clinical outcomes such as stent restenosis and stent thrombosis . in this review , we focus on the potential roles of ivus in stent optimization during pci and its clinical benefits . pci has been the fastest growing method for the treatment of ischemic cad over the past three decades . coronary stents have emerged as the predominant form of pci and are currently used in more than 90% of pci procedures . procedural success of pci is usually determined by visual estimation by the operator , and usually , angiographic success after pci is defined as the attainment of residual diameter stenosis of less than 30% , which is generally associated with at least a 20% improvement in diameter stenosis and relief of ischemia . however , such subjective estimation of the severity of coronary artery stenosis is thought to be of limited reliability . previous ivus studies have demonstrated that visual estimation or quantitative angiographic analyses of vessel dimension for stent deployment appear inaccurate [ 6 - 9 ] . the post - dilatation clinical comparative ( postit ) trial was designed to assess the achievement of optimal stent deployment by ivus , according to normal - to - high pressure balloon dilation after bare metal stent implantation . among 256 patients , only 14% of cases achieved optimal stent deployment with under 12-atmosphere pressure dilation and only 36% even with higher deployment pressures ( > 14 atmospheres ) . another ivus study with an early - generation des in 200 patients assessed stent expansion depending on the manufacturer 's compliance chart as a guideline . in that study , the des obtained only 75% of predicted minimal stent diameter and 66% of the predicted minimal stent area ( msa ) ( fig . angiographic success can not always be linked with optimal stent expansion , despite higher pressure balloon inflation during the stenting procedure . in turn , stent optimization using a high - pressure balloon without ivus guidance also has been associated with an increased risk of arterial perforation , probably secondary to vessel - balloon mismatch . thus , the operator should consider using ivus guidance for high - pressure balloon inflation during stent deployment . apposition of stent struts to the vessel wall is also an important facet of stent optimization . adequate stent expansion and adequate stent strut apposition have been reported to be important factors in reducing repeated revascularization due to stent restenosis or stent thrombosis . in the des era , incomplete stent apposition has been regarded as an important local factor in des failure , probably due to reduced drug delivery to the vessel wall [ 14 - 17 ] . in a recent report , incomplete stent apposition was significantly associated with vessel / stent mismatch rather than stent underexpansion immediately after stent implantation . thus , adequate stent sizing by ivus may be clinically important in preventing incomplete stent apposition and in optimizing initial stent deployment . ivus has been used to detect suboptimal results after apparently angiographically successful stent deployment in both the des and bare metal stent ( bms ) eras ( fig . ivus predictors that are associated with increased adverse outcomes include smaller msa , stent underexpansion , stent edge dissection , incomplete stent apposition , and incomplete lesion coverage [ 19 - 26 ] . in the bms era , a major problem after stent implantation was stent restenosis , and the main mechanism of this phenomenon was a smaller msa or stent underexpansion [ 21,26 - 30 ] . several studies in the bms era showed a beneficial effect of ivus guidance on post - procedural angiographic results and stent restenosis during long - term follow - up , resulting from a larger msa with a higher post - dilation balloon pressure . stent underexpansion , identified by ivus , can be treated with appropriate post - balloon dilation . ivus allows more aggressive intervention using a larger diameter balloon with confidence in terms of safety ; thus , bms implantation under ivus guidance can provide a bigger msa and more favorable clinical outcomes compared with angiography - guided pci . dess have led to a marked reduction in the rate of stent restenosis and the need for repeated revascularization compared with bmss . because of their efficacy , high - risk lesions and clinical conditions , including bifurcation lesions , long lesions , calcified lesions , left main disease , diabetes , and multivessel disease , are now being treated routinely with dess . thus , the risk of stent underexpansion , incomplete stent apposition , and incomplete lesion coverage increases and these suboptimal stent deployment conditions have been reported to be potent ivus predictors of stent restenosis and stent thrombosis , suggesting that stent implantation under ivus guidance still has a pivotal role even in the des era . an important aspect of ivus is determining appropriate reference segments that provide the landing zone for stent deployment . ivus examination typically reveals a considerable amount of plaque , even in segments of the vessel that appear " normal " on the angiogram , known as " reference vessel " disease . quantitative ivus studies have demonstrated that the segment chosen as the " normal " reference site for the calculation of angiographic percent stenosis has an average of 30 - 50% of its cross - sectional area occupied by plaque . by ivus , the definition of reference segment is a cross - sectional image adjacent to the lesion that has < 40% plaque burden . a previous study reported the association between clinical outcomes and longitudinal positioning of the stent in 162 consecutive patients with 180 lesions treated with sirolimus - eluting stent ( ses ) implantation . in that study , stepwise ivus criteria primarily targeting plaque burden < 50% were shown to be feasible and improved the rates of stent restenosis and target lesion revascularization ( tlr ) at 8 months follow - up . ivus is used frequently in pci , but the use of ivus can not be directly related to stent optimization . definitive guidelines for ivus - guided stent optimization are not available and it is still performed at the operator 's discretion . so , how do we perform stent optimization using ivus , and what ivus criteria are acceptable for current practice ? although many ivus criteria for stent optimization have been suggested , the basic concepts underlying them can be summarized briefly as minimizing the occurrence of ivus - related predictors of adverse events after pci , including stent underexpansion , incomplete stent apposition , edge dissection , and lesion undercoverage . stent underexpansion is defined as an area of inadequate stent expansion compared with the adjacent reference segments . however , a consensus definition of " adequate " expansion is still lacking . in the bms era , several randomized trials used various ivus criteria for stent deployment optimization , yielding mixed results . the first large multicenter study , called music ( multicenter ultrasound stenting in coronaries study ) , sought to define specific criteria for optimal stent deployment and demonstrate the feasibility and safety of ivus - guided stent optimization . in the music criteria , " adequate " expansion was defined as > 90% of the average reference cross - sectional area ( csa ) , or > 100% of a smaller reference csa with complete apposition and symmetric expansion . another large multicenter trial was avid ( angiographic versus ivus direct stent placement ) ; its defined ivus optimization required complete stent apposition with stent csa > 90% of the distal reference lumen area . the results of the avid trial showed a significant benefit of ivus guidance in vessels 2.5 - 3.5 mm in size and in saphenous vein graft pci . the tulip ( thrombotic activity evaluation and effects of ultrasound guidance in long intracoronary stent placement ) study also showed significant angiographic and clinical benefits of ivus guidance using criteria such as stent csa > distal reference lumen . in contrast to the results mentioned above , the opticus trial ( optimization with ivus to reduce stent restenosis ) using the music criteria for ivus guidance did not show a significant difference in 6- or 12-month clinical outcomes . however , in this study , only 56% of stents met all three music criteria . many other trials in the bms era used similar ivus criteria , and two meta - analyses showed better outcomes of ivus - guided pci than angiography - guided pci , especially in terms of in - stent restenosis and target vessel revascularization ( tvr ) , but not in mortality or myocardial infarction . in contrast to the bms era , for ivus - guided pci with des , few randomized studies showing clinical efficacy , preventing tvr or restenosis , or stent optimization under ivus guidance have been carried out . in the home des trial , optimal stent deployment was defined as complete apposition of the stent struts , no edge dissection , and adequate stent expansion , defined as either msa > 5.0 mm or > 90% of the distal reference lumen area . in that study , no significant benefit in terms of tvr or clinical events was reported . a similar finding was also noted in the avio ( angiography versus ivus optimization ) study in which optimal stent expansion was > 70% of the csa of the chosen balloon . increasing evidence indicates that ivus - guided pci may reduce the risk of stent thrombosis ( st ) . stent underexpansion was demonstrated to be one of the major causes of this disastrous complication , although the cause of st is multifactorial . in a substudy of the sirius trial , adequate patency was defined as a follow - up ivus msa > 4.0 mm . when the adequate post - interventional msa of sess was defined as > 5.0 mm , the positive predictive value of patency was 90% , but the optimal cutoff value of bms was defined as 6.5 mm . another study concerning ses failure also supported that msa < 5.5 mm and < 5.0 mm were the most important predictors of ses failure ( fig . even if ses had a considerably lower optimal msa threshold than bms , these studies showed that underexpansion remained the main cause of stent failure in dess ; at least a msa < 5.0 mm should be avoided in non - left main ( lm ) lesions . in lm lesions , optimal msa was reported in the main - compare ( revascularization for unprotected left main coronary artery stenosis : comparison of percutaneous coronary angioplasty versus surgical revascularization ) study to be > 8.7 mm to prevent tlr . edge dissection , which is complicated by lumen narrowing < 4 mm or dissection angle 60 , has been associated with an increased incidence of early st ; thus , additional stents may be needed to prevent st . however , a minor dissection , detected by ivus , may not be associated with an increased incidence of st . although no consensus exists on an optimal strategy , in minor dissection , careful observation without stenting can be helpful . overall , the results discussed above encourage ensuring good apposition of stent struts to the vessel wall , such that the stent struts are not surrounded by lumen , adequate stent expansion to obtain msa at least > 6.5 mm for bmss and > 5.0 mm for dess or msa > 90% of the distal reference lumen csa , and lack of major dissections , intramural hematomas , and geographic misses . numerous studies have evaluated the clinical benefits of ivus - guided pci compared with angiography - guided pci in the bms era [ 41 - 44 ] . the opticus trial showed no significant difference between ivus- and angiography - guided pci groups in terms of 6- and 12-month rates of death , myocardial infarction ( mi ) , and tlr in 550 patients meeting the music criteria . in contrast , the tulip study demonstrated favorable angiographic and clinical outcomes in patients with long coronary lesions ( > 20 mm ) treated with a bms ( > 3 mm ) under ivus guidance . in a meta - analysis of 2,193 patients from seven randomized trials , the rates of 6-month angiographic restenosis and target vessel revascularization were significantly lower in the ivus - guided pci group than the angiography - guided group ( 22% vs. 29% , p = 0.02 and 13% vs. 18% , p < 0.001 , respectively ) , with no difference in the rates of death ( 2.4% vs. 1.6% , p = 0.18 ) or mi ( 3.6% vs. 4.4% , p = 0.51 ) , consistent with a previous meta - analysis . to date , few studies have investigated the clinical benefits of des optimization under ivus guidance compared with that of bmss . the home des ivus ( long - term health outcome and mortality evaluation after invasive coronary treatment using drug - eluting stents with or without ivus guidance ) study was a randomized trial to investigate clinical outcomes of ivus - guided pci with dess . although the ivus - guided strategy resulted in the frequent use of adjunctive balloons and a larger size balloon with higher pressure , no significant difference was observed in major adverse cardiac events or stent thrombosis in the study . a similar retrospective study of ivus - guided stent optimization also showed no significant difference in the incidence of in - stent restenosis or neointimal volume between ivus- versus angiography - guided pci . conversely , a study with a propensity - matched analysis in 884 patients treated with dess showed a significant reduction in the stent thrombosis rate at both 30 days ( 0.5% vs. 1.4% , p = 0.046 ) and 12 months ( 0.7% vs. 2.0% , p = 0.014 ) in the ivus - guided pci group . recently , a large " real world " registry from two korean centers reported long - term outcomes of both ivus- and angiography - guided pci using bms or des implantation . in total , 8,371 patients who underwent coronary stenting under ivus guidance ( 4,627 patients ) or angiography guidance ( 3,744 patients ) were consecutively enrolled , and 3-year adverse clinical outcomes were compared between the groups using a cox regression model and propensity score matching . in the overall population , the 3-year adjusted incidence of mortality was significantly lower in the ivus - guided pci group compared with the angiography - guided pci group ( hazard ratio [ hr ] , 0.70 ; 95% confidence interval [ ci ] , 0.56 to 0.87 ; p = 0.001 ) ( fig . matched pairs of the overall population , the ivus - guided pci group also had a lower mortality risk ( hr , 0.71 ; 95% ci , 0.56 to 0.90 ; p = 0.005 ) . however , ivus - guided pci did not influence the rates of myocardial infarction , target - vessel revascularization , or stent thrombosis in the overall or in the 2,715 matched - pair populations . in the des subpopulation , ivus guidance significantly reduced the 3-year adjusted mortality rate ( hr , 0.55 ; 95% ci , 0.36 to 0.78 ; p = 0.001 ) , which was not the case in the bms subpopulation ( hr , 0.79 ; 95% ci , 0.59 to 1.05 ; p = 0.10 ) . a propensity score matching analysis of 201 matched patients from the main - compare study also demonstrated the importance of ivus - guided pci in unprotected left main disease . in this analysis , significantly lower incidence of 3-year mortality was noted in the ivus - guided pci group compared with the angiography - guided pci group ( 4.7% vs. 13.6% , p = 0.048 ) , but no significant difference was detected in the rates of tvr or mi . notably , this benefit was found only for des , and the benefit in mortality appeared to be primarily associated with reduced sudden cardiac death related to late stent thrombosis . similar findings were also observed in patients undergoing pci of non - left main bifurcations with dess . taken together , the benefit of ivus guidance contributed primarily to decreased rates of stent restenosis and repeated revascularization in the bms era , whereas reduction of the stent thrombosis rate with possible improvement in mortality have predominated in the des era . ivus can provide direct cross - sectional images as well as longitudinal images of the coronary vessel wall . it has also contributed to our understanding of mechanisms in coronary atherosclerotic plaques and provided real - time information at stented segments after coronary interventions . possible criteria for optimal stent deployment by ivus are complete stent apposition to the vessel wall , adequate stent expansion , and full lesion coverage without edge dissection ( fig . 5 ) . recent data suggest that ivus - guided pci may reduce long - term mortality when compared with angiography - guided pci , particularly after des implantation ; thus , the clinical importance of ivus - guided pci raised in the bms era persists in the des era . optimization of stent deployment by ivus during pci may be considered as a routine practice in daily pci , especially for complex lesion intervention .

the first case of tracheoesophageal fistula in a patient who sustained blunt thoracic trauma was reported by vinson . delayed treatment of esophageal injury is associated with increased morbidity such as mediastinitis . among all esophageal perforations , the reported incidence of traumatic esophageal rupture varies from 4% to 14% . esophageal rupture is usually reported by penetrating or iatrogenic injury caused during diagnostic or therapeutic interventions . in 1988 , beal et al . conducted a meta - analysis of esophageal perforation cases due to external blunt trauma and reported only 96 cases of which 5 cases were located distally . herein , we report a rare case of blunt lower esophageal injury presented with right hemopneumothorax after fall of heavy object over the patient chest . a 28-year old male patient presented at our trauma unit with history of fall of heavy object over the right side of chest and abdomen . clinical assessment showed that the patient was hemodynamically stable , fully conscious , and normothermic . chest wall was tender with mild skin contusion just to the right of sternum and mid chest . focused assessment with sonography in trauma ( fast ) was negative and the rest of the examination was normal . a 36f chest tube was inserted on the right side which drained 400cc dark stained fluid with food particles ( fig . computed tomography ( ct ) scan confirmed the presence of right hemopneumothorax , collapse of right lower lobe of lung , and normal mediastinum , diaphragm , and abdomen ( fig . 2 ) . a gastrografin esophagogram revealed large free esophageal perforation at right side with passage of contrast to the right pleura ( fig . immediate posterolateral thoracotomy was performed through the right 5th intercostal space which revealed a 4 cm longitudinal tear of lower esophagus 2 cm above the right hemidiaphragm with no active bleeding ( fig . a two layered - repair was performed , first continuous inverting pds 4 o followed by interrupted everting lembert sutures . the repair was checked for air leak under water ( with nasogastric tube bulled under vision to just above the repair position with air insufflation while lower esophagus was compressed ) . a follow - up gastrografin study on 7th postoperative day revealed free passage of contrast without leak ( fig . the patient was sent home in good general condition and was followed at the trauma outpatient clinic regularly . the present case report describes a unique location of the esophageal rupture after blunt trauma that happened on the right side of the chest . however , it has been speculated that instant dilatation of the esophagus could be possible due to pressurized air which pushes the cricopharyngeal sphincter . this high pressure might lead to esophageal rupture , if the esophagogastric sphincter remains closed . many factors contribute to the rarity of esophageal injury including the protective location of the esophagus deep in the posterior mediastinum , easy mobility ( lack of tethering ) , the rigid hard shell of human chest . blunt esophageal injuries are caused by an unusual increase in pressure of the esophageal lumen due to a strong blow to the epigastrium . distal esophageal rupture with spillage of upper gastrointestinal contents into the chest is most frequently encountered on the left side . though , esophageal rupture is rare , it causes severe morbidity and mortality up to 20% . treating esophageal injuries is further complicated by certain crucial considerations which include timing , extent of injury , containment and location . moreover , esophagus is anatomically divided into 3 parts , i.e. cervical ( upper ) , mediastinal ( middle ) and thoraco - abdominal ( lower ) and it does not have mesentery and serosal coating unlike most of the digestive tract , which makes it more complex . the level of the perforation of the thoracic esophagus determines the surgical approach ; a mid - esophageal perforation is approached through a right while a distal esophageal perforation is approached through a left thoracotomy . the gastrografin esophagogram was helpful in our case as it helped to identify the location of the injury and guided the surgical approach to the lower esophagus through the right side . prompt diagnosis and careful planning for surgical repair of esophageal injury should be the best approach to manage this potentially morbid injury . further , our case report addresses the need for organized approach to lower thoracic esophageal perforations with mini - thoracotomy on the side of associated pleural collection for the management of blunt esophageal rupture.the authors have no conflict of interest and no financial issues to disclose . the case report has been approved by the medical research center ( irb # 13296/13 ) , hamad medical corporation , doha , qatar .

despite numerous efforts , the search for identification of crucial ligands and receptors being involved in either promotion or inhibition of cell growth , progression and susceptibility to cytotoxic drugs of acute myeloid leukemia ( aml ) cells is still warranted . recently , tnf receptor family - related ligands were linked to pathogenesis of hematological malignancies , including aml 1 , 2 . two of these members , namely baff ( b cell activating factor of the tnf family ) and april ( a proliferation - inducing ligand ) , were shown recently to play crucial role in the development of autoreactive b cells and regulation of cell survival , growth and death 3 , 4 . interestingly , april and baff share two members of tnf superfamily receptors , namely b - cell maturation antigen ( bcma ) and transmembrane activator and calcium - modulating cyclophilin ligand interactor ( taci ) , while b - cell activating factor receptor ( baff - r ) appears to be the primary receptor for baff only 1 . to date , the anti - apoptotic activity of april and baff was demonstrated in b - lymphoma , multiple myeloma ( mm ) and chronic lymphocytic leukemia ( b - cll ) 5 - 7 . however , there were only limited studies on the role of april and baff in regulating apoptotic processes in aml . therefore , in the present study we aimed to determine whether baseline quantification of peripheral blood cd33 + blast cells expressing baff , april and their receptors in newly diagnosed aml patients could improve prognosis of treatment response . additionally , we aimed to analyze whether april and/or baff signaling could be involved in regulating leukemic cell sensitivity to chemotherapeutic drugs . peripheral blood from 24 patients with newly diagnosed aml ( diagnoses were established following the who classification system ) was provided by the department of hematology , medical university of bialystok . the samples were collected upon obtaining a written informed consent pursuant to the rules and tenets of the recently revised helsinki protocol . patient 's median age at the time of sample collection was 55 years and the range was 21 - 65 . blood counts and flow cytometry were performed in order to confirm the presence of blastic cells , whereas cytogenetic and molecular studies , including fish ( aml1/eto , cbf/ myh11 , mllt3-mll and frequently mutated genes flt3-itd , npm1 , cebpa ) were conducted with a view to determining the risk group , following the who recommendation . on this basis , it was established that all the patients involved in the study had a normal karyotype ( 46xx/46xy ) and none of them suffered from mutated core binding factor leukaemia ( cebpamut ) , mutated nucleophosmin ( npm1mut ) or internal tandem duplication of fms - like tyrosine kinase 3 ( flt3-itd ) . aml patients were treated with seven - day induction chemotherapy regimens corresponding to the standard therapy based on the polish adult leukaemia group : ( dac schedule ) 8 . after induction , the morphology response was evaluated ; twelve patients achieved complete remission ( cr ) after 1 induction and 12 were non - responders ( nr ) . the detailed characteristic of aml patients and treatment is listed in table 1 . peripheral blood mononuclear cells ( pbmcs ) , from patients before induction therapy , were isolated by using density gradient centrifugation . the cells were counted , cryopreserved and stored in liquid n2 for further analysis . in this study we used immunofluorescence staining for multicolor flow cytometry and confocal microscopy , combined bisulfite - restriction analysis ( cobra ) , quantitative pcr ( qpcr ) and in vitro stimulation . aml cells isolated from peripheral blood were used for staining and determination of april , baff and their receptor expression . first , we found significant differences ( fig.1a ) in frequencies of cd33 + blasts expressing april , baff , but not their mean fluorescence intensity ( mfi ) , among groups of patients with varying clinical responses to applied chemotherapy . non - responders ( nr ) showed elevated baseline frequencies of april- and baff - expressing cd33 + blasts as compared to patients who achieved complete remission ( cr ) ( 15.76%2.48 vs 34.16%6.53 , p=0.0343 and 40.87%6.3 vs 77.41%5.34 , p=0.0021 , respectively ) . surprisingly , however , there was no difference in plasma levels of april and baff between cr and nr patients ( data not shown ) . with regard to april and baff receptors , interestingly , we demonstrated here that the aml patients who did not respond to induction chemotherapy presented barely detectable frequencies ( below 1% ) of cd33+bcma+ cells . in contrast , in cr patients we found substantially elevated but still small frequencies of those cells ( 15.57%8.64 ) . on the other hand , we found no differences in the frequencies of aml blasts expressing taci ( p=0.4555 ) and baff - r ( p=0.3196 ) . moreover , we found that patients who further developed cr to induction chemotherapy presented with significantly elevated mfi of baff - r and taci , but not bcma on aml cells when compared nr ( 107.428.57 vs 21,847.78 , p=0.004 ; 132.532.9 vs 30.7613.67 , p=0.0062 ; 17.668.45 vs 3.290.50 p=0.1213 , respectively ) ( fig 1b ) . we next set out to confirm these findings by qpcr analysis of april , baff , bcma , baff - r and taci expression levels ( fig.1c ) . surprisingly , we found no differences in baff , baff - r and taci expression level in cd33 + aml cells ( p>0.05 ) . in some contrast , we observed a tendency ( at a threshold of statistical significance ) towards higher expression of april in nr when compared to cr patients ( 3.2841.77 vs 0.55470.17 , p=0.0745 ) . importantly , cr patients showed higher bcma expression when compared to nr ( 8.165.20 vs 0.120.03 , p=0.0127 respectively ) . next we confirmed membrane bcma expression on cd33 + aml blasts by immunofluorescence staining ( fig.1d ) . again , we demonstrated here that at baseline bcma expression on aml cells could have been detected only in cr , but not nr patients . interestingly , we observed significant negative correlation between the expression of baff , but not april , and bcma in aml cells ( r=-0.85 , p=0.00006 and r=0.02 , p=0.93 , respectively ) . next , we intended to analyze dna methylation status of promoter sequences of bcma gene . although we did not observe any differences in methylation levels with regard to the type of clinical response to the therapy ( data not shown ) , we found significant negative correlation between the level of methylation of bcma promoter and the frequencies of cd33+bcma+ cell ( p=0.0204 , r=0.5564 ) . finally , in order to directly evaluate the pro - survival effect of april and baff on cd33 + cells , we treated the blasts with cytosine arabinoside ( arac ) , a widely used dna - damaging drug , either in the presence or absence of recombinant human april and baff . as expected , arac significantly decreased frequencies of live cd33 + aml blasts ( fig.2a ) . notably , stimulation with april significantly reduced arac - related induction of cell death ( fig.2b ) in nr but not in cr patients . additionally , we observed a trend ( at a threshold of statistical significance ) to increase frequencies of viable cells after baff stimulation in presence of arac ( fig.2c ) . our results confirmed the previous observations suggesting a possible involvement of bcma in regulating apoptosis of aml cells and its usefulness as a potential marker that predict treatment response to induction chemotherapy . it should be noted that , bcma is now believed to enhance survival of b cells , especially at later stages of their differentiation 9 . on the other hand , early studies revealed that bcma knockout mice did not present with any specific phenotype 10 . subsequent experiments , however , proved that bcma deficiency led to impaired survival of long - lived bone marrow plasma cells 11 . moreover , a soluble form of bcma , which inhibited the proliferative activity of april in vitro , decreases tumor cell proliferation in nude mice . additionally , in xenograft models of lung and colon cancer , intratumoral delivery of soluble bcma was capable of significantly reducing tumor growth in mice , which had been treated with the soluble receptor 11 . it is likely , therefore , that bcma activation cause increased proliferation of aml cells what in turn may be associated with their increased sensitivity to chemotherapeutic drug , including commonly used arac . on the other hand , soluble bcma released from bcma cells may reduce the anti - apoptotic april and baff activity 12 . this hypothesis , however , did not explain why only a small subset of aml cells show bcma expression . to date , several studies indicated that aberrant dna methylation could also play important role in the progression of numerous human neoplasms , including aml 13 . interestingly , we found , significant negative correlation between the level of methylation of bcma promoter and the frequencies of cd33+bcma+ cell , suggesting possible contribution of methylation of bcma to the resistance of aml cells to the therapy . additionally , we did not observe any significant relationship between bcma methylation status and clinical features that may influence on analyzed gene methylation , namely : age , smoking ( data not shown ) . on the other hand , observed negative correlation between bcma and baff expression , suggests regulatory function of the latter . interestingly , we indicated that april shows protective effect on aml cells and reduces arac - related induction of cell death . similarly , bonci and colleagues showed that neutralization of april circuit resulted in marked induction of cell death in all aml samples , suggesting that april can act as a survival factor for aml cells . authors demonstrated also that the exogenous expression of april resulted in up - regulation of bcl-2 in cd34 + cells 2 . in contrast , april neutralization resulted in down - regulation of bcl-2 in primary aml cells 2 . indeed , the intensity of bcl-2 expression was found to correlate with cr rate , where a higher mfi was associated with a low cr rate after standard intensive chemotherapy 14 , indicating that april may protect aml from chemotherapeutic drugs through up - regulation of bcl-2 . in conclusion , we showed here clearly that april and baff are important players involved in regulation of apoptosis in cd33 + aml blasts . more importantly , the lack of bcma expression on aml cells may be useful in prediction of treatment response of myeloid leukemia . however , the exact function of bcma and regulation of its expression in aml blasts is still unknown and thus requires further investigation . l.b and a.e designed the experimental study , l.b , a.e and m.m wrote manuscript , l.b and j.k provided patients samples , a.e , k.g , g.s , p.m , u.r , and m.r performed experiments and analyzed data .

zeb1 ( also known in vertebrates as ef1 , zfhx1a , areb6 , tcf8 , and zfhep ) and zeb2 ( sip1 ) are the closely related human forms of a two - member family of e - box binding transcription factors that is conserved from worm to man ( for review , see . both proteins contain seven zinc fingers , four near the n - terminus and three near the c - terminus , and both can activate [ 24 ] and repress [ 5 , 6 ] target genes . zeb1 is encoded by the tcf8 ( also sometimes called zfhx1a or zeb1 ) gene . although tcf8 was cloned in 1991 and the gene that encodes zeb2 was cloned in 1999 , our understanding of the roles of the resultant proteins in normal and abnormal physiology is still emerging . both appear to promote cell migration during development and during cancer progression [ 914 ] , primarily by repressing the expression of the e - cadherin gene in epithelial cells [ 1 , 9 , 10 , 15 , 16 ] . however , zeb1 acts as a tumor suppressor in adult t - cell leukemia / lymphoma . interestingly , zeb1 is a key repressor of the p73 and p63 genes , members of the p53 gene family , and its binding to those genes promotes cell proliferation and prevents differentiation . zeb1 expression is limited in some contexts to proliferating cells by the rb - e2f1 complex . zeb1 also opposes fat accumulation in female mice and prevents uterine contraction during pregnancy , linking its actions to complex physiological events that are not related to cell migration or emt . despite zeb1 's importance in modulating development , cell proliferation , reproduction , and metabolism , the actual promoter has only been defined in chicken although promoter - reporter assays suggest that a comparable basal promoter region exists in humans 36 bp upstream of the translation start site . the gene is also modulated by a number of signal transduction pathways although the mechanisms remain undefined ( for review , see . while growth factors such as the tgf- family and igf-1 regulate tcf8 , steroid hormones are major inducers . notably , expression of zeb1 is induced by estrogen within one hour in chick oviduct . it is also regulated by estrogen in mouse pituitary [ 27 , 28 ] , in human myometrial cells , in mouse adipose tissue ( saykally , sandri , and sanders , manuscript in revision ) , and in human penile tissue and foreskin . this was shown to be at the transcriptional level , suggesting that tcf8 is a direct target for the estrogen receptor , although no estrogen response element(s ) has been identified as yet . zeb1 mrna is also induced by progesterone in the human t47d breast cancer cell line , in human myometrial cells , and in mouse uterus , indicating that expression of zeb1 is steroid - regulated in a variety of tissues . the goal of these studies was to assess whether androgen receptor ( ar ) directly regulates tcf8 . this question is particularly relevant as zeb1 expression is high in aggressive prostate cancer ( pca ) cell lines and tissues and as it promotes phenotypic changes consistent with epithelial to mesenchymal transitions ( emt ) in those lines [ 26 , 31 ] . this raises the possibility that zeb1 mediates at least some of the effects of ar in promoting pca progression . our preliminary results showing that zeb1 mrna levels increase in response to androgen treatment in the 22rv1 and pc3/ar prostate cancer cell lines have been published [ 32 , 33 ] . this study extends those observations to show that androgen affects the transcriptional activity of tcf8 through binding or tethering of ar to this gene . treatment of the pc-3/ar prostate cancer cell line , which overexpresses ar , with dihydrotestosterone ( dht ) induced endogenous zeb1 mrna and protein , confirming our observations that androgen increases zeb1 mrna and showing for the first time that this is reflected in zeb1 protein levels . inspection of the proximal ~1000 bp of 5-flanking dna revealed two potential androgen response elements ( ares ) . characterization of these sites revealed that both are required to confer androgen responsiveness to tcf8 and that both bind ar . this raises the possibility that zeb1 serves as a direct intermediary in at least some androgen signal transduction pathways . the lncap , c4 , c4 - 2 , and c4 - 2b cell lines were purchased from viromed laboratories ( minnetonka , mn ) and were maintained in t - medium ( invitrogen , carlsbad , calif , usa ) with 10% fbs . dihydrotestosterone ( dht , d-7149 from sigma chemical co. ) dissolved in 95% ethanol was added as indicated . transient transfection assays were performed in pc-3/ar cells using lipofectamine with the lipofectamine 2000 protocol ( invitrogen ) . transfections were done on the lncap lines using expressfect ( denville scientific , metuchen , nj , usa ) . -galactosidase assays were performed according to the manufacturer 's instructions using the galacto - star system ( applied biosystems , foster city , calif , usa ) , with chemiluminescence measured in a standard luminometer at 1.0 second / tube . total rna from cultured cell lines was isolated using trizol reagent ( invitrogen ) according to the manufacturer 's protocol . two g aliquots of mrna were reverse transcribed with mmlv reverse transcriptase ( invitrogen ) to create cdna templates for real - time reverse transcriptase pcr ( qpcr ) . for figure 1 , zeb1 and rpl32 mrna were measured simultaneously using taqman qpcr in 96-well plates on an icycler ( biorad , hercules , calif , usa ) using the probes and primers designated in table 1 . each primer was dual labeled with a fluorescent dye at its 5 end and a fluorescent quencher molecule at its 3 end . the emission spectrum for each dye is different , which allows the discrimination of both probes in the same reaction . for zeb1 , the fluorophore was 6-carboxy-4,7,2,7-tetrachlorofluorescein ( tet ) and the quencher was 6-carboxy - n , n , n , n-tetramethylrhodamine ( tamra ) . for rpl32 , the fluorophore was 6-carboxyfluorescein ( 6-fam ) and the quencher was tamra . both probes and primer sets were used in each reaction with the following optimized multiplex pcr protocol : 12.5 l iq supermix ( biorad ) , 2.5 l 10 mm dntps , 2 l 50 mm mgcl2 , 0.5 l itaq ( biorad ) , and 0.5 l cdna template . cycling parameters were as follows : 1 cycle at 95c for 3 minutes , followed by 60 cycles at 95c for 10 seconds and 55c for 1 minute . in figure 7(a ) , sybr green qpcr was done to quantitate the zeb1 and gapdh mrna using the primers in table 1 in reactions as follows : 12.5 l iq sybr green supermix ( biorad , cat # 1708885 ) , 2 l primer mix ( 10 m each ) , 2 l template ( 100 ng/l ) , and 8.5 l h2o . this reaction was prepared as a 3x cocktail and distributed in triplicate into a 96-well plate . qpcr was performed using the biorad icycler with an annealing temperature of 61c for both cdnas . protein extracts were harvested from pc-3/ar cells using the readyprep protein extraction kit ( biorad ) according to the manufacturer 's protocol . protein concentrations were determined by bradford assay ( biorad ) , and 100 mg of nuclear extract was loaded per well on a 420% readygel tris - hcl ( biorad ) . proteins were run at 60 mamp for 45 min and transferred to a polyvinyldifluoride ( pvdf ) membrane for blotting with a zeb1 antibody and a control -tubulin antibody ( santa cruz biotechnology , santa cruz , ca , e-20 and h-300 , resp . ) followed by horseradish peroxidase - conjugated secondary antibody ( sigma - aldrich , st . blots were developed with nbt / bcip solution ( pierce , rockford , ill , usa ) . a 974 bp region of dna spanning 982 to 9 relative to the translation start site of tcf8 was cloned by pcr from human genomic dna ( clontech , palo alto , ca ) using the forward primer 5-tggcctgtggataccttagc-3 ( 982 to 963 ) and reverse primer 5-cgcttgtgtctaaatgctcg-3 ( 9 to 28 ) into the pbluetopo -galactosidase reporter vector ( invitrogen ) , and the insert was sequenced . site - directed mutagenesis was carried out on pbluezeb974 using the quikchange mutagenesis kit ( stratagene ) , following the manufacturer 's suggested protocol with the oligomers listed in table 1 and their corresponding complimentary oligomers . to create the double - mutation , the downstream are was initially mutated , and this construct was then used as the template for another round of mutation using the upstream are primers . nuclear protein extracts were harvested from pc-3/ar cells treated with 5 nm dht using the readyprep protein extraction kit ( biorad ) according to the manufacturer 's protocol . gmsas were performed as described using 10 g of nuclear protein and 80,000 cpm p - end - labeled oligomer as indicated per lane . the sequences of the top strands are listed in table 1 with the are half - sites underlined . antibody supershift reactions were performed using a 0.1 mg / ml final concentration of -ar ( a2281x , u.s . gmsas were placed against film for 48 h at 80c prior to autoradiographic development . nuclear run on and reporter assays indicate that tcf8 is induced at the transcriptional level by both estrogen and progesterone , respectively . because progesterone receptor and ar recognize many of the same binding sites ( for recent update , see , it seemed plausible that tcf8 might be a target gene for ar , especially as androgen induces expression of zeb1 mrna [ 32 , 33 ] . to extend these observations , the response of endogenous tcf8 to dihydrotestosterone ( dht ) , an androgen that can not be aromatized to estrogen , was examined . human pc-3/ar cells were treated with doses of dht from 1 to 100 nm for 24 hours ( figure 1 ) . total rna was harvested and subjected to real - time taqman pcr ( qpcr ) for expression of zeb1 mrna using the 60s ribosomal protein l32 ( rpl32 ) housekeeping gene as a control . as expected , zeb1 mrna was induced about 4-fold by low doses of dht but did not respond to doses over 7 nm and was significantly repressed by 100 nm dht . nonetheless , these data confirm our data in 22rv1 prostate cancer cells that endogenous zeb1 mrna levels are increased by androgen . to ascertain whether the increase in zeb1 mrna was mirrored by an increase in protein , these blots revealed that zeb1 levels do indeed increase from essentially undetectable in response to dht treatment . thus , not only are zeb1 mrna levels increased by estrogen and progesterone , but they are also regulated by androgen and this is reflected by an increase in zeb1 . this suggests that zeb1 plays an important role in androgen signaling although no androgen - responsive zeb1 target genes have been identified as yet . to ascertain whether tcf8 contains any obvious ares , its sequence was inspected and two putative ares were identified from 944 to 930 and from 140 to 126 relative to the start site of translation . the start site of transcription has only been experimentally determined in chicken although it is predicted in human to be 36 nucleotides upstream of the translation start site . to investigate whether these potential ares are functional , a 974 bp genomic fragment ( 982 to 9 relative to the translation start site ) was subcloned into a -galactosidase reporter vector ( called pbluezeb974 ) , and this plasmid was tested in transfection assays with increasing amounts of dht ( figure 3(a ) ) . this promoter construct responded to low doses of dht but not to higher doses , confirming the results with the endogenous gene ( figure 1 ) . in contrast , the mmtv promoter was induced by all doses of dht ( figure 3(b ) ) including doses up to 100 nm ( data not shown ) , suggesting that lack of induction by concentrations higher than 5 nm is selective for the tcf8 promoter . because the fold induction of the tcf8 promoter construct by dht equals or exceeds that of the endogenous gene , all of the effects of androgen are likely at the level of transcription . these results also demonstrate that this 974 bp region is sufficient for the induction of tcf8 by androgen . in order to determine whether the two putative ares at 944 and 140 mediate the effects of androgen , point mutations in the critical ar binding residues were introduced into each separately and together ( figure 4(a ) ) . transient transfections of these plasmids were performed in pc-3/ar cells treated with or without dht ( figure 4(b ) ) . the data indicate that mutation of either putative are completely abolished the responsiveness of tcf8 to androgen , confirming that these are in fact authentic ares . to ascertain whether , as predicted , ar binds to the ares at 944 and 140 , gmsas were performed using pc-3/ar cell nuclear proteins extracted from cells treated with 5 nm dht ( figure 5 ) . as is typical with gmsas using crude nuclear proteins extracts with ares [ 37 , 38 ] , multiple bands were observed , all of which are competed with wild - type ( wt ) self - oligomers ( lane 3 , figures 5(a ) and 5(b ) ) and an are consensus oligomer ( lane 5 ) but not with mutated ( mt ) self ( lane 4 ) or consensus ( lane 6 ) are oligomers . the addition of an ar antibody ( lane 7 ) caused a significant supershift , whereas preimmune serum did not ( not shown ) , verifying that the binding complex for each are contains ar . the demonstration that tcf8 is regulated by androgen , the high expression of zeb1 mrna in aggressive pca cell lines , zeb1 's role in pca cell migration [ 26 , 31 ] , zeb1 's correlation with gleason grade in human pca samples , and the increased expression of zeb1 in primary pca and associated bone metastases raises the possibility that aberrant expression of tcf8 might promote pca metastasis . this concept is supported by the observation that expression of zeb1 becomes estrogen independent in aggressive endometrial and ovarian carcinomas . to examine whether zeb1 expression becomes independent of androgen during pca progression , the lncap cell line is an androgen - responsive , prostate - specific antigen ( psa)-secreting , nonmetastatic human prostate cancer cell line established from a lymph node metastasis . the lncap c4 - 2 and c4 - 2b lines are two derivatives that have metastatic potential . they are androgen independent for growth in castrated hosts but still have androgen - responsive ar . we hypothesized that zeb1 expression increases independently of androgen in these metastatic lines and contributes to androgen - independent pca cell proliferation , comparable to what occurs with zeb1 expression in advanced ovarian and endometrial cancers . if true , this inappropriate expression of zeb1 might contribute to the androgen insensitivity observed in advanced pca . in order to examine the relative androgen responsiveness of the cell lines , the pbluezeb974 plasmid was transfected into the lncap ( figure 6(a ) ) , c4 ( figure 6(b ) ) , c4 - 2 ( figure 6(c ) ) , or c4 - 2b ( figure 6(d ) ) cell lines with increasing amounts of dht . in all cases , expression of the tcf8 promoter was strongly induced by dht in a dose - dependent manner , and the level of expression did not correlate with metastatic potential . however , in contrast to what was observed with the pc-3/ar cells ( figure 1 ) , high concentrations also induced expression . as was seen with the pc-3/ar line , thus , the tcf8 promoter is regulated by androgen in lncap cell lines and that regulation is dependent upon the two ares . in order to determine whether endogenous tcf8 was induced by dht in these cell lines and whether it was affected by the metastatic potential of the cells , the four lines were incubated with or without 5 nm dht , the rna was isolated , and subjected to qpcr ( figure 7(a ) ) . surprisingly , tcf8 was not induced by dht in any of the lines even though the tcf8 promoter construct was in cells from the same experiments ( figure 7(b ) ) . these data indicate that endogenous tcf8 is differentially regulated by androgen in the pc-3 and lncap cell lines even though the isolated promoter is highly induced in both . evidence has accumulated from cell lines and human samples to suggest that zeb1 plays a role in the progression of pca [ 14 , 26 , 31 , 39 ] . as zeb1 mrna is induced by both estrogen and progesterone , it seemed plausible that it might also be regulated by androgen , especially as ar and pr can bind to the same regulatory element ( for review , see ) . our preliminary studies revealed that it was indeed regulated by androgen in both pc3/ar and 22rv1 prostate cancer cell lines [ 32 , 33 ] . these studies extend those observations and show that dht induces endogenous zeb1 mrna , but over a fairly small range of concentrations in the pc3/ar cells ( figure 1 ) . a similar dose response was seen with a tcf8 promoter construct but not with the mmtv promoter ( figure 3 ) . in contrast , this same promoter reporter vector was responsive to a wide range of androgen concentrations in lncap cell lines ( figure 6 ) indicating that this restriction is cell type specific , selective to the tcf8 promoter , and not a consequence of posttranscriptional regulation . the reason for the restricted dose responsiveness of tcf8 to androgen in pc3/ar cells remains to be elucidated . nonetheless , our data indicate that androgen induces the transcription of tcf8 4- to 9-fold in pc-3 cells , and the effect is even greater at the protein level as little basal expression could be detected ( figure 2 ) . our data also show for the first time that the androgen induces zeb1 protein as well as the mrna ( figure 2 ) . transient transfection assays with tcf8 promoter constructs revealed that both putative ares ( 944 and 140 relative to the translation start site ) are functional and are required for induction ( figure 4 ) . as neither of these are perfect ares and as they are about 800 bp apart , it may be that a looping mechanism brings the two ares together to provide sufficient affinity to get induction by androgen . a similar mechanism occurs in the psa promoter , where an upstream enhancer containing an are loops to interact with two promoter - proximal ares at 400 and 170 . these data support the concept that androgen elicits a transcriptional cascade through the induction of tcf8 . this is also supported by a recent paper showing that ar induces expression of zeb1 in triple negative breast cancer cells . furthermore , treatment of these cells with the antiandrogen bicalutamide reduces zeb1 protein expression although no mechanism was provided . however , it was somewhat surprising that androgen does not induce endogenous tcf8 in the androgen - responsive lncap cell line derivatives ( figure 7 ) , even though the promoter construct is responsive ( figure 6 ) . one explanation may be that epigenetic silencing of the tcf8 locus occurs in some cell types . this was shown to be the case in adult t - cell leukemia and lymphoma cells , where epigenetic silencing of tcf8 appears to contribute to malignancy . another possibility is that the tcf8 gene is actually induced by androgen in lncap cell lines , but the mrna is rapidly degraded by micrornas ( mirnas ) . considerable evidence indicates that there is a reciprocal relationship between zeb1 and several mirnas , most notably those of the mir-200 family ( for review , see . the zeb1 3-utr contains eight seed sequences for the mir-200 family , and expression of zeb1 mrna is inversely correlated with expression of the mir-200 family . moreover , lncap cells have extremely high levels of mir-200c , about 1,420-fold that of normal prostate fibroblasts . additional support for this hypothesis comes from the inverse relationship seen between mir-200 and zeb1 in a different pc3 cell line derivative . thus , androgen may in fact be inducing zeb1 mrna in lncap cells , but it is rapidly degraded by mir-200c . although zeb1 is regulated by estrogen , progesterone , and androgen [ 2 , 12 , 21 , 27 , 28 ] , only a couple of publications address its normal role in mammalian reproduction [ 12 , 21 ] . . showed that zeb1 is expressed in normal virgin and pregnant mouse myometrium and stroma , but not in epithelial cells , even though those cells also contain estrogen and progesterone receptors . this supports our observations that the presence of steroid receptors is not sufficient to induce tcf8 in all cell types ( this manuscript and ) . in the pregnant uterus interestingly , the mir-200 family is induced at term , repressing zeb1 expression and allowing parturition . an ar - dependent signal from the urogenital mesenchyme is required during prostate development ( for review , see ) . as zeb1 expression in normal cells is primarily mesenchymal [ 12 , 40 ] , it may be that zeb1 mediates androgen 's actions during the development of the prostate and in some aspects of normal male reproductive function . the contribution that zeb1 makes to the physiological and pathological signaling pathways triggered by androgen remains to be established . in addition to demonstrating that tcf8 is regulated at the transcriptional level by androgen , the data herein show that it is a direct target for ar and identify its specific binding sites . this is an important contribution as very few regulatory elements have been characterized in tcf8 , although a number have been proposed ( for review , see ) including two progesterone response elements that do not correspond to our proposed ares . in fact , not even the transcription start site has been experimentally defined in mammals . characterization of the normal regulation of tcf8 and expression of zeb1 is essential for understanding its aberrant expression in various carcinomas , including pca . these data define a previously uncharacterized mechanism for the regulation of tcf8 and provide the basis for additional studies defining its function in the male . the tcf8 gene is a direct target for ar , which binds to two ares in the 1000 bp proximal to the translation start site . the increase in expression of this gene is reflected by an increase in zeb1 protein . however , expression of tcf8 is silenced in some reproductive cell lines including the lncap pca cell line by mechanisms that remain to be elucidated but that may involve epigenetic silencing or the mir-200 family of micrornas .

it is the next step allowing for improved cosmetic effects , potentially faster recovery , reduction of the number of integument incisions , which reduce postoperative pain , and further reduction of morbidity , such as wound suppuration and incision site herniation . single access laparoscopy was recommended nomenclature less ( laparoendoscopic single - site surgery on a multispecialty working group meeting . the first reported single access laparoscopic approach for nephrectomy in an adult human patient , which used three traditional 5 mm trocars inserted adjacent to each other through a single incision at the umbilicus , was presented in 2007 by raman . the next article presented in 2007 by ponsky employed a gelport with three trocars inserted through it using only one incision . notes ( natural orifice translumenal endoscopic surgery ) is an exciting new approach to minimally invasive surgery ; however , significant technical barriers to these techniques have hindered widespread clinical use . however , to date , this type of surgery has not been approved in children . we have been using multi - channel 5 - 12 mm sils ports manufactured by covidien. to insert the port into the abdominal cavity , a semicircular incision is made below the umbilicus , similarly as it is done when creating access using the hasson technique . the modification consists in fitting the instruments with articulating working heads , which prevents clashing of the instruments with the optical system . in this case report we would like to present our initial experience with single port laparoscopic nephrectomy and nephroureterectomy in pediatric patients with left ( four patients ) and right ( three patients ) nonfunctioning kidneys . although transumbilical single access nephrectomies have been reported in adults , to our knowledge there are only three reports on pediatric nephrectomy and one on nephroureterectomy in the published studies . the first reports employing this technique for nephrectomy in a child were published in 2009 by johnson [ 7 , 8 , 9 ] . three patients , aged three , six , and 10 years , were followed up due to a multicystic dysplastic kidney , which did not demonstrate involution . within the two years before surgery the renal scan revealed 100% split renal function on one side and a nonfunctioning kidney on the other side . a left nephrectomy in one patient , and right nephrectomy in the other , were planned . the next two patients , 6- and 8-year old girls , were referred to our department due to recurrent urinary tract infections . the ultrasound examination showed a small left kidney in both patients with thin , 3 mm parenchyma and dilated calices and pelvis . the next two children 6 and 9-year old were admitted to our department due to giant hydronephrosis . the renal scan revealed 100% split renal function on the other side and nonfunctioning hydronephrotic kidneys . under general anesthesia , the patients were placed in the supine position and a urethral foley catheter was inserted . subsequently , a semicircular 2.5 - 3 cm incision was made below the umbilicus , similarly as it is done while achieving access to the peritoneum using the hasson technique . we have been using multi - channel 5 - 12 mm sils ports manufactured by covidien. to insert the port into the abdominal cavity we employed two forceps . following the insertion of the multi - channel port , the insufflator was attached and pneumoperitoneum was achieved to the value of 10 - 12 mmhg . subsequently , the patient was placed in the flank position with the kidney to be nephrectomized upwards . mobilization of the left or right colon using a dissector and scissors with articulating working heads ( endo dissect and endo shear , covidien ) allowed for identification of the kidneys and renal hilum ( fig . the kidneys were retracted anterolaterally with the help of a flexible grasper and the hilar vessels were exposed . after complete mobilization in the three patients with multicystic dysplastic kidneys , the hypoplastic renal artery , renal vein , and ureter were sealed using liga - sure manufactured by covidien . in the remaining patients , the vessels were clipped using a 5 mm rigid vascular stapler ( endo gia vascular , covidien ) and the ureter proximal to the bladder was sealed using liga - sure . the ureters were followed to the distal part of the iliac vessels and divided close to the bladder in children with vur . there was no bleeding under the 5 mmhg intraperitoneal pressure , hence , it was decided that there was no need for drain insertion in any patient . the kidneys were removed through the infraumbilical incisions using a grasper simultaneously with the sils port . the fascia and the subcutaneous tissue were sutured with 3 - 0 vicryl ( ethicon ) and the skin was closed with subcuticular stitches 4 - 0 vicryl . the skin around the incision was injected with 0.5% solution of bupivacaine in general doses appropriate for weight to achieve local anesthesia . all the nephrectomies were successfully performed via a single port within 50 - 90 minutes with no blood loss . none of the procedures required conversion to open surgery , placement of additional ports or blood transfusion . oral feeding was started on the first postoperative day ; on the second day , the patients were mobilized and they were released on the third day . in all the cases , surgical wounds healed by primary adhesions and were hidden in the umbilicus . there were no long - term complications identified duirng the follow - up after 4 months . pathology revealed multicystic renal dysplasia in the three patients , with cysts up to 2.5 cm in diameter . the diagnosis of the next four patients was post - inflammatory kidney destruction due to endstage reflux nephropathy or giant hydronephrosis . nevertheless , there have been efforts to further reduce its invasiveness and access - related complications . each additional port increases the potential morbidity from internal organ damage and bleeding from abdominal integument vessels and also increases postoperative pain and the risk of wound infections or herniation in the site of incision . in videoscopic surgery in adults , the search for a method that would allow for minimizing scars has culminated in the development of notes , where the surgical procedure is performed through an approach that takes advantage of natural body orifices , e.g. the stomach , rectum , vaginal vault or urinary bladder . however , to date this type of surgery has not been approved in children . single port access surgery has been successfully used for laparoscopic cholecystectomy , appendectomy , and varicocelectomy [ 6 , 13 , 14 ] . the first single port nephrectomy in an adult patient by raman et al . took place in may 2007 . performing this procedure using the single port technique is quite different from standard laparoscopy and requires a new set of skills . the greatest difficulty in surgeries employing this technique is posed by the small distance between the instruments that are inserted into the abdominal cavity as well as their parallel positioning . single port surgery involves multiple technical challenges different from classic laparoscopy , including loss of triangulation , challenging work angle , limitation in instrumentation , instrument crowding and clashing , crossing instruments , and obscuring the line of vision . development of new instrumentation with articulating heads and four channel ports has allowed for performing laparoscopic procedures employing a single incision of the abdominal integument while minimizing its drawbacks . flexible instruments help overcome the problems during the dissection that arise from the closeness of the tips of the straight instruments . our surgeon used flexible instruments in one hand because it was easier than either using them in both hands or crossing the instruments in a counterintuitive fashion . however , no complete reduction of clashing of the instruments and the optical system , which hinders tissue dissection or suturing , has been achieved . for this reason , some authors recommended optical system equipped with built - in digital cameras , not only in view of the integration of the camera and laparoscope , which results in the absence of the camera head at the extra - abdominal end of the laparoscope . very helpful we try to resolve this problem by keeping the camera as far back as possible during the operation , using the zoom of the camera and a 300 lens . we have observed that these two solutions limit the clashing of the laparoscope and instruments . we have found only three papers that reported on experiences using the single port access technique in pediatric nephrectomy or nephroureterectomy [ 7 , 8 , 9 ] . less represents a novel approach to major urological procedure and underscores the evolution to progressively fewer incisions required to perform surgery . except the aesthetic advantages , it has been proposed that patients may experience less perioperative pain with fewer port site - related complications such as hernia , hemorrhage or healing complications . however , due to lack of comparative studies between these new laparoscopic techniques and standard laparoscopy , it is still under debate [ 16 , 17 ] . after using this technique in the simplest urological procedures , such as varicocelectomies , abdominal cavity revision in patients with non - palpable testis syndrome and orchiectomies in patients with dysplastic testis , we employed these procedures for nephrectomy and nephroureterectomy . compared with classic transperitoneal laparoscopic nephrectomy , we found that the benefits of single - port - access nephrectomy include improved cosmetic effects , reduction in iatrogenic visceral and vascular injuries resulting from port placement , and a lower risk of potential wound healing complications . in order to minimize the drawbacks of this technique the time needed to perform a procedure using single port access is similar to that required by classic laparoscopic surgery . we would like to demonstrate the feasibility and efficacy of this technique in the pediatric population and advantages of this technique . nevertheless , wide studies are still needed to compare less to the classic laparoscopic procedures to determine if there truly may be any real benefit to this approach .

rheumatoid arthritis ( ra ) is the most common disease of the inflammatory joint diseases , afflicting about 1% of the world population . the disease can have a very aggressive course and poor outcome as inferred by the analysis of its social impact ( after 10 years of disease duration , more than 50% of ra patients are unable to perform professional activities ) and life expectancy diminishes 10 years due to disease activity and associated comorbidities . ra is a chronic systemic inflammatory disease characterized by synovial hyperplasia caused by a large proliferative cellular infiltrate of leukocytes , high expression levels of proinflammatory cytokines , and consequent erosion of joint cartilage and bone . the therapeutic approach of ra has been revolutionized in the last decade with the discovery of specific targeted treatments . however , despite all available therapeutic options , ra remains a progressive , destructive , and debilitating disease with only 20% of patients reaching remission . anakinra , an antagonist of interleukin ( il)-1 , was approved for ra treatment in the last decade . however , the real impact on disease activity has been shown in practice to be lower than what was anticipated from clinical trial results , casting doubts on the role of il-1 as a therapeutic target . nonetheless , we have previously reported increased levels of il-1 in very recent onset arthritis and in the synovial fluid of established ra patients . this observation could be explained by the activation of caspase-1 that we also have observed both in early and established ra patients . therefore , it is possible that il-1 plays an important role in early rather than late stages of the disease and that pathways regulating this cytokine , such as caspase-1 and nf-b activation , can potentially constitute promising therapeutic targets for specific drugs . the effect might be further boosted if an inhibitory effect on tumour necrosis factor ( tnf ) can also be achieved . based on the results of a recent drug screen for compounds that simultaneously inhibit il-1 and tnf secretion , we chose gambogic acid as a promising therapeutic candidate for the treatment of arthritis . gambogic acid is a polyprenylated xanthone abundant in resin derived from garcinia hanburyi and g. morella and is used in southeast asia complementary and alternative medicine . recent studies showed that gambogic acid could inhibit the growth of a wide range of tumour cells . our aim in this study was to investigate whether gambogic acid administration was able to attenuate inflammation in a rat model of antigen - induced arthritis ( aia ) . all experiments were approved by the animal user and ethical committees at the instituto de medicina molecular , according to the portuguese law and the european recommendations . thp-1 cells were stimulated for 6 hours with 4% pfa - fixed dh5 escherichia coli ( e. coli ) at a multiplicity of infection ( moi ) of 20 bacterial cells per thp-1 cell , 1 hour after incubation with gambogic acid . cell supernatants were collected and il-1 and tnf cytokines were quantified by enzyme - linked immunosorbent assay ( elisa ) ( r&d systems , usa ) according to the provider 's instructions . female wistar aia rats were purchased from charles river laboratories international ( ma , usa ) and maintained under specific pathogen - free ( spf ) conditions . animals were inoculated under isoflurane anesthesia by subcutaneous injection of complete freund 's adjuvant ( cfa ) containing mycobacterium butyricum in the rat right pad which leads to a profound systemic inflammation resulting in severe joint swelling and destruction . gambogic acid was administrated at a dose of 4 g / g body weight every day . drugs and vehicle control ( dimethyl sulfoxide , dmso ) were dissolved in normal saline solution and injected intraperitoneally to aia rats after 4 days ( early treatment group , n = 10 ) and after 11 days ( late treatment group , n = 5 ) of disease induction , when arthritis was already present . healthy nonarthritic ( n = 10 ) and vehicle - injected ( n = 10 ) animals were used as controls for comparison . the inflammatory score , ankle perimeter , and body weight were measured during the time of treatment . inflammatory signs were evaluated through the counting of the score of each joint in a scale of 03 ( 0 : absence , 1 : erythema , 2 : erythema and swelling , and 3 : deformities and functional impairment ) . the total score of each animal was defined as the sum of the partial scores of each affected joint . rats were sacrificed after 19 days of disease evolution and paw samples were collected for histological and immunohistochemical evaluation . for histopathological observation , paws , lungs , livers , kidneys , spleens , and pancreas samples were collected at the time of sacrifice . samples were fixed immediately in 10% neutral buffered formalin solution and then dehydrated using increased ethanol concentrations ( 70% , 96% , and 100% ) . samples were next embedded in paraffin , sectioned , and stained with hematoxylin and eosin for morphological examination . paws were also used for immunohistochemical staining with ki67 antibody , a cellular proliferation marker . tissue sections were incubated with primary antibody against rat polyclonal ki67 ( abcam , uk ) and with envision + ( dako , denmark ) . colour was developed in solution containing diaminobenzadine - tetrahydrochloride ( sigma , usa ) , 0.5% h2o2 in phosphate - buffered saline buffer ( ph 7.6 ) . all images were acquired using a leica dm 2500 ( leica microsystems , germany ) microscope equipped with a colour camera . data regarding the degree of proliferation of synovial cells was scored from 03 ( 0 : fewer than three layers , 1 : three to four layers , 2 : five to six layers , and 3 : more than six layers ) . lymphoid cell infiltration was scored from 03 ( 0 : none to diffuse infiltration , 1 : lymphoid cell aggregate , 2 : lymphoid follicles , and 3 : lymphoid follicles with germinal center formation ) . thp1 ( atcc tib-202 ) macrophage - like cell line and thp1/nf-b reporter cell line were cultured in r10-rpmi media 1640 supplemented with 10% ( v / v ) fetal bovine serum , 1% ( v / v ) penicillin - streptomycin , 1% ( v / v ) pyruvate , 1% ( v / v ) l - glutamine , 1% ( v / v ) nonessential amino acids , 1% ( v / v ) hepes buffer , and 2-mercaptoethanol to a final concentration of 0.05 m , as recommended by the american tissue culture collection ( atcc ) . cells were cultured at 250.000 cells / ml , incubated with 10 m of gambogic acid for 1 h at 37c 5% co2 , and then stimulated with pfa - fixed e. coli ( 20 e. coli per cell ) for 8 h and 24 h at 37c 5% co2 . simultaneously , nonstimulated negative control cells were also cultured at the same density as the stimulated population for comparison . caspase-1 activity was measured in thp1 macrophage - like cell line using the carboxyfluorescein flica detection kit for caspase assay ( immunochemistry technologies , llc , usa ) following the reagent instructions . briefly , cells from the different assays were protected from light exposure while incubated for 1 hour at 37c with 30x flica solution at a 1 : 30 ratio . lentiviral particles carrying a nf-b - responsive gfp - expressing reporter gene ( cignal lenti reporters , sabiosciences , usa ) were used to infect thp-1 cells and to establish a stable cell line . all samples were analyzed by flow cytometry using a facs calibur ( bd biosciences , usa ) . statistical differences were determined with nonparametric kruskal - wallis and mann - whitney tests using graphpad prism ( graphpad , usa ) . to study the effect of this drug on the inhibition of il-1 and tnf secretion , we treated the human thp-1 macrophage - like cell line with growing concentrations of gambogic acid for 1 hour before challenging them with pfa - fixed e. coli for 6 hours . the conditioned media was then probed for the secretion of either il-1 or tnf using elisa . gambogic acid significantly inhibited the secretion of both cytokines over a wide range of concentrations ( figure 1 ) , confirming the previously reported effect of gambogic acid in blocking the secretion of these cytokines and validating our earlier findings . pro - il-1 and tnf both depend on nf-b activation for the transcription of their respective mrnas . we therefore tested the effect of gambogic acid on these key pathways . to investigate the effect of this drug in the activation of nf-b , we used an nf-b reporter cell line created by stably infecting thp-1 cells with a commercial lentiviral gfp reporter under the control of a minimal cmv promoter and tandem repeats of the nf-b transcriptional response element ( tre ) . gambogic acid was able to suppress nf-b reporter activation upon e. coli stimulation in comparison with cells that were also stimulated but did not receive treatment ( figure 2(a ) ) . to test the effect of this drug on caspase-1 processing and activation , we used a caspase-1 fluorescent substrate and measured relative active caspase-1 levels using facs . also in this setting , gambogic acid significantly decreased the activation of caspase-1 ( figure 2(b ) ) . to study the anti - inflammatory properties of gambogic acid in vivo , aia rats were treated daily with 4 g / g body weight of gambogic acid intraperitoneally after the disease had already become symptomatic . we started the treatment after 4 days of disease induction ( early treatment group ) and after 11 days of disease induction ( late treatment group ) . the inflammatory score and ankle perimeter were evaluated during the period of treatment . as shown in figure 3 , by the 4th day , all induced animals already presented with arthritis . after 6 days of treatment , the vehicle - injected group increased sharply the inflammatory manifestations , whereas , in gambogic acid - treated rats , there was minimal inflammatory activity or even complete abrogation of arthritis manifestations . in the late treatment group , drug administration was started after 11 days of disease evolution , when animals presented a mean inflammatory score of 6 . also in this group , by the second day of treatment with gambogic acid , the inflammatory manifestations started to significantly decrease over time . this result shows that this drug has anti - inflammatory effects even when administrated in a later phase of arthritis . after 15 ( early treatment group ) and 8 ( late treatment group ) days of treatment , gambogic acid showed significant anti - inflammatory effects , as assessed by the evaluation of the inflammatory score ( figure 4 ) and ankle perimeter ( p = 0.0126 in early group and p = 0.0126 in late group versus untreated animals ) . we have also tested 2 g / g body weight subcutaneously every day . with this regimen , in the early treatment group , the anti - inflammatory effects were significant ( p = 0.0048 ) but not as dramatic as the 4 g / g body weight intraperitoneally , and , in the late treatment group , the drug had no efficacy . in addition , we have observed that a subset of animals kept a low inflammatory score , even after stopping the administration of the drug at day 13 . we were able to observe this anti - inflammatory effect in the absence of drug until day 21 ( data not shown ) . of note , in some of the intraperitoneally treated animals , we have observed decreased body weight and ascites , in accordance with previous literature reports . during autopsy , we , together with a veterinary , have observed macroscopically internal organs that showed no alterations when comparing gambogic acid - treated , vehicle - treated , and healthy nonarthritic rats . additionally , we have observed sections of internal organs collected at the time of sacrifice by histology , which have also shown no alterations comparing all experimental groups of animals . specifically , in the case of the spleen , which in some ra patients and in animal models of arthritis present splenomegaly , we have observed that gambogic acid - treated and vehicle - treated rats showed spleen hyperplasia , with increased cellularity , compared with healthy nonarthritic rats ( see supplemental figure s1 in supplementary material available online at http://dx.doi.org/10.1155/2014/195327 ) . this result might be explained by the fact that gambogic acid is not modulating the lymphocyte development but is instead targeting downstream caspase-1 and nf-b activation . to evaluate the infiltration of immune cells within joints in aia rats , joint tissue sections were stained with hematoxylin and eosin . the histological evaluation shown in figure 5 revealed that rats treated with gambogic acid had a normal joint structure with complete abrogation of the inflammatory infiltrate ( p < 0.0001 in early treatment group versus untreated animals ) . in contrast , vehicle - treated rats exhibited infiltration of inflammatory cells , bone invasion and erosions ( figure 5 ) . we also studied the levels of proliferation of immune cells by staining joint tissue sections with ki67 . the immunohistochemical results revealed that rats treated with gambogic acid presented reduced proliferation of immune cells within joints ( p = 0.0098 versus untreated animals ) . moreover , treatment with gambogic acid prevented cartilage and bone damage ( figure 5 ) . our results demonstrated that treatment with gambogic acid protected wistar aia rats from arthritis development with a complete abrogation of joint immune cellular infiltration and proliferation , preventing cartilage and bone damage . our laboratory has used a thp-1 macrophage - like cell line to screen 2320 drugs for those that simultaneously inhibit il-1 and tnf secretion . we have selected gambogic acid as a promising therapeutic candidate for the inhibition of both il-1 and tnf secretion , due to the reduction in caspase-1 and nf-b activation , and consequently for the treatment of arthritis . previous reports have demonstrated that gambogic acid can inhibit the growth of a wide variety of tumour cell lines , possibly due to its ability to induce apoptosis via the transferrin receptor ( tfr1 ) . additionally , recent data have shown that this drug can inhibit nf-b signalling pathway in human leukemia cancer cells and in a noncancerigenous macrophagic cell line also via tfr1 . therefore , the anti - inflammatory effects of gambogic acid appear to be mediated by the inhibition of nf-b activation pathway which in turn leads to the silencing of most of the inflammatory genes . in fact , the inhibition of nf-b in animal models has shown the ability to inhibit inflammatory arthritis , demonstrating that nf-b may be an important therapeutic target in ra [ 1719 ] . indeed , nf-b participates in the transcription of the genes encoding many proinflammatory cytokines and chemokines , in the regulation of the different immune cells , and in the regulation of the expression of adhesion molecules and matrix mmps [ 2022 ] . as recently reviewed , the ikb kinase ikk is essential for the inflammatory cytokine - induced activation of nf-b . importantly , it has already been reported that gambogic acid is able to inhibit ikk activity . in our study , we demonstrated that the anti - inflammatory properties of this drug in aia rats might not only be related with its ability to suppress the activation of nf-b but also to its effect on inhibiting caspase-1 activation . interestingly , joosten et al . showed uncoupled activities of il-1 and tnf in joint swelling and ongoing cartilage destruction . also , it has been reported that there are different time - dependent roles for il-1 and tnf in the various stages of collagen - induced arthritis . in ra , the inflamed synovium expands into and destroys the underlying cartilage and bone , resulting in irreversible erosion of the bone and in the loss of normal joint architecture leading to disability . the inhibitory effect of gambogic acid in cellular infiltration and proliferation as well as in bone erosions can thus prove to be of interest to prevent and treat structural joint destruction induced by ra . in addition , administration of gambogic acid to aia rats tested a therapeutic strategy , since it started in the early phase of arthritis , after 4 days of disease induction , with all animals already displaying signs of inflammation . to further evaluate the effect of the compound in a latter phase of the disease , gambogic acid was also administrated after 11 days of disease induction , when the animals displayed a high inflammatory score . thus , gambogic acid treatment is effective when administrated both in the early and more established phase of arthritis , which is relevant to the possible clinical implications of our findings . in conclusion , we have found that wistar aia rats can be effectively treated by gambogic acid . the effects of this drug probably rely on the inhibition of il-1 and tnf secretion , possibly explained by its ability to downregulate caspase-1 and nf-b activation and by blocking synovial hyperplasia due to its significant antiproliferative properties . these results support our initial hypothesis that a double inhibition of il-1 and tnf could be effective in the treatment of inflammatory diseases , such as ra , and further indicate that the antiproliferative properties of gambogic acid may prove essential for an effective clinical control and to induce early remission in ra patients . based on our observations indicating possible toxicity in some of the regimens used , we suggest that gambogic acid might not be suitable to directly enter phase i clinical trials , but it can certainly serve as a prototype drug to search for derivate compounds with similar effects on inflammatory mediators and cell proliferation and a more favourable safety profile .

during gestation , products of the endocannabinoid system in the placenta are crucial for maintenance of pregnancy and for both the onset and progression of labor . the endogenous ligands for this system , the endocannabinoids , are formed from membrane phospholipids . two well characterised endocannabinoids are 2-acylglycerol ( 2ag ) and anandamide ( aea ) . these molecules are not stored within cells but are synthesised and released in response to increased substrate availability and synthase activity . 2ag and aea bind to the g - coupled protein receptors , cannabinoid receptors 1 and 2 ( cb1 and cb2 ) . the loss of the cb receptors has been linked to preterm delivery which accounts for 518% of pregnancies worldwide [ 3 , 4 ] . aea is believed to be formed by a two - step catalysis in which arachidonic acid is transferred to a phospholipid precursor , phosphatidylethanolamine , by n - acyltransferases ( nats ) to form n - arachidonoyl phosphatidylethanolamine ( nape ) . diacylglycerol is formed from phosphoinositides by the action of an important enzyme , phospholipase c ( plc ) . the liberation of arachidonic acid occurs via the catalysis of enzyme phospholipase a2 or indirectly by the catalyses of plc and dagl and monoacyl glycerol lipase enzymes ( magl , protein coded by mgll ) . pgh2 is further converted to other key downstream molecules / metabolites of the eicosanoid family ( prostaglandin glycerol esters , prostanoids , and prostamides ) that have been studied for their instrumental roles in the aetiology of labor , namely , txa2 , pgd2 , pge2 , pgi2 and pgf2a , and so forth ( figure 1 ) . ptgs2 is crucial for its multifactorial role , as it also converts anandamide to pgh2-e and 2ag to pgh2-g . in rat placenta , 2ag and aea play important roles in fetoplacental development and aberrant cannabinoid signalling can cause pregnancy complications [ 10 , 11 ] . this paper focuses on the endocannabinoid system from mid to late gestation leading up to the period just prior to labor . distorted expression of genes and their metabolites of the endocannabinoid system can result in spontaneous miscarriage and poor pregnancy outcomes such as preterm birth . recently it has been suggested that the endocannabinoid profiles can serve as biomarkers in a clinical setting to identify and/or predict spontaneous preterm labor . to date , limited studies have reported on placental gene expression of these enzymes from mid pregnancy to labor . the aim of this study is to compare the expression changes of eight enzymes involved in the endocannabinoid system from rat placenta taken at 4 gestational stages e14.25 , e15.25 , e17.25 , and e20 . the hypothesis of this study was that endocannabinoid enzyme expression would change ( either increase or decrease ) throughout gestation , in support of a normal gestation . although placental factors can not be used directly as biomarker(s ) to predict and/or identify spontaneous labor onset in a clinical setting , as placental biopsies will not be available prior to labor , the study may be able to provide insight into what factors need further investigation in biological fluids that can be quantified during pregnancy . genes were selected based on their involvement in the endocannabinoid system ( as shown in figure 1 ) , specifically genes for enzymes that are involved in the synthesis of the molecules pgh2 , aea , and 2ag and arachidonic acid , namely , plc , dagl , mgll , nat , faah , pld , ptgs1 , and ptgs2 . the expression of these genes , except ptgs1 , was analyzed using microarray on a first cohort of rat placental samples . a second cohort of independent samples was then used for biological replication as validation by qpcr as shown in figure 2 . all animal experiments were performed with the approval of the school of biomedical sciences ' animal ethics committee of monash university . experiments were carried out in accordance with the national health and medical research council of australia healthy dams were allowed to adapt to the animal house for one week . they were fed a standard chow diet ( 20% protein , 68% carbohydrate , and 12% fat ) and water ad libitum in a light controlled environment ( 12 h light / dark cycle ) throughout the study . female rats were time - mated for 3 h with male sprague dawley rats , to reduce variability of gestational age among the offspring and to maximize the accuracy in staging of gestation . dams were individually housed after mating . both cohort samples ( cohorts 1 and 2 ) were fed and treated in the same fashion to maintain consistency . p / l , vic , australia ) and humanely killed at embryonic days ( e ) 14.25 , 15.25 , 17.25 , and 20 ( n = 6 per gestational age ) . whole placentae were collected from each of the pregnant dams , weighed , and snap - frozen in liquid nitrogen . tissues were stored at 80c until being processed and analyzed . using a mortar and pestle , total rna was extracted from 30 mg of pulverized frozen placental tissue using the allprep dna / rna mini kit ( qiagen ) as per manufacturers ' instructions . total rna was quantified via nanodrop nd-1000 spectrophotometer ( thermo scientific , de , usa ) . rna integrity was verified using an agilent 2100 bioanalyzer ( vic , australia ) , by rin ( rna integrity number ) score measurements prior to the analysis . rna samples that fulfilled the following criteria were selected for microarray analysis : ( i ) rin > 8.5 ; ( ii ) 260/280 ratio > 2 ; ( iii ) 260 : 230 > 1 . all rin scores were greater than 8.7 . for the microarray analysis , 500 ng of total rna was converted to double stranded cdna and this was used to generate biotinylated crna probes using the illumina totalprep rna amplification kit . biotin - labelled crna were then hybridized to illumina ratref-12 expression beadchip ( san diego , ca , usa ) . array experiment readout was deposited on arrayexpress ( arrayexpress accession number e - mtab-1987 ) . overall microarray results were previously published by our group . for this study , expression of the 7 enzymes faah , mgll , plcd4 , pld1 , nat1 , dagl , and ptgs2 in the endocannabinoid system was analyzed . qpcr was used to confirm and validate the expression of 8 genes , faah , mgll , plcd4 , pld1 , nat1 , dagl , ptgs1 ( data not shown , not carried out in microarray ) , and ptgs2 in an independent cohort on an abi stepone pcr machine . all primers were designed specifically for rat sequences only spanning intron junctions , to eliminate amplification of genomic dna . supplementary figure s2 ( see supplementary material available online at http://dx.doi.org/10.1155/2015/850471 ) displays the amplification products of the various genes using these primers displayed in table 1 . -actin was used as the endogenous control gene and relative expression was calculated using the ct method . of the 7 endocannabinoid enzymes involved in the metabolism of endocannabinoids , 4 ( ptgs2 , mgll , plcd4 , and pld1 ) were differentially expressed in cohort 2 samples by qpcr . cohort 1 samples showed differential expression of ptgs2 , mgll , and plcd4 but not pld1 ( figure 3 ) . ptgs2 showed a marked increase in expression between e17.25 and e20 of fold change of > 2.5 ( p < 0.001 ) , mgll showed consistent 3-fold increase from e15.25 to e17.25 ( p < 0.05 ; qpcr and < 0.001 ; microarray ) , and plcd4 showed a gradual decrease in expression from e14.25 to e15.25 ( fold change approximately 2 , p < 0.05 ) and e17.25 with expression remaining relatively low at e20 just prior to labor onset . a gene expression pattern for these 3 genes ( ptgs2 , mgll , and plcd4 ) is shown in figure 4 . the gene expressions of 8 enzyme genes were carried out , out of which nat1 , dagl , and faah expression remained relatively stable throughout gestation in both cohorts 1 ( microarray ) and 2 ( qpcr ) as shown in figure 3 . ptgs1 ( data not shown ) was only studied by qpcr and showed no differential expression ( constitutive expression ) , consistent with literature . interestingly the expression of other genes in the system that were stable over the course of gestation included the phospholipases plcb3 , plcd3 , plcl3 , plcz1 , pld2 and pld3 , and nat2 and nat3 ; the genes for phospholipase 2 isotypes pla2g15 , pla2g2d , pla2g2e , pla2g3 , and pla2g5 ; and genes for cannabinoid receptors cb1 and cb2 , that is , cnr1 and cnr2 ( data not shown ) . this study is the first to highlight differential expression of 4 genes ( plcd4 , ptgs2 , pld1 , and mgll ) in rat placenta from mid - late gestation to labor onset . our study expands on a previous study in rat placenta that has focused on expression of cb1 and cb2 endocannabinoid receptor , genes ( cnr1 and cnr2 ) , and some genes of the endocannabinoid system . most other studies of placenta from normal and pathological pregnancies have focused on the downstream prostaglandins ( particularly pgh2 ) as an end product , some with the involvement of ptgs1 and ptgs2 , during the onset of labor . consistent with previous literature dagl and faah expression and ptgs1 and nat1 expression remained stable throughout the four gestational ages . ptgs1 and ptgs2 have been widely studied in placentae of women and animals models for decades . here we observe that expression of ptgs2 in whole rat placenta in both cohorts remained stable throughout e14.25 , e15.25 , and e17.25 but was significantly upregulated at e20 , just before the onset of labor , consistent with previous studies [ 16 , 1921 ] . since ptgs1 and ptgs2 are directly involved in prostaglandin syntheses , any differential or aberrant expression of these two enzymes may have a dramatic effect in prostaglandin levels . we also know that ptgs2 is an inducible gene , which may be triggered to initiate the labor process . however it can not be determined conclusively whether the increase of ptgs2 leads to prostaglandin production , which then triggers the labor process ( which is the ongoing hypothesis in various animal studies ) . in fact , some studies have suggested that pgs originating in the fetal membranes are a trigger for the onset of normal labor . this sudden observed spike in ptgs2 expression further studies on ptgs2 in mammalian pregnancy and labor onset are required given its variable expression and vital role in the biosynthesis of prostaglandins . ptgs1 on the other hand is a constitutively expressed gene and by qpcr demonstrated that its expression remained relatively stable throughout mid to late gestation ( data not shown ) . ptgs2 is important in the endocannabinoid system and there is now evidence to show that factors ( pge2 , ptgs2 , etc . ) involved in this pathway in placenta may have a role in ovulation , implantation , and decidualization [ 22 , 23 ] and abnormal expression may result in spontaneous miscarriage , placental abruptions , and poor pregnancy outcomes [ 24 , 25 ] . prostaglandins increase myometrial sensitivity and ptgs2 is especially associated with changing myometrial contractility during pregnancy . in a clinical setting , selective ptgs2 inhibition is used to delay premature onset of labor . although nonsteroidal anti - inflammatory drugs ( nsaids ) , which inhibit ptgs2 , are usually successful in suppressing preterm labor or prolonging pregnancy in animal and human studies , the nsaids have adverse effects on fetal physiology and development , including renal defects and cardiomyopathies [ 5 , 26 , 27 ] . fluctuations in gene expression and enzyme concentrations and metabolite production may serve as useful indicators of labor onset . these factors , their metabolites , and substrates may then be further investigated in biological fluids to study their potential as biomarkers for early detection of preterm birth and hence pregnancies where there is a risk of preterm delivery to be captured early . ptgs2 regulation occurs at many levels and is affected by transcription factors , activator proteins , and chromatin remodelling . moreover , the endocannabinoid system has come under scrutiny given that several micrornas such as mir26 , mir146 , and mir10 are responsible for its gene regulation [ 28 , 29 ] . these have emerged as regulators of not only physiological processes of pregnancy and labor , such as uterine contractions [ 3032 ] . a future study to link gene regulation patterns and also , other prostaglandin and thromboxane molecules should be investigated further to determine their roles in pregnancy and the inflammatory process . plcd4 expression was relatively high at e14.25 and gradually decreased at e15.25 , e17.25 and further decreased at e20 . the results perhaps indicate that the product of this enzyme , diacylglycerol , is reduced and that plcd4 may be necessary to control levels of diacylglycerol and subsequently 2ag and pgh2-g . this control mechanism could be an important player in the process of labor and hence this study should be further expanded in preterm samples as there is a paucity of data with regard to the role of plcd4 in pregnancy . apart from identifying the presence of the gene transcripts in myometrium from pregnant versus nonpregnant rats , plcd4 , unlike phospholipase a [ 16 , 33 ] , has not been studied for its role in placenta especially with its link to labor and parturition . nevertheless plcd4 can be further investigated in human samples as a potential biomarker for preterm deliveries in perhaps other gestational tissues , including membranes and/or biological fluids , and a reduced level of the enzyme could be a useful prognostic for impending preterm birth . another gene to be highlighted in this study for its marked gene expression change from e15.25 to e17.25 is mgll where a 2-fold increase was observed in both cohorts ( e15.25 to e17.25 ) . an increase of arachidonic acid at e17.25 and then a subsequent increase in expression of ptgs2 at e20 are likely to increase arachidonic acid derived pgh2 . interestingly , when plcd4 expression is at its lowest level , then ptgs2 expression is at its highest level , just prior to labor . a relationship between the 3 genes ptgs2 , mgll , and plcd4 and time points of gestation is shown in figure 4 . from qpcr results on cohort 2 samples , interestingly microarray signals were below the level to detect change as it has a very low expression level overall . steady expression of faah across gestation observed in the present study is consistent with the findings of mijovic et al . in rat placenta . in both qpcr and microarray , dagl and nat1 expression were both observed to remain stable in mid to late gestation , indicating their constitutive expression during this time window likely maintaining the function of their by - products of metabolism nape ( precursor for anandamide ) and 2ag , respectively , throughout pregnancy . interestingly the expressions of other enzyme genes , in microarray , in the system that did not change during the gestation window are nat3 , plcb3 , plcd3 , plcl3 , plcz1 , pld2 , pld3 , pla2g15 , pla2g2d , pla2g2e , pla2g3 , and pla2g5 ( data not shown ) . other related genes from microarray data are shown in supplementary figure s1 . from the results of this study , summarized in figure 5 , increasing gestational age is associated with increasing synthesis of pgh-2 ( and downstream metabolites , prostanoids , and prostamides ) from aa and aea , respectively ( figure 5 ) . on the other hand both aea and aa are metabolites of pld and magl , respectively , and genes for these two enzymes show upregulation from mid to late gestation . this is complemented by the study by fonseca et al . , 2012 , which shows that aea levels in the placenta gradually increased reaching maximum level on day 19 of gestation , while expression remained lower on days 14 and 16 . the downregulation in plcd4 may indicate a control mechanism that reduces the synthesis of 2ag and hence pgh2-g and its derivatives . there is a paucity of data pertaining to the formation of glycerol esters in late stage pregnancy and labor . this shift in gene regulation away from 2ag metabolism may be key in studying and choosing the metabolites for prediction of preterm birth . furthermore , it is predicted that increases in arachidonic acid concentration would arise by 2ag and aea metabolism , prior to pgh2 formation by ptgs2 . the data provides an opportunity to further elucidate functional pathways that may be of significance in late placental function in both normal and pathological pregnancies . from the results of this study , late gestation is associated with the synthesis of pgh-2 ( and their downstream metabolites , prostanoids , and prostamides ) from aa and aea , respectively . the formation of prostaglandin glycerol esters may be downregulated as gestation progresses . in this study , we show that the genes for pld and magl , which catalyse the synthesis of both aea and aa , are upregulated from mid to late gestation , while the plcd4 expression is downregulated . this may indicate a regulatory mechanism that reduces the synthesis of 2ag and hence pgh2-g and its derivatives . this shift in gene regulation away from 2ag metabolism could be a key pathway for the identification of metabolites for the prediction of preterm birth .

chronic prostatitis is a common disease among men , and the incidence is gradually increasing . due to the complicated condition of patients , the suboptimal effect of treatment , and the high rate of relapse although a many studies have been done on this topic , the pathogenesis and pathophysiologic alterations and the best treatment are still unknown . recent studies have shown that the main problem of chronic prostatitis is that medicine can not fully enter the prostate tissue , which limits the efficiency of the treatment . similar to the blood - brain barrier , the blood - prostate barrier protects the prostate tissue and has the function of permselectivity . in multicellular organisms , tight junctions ( tjs ) the changes of the tjs proteins can affect the blood - brain barrier permeability . in studies on ultrasound microbubbles changing the blood - brain barrier , researchers found that the main mechanism of ultrasound microbubbles affecting the blood - brain barrier permeability is changing the expression level of tjs proteins . we hypothesize that this is also the main mechanism by which ultrasound microbubbles change the blood - prostate barrier permeability . at present , there are few relevant research reports and the topic deserves further study . tjs are responsible for the regulation of transmembrane transport of ions and solutes ( the fence function ) and maintenance of cell polarity ( the barrier function ) . studies have shown that the tjs are ubiquitous in the blood - brain barrier , the enteric epithelial barrier , and the blood - testis barrier , and significantly regulate the barriers functions . an experiment has shown that the tjs also exit in prostate tissue , but did not indicate the relationship between the tjs and the blood - prostate barrier . a recent study has shown that ultrasound microbubbles may enhance blood - prostate barrier permeability through opening the tjs . the tight junction is a fundamental junctional complex , composed of transmembrane proteins , cytosolic proteins , and cytoskeletal proteins . the transmembrane proteins include 3 kinds of integral membrane proteins : occludin , claudins , and junctional adhesive molecule ( jam ) . occludin and claudin may play a primary role in regulating the barrier function and claudins are even more important . the claudins are the essential component of tjs , and they are prevalent in the epithelia and endothelia . some claudins create paracellular pores that are the basis for paracellular permeability , allowing small ions and molecules to pass through . this function is achieved by the protein kinase pathway pka or pkc acts on the specific amino acid sites of claudins . tj function is decreased by closed phosphorylation , indicating that various physiological or pathological factors may influence the permeability of tjs by changing the expression level of the claudins . identified claudin-1 and claudin-2 from chicken liver and found that over - expression of these 2 proteins also creates a tight junction between the fibroblasts , demonstrating their importance in the assembly of tjs . in recent years , many tests have indicated that claudins are very important to the blood - brain barrier . confirmed that there is rich expression of claudin-3 and claudin-5 in the brain microvascular endothelial circulation system and that claudin-5 is the major transmembrane protein of the blood - brain barrier . a study using a c6 glioma model indicated that the dysfunction of tjs enhances the permeability of the blood - tumor barrier , probably by the direct invasion of c6 glioma cells , which reduces the expression level of claudin-5 in the endothelial cells . it has been proposed that both over - expressing and ablating expression of claudins improve biofilm barrier permeability . all of these studies suggest that it is possible to regulate biofilm barrier permeability by changing the expression level of claudins . tjs exist in rat prostatic epithelial cells , and the expression levels of claudin-1,-3,-4,-5,-7,-8,-10 in the prostate are significantly higher than in the kidney . it was previously demonstrated that claudin-1 , -3 , -4 , -7 are expressed in rat and human prostate tissue , indicating that these claudins may play the same role in the rat and human prostate barrier . therefore , it may be the key to treatment of chronic prostatitis , solving the problem of how to overcome the barrier properties of tight junctions and help move drugs into the prostate tissue . therefore , we suggest that : 1 ) claudins are important regulators of the barrier function of tjs ; and 2 ) claudins not only expressed in the blood - brain barrier , but also in rat and human prostate tissue . we propose this hypothesis : opening the tight junctions ( tjs ) may be a key to enhancing the permeability of the blood - prostate barrier . we plan to observe the expression of claudins in rat prostate to determine the changes occurring in blood - prostate permeability in future studies . as mentioned above , the blood - prostate barrier could be the major factor leading to the failure of treatment of chronic prostatitis , but the active component of the blood - prostate is still unknown . some researchers consider that vascular endothelial cells and basement membrane comprise the barrier , but epithelial cells are deemed essential by others . it is certain that , similar to the blood - brain barrier , the blood - prostate barrier may be a key point in treatment of chronic prostatitis . the ultrasonic contrast agent microbubble is a new kind of contrast agent ; it not only enhances ultrasound imaging , but also increases the permeability of local organizations , thus improving the effective drug concentration . some researchers have used ultrasound microbubble to enhance the blood - prostate barrier permeability , and indicated the possible mechanism the ultrasound microbubble opens the tight junctions of vascular endothelial cells and epithelial cells of prostate tissue , then leads evans blue ( eb ) dye into the glandular epithelium cells . therefore , we consider that the tight junctions may be the main regulator of blood - prostate permeability . to elucidate the molecular biology basis of the prostate tissue that limits the penetration of drugs into the prostate , and further understand the mechanism by which ultrasound microbubble enhances the prostate permeability , we plan to start with the expression level of tight junctions proteins , and observe changes in the expression of tjs proteins after the ultrasound microbubble acts on the prostate . we propose to intravenously inject the ultrasound contrast agent microbubbles , and then make use of sonoporation induced by ultrasonic cavitation to open the blood - prostate barrier , and observe the relationship of the expression level of tight junction proteins and the level of the opening of the blood - prostate barrier . to achieve this aim , we will establish a rat model with cp in future studies , including a normal control group ( nc group ) , chronic prostatitis model group ( cp group ) , microbubble - only group ( mb group ) , ultrasound - only group ( us group ) , and microbubble - enhanced therapeutic ultrasound group ( meus group , and different intensity ultrasound subgroups ) , and inject the microbubbles and evans blue ( eb ) , then observe the prostate tissue ultrastructure ( e.g. , vascular endothelial cells , basement membrane , basal cells , epithelial cells ) after ultrasonic irradiation , and then test the concentration of eb in the prostate tissue by fluorescence spectrophotometry and analyze the expression level of the tjs proteins by immunohistochemistry and western blot . then we will make the following comparisons : 1 ) compare the expression level of the tj proteins between the nc group and cp group ; 2 ) compare the concentration of eb in the prostate tissue and the expression level of the tjs proteins underlying the different intensity ultrasound ; and 3 ) compare the expression level of the tjs proteins between the mb group , us group , and the meus group . if the difference in expression level of tj proteins in the nc group and cp group are statistically significant , it suggests that chronic inflammation could alter the blood - prostate barrier . if the degree of changes of the concentration of eb is consistent with the expression level of tjs proteins , then it indicates that the change of the blood - prostate is consistent with tjs . if the differences in expression level of the tjs proteins in the mb group , us group , and meus group are statistically significant , together with the result of the eb in the prostate tissue and the expression level of the tjs proteins underlying the different intensity ultrasound , it would suggest that the ultrasound microbubble enhances the permeability of the blood - prostate barrier by changing the expression level of the tjs proteins . moreover , it is known that the tjs proteins could regulate the permeability of the tjs , which means the ultrasound microbubble opens the blood - prostate barrier by opening the tight junctions . we hope to prove our hypothesis that opening the tight junctions may be a key to enhancing the permeability of the blood - prostate barrier . it is difficult to completely cure chronic prostatitis , mostly because the blood - prostate barrier limits the passage of drugs into the prostate tissue , which therefore fails to form an effective drug concentration in the prostate . however , ultrasound microbubble technology can increase blood - prostate barrier permeability , and at the same time improve the effective drug concentration . therefore , we plan to start with the expression level of tight junctions proteins to explore the mechanism , and study it through the above experimental procedure and method , which may elucidate the mechanism of enhancing the blood - prostate barrier permeability and determine the composition of blood - prostate barrier . this will provide theoretical and methodological foundations to guide development of a new treatment for chronic prostatitis . at present , much research has been devoted to investigating tight junction protein regulation of the blood - brain barrier , but few have studied the blood - prostate barrier , which is a topic deserving further study .

guz ten lokalizuje si gwnie w jelitach ( z predyspozycj do okolicy krtniczo - ktniczej ) , w wzach chonnych krezki i przestrzeni pozaotrzewnowej . objawy kliniczne choroby s niespecyficzne : ble brzucha , wymioty , krwawienie z przewodu pokarmowego , objawy ostrego brzucha sugerujce zapalenie wyrostka robaczkowego lub wgobienie jelitowe . w badaniu ultrasonograficznym jamy brzusznej choniak burkitta moe mie rn manifestacj , co wie si z punktem wyjcia zmiany . badania ultrasonograficzne jamy brzusznej oraz przewodu pokarmowego wykonano aparatem siemens , stosujc gowic convex 3,55 mhz w badaniu ultrasonograficznym w analizowanej grupie 15 pacjentw u 3 ( 20% ) patologiczna masa zlokalizowana bya w cianie odka , u 10 ( 67% ) w okolicy krtniczo - ktniczej , u 2 ( 13% ) proces by rozsiany w jamie brzusznej . u 12 pacjentw z rozpoznanym nieziarniczym choniakiem burkitta w lokalizacji pozaodkowej stwierdzono rn morfologi zmian w obrazie ultrasonograficznym . dokadna ocena w badaniu ultrasonograficznym wszystkich narzdw jamy brzusznej gowicami convex i liniow zwiksza szanse prawidowego rozpoznania . burkitt 's lymphoma accounts for 2550% of non - hodgkin lymphomas that occur in childhood . it occurs endemically in african children in whom it is localized mainly in the mandibular and maxillary bones . the abdominal form of burkitt 's lymphoma is more frequently found in non - african children . the extranodal localization of lymphoma is usually the small intestine , with a tendency to occur in the ileocecal region . the gastric wall is a very rare site of origin in children . within the abdominal cavity , patients seek medical advice due to non - specific symptoms of abdominal pain , gastrointestinal bleeding and anemia or with symptoms of acute abdomen suggestive of appendicitis , bowel obstruction or intestinal intussusception . the ultrasound ( us ) presentation of gastrointestinal non - hodgkin lymphoma is ambiguous and should be differentiated from other pathologies , such as inflammatory lesions in the intestine , appendicitis or intestinal intramural hematoma . burkitt 's lymphoma that originates from lymph follicles of the terminal ileum submucosa spreads along the wall , which on us examination is manifested by fragmentary thickening with low echogenicity and a blurred layered structure . wall infiltration may be asymmetrical and the bowel lumen may be normal or narrowed . single or multifocal and oval areas of low echogenicity surrounded by hyperechoic mesentery are characteristic of abdominal lymph node involvement by lymphoma . in physiological conditions , there is no lymphatic tissue in the gastric mucosa . a chronic inflammatory process , e.g. infection with helicobacter pylori , may facilitate the development of the lymphatic tissue , which , in turn , may transform into a neoplasm . in the adult population , non - hodgkin lymphomas of the stomach are usually of the malt type ( mucosa - associated lymphoid tissue ) . in children however , burkitt 's lymphoma may be highly malignant . depending on the extent of the lesion , us examination helps to assess the thickening of individual layers of the gastric wall , which is usually of low echogenicity , with or without a preserved layered structure . the initial diagnosis of the character of the infiltration in the gastrointestinal tract may be determined on the basis of ultrasound presentation . from 2000 to 2012 , 15 pediatric patients ( one girl and 14 boys ) were diagnosed and treated for abdominal burkitt 's non - hodgkin lymphoma in the department of pediatric radiology and in the department of pediatric hematology and oncology , medical university of lublin , poland . the patients were aged between 2 and 17 ( mean age was 9 years and 6 months ) . the clinical symptoms of abdominal pain , weight loss , vomiting and general malaise prevailed . additionally , 2 patients presented with yellowed skin and 2 had gastrointestinal bleeding and anemia . in one case , the only symptom of the neoplasm was an enlarged lymph node localized on the medial surface of the right thigh . ultrasound examinations were conducted with the use of a siemens scanner with a convex transducer of 3.55 mhz and a high - frequency linear array transducer of l4 7.5 mhz . the following modes were used : b - mode , color and power doppler as well as tissue harmonic imaging ( thi ) . the gastrointestinal tract was assessed using an ultrasound set - up for organs located superficially ( set - up small parts ) . during the diagnostic process , all patients underwent laboratory tests , biochemical examinations ( ldh level ) , bone marrow examination as well as abdominal and pelvic computed tomography . ultrasound examinations conducted in the group of 15 patients revealed pathological masses localized in the gastric wall in 3 patients ( 20% ) , in the ileocecal region in 10 patients ( 67% ) and a disseminated process in 2 patients ( 13% ) . in the group of 3 patients in which the lesion originated from the gastric wall , the body of the stomach and prepyloric region were involved . in 2 patients , a pathological mass with heterogeneous echogenicity protruded into the gastric lumen and narrowed it considerably ; in one patient we observed polycyclic margins of a lesion of low echogenicity that extended beyond the gastric wall . in 2 patients , the size of the lesions ranged from 63 56 to 94 101 mm . gastroscopy , which was conducted in these patients , confirmed the presence of an infiltration in the gastric wall . in a histopathological examination of the collected samples , burkitt 's lymphoma was diagnosed in 2 patients and malt lymphoma in one patient . the urease test for us examination , epigastric region . a low echogenicity mass originating from the gastric wall . intestinal dilation us examination , right lumbar region . a low echogenicity tissue area with calcifications a lymph node affected by lymphoma in 12 patients with a diagnosed burkitt 's non - hodgkin lymphoma in an extragastric localization , differences in the morphology of the lesions were observed in us images and the patients were thus divided into three groups : group i in 2 patients ( 17% ) , a pattern was observed in us images in the subhepatic region , which is characteristic of ileocecal intussusception ; in the center , the intussusception had low echogenicity and the lymph nodes were round.group ii in 8 patients ( 66% ) , us examination revealed well - circumscribed areas of low echogenicity that were connected to the adjacent intestinal loops . in one patient , the lesion was extensive and reached the hepatic hilum , which caused bile duct dilation . in all patients , the intestinal wall was 923 mm thick , presented low echogenicity and lacked any layered structure . one patient manifested a dilated intestinal lumen and in all remaining cases , the lumen was narrow but did not cause obstruction . in 4 patients , power doppler demonstrated the rich vascularity of the altered intestinal wall.group iii in 2 patients ( 17% ) , us examination revealed numerous lymph nodes in the epigastric and umbilical regions , ranging in size from 821 mm , with low echogenicity and without a vascular hilum . group i in 2 patients ( 17% ) , a target pattern was observed in us images in the subhepatic region , which is characteristic of ileocecal intussusception ; in the center , the intussusception had low echogenicity and the lymph nodes were round . group ii in 8 patients ( 66% ) , us examination revealed well - circumscribed areas of low echogenicity that were connected to the adjacent intestinal loops . in one patient , the lesion was extensive and reached the hepatic hilum , which caused bile duct dilation . in all patients , the intestinal wall was 923 mm thick , presented low echogenicity and lacked any layered structure . one patient manifested a dilated intestinal lumen and in all remaining cases , the lumen was narrow but did not cause obstruction . in 4 patients , group iii in 2 patients ( 17% ) , us examination revealed numerous lymph nodes in the epigastric and umbilical regions , ranging in size from 821 mm , with low echogenicity and without a vascular hilum . additionally , in 2 patients , fluid was present between the intestinal loops and in the vesicorectal space . the final diagnosis was established on the basis of a histopathological examination of the sample collected intraoperatively in 8 patients and during gastroscopy in 3 patients . in a further 3 cases , a diagnosis was made based on the clinical picture and changes in the bone marrow . five patients with lesions in the ileocecal region qualified for surgical treatment due to acute stomach . in 2 cases , the process was disseminated at the time of admission to hospital . the disease is currently in remission and these patients are undergoing periodic follow - up examinations . malignant non - hodgkin lymphomas constitute a diverse group of neoplasms of the lymphatic system . in children , the abdominal form is dominant , i.e. burkitt 's lymphoma , with the tendency to occur in the ileocecal region in 2550% of cases . neoplasms of this type are highly malignant ; they may double their mass within 24 hours . due to their non - specific clinical and radiological signs , the diagnosis of gastrointestinal lymphomas in children is difficult . this is because the common and most frequent abdominal symptoms , such as abdominal pain , loss of appetite , flatulence , diarrhea , constipation , hepato- and splenomegaly , are usually associated with infectious diseases ( e.g. appendicitis ) rather than oncological causes . the process of conducting abdominal us examinations in frequently agitated children may also be challenging and may result in overlooking lesions in the intestinal loops . such an assessment , however , is essential since the gastrointestinal tract , and the ileocecal region in particular , is the most common localization of extranodal non - hodgkin lymphomas . during the examination , one should assess the thickness of the intestinal wall , the presence or absence of the layered wall structure and the degree of intestinal dilation . such a manifestation of lymphoma was observed in 5 patients in our study . depending on the degree of wall thickening and exophytic proliferation , lesions may narrow the lumen of the gastrointestinal tract ( to various degrees ) or cause its compression , leading to obstruction . in the case of circular wall thickening , however , the presence or even the dilation of the intestinal lumen is apparent . in certain patients , the first sign of lymphoma may be ileocecal intussusception with tumor acting as its front . in our patients , intestinal intussusception frequently occurs in young children and is called spontaneous intussusception and is associated with peristalsis , elongation of the intestinal mesentery or lymph node enlargement . in older children , intussusception may be caused by the presence of pathological lesions , such as lymphoma , or intestinal polyps . in one girl of the analyzed group who was diagnosed with lymphoma , numerous polyps of the large bowel were also observed and confirmed endoscopically . persisting non - specific abdominal pain in children requires differential diagnosis between inflammatory and neoplastic diseases . in each child with abdominal symptoms , abdominal us examination is the first test , and provides physicians with information that is valuable in the diagnostic and therapeutic process . the application of high - frequency transducers enables the assessment of the gastrointestinal wall , the degree and extent of its thickening , and whether or not the lymph nodes are enlarged . a thorough us assessment of all abdominal organs conducted using convex and linear probes increases the chances of establishing an adequate diagnosis . the authors do not report any financial or personal links with other persons or organizations , which might affect negatively the content of this publication and/or claim authorship rights to this publication .

copy number variation clustered regularly interspaced short palindromic repeats human leukocyte antigen - a inhibitor of nuclear factor kappa - b kinase subunit kirsten rat sarcoma viral oncogene homolog mammalian target of rapamycin nuclear factor kappa b non - small cell lung cancer programmed death ligand 1 phosphoinositide-3-kinase phosphatase and tensin homolog squamous cell carcinoma sex - determining region y - box 2 signal transducer and activator of transcription the cancer genome atlas squamous cell carcinoma ( scc ) of the lung is the second most common subtype of lung cancer , with a 5-year survival rate of only 15% . the poor prognosis associated with scc results from ineffective standard of care chemotherapy and a lack of alternative targeted therapies . targeted therapies that have positive responses in adenocarcinoma , the other major subtype of non - small cell lung cancer ( nsclc ) , are often inadequate or contraindicated for scc . there is an urgent need to identify the genes and pathways driving scc , which should represent new therapeutic targets . numerous studies have identified genetic alterations in lung adenocarcinoma , but only recently has lung scc been extensively explored at the genomic level ( figure 1 ) . in 2012 , the cancer genome atlas ( tcga ) research network sequenced 178 human sccs . this study identified therapeutically targetable mutations in lung scc and also highlighted genetic distinctions between scc and adenocarcinoma . this altered genomic spectrum in scc may explain why targeted therapies for adenocarcinomas have historically failed in scc patients . this makes it challenging to distinguish driver mutations , which represent promising therapeutic targets , from passenger mutations , which are simply carried along for the ride . mouse models of adenocarcinoma and small cell lung cancer have shown that although mouse tumors have fewer genetic alterations than their human counterparts , they exhibit similar gene expression signatures and acquire key genomic changes found in the human disease . mouse models for lung scc should thus provide a genetic filter to pinpoint key pathways driving the disease . there has been an explosion of mouse models for lung scc over the last 7 years . one of the first models generated involved homozygous inactivation of liver kinase b1 ( lkb1 ) in the context of kirsten rat sarcoma viral oncogene homolog ( kras)-driven lung tumors . a second mouse model was generated by a knock - in of kinase dead inhibitor of nuclear factor kappa - b kinase subunit ( ikk ) , which led exclusively to squamous tumors . recently , a conditional genetic model involving simultaneous loss of lkb1 and phosphatase and tensin homolog ( pten ) was developed . this model exclusively generated squamous lung tumors and is a major step forward in the field . in our recent work , we used a rapid , reverse - genetic approach to deliver combinations of clinically relevant genetic alterations specifically to the mouse lung using lentiviruses . bicistronic lentiviruses were delivered specifically to the mouse lung using intranasal inhalation , allowing constitutive expression of 2 genes driven by 2 separate promoters . using this approach we discovered that expression of sox2 together with loss of lkb1 led to lung scc with a latency of 57 months . these tumors highly resembled human scc in terms of histopathology and expression of scc biomarkers including keratin-5 , keratin-14 , and tumor protein p63 ( tp63 ) . this is clinically important because sox2 is one of the most commonly overexpressed and amplified genes in lung scc , but until now has not been demonstrated as a driver of squamous lung tumors . our approach could easily be adapted to test the role of candidate genes or gene combinations that drive lung scc in a swift and relatively inexpensive manner . with the advent of clustered regularly interspaced short palindromic repeats ( crispr ) technology , this approach is amenable to altering combinations of genes without the time and expense of traditional genetic engineering . prominent large - scale genomic analyses and clinically relevant mouse models of lung cancer subtypes are depicted . mouse models of squamous cell lung cancer have lagged behind those for adenocarcinoma and small cell lung cancer , but a recent explosion of mouse squamous lung tumor models has suggested new therapeutically relevant targets . alk , anaplastic lymphoma kinase ; braf , v - raf murine sarcoma viral onco homolog b1 ; cnv , copy number variation ; egfr , epidermal growth factor receptor ; eml4 , echinoderm microtubule associated protein like 4 ; fgf9 , fibroblast growth factor 9 ; fgfr2 , fibroblast growth factor receptor 2 ; ikk , inhibitor of nuclear factor kappa - b kinase subunit ; kras , kirsten rat sarcoma viral oncogene homolog ; lkb , liver kinase b1 ; c - myc , avian myelocytomatosis viral oncogene homolog ; pik3ca , phosphatidylinositol 4,5-biphosphate3-kinase catalytic subunit ; p130 , retinoblastoma - like 2 ; p53 , tumor protein p53 ; pten , phosphatase and tensin homolog ; rb , retinoblastoma protein ; sox2 , sex - determining region y - box2 ; spc , surfactant protein c ; tcga , the cancer genome atlas . prominent large - scale genomic analyses and clinically relevant mouse models of lung cancer subtypes are depicted . mouse models of squamous cell lung cancer have lagged behind those for adenocarcinoma and small cell lung cancer , but a recent explosion of mouse squamous lung tumor models has suggested new therapeutically relevant targets . alk , anaplastic lymphoma kinase ; braf , v - raf murine sarcoma viral onco homolog b1 ; cnv , copy number variation ; egfr , epidermal growth factor receptor ; eml4 , echinoderm microtubule associated protein like 4 ; fgf9 , fibroblast growth factor 9 ; fgfr2 , fibroblast growth factor receptor 2 ; ikk , inhibitor of nuclear factor kappa - b kinase subunit ; kras , kirsten rat sarcoma viral oncogene homolog ; lkb , liver kinase b1 ; c - myc , avian myelocytomatosis viral oncogene homolog ; pik3ca , phosphatidylinositol 4,5-biphosphate3-kinase catalytic subunit ; p130 , retinoblastoma - like 2 ; p53 , tumor protein p53 ; pten , phosphatase and tensin homolog ; rb , retinoblastoma protein ; sox2 , sex - determining region y - box2 ; spc , surfactant protein c ; tcga , the cancer genome atlas . the mouse models highlighted here have identified therapeutically relevant pathways that are active in scc phosphoinositide-3-kinase ( ptdins-3k)/mammalian target of rapamycin ( mtor ) , janus kinase ( jak)/signal transducer and activator of transcription ( stat ) , nuclear factor kappa b ( nf-b ) , and programmed death ligand 1 ( pd - l1)thus underscoring the usefulness of in vivo models . squamous tumors generated in both the pten;lkb1 loss and lenti - sox2;lkb1 models exhibit activation of the mtor pathway . interestingly , immune - related and inflammatory pathways are commonly altered across all mouse models of lung scc : activated stat3 pathway in the lenti - sox2;lkb1 mice , activated nf-b pathway in the kinase dead ikk knock - in mice , and an increase in immune checkpoint molecule pdl1 in the pten;lkb1 loss mice . these findings are clinically relevant as genomic studies of human scc reveal somatic alterations in genes involved in the immune response , including human leukocyte antigen - a ( hla - a ) . in addition , immunomodulatory drugs are exhibiting impressive response rates in clinical trials . although it is currently not clear how to predict which patients will achieve durable responses to immunotherapies , these mouse models may serve as tools to decipher the mechanisms that dictate therapeutic response . advances in mouse models of squamous lung tumors have highlighted many provocative but unanswered questions . it is unclear whether sccs arise from basal cells or whether the transforming events promote basal cell differentiation . this should be explored by expressing candidate genes that drive lung scc in specific cell types of the lung . in addition , world health organization classification ( 2004 ) recognizes 4 variants or subtypes of scc that can be stratified by gene expression . given that each tumor subtype has different survival outcomes for patients , these subtypes need to be modeled independently to understand how they impact treatment response . finally , for efficient translation of results obtained from mice to the clinic , it is important to establish paradigms for preclinical testing . clinically relevant mouse models need to be harnessed to investigate both responsiveness and resistance to targeted therapy . high - throughput genome sequencing has provided a global view of the genetic alterations associated with lung scc . mouse studies have implicated potential new drug targets for scc that need to be investigated in the preclinical setting . models such as these will ultimately impact the lives of patients suffering from this life - threatening disease .

the evolution of pathogens and their hosts is often interpreted as an arms race : while hosts have developed multiple mechanisms to protect themselves , pathogens have generated diverse strategies to evade their hosts ' defenses . but pathogens and hosts have also evolved mechanisms that allow a mutualistic coexistence . during their coevolution , a relationship between pathogen and host has been established based on intricate and specialized molecular interactions . one of the possible outcomes of this long - standing and close relationship is the exchange of genetic material . horizontal or lateral gene transfer ( ht ) is the nonsexual movement of genetic information between two organisms . these sequences can be modified and adapted ( i.e. , coopted , domesticated ) during the evolution of the recipient species to improve their own survival . when ht occurs between hosts and their pathogens ( in one direction or the other ) , the acquired sequences can be coopted to affect how the two organisms interact with each other . pathogens have developed an impressive array of strategies to avoid host defenses , including the interference or disruption of the host defensive mechanisms and signaling cascades . for instance , vertebrate viruses can avoid detection and elimination by the host immune response by obstructing antigen presentation , blocking apoptosis , disrupting complement cascades , and mimicking or modulating cytokines and their receptors , among others [ 2 , 3 ] . i will discuss how several of these strategies have been achieved through the acquisition and domestication of host genes . hosts , in turn , can protect themselves from the deleterious effects of infections by two approaches : resistance and tolerance [ 4 , 5 ] . resistant traits reduce damage by limiting the pathogen growth and , therefore , the extent of the infection , sometimes eliminating the pathogen temporarily . many pathogens evolve very quickly , developing novel or improved infection strategies ; this , in turn , has driven a rapid diversification of many host defense proteins . for instance , mitochondrial antiviral signaling proteins ( mavs ) have evolved under strong positive selection in primates , driven by viral antagonism . rapid evolution of host proteins can be particularly important for counteracting molecular mimicry of host molecules , a mechanism developed by some pathogens ( usually by coopting horizontally transferred host defense genes ) to evade the host protective response . in addition , resistance can also be achieved by hosts acquisition and domestication of genes from their parasites , and several examples will be discussed . in contrast , tolerance is attained by diminishing the pathogenic consequences of infection without reducing or eliminating the pathogen [ 4 , 5 ] . although resistance and tolerance can have similar short - term consequences , they have different long - term dynamics : resistance traits often imply physiological costs for the host ; thus , their benefits decrease as the risk of infection diminishes in the population . therefore , fixation of complete resistance traits is unlikely to occur . in contrast , tolerance does not reduce the infection and represents an advantage as pathogens multiply . consequently , tolerance traits can spread in the population until they become fixed , resulting in evolutionarily stable associations between hosts and parasites . it has been postulated that mutualism could evolve from parasitism due to natural selection for host tolerance . moreover , the interaction between the two organisms could be beneficial for both in some circumstances , while disadvantageous to either host or parasite in others . ht can also contribute to this conditional mutualism by providing novel genes to hosts or parasites that allow them to survive without a costly toll on their partners , at least under certain conditions , and several examples will be presented . i will start by reviewing the extent of ht in general and the possible role of pathogens as vectors of ht . i will later discuss ht from parasites to hosts and from hosts to parasites , including representative cases of domestication of the acquired sequences ( figure 1 ) . the studies presented here reveal that ht has been a source of novel genetic material that has shaped the interactions ( whether purely parasitic or conditional mutualistic ) and evolution of many organisms . ht is often observed in prokaryotes , as well as in viruses , and has played an important role in their evolution . through ht , they can incorporate dna from both related and unrelated organisms and adapt to novel environmental conditions , including the infectious way of life [ 1 , 9 , 10 ] . in fact , ht was first discovered by the ability of bacteria to incorporate drug resistance genes from other organisms ; since then , numerous studies have found that horizontally transferred ( htd ) sequences constitute a substantial part of the genome of bacteria and archaea , blurring the boundaries between species [ 1113 ] . prokaryotes can not only exchange genetic material with other prokaryotes and viruses with viruses , but also between them and with eukaryotes . however , there are few reports of eukaryote - to - prokaryote ht . prokaryotes ( as well as viruses ) are unable to process introns , which posses a difficulty for the domestication of many eukaryotic genes [ 1416 ] . it has also been postulated that , being more abundant and diverse than eukaryotes , prokaryotes might have more opportunities for ht and more functionally diverse genes to offer to other organisms . compared to prokaryotes , ht has been considered a rare event in eukaryotes . an exceptional phenomenon is that of introgression in animals and plants as a result of hybridization between related species : when hybrids survive and reproduce , they may transfer sequences from one species into the other [ 17 , 18 ] . however , in general , integration of htd sequences in the germ line can be troublesome in eukaryotes with confined and sheltered reproductive systems [ 1 , 19 ] . furthermore , fixation of foreign genes depends on the selective forces operating on the novel acquired sequences and on other factors affecting the population dynamics of each species [ 15 , 2022 ] . nevertheless , there are multiple examples of ht in eukaryotes [ 1 , 19 , 20 ] . apart from the well - known relocation of genes of endosymbiotic organelles ( mitochondria , plastids ) to the eukaryotic nucleus , ht is relatively frequent among plant mitochondria and in microbial eukaryotes [ 1 , 20 , 23 ] . in protists , especially in those with phagotrophic lifestyles , ht is quite common , and most transferred sequences are of bacterial origin [ 20 , 24 ] . this is not surprising since the most common sources of foreign dna are the organisms in close contact with eukaryotes ( particularly bacteria , but also other pathogens and parasites ) . bacteria are very diverse and can provide genes for novel functions that might allow eukaryotes to colonize novel environments [ 15 , 20 ] . in contrast with protists , ht appears to be a rare event in animals and fungi [ 20 , 2527 ] . studies of transposable elements ( which constitute a large part of eukaryotic genomes ) have shown that ht has occurred during the evolution of larger animals more often than previously expected . once transferred , transposable elements can take advantage of their inherent ability to mobilize and integrate into chromosomes , increasing their probability of fixation with respect to other htd dna sequences [ 17 , 19 , 29 ] . ht of transposable elements has been observed between closely related species [ 3033 ] , but also across distantly related species [ 34 , 35 ] and even distant locations : such is the case of several transposable element families , which have been found in diverse tetrapods of different continents and even in invertebrates [ 22 , 3640 ] . moreover , the data suggest that each of the transposable element families found in tetrapods have undergone not one but multiple ht events . although many studies have shown that ht in vertebrates is more common than previously thought , it is unclear how ht of homologous sequences could occur multiple times in distant species . one of the hypotheses is that pathogens might act as vectors capable of capturing host sequences and donating them to other hosts [ 17 , 19 ] . this hypothesis is supported by several observations ; for instance , several of the transposable elements found in tetrapods have also been observed in several parasites [ 37 , 39 ] , such as trypanosomes . these parasitic protozoa can not only capture but also donate sequences to their hosts : ht of trypanosome sequences has been reported in infected human beings , who vertically transmitted them to their children . in the genus drosophila , a parasitic mite appears to be responsible for the ht of p elements among different species . pathogens such as bacteria and viruses could also play a key role as ht vectors ; for instance , wolbachia ( an intracellular parasitic bacteria that is horizontally transmitted ) can donate genetic material to the nuclear genome of its insect and nematode hosts [ 44 , 45 ] . moreover , wolbachia can be infected by bacteriophages , and it has been suggested that these viruses might mediate ht among intracellular bacteria . viruses might also mediate the ht of transposable elements from lepidopteran hosts to their parasitic wasps [ 19 , 46 ] . in fact , viruses are potential ht vectors not only among vertebrates , but also in other eukaryotes and prokaryotes . the genomes of large dna viruses , such as poxviruses , contain many genes derived from their bacterial and eukaryotic hosts [ 9 , 10 ] ( including transposable elements ) . poxviruses also seem to have changed hosts recurrently during their evolution , broadening their opportunities to transfer sequences among distantly related species . for instance , a transposable element related to snake sequences has been found in the genome of a taterapox virus isolated from a rodent . therefore , poxviruses may be good ht vectors due to their large genomes and low host and cell specificity . other potential vectors are double - stranded rna viruses , which are likely the donors of multiple sequences found in the nuclear genome of very diverse eukaryotes , including plants , fungi , protozoa , arthropods , and nematodes [ 5052 ] . regardless of the frequency of ht events in diverse species , the fact is that many of them are deleterious , are not transmitted , or do not succeed in the populations . . regardless of their mobile or nonmobile nature , horizontally transferred sequences can carry new coding or regulatory sequences to the recipient organisms , upon which natural selection can operate . therefore , htd sequences have the potential of providing novel functions ( or improved versions of existing ones ) that turn out to be beneficial for the receivers . indeed , htd sequences have at times been domesticated ( for instance , for protective or pathogenic functions in the case of hosts and parasites ) and , thus , successfully maintained in the genome of the recipient species . the first class includes htd genes that were acquired from other pathogens and provided functions related to infectivity . such is the case of the transfer of fungal genes , or even entire extranumerary chromosomes , that converted nonpathogenic fungi into pathogens [ 27 , 54 ] . the htd genes that contributed to the adaptation of nematodes to plant parasitism are another example [ 35 , 55 ] . physical proximity obviously facilitates ht , although it does not guarantee that the inserted sequence will be retained in the population . but modifications of host genes can provide selective advantages and drive their fixation in the pathogen population , especially when such adaptations serve to elude or counteract the host defenses . for instance , there are several reports of bacterial sequences of eukaryotic origin [ 5658 ] . some of these sequences might have been coopted by bacteria to their advantage against their host . as previously discussed , several parasitic animals contain transposable elements likely of host origin [ 41 , 43 ] . ht from host to parasitic plants can also occur , affecting not only mitochondrial genes , but also nuclear sequences [ 5961 ] . for instance , the parasitic flowering plant rafflesia cantleyi expresses several genes ( mostly nuclear ) that were likely acquired from their hosts , which are also flowering plants . in addition , many vertically inherited genes in the rafflesia genome display codon - usage properties that are more similar to those of the host genes than to sequences of related species . therefore , while the close host - parasite relationship could facilitate ht , maintenance of the transferred genes may have been favored by codon - usage convergence between rafflesia and its host . ht of host genes has been particularly common in nucleocytoplasmic large dna viruses , such as poxviruses and herpesviruses [ 2 , 3 ] . these viruses are large enough to allow the incorporation of foreign genes . however , the number and size of the insertions are limited by the packaging restrictions of these viruses ; in addition , htd sequences can be removed over time by deletions ( which occur at a high rate in viruses ) . nevertheless , large dna viruses have been quite proficient at domesticating host genes for eluding multiple aspects of the immune response [ 2 , 6265 ] . poxviruses are particularly prone to exchange genes with their hosts [ 9 , 14 , 47 , 48 , 66 ] . within this viral group ( which includes the vaccinia and smallpox viruses ) , many of the host - derived genes vary among different genera . this suggests that ht may have contributed to poxvirus diversification and adaptation to diverse hosts [ 14 , 65 ] . host - derived genes tend to be located at the two ends of the poxviral linear genome , along with poxviral genes involved in limiting the host antiviral response ; these extremes seem more recombinogenic than the central region , which contains conserved and essential genes [ 14 , 47 ] . in an extensive analysis , hughes and friedman characterized 16 poxviral gene families that were acquired from their hosts . these findings have been further expanded in other studies [ 6669 ] , supporting the existence of many host homologues in the genomes of poxviruses . other studies have also reported numerous ht events from hosts to herpesviruses during evolution [ 2 , 7073 ] . the genomes of poxviruses and herpesviruses contain several host homologues coding for proteins that modulate the immune response . many of the coopted htd genes code for proteins that interact with host ligands or receptors , targeting cytokines , and innate immune mechanisms [ 3 , 6365 ] . for instance , homologues of chemokines and chemokine receptors are found in both poxviruses and herpesviruses [ 2 , 62 , 73 ] . the host versions of these proteins recruit leukocytes to the sites of infection and , thus , are key components of the antiviral defense pathways . to oppose their effects , viruses express proteins that mimic these components and interfere with their normal functions . one of the best - studied examples is the ht of mammalian interleukin-10 family members ( il-19 , il-20 , and il-24 ) to viruses . in human epstein - barr virus ( a herpesvirus ) , a viral homologue was identified as an agonist with impaired binding to the host il-10 receptor ; moreover , it contributes to the downregulation of the host immune response during early infection [ 75 , 76 ] . homologues of il-10 family members have also been found in other herpesviruses ( such as the cytomegalovirus ) , as well as in poxviruses . studies in the latter have shown that il-10 family homologues also have immunosuppressive properties , because they contribute to delaying the development of acquired immunity to orf virus in humans and to vaccinia virus in mice [ 78 , 79 ] . in addition , phylogenetic studies have shown that il-10 homologues were acquired multiple times in poxviruses by independent ht events [ 48 , 66 , 80 ] . the recurrent ht of these host genes to poxviruses and herpesviruses further supports the idea that the acquisition of il-10 family members provided a selective advantage to them . other examples of genes of the host defense machinery that have been horizontally transferred to poxviruses include those coding for proteins of the mhc class i , other interleukins and their receptors and the interferon gamma receptor [ 9 , 65 ] . although in some cases they interfere with the normal action of homologous proteins in the host , in other cases the acquired genes have adopted new immunomodulatory functions [ 65 , 81 ] . poxviral genomes have also incorporated host sequences for serine protease inhibitors ( serpins , involved in inflammation control ) , glutaredoxin ( which has antiapoptotic effects under oxidative stress ) , and glutathione peroxidase , [ 9 , 14 , 66 ] . in the host , glutathione peroxidase has a protective role against oxidative stress , a function that has been adapted by certain poxviruses to protect themselves and the infected cells from the oxidative damage that results from the immune response . in all these examples , host - to - virus htd genes enhance viral survival and propagation by interfering with the immune response ; however , other host - derived genes adopted by viruses contribute to control their own infectivity . such is the case of the interleukin-24 ( il-24 ) gene homolog found in the yaba - like disease virus ( a poxvirus ) , which appears to reduce its virulence upon infection . another example found in poxviruses is that of homologues of the mammalian schlafen gene family . these genes are expressed in cells of the host immune system and were horizontally transferred to orthopoxviruses , probably from rodents [ 67 , 84 ] . studies of the schlafen viral homologue in a recombinant viral model suggested that the gene product might contribute to decrease virulence . although little is known about the function of the schlafen genes in the host , functional studies of viral schlafen copies and of the interactions between the host and viral homologues may be instrumental for unveiling the contribution of this gene family to the immune response . similarly , studies of other htd genes in both hosts and pathogens might provide complementary insights into diverse aspects of immunology and infectivity . in addition , htd genes involved in modulating immunity and virulence constitute potential therapeutic targets [ 3 , 64 , 65 , 86 , 87 ] . the existence of host - to - virus htd genes that restrain their infectivity supports the notion that the evolution of hosts and viruses , like other organisms considered pathogens , can not simply be viewed as an arms race . for example , upon infection , herpesviruses remain usually latent during the host lifetime and only become reactivated and pathogenic under particular conditions . coevolution with their hosts and their immune response has allowed these viruses to persist with little cost to animals . in the course of this evolution , herpesviruses have horizontally acquired certain vertebrate genes and coopted them to evade the immune response [ 73 , 75 , 76 ] . moreover , during latency , herpesviruses can also modulate the defenses against other diseases , providing immunity against some secondary infections in mice [ 88 , 89 ] . therefore , herpesviruses could be considered as part of our normal defensive response , with both beneficial and detrimental consequences . through evolution , they have acquired some advantageous sequences from their hosts , but they have also evolved mechanisms to modulate the immune system that benefit both parts . in summary , htd genes from host to pathogens can be domesticated by the latter to regulate not only the host defense response , but also their infectivity . consequently , these genes have contributed to the coevolution between pathogens and their hosts . on one hand , ht provides new tools to the pathogen to ensure its propagation and even to occupy new niches . on the other , genes that originally contributed to the host defensive response may become instruments to elude it , leaving the host exposed to the pathogen attack . thus , it has been postulated that ht of host defense genes to viruses and other pathogens may have been counteracted through rapid evolution of the original copies in the host . the ability of pathogens to exploit the similarities of their gene products with their host homologues to elude the defense response may have contributed to drive the rapid diversification of host defense genes in mammals . recently several reports have brought light into the relevance of ht from pathogens to their hosts , even to those species with a sheltered germ line ( such as vertebrates ) . as previously discussed , there are several reports of animals bearing certain sequences that are more similar to sequences of their parasites than to those of related species ; these sequences were likely transferred from the parasites to their hosts . the sequence donors include eukaryotic parasites ( such as trypanosomes [ 42 , 43 ] ) , prokaryotic pathogens ( such as wolbachia [ 44 , 45 ] ) , and viruses ( discussed below ) . in plants , several data support the existence of ht of mitochondrial genes from parasitic to host flowering plants as well as ht of genes of pathogenic bacteria to their host plants [ 90 , 91 ] . in some cases , the sequences acquired by ht have been modified , adopting novel functions in the recipient hosts . in fact , several studies of coevolution of diverse viruses and their hosts have arrived to the same conclusion : viral sequences have been the likely origin of many host defense mechanisms ( reviewed in ) . this is not surprising if we consider that some viruses can provide resistance to their prokaryote or eukaryote hosts against secondary infections [ 88 , 92 ] . the intimate relationship between phages and bacteria facilitates ht , and the advantageous nature of antiviral sequences promotes their retention and selection in the host . an example is found in restriction - modification systems present in prokaryotes as major defenses against viral attacks . some of them have also been found in prophages , suggesting a viral origin of the restriction - modification genes [ 92 , 93 ] . in addition , bacteria and archaea also have virus resistance mechanisms based on the incorporation of viral sequences : the clusters of regularly interspaced palindromic repeats ( crisps ) . these loci integrate small segments of bacteriophage and plasmid dna to generate rna - mediated defenses against viral and plasmidic invasions [ 92 , 94 , 95 ] . it has been proposed that the widespread ht of double - stranded rna viral sequences to several eukaryotic groups generated novel functional genes , some of which could be involved in protection against exogenous viral infections [ 5052 ] . in many eukaryotes , an antiviral defensive response with possible viral origin is that provided by small rnas , which interfere or silence viral gene expression . studies of c. elegans small rnas suggest they were originated from transposable elements and endogenous retroviral sequences . in vertebrates , retroviruses constitute a major source of exogenous sequences ( up to 8% of the human genome ) , because they can integrate into their hosts genomes as proviruses , occasionally , reach the germline of the infected animals , and get passed on to the next generations . consequently , retroviral sequences , as well as dna from other endogenous viral elements , have been coopted numerous times by the hosts . in some cases , they have been domesticated to interfere with molecular interactions required for virus integrity and infectivity , including viral - host interfaces . the best - known example is the friend - virus - susceptibility-1 gene ( fv1 ) , a retrovirus - resistance gene found in mice . it is a domesticated version of the gag gene of the muerv - l retrovirus [ 98100 ] . other retroviral genes coopted by their hosts to serve as antiviral defenses include fv4 , rcmf , and rmcf2 . all these genes are derived from the env genes of endogenous retroviruses and are found in mice , in which they interfere with the activity of viral receptors and with viral entry [ 99 , 101 ] . there are several alleles of this gene in mice , and the allele that provides the most resistance has the highest levels of expression . it turns out that this elevated expression is due to the intronic insertion of a transcriptional enhancer of retroviral origin [ 99 , 102 ] . therefore , not only viral genes , but also regulatory sequences can be coopted by hosts for antiviral purposes . at a larger scale , villarreal [ 92 , 103 ] has proposed that , not only individual genes , but also coordinated processes coded by multiple genes in viruses have been the likely origin of the vertebrate adaptive immune system . this hypothesis is based on several observations : first , the emergence of a complex immune system in jawed vertebrates ; second , the large viral colonization and genome expansion occurred in teleost fish , compared with those of their presumed ancestors ; third , the presence in viruses of elements or networks that resemble several components of the adaptive immune response ( for instance , t - cell receptors have several features that are similar to those of viral receptors ) [ 92 , 103 , 104 ] . therefore , ht and domestication of viral sequences could have originated the basal components of a highly complex adaptive immune system in the emergent vertebrates . the tight physical and evolutionary relationship between pathogens and their hosts facilitates ht of dna sequences . although the acquisition of host genes by viruses , prokaryotes , and other parasites has been recognized for a long time , the relevance of ht in the opposite direction , from pathogens to their hosts ( and especially to those that seemed more protected from germ line insertions , such as vertebrates ) , has been only acknowledged recently . however , one thing is the transfer of a dna sequence and another is its spread and maintenance in the population , which depend on the selective forces operating on the foreign sequence . any htd sequence that provides a selective advantage to the host against the pathogen or vice versa , or is able to benefit the coexistence of both , is more likely to be retained than a sequence that does not provide any competitive advantage . htd genes have been coopted by pathogens to modulate the host defense response and ensure their survival and multiplication , and have been domesticated by hosts to improve their protection against deleterious infections . in addition therefore , horizontal gene transfer can be an important factor in the evolution of hosts , pathogens , and the ways they interact with each other .

the prevalence of diabetes mellitus ( dm ) and prediabetes is rapidly increasing worldwide , especially in the developing countries . as a result , both the increase in medical costs and the decrease in the quality of life are becoming formidable challenges . the early diagnosis is of fundamental importance to the management of diabetes and prevention of diabetes - related complications . nevertheless , the majority of patients with type 2 diabetes are asymptomatic , and over 30% of type 2 diabetic patients have not been diagnosed until systemic complications occurred . therefore , screening for diabetes , in particular among high - risk population , is essential for achieving optimal outcomes of diabetes intervention . to date , the conventional approaches to screening for diabetes include evaluation of fasting plasma glucose ( fpg ) , hemoglobin a1c ( hba1c ) , and fpg in combination with 2-h plasma glucose ( 2 h - pg ) after a 75 g oral glucose tolerance test ( ogtt ) . however , fpg alone has not been sensitive enough in detecting postprandial glycemic excursions , while hba1c has not been recommended for diabetes screening in china because of several issues . for example , diabetes diagnosis based on hba1c ( i.e. , hba1c 6.5% ) was reported to result in a significant underestimation of the prevalence of diabetes in both community and hospital based diabetes screening patients in china . furthermore , hba1c measurement is laboratory - dependent and relatively expensive , which limit its application in population - based screening , especially in low - income regions . ogtt has been used as the gold standard test for the diagnosis of diabetes , which involves venipuncture for blood sampling during fasting and at 2-h after oral ingestion of 75 g glucose and is reported as the more commonly used approach in china . however , the compliance of a standard ogtt is often poor , mostly due to the addition of venipuncture and blood sampling at 2-h after oral glucose . therefore , alternative methods that are reliable , noninvasive , economical , and convenient are needed for diabetes screening . the measurement of postprandial urine glucose ( pug ) appears to be an attractive option , which reflects the glycemic excursions in excess of the renal glucose threshold . for example , semiquantitative measurement using reagent strips is frequently used to monitor glycosuria and is recommended by the international diabetes federation when blood glucose monitoring may not be accessible or affordable . quantitative measurement of pug allows more precise assessment on the average elevation of postprandial glucose . although it is limited by retrospective and indirect interpretation , we have shown that quantitative monitoring of urine glucose has comparable efficacy to blood glucose on glycemic control . however , its value in diabetes screening has not been reported . in this study , we evaluated the performance of fpg combined with 2 h - pug in diabetes screening in chinese high - risk population , with the measurements of fpg and 2 h - pg after 75 g oral glucose challenge as reference tests . a total of 1035 subjects with high - risk factors of diabetes were recruited by advertisement from eight community health service centres in nanjing , china , from september 2010 to september 2011 with a nonresponse rate of 12.2% . eligibility of subjects was assessed through a face - to - face interview , according to the 2010 guidelines for prevention and treatment of type 2 diabetes in china . risk factors for prediabetes and diabetes include the following : ( 1 ) previously reported impaired fasting glucose ( ifg ) and/or impaired glucose tolerance ( igt ) ; ( 2 ) being older than 40 years at the entry of the study ; ( 3 ) body mass index ( bmi ) 24 kg / m and/or the waist circumference 90 cm in males or 85 cm in females ; ( 4 ) having a family history of diabetes in first - degree relatives ( i.e. , parents or siblings ) ; ( 5 ) having an ethnic background of han ; ( 6 ) giving birth to a baby weighing more than 4 kg or being diagnosed with gestational diabetes ; ( 7 ) hypertension ( blood pressure 140/90 mmhg ) or being treated for high blood pressure ; ( 8) high - density lipoprotein cholesterol below 0.91 mmol / l , or triglyceride above 2.22 mmol / l , or taking lipid - regulating drugs ; ( 9 ) having a history of cardio - cerebro - vascular diseases or being physically inactive ( < 150 min / week ) ; ( 10 ) having a history of transient hyperglycemia induced by steroid ; and ( 11 ) presence of polycystic ovary syndrome in females . subjects who presented one or more high - risk factors at the time of interview were considered eligible but were excluded if they met one of the following criteria : ( 1 ) unstable health conditions ( i.e. , unstable angina pectoris , blood pressure > 200/100 mmhg , and infections ) ; ( 2 ) severe psychiatric disturbance ; ( 3 ) cancer on radio- and/or chemo - therapy ; ( 4 ) taking medications which may potentially affect blood and/or urine glucose levels ; or ( 5 ) suffering from chronic kidney disease , urinal tract infection , or benign prostate hyperplasia . the protocol was approved by the research ethics committee of zhongda hospital and was conducted in accordance with the principles of the declaration of helsinki as revised in 2000 . after at least 10 h of overnight fast , subjects ingested 75 g anhydrous glucose dissolved in water to a volume of 250 ml within 5 min . fasting venous blood was sampled for the measurements of fpg and hba1c . at 2-h after oral glucose , venous , and urine samples were collected for the measurements of 2 h - pg and 2 h - pug . the definition of glycemic status was based on the measurements of fpg and 2 h - pg after ogtt , according to the cut - offs of american diabetes association . briefly , diagnosis of diabetes is based on fpg 7.0 mmol / l and/or 2 h - pg 11.1 prediabetes include igt ( i.e. , 2 h - pg 7.8 mmol / l and 11.0 mmol / l ) and/or ifg ( i.e. , fpg 5.6 mmol / l and 6.9 mmol / l ) . normal glucose tolerance ( ngt ) is defined based on fpg < 5.6 mmol / l and 2 h - pg < 7.8 these were used as the references to evaluate the performance of fpg combined with 2 h - pug in detecting both glucose abnormalities and diabetes . fpg and 2 h - pg were measured by glucose oxidase method using an automatic chemistry analyzer ( synchron lx-20 , bechman coulter inc . , hba1c was determined by high - performance liquid chromatography ( hplc , d-10 , bio - rad inc . , urine glucose concentrations were assayed using a quantitative urine meter ( ug-201-h , tanita corporation , tokyo , japan ) . the high - sensitive amperometric glucose sensor allows accurate measurement in the range of 0111.1 mmol / l with a rapid response time of 6 s. all analyses were performed using spss 17.0 ( spss inc . , a sample size of 460 subjects was calculated to have 81% power ( at = 0.05 ) to detect a 30% difference in sensitivity between two diagnostic tests , assuming the inter - test discordant proportion of 50% and the prevalence of diseased individual of 10% . the 1035 subjects were recruited to the trial due to overestimation of the nonresponse and dropout rates in the community environment . because 2 h - pug data were not normally distributed , spearman 's correlation was used to assess the relationships between 2 h - pug and other glycemic variables , including fpg , 2 h - pg , and hba1c . wallis test was first performed to compare the differences among subjects with different glycemic status , followed by mann - whitney u - test for subgroup comparisons between diabetic subjects and subjects with isolated impaired fasting glucose ( iifg ) , isolated impaired glucose tolerance ( iigt ) , ifg and igt , and ngt . to develop a new method that combines fpg and 2 h - pug for glycemic screening , youden indexes with various cut - offs were calculated based on the receiver operating characteristic ( roc ) curve analysis . cut - off values of 2 h - pug to predict the 2 h - pg of 7.8 mmol / l and 11.1 mmol / l were determined to provide the largest youden indexes . mcnemar 's test was used to compare different ratios . the likelihood ratio ( calculated as sensitivity/[1 specificity ] ) was calculated to estimate the odds of having glucose abnormalities and diabetes , based on the screening values of fpg and 2 h - pug . a total of 1035 subjects with high - risk factors of diabetes were recruited by advertisement from eight community health service centres in nanjing , china , from september 2010 to september 2011 with a nonresponse rate of 12.2% . eligibility of subjects was assessed through a face - to - face interview , according to the 2010 guidelines for prevention and treatment of type 2 diabetes in china . risk factors for prediabetes and diabetes include the following : ( 1 ) previously reported impaired fasting glucose ( ifg ) and/or impaired glucose tolerance ( igt ) ; ( 2 ) being older than 40 years at the entry of the study ; ( 3 ) body mass index ( bmi ) 24 kg / m and/or the waist circumference 90 cm in males or 85 cm in females ; ( 4 ) having a family history of diabetes in first - degree relatives ( i.e. , parents or siblings ) ; ( 5 ) having an ethnic background of han ; ( 6 ) giving birth to a baby weighing more than 4 kg or being diagnosed with gestational diabetes ; ( 7 ) hypertension ( blood pressure 140/90 mmhg ) or being treated for high blood pressure ; ( 8) high - density lipoprotein cholesterol below 0.91 mmol / l , or triglyceride above 2.22 mmol / l , or taking lipid - regulating drugs ; ( 9 ) having a history of cardio - cerebro - vascular diseases or being physically inactive ( < 150 min / week ) ; ( 10 ) having a history of transient hyperglycemia induced by steroid ; and ( 11 ) presence of polycystic ovary syndrome in females . subjects who presented one or more high - risk factors at the time of interview were considered eligible but were excluded if they met one of the following criteria : ( 1 ) unstable health conditions ( i.e. , unstable angina pectoris , blood pressure > 200/100 mmhg , and infections ) ; ( 2 ) severe psychiatric disturbance ; ( 3 ) cancer on radio- and/or chemo - therapy ; ( 4 ) taking medications which may potentially affect blood and/or urine glucose levels ; or ( 5 ) suffering from chronic kidney disease , urinal tract infection , or benign prostate hyperplasia . the protocol was approved by the research ethics committee of zhongda hospital and was conducted in accordance with the principles of the declaration of helsinki as revised in 2000 . after at least 10 h of overnight fast , subjects ingested 75 g anhydrous glucose dissolved in water to a volume of 250 ml within 5 min . fasting venous blood was sampled for the measurements of fpg and hba1c . at 2-h after oral glucose , venous , and urine samples were collected for the measurements of 2 h - pg and 2 h - pug . the definition of glycemic status was based on the measurements of fpg and 2 h - pg after ogtt , according to the cut - offs of american diabetes association . briefly , diagnosis of diabetes is based on fpg 7.0 mmol / l and/or 2 h - pg 11.1 prediabetes include igt ( i.e. , 2 h - pg 7.8 mmol / l and 11.0 mmol / l ) and/or ifg ( i.e. , fpg 5.6 mmol / l and 6.9 mmol / l ) . normal glucose tolerance ( ngt ) is defined based on fpg < 5.6 mmol / l and 2 h - pg < 7.8 these were used as the references to evaluate the performance of fpg combined with 2 h - pug in detecting both glucose abnormalities and diabetes . fpg and 2 h - pg were measured by glucose oxidase method using an automatic chemistry analyzer ( synchron lx-20 , bechman coulter inc . , hba1c was determined by high - performance liquid chromatography ( hplc , d-10 , bio - rad inc . , urine glucose concentrations were assayed using a quantitative urine meter ( ug-201-h , tanita corporation , tokyo , japan ) . all analyses were performed using spss 17.0 ( spss inc . , chicago , il , usa ) . the data are presented as mean standard deviation ( sd ) . a p < 0.05 was considered statistically significant . a sample size of 460 subjects was calculated to have 81% power ( at = 0.05 ) to detect a 30% difference in sensitivity between two diagnostic tests , assuming the inter - test discordant proportion of 50% and the prevalence of diseased individual of 10% . the 1035 subjects were recruited to the trial due to overestimation of the nonresponse and dropout rates in the community environment . because 2 h - pug data were not normally distributed , spearman 's correlation was used to assess the relationships between 2 h - pug and other glycemic variables , including fpg , 2 h - pg , and hba1c . wallis test was first performed to compare the differences among subjects with different glycemic status , followed by mann - whitney u - test for subgroup comparisons between diabetic subjects and subjects with isolated impaired fasting glucose ( iifg ) , isolated impaired glucose tolerance ( iigt ) , ifg and igt , and ngt . to develop a new method that combines fpg and 2 h - pug for glycemic screening , youden indexes with various cut - offs were calculated based on the receiver operating characteristic ( roc ) curve analysis . cut - off values of 2 h - pug to predict the 2 h - pg of 7.8 mmol / l and 11.1 mmol / l were determined to provide the largest youden indexes . mcnemar 's test was used to compare different ratios . the likelihood ratio ( calculated as sensitivity/[1 specificity ] ) was calculated to estimate the odds of having glucose abnormalities and diabetes , based on the screening values of fpg and 2 h - pug . nine hundred and nine subjects ( 87.8% of the total number ) completed the study and were included in the analysis . bmi , waist - hip ratio ( whr ) , systolic blood pressure ( sbp ) , diastolic blood pressure ( dbp ) , and 2 h - pug were significantly higher in males than those in females without significant difference in fpg , 2 h - pg , or hba1c [ table 1 ] . demographic , anthropometric and metabolic parameters of the study participants data are shown as a mean sd . * f values ; z values sd : standard deviation ; bmi : body mass index ; sbp : systolic blood pressure ; dbp : diastolic blood pressure ; fpg : fasting plasma glucose . according to the predefined criteria , 909 subjects were categorized into five groups : ( 1 ) 156 ( 17.2% ) subjects in newly diagnosed diabetes mellitus ( nddm ) group ; ( 2 ) 92 ( 10.1% ) in iigt group ; ( 3 ) 138 ( 15.2% ) in iifg group ; ( 4 ) 74 ( 8.1% ) in igt and ifg group ; and ( 5 ) 449 ( 49.4% ) in ngt group . subjects with glucose abnormalities in addition , they had significantly higher bmi , whr , sbp , dbp , and hba1c than ngt group ( all p < 0.05 ) . 2 h - pug was highest in the nddm group , and higher in the iigt and igt and ifg groups than the iifg and ngt groups ( all p < 0.05 ) without significant difference between the iifg and ngt groups [ table 2 ] . demographic , anthropometric and metabolic parameters in nddm , iigt , iifg , igt and ifg , and ngt groups data are shown as mean sd , median ( maximum , minimum ) , or n ( % ) .*p<0.001 , p<0.01 , p<0.05 , p>0.05 , compared with ngt group by lsd . mmol / l and fpg < 5.6 mmol / l ; nddm : newly diagnosed diabetes mellitus , represents 2-h plasma glucose 11.1 mmol / l ; iigt : isolated impaired glucose tolerance , represents 2-h plasma glucose between 7.8 mmol / l and 11.0 mmol / l only ; iifg : isolated impaired fasting glucose , represents fpg between 5.6 mmol / l and 6.9 mmol / l only ; igt : impaired glucose tolerance , represents 2-h plasma glucose between 7.8 mmol / l and 11.0 mmol / l ; ifg : impaired fasting glucose , represents fpg between 5.6 mmol / l and 6.9 mmol / l ; lsd : least significant difference ; sd : standard deviation ; bmi : body mass index ; sbp : systolic blood pressure ; dbp : diastolic blood pressure ; fpg : fasting plasma glucose . h - pug were positively related to those of fpg ( r = 0.428 , p < 0.001 ) , 2 h - pg ( r = 0.551 , p < 0.001 ) , and hba1c ( r = 0.467 , p < 0.001 ) among 909 subjects . similar but weaker relationships of 2 h - pug with fpg , 2 h - pg , and hba1c ( r = 0.16 , 0.20 , and 0.34 , respectively , all p < 0.05 ) were observed in subjects with nddm . roc curves shown in figure 1 represent the diagnostic accuracy of 2 h - pug for the estimation of 2 h - pg 7.8 mmol / l and 2 h - pg 11.1 the areas under the curves were 0.772 ( 95% confidence interval [ ci ] : 0.7380.806 ) and 0.885 ( 95% ci : 0.8500.921 ) , respectively , suggesting a good accuracy of 2 h - pug in predicting 2 h - pg . youden indexes for both were calculated to determine the optimal cut - off values of 2 h - pug . 2 h - pug of 5.6 mmol / l gave an optimal sensitivity of 54.4% and specificity of 89.7% to predict 2 h - pg of 7.8 2 h - pug of 7.5 mmol / l gave an optimal sensitivity of 76.2% and specificity of 89.4% in the estimation of 2 h - pg of 11.1 receiver operating characteristic curve of 2-h postprandial urine glucose test to detect a 2-h plasma glucose 7.8 mmol / l ( a ) and a 2 h - plasma glucose 11.1 mmol / l ( b ) . mmol / l ) had a sensitivity of 84.1% ( 387/460 ) and a specificity of 86.6% ( 387/447 ) for detecting glucose abnormalities . its sensitivity is significantly higher than that of fpg alone ( fpg 5.6 mmol / l ; sensitivity : 73.7% [ 339/460 ] ) ( = 15.05 , p < 0.001 ) . by applying these paired values of fpg and 2 h - pug in glycemia screening , 50.8% ( 462/909 ) subjects would be found normal and exempt from entailing a standard 75 g ogtt . the combined measurements of fpg ( 7.0 mmol / l ) and 2 h - pug ( 7.5 mmol / l ) also had a better sensitivity ( 82.7% [ 129/156 ] ) to identify diabetic patients relative to fpg alone ( fbg 7.0 mmol / l ; sensitivity : 48.1% [ 75/156 ] ) ( = 41.29 , p < 0.001 ) , with a specificity of 90.4% ( 681/753 ) . subjects with diabetes or abnormal glucose tolerance had increased likelihood of having abnormal fpg and 2 h - pug ( i.e. , 5.6 mmol / l ) , and decreased likelihood of having normal fpg and 2 h - pug ( i.e. , 5.6 positive likelihood ratio for fpg and 2 h - pug in subjects with glucose abnormalities and diabetes normal represents 2-h plasma glucose < 7.8 mmol / l and fpg lr : sensitivity/(1specificity ) , which is used for assessing the value of performing a diagnostic test . they use the sensitivity and specificity of the test to determine whether a test result usefully changes the probability that a condition exists . fpg : fasting plasma glucose ; 2 h - pug : 2-h postprandial urine glucose ; igt : impaired glucose tolerance ; ogtt : oral glucose tolerance test ; lr : likelihood ratio . nine hundred and nine subjects ( 87.8% of the total number ) completed the study and were included in the analysis . bmi , waist - hip ratio ( whr ) , systolic blood pressure ( sbp ) , diastolic blood pressure ( dbp ) , and 2 h - pug were significantly higher in males than those in females without significant difference in fpg , 2 h - pg , or hba1c [ table 1 ] . demographic , anthropometric and metabolic parameters of the study participants data are shown as a mean sd . * f values ; z values sd : standard deviation ; bmi : body mass index ; sbp : systolic blood pressure ; dbp : diastolic blood pressure ; fpg : fasting plasma glucose . according to the predefined criteria , 909 subjects were categorized into five groups : ( 1 ) 156 ( 17.2% ) subjects in newly diagnosed diabetes mellitus ( nddm ) group ; ( 2 ) 92 ( 10.1% ) in iigt group ; ( 3 ) 138 ( 15.2% ) in iifg group ; ( 4 ) 74 ( 8.1% ) in igt and ifg group ; and ( 5 ) 449 ( 49.4% ) in ngt group . subjects with glucose abnormalities were older than those with ngt ( p < 0.05 ) . in addition , they had significantly higher bmi , whr , sbp , dbp , and hba1c than ngt group ( all p < 0.05 ) . 2 h - pug was highest in the nddm group , and higher in the iigt and igt and ifg groups than the iifg and ngt groups ( all p < 0.05 ) without significant difference between the iifg and ngt groups [ table 2 ] . demographic , anthropometric and metabolic parameters in nddm , iigt , iifg , igt and ifg , and ngt groups data are shown as mean sd , median ( maximum , minimum ) , or n ( % ) .*p<0.001 , p<0.01 , p<0.05 , p>0.05 , compared with ngt group by lsd . mmol / l and fpg < 5.6 mmol / l ; nddm : newly diagnosed diabetes mellitus , represents 2-h plasma glucose 11.1 mmol / l ; iigt : isolated impaired glucose tolerance , represents 2-h plasma glucose between 7.8 mmol / l and 11.0 mmol / l only ; iifg : isolated impaired fasting glucose , represents fpg between 5.6 mmol / l and 6.9 mmol / l only ; igt : impaired glucose tolerance , represents 2-h plasma glucose between 7.8 mmol / l and 11.0 mmol / l ; ifg : impaired fasting glucose , represents fpg between 5.6 mmol / l and 6.9 mmol / l ; lsd : least significant difference ; sd : standard deviation ; bmi : body mass index ; sbp : systolic blood pressure ; dbp : diastolic blood pressure ; fpg : fasting plasma glucose . the concentrations of 2 h - pug were positively related to those of fpg ( r = 0.428 , p < 0.001 ) , 2 h - pg ( r = 0.551 , p < 0.001 ) , and hba1c ( r = 0.467 , p < 0.001 ) among 909 subjects . similar but weaker relationships of 2 h - pug with fpg , 2 h - pg , and hba1c ( r = 0.16 , 0.20 , and 0.34 , respectively , all p < 0.05 ) were observed in subjects with nddm . roc curves shown in figure 1 represent the diagnostic accuracy of 2 h - pug for the estimation of 2 h - pg 7.8 mmol / l and 2 h - pg 11.1 the areas under the curves were 0.772 ( 95% confidence interval [ ci ] : 0.7380.806 ) and 0.885 ( 95% ci : 0.8500.921 ) , respectively , suggesting a good accuracy of 2 h - pug in predicting 2 h - pg . youden indexes for both were calculated to determine the optimal cut - off values of 2 h - pug . 2 h - pug of 5.6 mmol / l gave an optimal sensitivity of 54.4% and specificity of 89.7% to predict 2 h - pg of 7.8 2 h - pug of 7.5 mmol / l gave an optimal sensitivity of 76.2% and specificity of 89.4% in the estimation of 2 h - pg of 11.1 receiver operating characteristic curve of 2-h postprandial urine glucose test to detect a 2-h plasma glucose 7.8 mmol / l ( a ) and a 2 h - plasma glucose 11.1 mmol / l ( b ) . the combined utilization of fpg ( 5.6 mmol / l ) and 2 h - pug ( 5.6 mmol / l ) had a sensitivity of 84.1% ( 387/460 ) and a specificity of 86.6% ( 387/447 ) for detecting glucose abnormalities . mmol / l ; sensitivity : 73.7% [ 339/460 ] ) ( = 15.05 , p < 0.001 ) . by applying these paired values of fpg and 2 h - pug in glycemia screening , 50.8% ( 462/909 ) subjects would be found normal and exempt from entailing a standard 75 g ogtt . the combined measurements of fpg ( 7.0 mmol / l ) and 2 h - pug ( 7.5 mmol / l ) also had a better sensitivity ( 82.7% [ 129/156 ] ) to identify diabetic patients relative to fpg alone ( fbg 7.0 mmol / l ; sensitivity : 48.1% [ 75/156 ] ) ( = 41.29 , p < 0.001 ) , with a specificity of 90.4% ( 681/753 ) . subjects with diabetes or abnormal glucose tolerance had increased likelihood of having abnormal fpg and 2 h - pug ( i.e. , 5.6 mmol / l ) , and decreased likelihood of having normal fpg and 2 h - pug ( i.e. , 5.6 mmol / l ) [ table 3 ] . positive likelihood ratio for fpg and 2 h - pug in subjects with glucose abnormalities and diabetes normal represents 2-h plasma glucose < 7.8 mmol / l and fpg lr : sensitivity/(1specificity ) , which is used for assessing the value of performing a diagnostic test . they use the sensitivity and specificity of the test to determine whether a test result usefully changes the probability that a condition exists . fpg : fasting plasma glucose ; 2 h - pug : 2-h postprandial urine glucose ; igt : impaired glucose tolerance ; ogtt : oral glucose tolerance test ; lr : likelihood ratio . in this community - based , cross - sectional study , we showed that in chinese high - risk population , the prevalences of diabetes and prediabetes approximated 17.2% and 33.4% , both of which were higher than those reported in general chinese adults . the high prevalence of impaired glucose metabolism in this high - risk population suggested that timely screening for diabetes is demanding . we have also demonstrated the diagnostic value of quantitative measurement of 2 h - pug in combination with the measurement of fpg . despite a relatively low specificity , 2 h - pug combined with fpg has substantially improved the sensitivity in detecting both glucose abnormalities and diabetes and may represent a promising alternative for diabetes screening . a large proportion of patients with prediabetes or diabetes are asymptomatic and the latter is likely to reflect that the present tools for diabetes screening are suboptimal . for example , assessment of diabetes risk scores is economical , but it is largely dependent on self - report , such that the demographic , behavioral , and medical information are subjective . therefore , it may be simply used as an initial assessment tool to identify the high - risk individuals . although a 75 g ogtt can most acutely confirm the diagnosis of diabetes and prediabetes , it has not been recommended as a routine test for diabetes screening due to the unsatisfactory compliance . recently , hba1c being > 6.5% has been advocated as a diagnostic criterion of dm while levels ranging from 5.7% to 6.4% are considered risky . however , there is evidence that hba1c - based diagnosis of diabetes may lead to a significant underestimation in the chinese population . in addition , hba1c measurement is laboratory - dependent and not inexpensive . in this study , although hba1c levels were greater than 6.5% in nddm , but not in these with ifg or igt , application of hba1c from 5.7% to 6.4% to predict igt and/or ifg only yielded a sensitivity of 63.8% and specificity of 53.9% ( data not shown ) . therefore , hba1c measurement did not seem to be an optimal method for diabetes screening . as a result , fpg test is frequently recommended . unfortunately , as previously reported , we showed that when fpg test alone was performed , subjects with iigt and nearly half of the nddm patients would be undiagnosed . in this study , we found that fpg combined with 2 h - pug resulted in a substantial improvement in the efficacy of glycemic screening , compared to the measurement of fpg alone . although the diagnostic specificity by these paired values was relatively low , the combined use of fpg and 2 h - pug narrowed the suspected subjects who would otherwise require a standard 75 g ogtt for diagnosis . as a result in addition , the measurement of 2 h - pug entails a minimal cost and does not cause any discomfort . previously , the measurement of urine glucose in diabetes screening has been largely overlooked , probably because it was limited by semiquantitative , retrospective , and indirect interpretations , although it was reinstated in guidelines , drug benefit programs , and educational programs . in contrast to the use of traditional urine dipsticks , we were able to precisely quantity 2 h - pug in this study by a highly sensitive urine meter . the occurrence of glycosuria is known to be a result of plasma glucose concentrations in excess of the renal threshold for glucose and is , therefore , indicative of the magnitude of hyperglycemia . in line with this concept , we have demonstrated that 2 h - pug was related directly with fpg , 2 h - pg , and hba1c . furthermore , subjects with glucose intolerance or diabetes had increased the likelihood of abnormally high fpg and 2 h - pug and decreased the likelihood of normal fpg and 2 h - pug . we have previously shown that self - monitoring of urine glucose in noninsulin - treated type 2 patients has comparable efficacy on glycemic control , and facilitates better compliance than self - monitoring on blood glucose without increasing the risk of hypoglycemia . interestingly , the relationships between the 2 h - pug and the glycemic variables appeared to be weaker in patients with nddm compared with the whole study population , which may be accounted for by the smaller sample size and the wider range of renal threshold for glucose in subjects with nddm . therefore , quantification of urine glucose in conjunction with evaluation of the renal threshold is warranted for further evaluation . alternatively , it might suggest that 2 h - pug is more sensitive in reflecting glycemic excursions in prediabetes , which would support the use of 2 h - pug for diabetes screening first , the study subjects were recruited from limited community health service centers in one chinese city , which might cause selective bias . second , the definition of the glycemic status was based on the measurement of fpg and 2 h - pg after a 75 g oral glucose challenge while a repeated 2 h - ogtt for confirmation was not performed , due to the poor compliance . third , the renal glucose threshold was not evaluated in the study , the variation of which may affect the urine glucose levels and prediction of glycemic status . however , subjects who were considered to potentially have a renal impairment at the interview were excluded . fourth , we did not collect multiple urine samples after oral glucose administration in this study . future research is warranted to investigate whether 2-h urine sample represents the best timing for the assessment of postprandial glycemic excursions . , our study has demonstrated that as an easy and economical approach , fpg combined with 2 h - pug has a high sensitivity in detecting glucose abnormalities metabolism , whereby narrowing down the target population and saving unnecessarily performed ogtt . the combined measurements of fpg and 2 h - pug may not replace the diagnostic value of a 2 h - ogtt , but represents a promising alternative for diabetes screening , particularly in low - income regions . this study was funded by the key program of jiangsu natural science foundation ( bk 2010087 ) and sponsored by the san chuang joint project of nanjing new and high technology industry development zone . this study was funded by the key program of jiangsu natural science foundation ( bk 2010087 ) and sponsored by the san chuang joint project of nanjing new and high technology industry development zone .

most common chronic diseases are the result of interactions between multiple genetic variants and environmental factors . despite significant advances in the last decade in the understanding of our genome , there are substantial limitations in epidemiological and analytic approaches to studying the effects of genetic determinants of common chronic diseases , e.g. , metabolic syndrome ( ms ) , type 2 diabetes mellitus ( t2 dm ) , etc . family history has been shown to be a risk factor for a majority of chronic diseases of public health significance , including cardiovascular disease ( cvd ) , t2 dm , etc . family history of specific diseases reflects the consequences of genetic susceptibility , shared environment and common behaviors . family history has been recognized in clinical medicine as an important , yet non - modifiable , disease risk factor that when present might influence the probability of a suspected diagnosis . however , collection and interpretation of family history has rarely been applied in the practice of preventive medicine to assess disease risk and influence early detection and prevention strategies . professional guidelines usually include family history to assess health risk , initiate interventions , and motivate behavioral changes . the advantages of family history over other genomic tools include a lower cost , greater acceptability , and a reflection of shared genetic and environmental factors . however , the utility of family history in public health has been poorly explored . family history of diabetes is not only a risk factor for the disease but is also positively associated with risk awareness and risk - reducing behaviors . it may provide a useful screening tool for detection and prevention of diabetes . in a recent study among the adult chinese , it was found that sufficient physical activity and negative family history of diabetes might jointly reduce the risk of developing hyperglycemia and t2 dm . in the us population , family history of diabetes showed significant , independent , and graded association with the prevalence of diabetes . this association not only highlights the importance of shared genes and environment in diabetes but also opens the possibility of formally adding family history to public health strategies aimed at detecting and preventing the disease . in another study on a nationally representative sample of us adults without diabetes , family history of diabetes showed a significant , independent association with ms and its traits . this association supports the idea that shared genes and environment contribute to the expression of complex traits such as diabetes and ms . ms is defined as a constellation of several cvd risk factors including genetic and environmental factors that is responsible for ultimate predisposition of the disease . however , studies related to the effect of positive family history of diabetes on ms are sadly lacking in india . in one of the two recent studies from india , it was found that apparently healthy individuals with family history of t2 dm had higher anthropometric values ( body mass index [ bmi ] , waist - hip ratio [ whr ] ) and lower physical fitness than their counterparts . in the other study , multiple logistic regression analysis showed significant association of diabetes with family history of diabetes along with other risk factors . but association between positive family history of diabetes and ms is not known to best of our knowledge from this part of the world . the present study was therefore undertaken to find out whether there exists any significant association between family history of t2 dm and prevalence of ms among the adult asian indians . the present community based cross - sectional study comprised adults ( 30 years ) living in and around kolkata ( erstwhile calcutta ) , india . a total of 448 individuals ( 257 males and 191 females ) participated in the study . demographic profiles including name , date of birth , occupation , etc . , were obtained from participants using a schedule . family history of t2 dm was obtained from each subject and classified into three groups , viz . 1=both parents affected , 2=parent and/or siblings affected and 3=none or no family history for t2 dm . the institutional ethics committee ( iec ) of the human genetic engineering research center anthropometric measures namely height , weight , circumferences of waist ( wc ) and hip were obtained using standard techniques . height and weight ( in light clothing ) was measured to the nearest 0.1 cm and 0.5 kg , respectively . waist and hip circumferences were measured to the nearest 0.1 cm using an inelastic tape . the minimum wc was measured at the level of natural waist , which was the narrowest part of the torso . the body mass index ( bmi in kg / m ) ; waist - hip ratio ( whr ) and tc : left arm systolic ( sbp ) and diastolic ( dbp ) blood pressure measurements were twice taken using sphygmomanometer and stethoscope and were averaged for analyses . a third measurement was taken only when the differences between the two measurements were > 5 mmhg . prior medical records for blood pressure were also taken into consideration . to obtain blood pressure , each participant was requested to seat at least 5 minutes in complete relaxed mood on a chair and was also requested not to change his posture during that relaxation period . a fasting blood sample ( ~7 ml ) was collected from each subject for the determination of metabolic profiles . estimation of total cholesterol ( tc ) , triglyceride ( tg ) , high - density lipoprotein cholesterol ( hdlc ) and fasting blood glucose ( fbg ) was carried out on separated plasma using a semi auto - analyzer ( erbachem-5v2 , transasia biomedical ltd . , very low - density lipoprotein cholesterol ( vldlc ) was calculated as tg/5 . however , in case of tg > 400 mg / dl vldlc was estimated separately using the semi auto - analyzer . low - density lipoprotein cholesterol ( ldlc ) was estimated using standard formula ldlc = tc ( hdlc+vldlc ) . wc ( cm ) : male>90 , female>80 ; hdlc ( mg / dl ) : male<40 , female < 50 ; blood pressures ( mmhg ) : sbp>130 and/or dbp>85 ; fasting blood glucose ( mg / dl ) : > 100 . differences in means of the studied variables between the three groups were undertaken using analysis of variance ( anova ) with scheffe 's posthoc test to determine which two family history groups were different from each other . contingency chi - square test was performed to find out the differences in accordance with family history on prevalence of ms as well as its confounding factors and illustrated by means of bar diagram . all statistical analyses were performed using spss ( pc + version 10.0 ) . a statistical significance ( two tailed ) was set at p<0.05 . the present community based cross - sectional study comprised adults ( 30 years ) living in and around kolkata ( erstwhile calcutta ) , india . a total of 448 individuals ( 257 males and 191 females ) participated in the study . demographic profiles including name , date of birth , occupation , etc . , were obtained from participants using a schedule . family history of t2 dm was obtained from each subject and classified into three groups , viz . 1=both parents affected , 2=parent and/or siblings affected and 3=none or no family history for t2 dm . the institutional ethics committee ( iec ) of the human genetic engineering research center anthropometric measures namely height , weight , circumferences of waist ( wc ) and hip were obtained using standard techniques . height and weight ( in light clothing ) was measured to the nearest 0.1 cm and 0.5 kg , respectively . waist and hip circumferences were measured to the nearest 0.1 cm using an inelastic tape . the minimum wc was measured at the level of natural waist , which was the narrowest part of the torso . the body mass index ( bmi in kg / m ) ; waist - hip ratio ( whr ) and tc : left arm systolic ( sbp ) and diastolic ( dbp ) blood pressure measurements were twice taken using sphygmomanometer and stethoscope and were averaged for analyses . a third measurement was taken only when the differences between the two measurements were > 5 mmhg . prior medical records for blood pressure were also taken into consideration . to obtain blood pressure , each participant was requested to seat at least 5 minutes in complete relaxed mood on a chair and was also requested not to change his posture during that relaxation period . a fasting blood sample ( ~7 ml ) was collected from each subject for the determination of metabolic profiles . estimation of total cholesterol ( tc ) , triglyceride ( tg ) , high - density lipoprotein cholesterol ( hdlc ) and fasting blood glucose ( fbg ) was carried out on separated plasma using a semi auto - analyzer ( erbachem-5v2 , transasia biomedical ltd . , very low - density lipoprotein cholesterol ( vldlc ) was calculated as tg/5 . however , in case of tg > 400 mg / dl vldlc was estimated separately using the semi auto - analyzer . low - density lipoprotein cholesterol ( ldlc ) was estimated using standard formula ldlc = tc ( hdlc+vldlc ) . wc ( cm ) : male>90 , female>80 ; hdlc ( mg / dl ) : male<40 , female < 50 ; blood pressures ( mmhg ) : sbp>130 and/or dbp>85 ; fasting blood glucose ( mg / dl ) : > 100 . differences in means of the studied variables between the three groups were undertaken using analysis of variance ( anova ) with scheffe 's posthoc test to determine which two family history groups were different from each other . contingency chi - square test was performed to find out the differences in accordance with family history on prevalence of ms as well as its confounding factors and illustrated by means of bar diagram . all statistical analyses were performed using spss ( pc + version 10.0 ) . a statistical significance ( two tailed ) was set at p<0.05 . the descriptive statistics of the study population is presented in table 1 . in the present study , it was found that the prevalence of ms was 29.2% . anova with scheffe 's posthoc test revealed that individuals with either or both of their parents affected with t2 dm had significantly ( p<0.001 ) higher bmi , wc , whr , sbp , dbp and fbg ( p=0.035 ) than individuals having no family history of t2 dm . descriptive statistics of the study population the prevalence of ms by family history of diabetes is presented in table 2 . it was observed that there was a highly significant difference ( p<0.0001 ) between individuals having ms with and without family history . prevalence of metabolic syndrome by family history of type 2 diabetes mellitus the difference in family history of diabetes by confounding factors satisfying the criteria for ms is presented in table 3 . it was found that individuals with positive family history had significantly higher ( p<0.001 ) prevalence of satisfying more ms criteria than their counterparts . family history of t2 dm and metabolic syndrome phenotypes it was also found that females with positive family history had relatively higher prevalence of ms than males , whereas the prevalence was similar among males and females with no family history of t2 dm as illustrated in figure 1 . evidence suggests that family history by itself is most useful for predicting disease when there are multiple family members affected , the relationship among relatives is close , and disease is premature , that is , it occurs at younger ages than would be expected . it has been mentioned that family history information in combination with other known risk factors could be used to provide more personalized information about our risk for common diseases . yang et al . , further suggested that adding family history of diabetes could provide significant improvements in detecting undiagnosed diabetes ; however , it needs further validation . in an another study , it was found that not only the adults , even the youths with a positive family history showed signs of increased risk for these conditions which indicates the importance of family history approach to screening for children at risk of diabetes and cardiovascular disease . in addition to risk assessment , family history information can be used to personalize health messages , which are potentially more effective in promoting healthy lifestyles than standardized health messages . the accuracy of self - reported family history of diabetes and hypertension is strongly influenced by the accuracy of self - reported personal health status of relatives . raising awareness of personal health status is crucial to ensure the utility of family history for the assessment of risk and disease prevention . having a family history of a disease increases its salience and does not change one 's perceived ability to prevent the disease . to establish family history as a public health tool , it needs to be evaluated within the acce ( analytical validity ; clinical validity ; clinical utility ; and ethical , legal , and social issues ) framework . these advances will help realize the potential of family history as a public health tool . in the present study , it was found that individuals with positive family history of diabetes had significantly higher prevalence of ms and its confounding factors as compared to their counterparts . it suggests that family history could be used as a tool for genomic studies among the asian indians . it is , however , important to mention that in developing countries including india , a large section of the community remains undiagnosed and therefore accuracy of self - reported family history could be challenging . the major limitation of the study was the cross - sectional nature , and moreover , it was performed on a relatively small sample size and , therefore , is not representative of the asian indian population . large - scale , nationally representative data , which is actually lacking in india , would have provided a better insight into the role of family history for the early diagnosis of chronic diseases , e.g. , ms . it may also be argued that population screening would be of substantial importance for further research as family history may play a vital role in better understanding the etiological factors related to such complex traits . the present study indicates the importance of positive family history associated with adverse cvd risk factors like ms among the adult asian indians . family history , thus , could be used as a tool for genomic studies in order to understand the underlying shared gene - environment interrelation associated with complex traits . this in turn would help to develop comprehensive risk stratification protocol for better assessment and successful management of cvd risk factors in this part of the world .

myoepithelioma , also known as myoepithelial adenoma and benign myoepithelial tumour is a neoplasm mainly constituted by ectodermally derived contractile cells that act as smooth muscle cells and are named myoepithelial cells . these tumours can present several architectural patterns , which are non - myxoid ( solid ) , myxoid ( pleomorphic adenoma - like ) , reticular ( canalicular - like ) and mixed . in that way , myoepitheliomas that arise in the oral cavity are very rare , accounting for 1.5% of all tumours in the major and minor salivary glands and representing 2.2% and 5.7% , respectively of all benign major and minor salivary gland tumours . the lesions are generally asymptomatic and may increase slowly in size over a period of several months or years . the myoepithelial cells may be encountered in several human organs , especially in the salivary glands . thus , the greatest part of myoepitheliomas develop in the major salivary glands , while they are uncommon in the other parts of body , such as soft tissues and the carpal tunnel [ 3,6 - 8 ] . finally , one of the most frequent salivary gland - myoepitheliomas , was found in the parotid gland ( 50% ) , in the sublingual gland ( 33% ) , and in the submandibular gland ( 13% ) . myoepitheliomas and pleomorfic adenomas have very similar clinical outcomes , which makes this distinction not important for the patients . nevertheless , pathologists must analyze the lesions carefully in order to differentiate them from other malignant neoplasms that have analogous histological features . this case report sought to demonstrate a myoepithelioma arising from the soft palate , as well as its respective histological analysis and clinical management giving special attention to the possible differential diagnosis of this tumour . a 42 year old black woman , smoker and nonalcoholic , presented with a 6 months history of asymptomatic mass in the oral cavity . on the clinical examination , a firm and rubbery nodular ovoid in shape with a well delimited border lesion measuring 1.5 cm in diameter was palpated at the left side of soft palate . an elevated maxillary torus could also be noticed in the anterior part of the hard palate ( figure 1a ) . a = photograph showing clinical appearance of the myoepithelioma located on the left side of the soft palate . an elevated maxillary torus can also be seen in the anterior part of the hard palate . the lesion can be seen in the submucosa and recovered by a stratified oral squamous epithelium ( hematoxylin and eosin stain , original magnification x25 ) . c = photomicrograph showing the myxoid pattern of the tumour ( hematoxylin and eosin stain , original magnification x100 ) . d = high power photomicrograph showing the types of tumour cells encountered : spindle , plasmacytoid and epithelioid ( hematoxylin and eosin stain , original magnification x400 ) . an incisional biopsy of soft palate lesion was carried out with a surgical punch ( 8 mm in diameter and 7 mm in depth ) and was submitted for histological evaluation under the clinical hypotheses of salivary gland tumour . the publication of this case report was approved by the ethical committee of the school of dentistry of the university of so paulo , brazil . after this , 5 m thick sections were obtained and stained with hematoxylin and eosin for histological analysis . the histological examination revealed the presence of tumour originating from a minor salivary gland and covered by a stratified squamous oral epithelium ( figure 1b ) . the tumour cells were arranged in order to form a myxoid pattern ( figure 1c ) and , individually , small and/or medium spindle - shaped cells with predominantly round or ovoid nuclei , as well as epithelioid and palsmocytoid cells were noted ( figure 1d ) . the stroma was myxomatous and no ductal or syringomatous epithelial structures were observed ( figures 1b and 1c ) . to confirm the myoepithelial nature of neoplastic cells the immunohistochemical reactions for smooth - muscle actin following the histological diagnosis of myoepithelioma , the lesion was surgically removed along with accessory minor salivary glands of the palate and the histological diagnosis was reconfirmed . the follow - up of one year showed no signs and symptoms of reccurrence . distinct cell morphologies have been recognized in the myoepitheliomas . the tumour may be composed by spindle cells , which are arranged in interlacing fascicles that show stroma - like appearance , by plasmacytoid cells that are polygonal cells with eccentric nuclei , or even by epithelioid cells arranged in the nests or cords . some myoepitheliomas can also show clear polygonal cells with abundant and optically clear cytoplasm . plasmacytoid cells are distinguishable by their dense , nongranular or hyaline , abundant eosinophilic cytoplasm , whereas epithelioid cells are round to polygonal cells , with centrally located nuclei and a variable amount of eosinophilic cytoplasm . finally , clear cells contain large amounts of the glycogen but are devoid of mucin or fat . these different types of cells may be arranged in several architectural patterns , which are non - myxoid ( solid ) , myxoid ( pleomorphic adenoma - like ) , reticular ( canalicular - like ) and mixed . benign and malignant tumours such as pleomorphic adenoma , adenocarcinoma , nerve sheath tumour , fibrous hystiocytoma , nodular fasciitis , synovial sarcoma , leiomyoma , leiomyosarcoma , hemangiopericytoma , solitary fibrous tumour and paraganglioma may be cited . in that way clinical appearance of the lesions mentioned above can share similarity with myoepitheliomas and the only safe way to differentiate them is by the means of histological evaluation . myoepitheliomas present rare mitoses and absence of nuclear and cellular pleomorphism as well as a non - infiltrative growth typical of a benign tumour . the tumour cells are also generally positive for smooth muscle actin ( sma ) . in the case in addition , tumour showed cytokeratins and s100 protein positivity , which may help the distinction between this lesion and nodular fasciitis , leiomyoma and leiomyosarcoma , once these tumours normally do not express these proteins . besides , the competent observation of the tumour morphology is very important to perform the diagnosis . as mentioned previously , the predominant type of myoepithelial cell in the myoepithelioma may be spindled , epithelioid , clear or plasmacytoid , and one single tumour may also present two or more histological patterns . accordingly , peripheral nerve sheath tumours should participate in the differential diagnosis of spindle cell variant of myoepithelioma , whilst the clear cell myoepithelioma should be correctly discerned from the clear cell adenocarcinoma and mucoepidermoid carcinomas . myoepitheliomas may be considered as a variant of pleomorphic adenoma in which glanduloductal differentiation is entirely or virtually absent . thus , the architectural patterns and the cellular differentiation in myoepitheliomas are equally apparent to that of the nonluminal portions of pleomorphic adenomas . in addition to these features , sciubba and brannon reported the absence of chondromyxoid or chondroid foci as characteristics of myoepitheliomas . in fact , no more than 5 to 10 percent of myoepitheliomas may be composed of ducts . other authors suggest that no more than one duct every medium to high power field ( x200 - 400 ) or no more than one small cluster of ducts is acceptable in these lesions . moreover , the myoepithelial cells that compose myoepitheliomas are also found in many organs ( mainly the salivary glands ) , and thus are the major components of salivary gland tumours . the tumours that arise from the salivary glands comprehend the already mentioned pleomorphic adenoma , adenoid - cystic carcinoma and epithelial - myoepithelial carcinoma of intercalated duct origin . although these lesions hold the myoepithelial cells as their main neoplastic cells , their clinical management may vary consistently . as a general rule , adenoid - cystic carcinoma , epithelial - myoepithelial carcinoma and mucoepidermoid carcinoma demand surgical excision with postoperative intensity - modulated radiotherapy with or without chemotherapy . on the other hand , the accepted treatment of myoepitheliomas as well as for other benign tumours is the simple surgical excision . these tumours do not present high levels of the recurrence and this clinical case did not show any sign of recurrence after a one year follow - up . the myoepithelioma should be carefully distinguished from the other soft tissue tumours , especially those arising from salivary glands , such as pleomorphic adenoma and adenoid - cystic carcinoma .

agents that selectively associate with cell surface receptors overexpressed on tumor cells can be used to deliver therapeutics . this strategy is referred to here as active to distinguish it from other forms of targeting ( e.g. , where the agent is intended to directly cause a therapeutic effect by binding cell surface receptor or is designed to target intracellular pathways upregulated in cancer cells ) . the most widely investigated active targeting agents are humanized monoclonal antibodies ( hmabs ) , but mabs in general have limitations . first , they have poor permeation into solid tumors , only a few mabs enter cells , most do not , and even cell - permeable mabs may not reach the intracellular target . moreover , mabs can have undesirable immunogenic effects , circulation times , and they are further restricted by cost and shelf life issues . active targeting via small molecules that selectively bind to receptors on tumor cells can have advantages relative to hmabs with respect to cell internalization and affordability ; however , relatively few small molecule targeting entities are known . folic acid and arg - gly - asp peptidomimetics are probably the most widely appreciated examples , but there are not many more besides these . there are no clinically approved small molecule active targeting agents for delivering therapeutics to breast cancer . trkc , a cell surface receptor , and its natural ligand neurotrophin-3 were reported to play an essential role in breast cancer growth and metastasis ; suppression of trkc expression in highly metastatic mammary carcinoma cells inhibited their growth in vitro and their ability to metastasize from the mammary gland to the lung in vivo . this paper features a novel molecular fragment a ( figure 1 ) for active targeting of breast cancer types that overexpress trkc . trkc - targeting molecules 1 , containing fragments a ( blue in figure 1 ) , elicit only weak functional effects . one fragment a is not enough to bind trkc adequately , but two joined as shown give good affinity . the scaffold part in molecules 1 ( shown in black ) supports the two trkc - binding entities and the bodipy cargoes ( colored purple and red here ) . the bodipy dyes are similar , except that those without iodines are highly fluorescent , while ones with are only weakly fluorescent and act as sensitizers for singlet oxygen production . thus , compound 1-f ( f = fluorescent ) is designed for cell imaging and histology , while 1-pdt is intended for use in pdt . ( a ) mab conjugates have limited cell permeabilities , but ( b ) many small molecule conjugates can . ( c ) structures of the targeted compounds featured in this work , 1-f and 1-pdt , and the parent iodinated bodipy , singlet oxygen is very reactive , half - life 1 ns to 1 s , hence the effect of pdt agents illuminated in an aerobic environment is to generate o2 that kills cells in a highly localized area . some boron dipyrromethene ( bodipy ) based systems can have excellent attributes for pdt with high extinction coefficients , favorable light - to - dark toxicity ratios , high antitumor efficacies in vivo , and good body clearance , but they are not inherently inclined to localized in tumors ; accumulation of the sensitizers in tumors is important for pdt . experiments described in this paper were undertaken to explore the effects of coupling molecular fragments a that can bind trkc receptors expressed on breast cancer cells and deliver a bodipy - based pdt agent to them . as far as we are aware , there are no other agents , in the clinic or in the literature , on experimental modalities that actively target trkc+ with a small molecular fragment that binds this receptor conjugated to a therapeutic . we hypothesized that agent 1-f could be used to stain tissue that express trkc+ ( e.g. , from biopsies ) and trkc+ tumors and metastases during surgeries . in histochemistry on a commercial array of human breast cancer sections using 1-f , all 36 malignant tissues showed evidence for expression of trkc ( 23% with unambiguous staining in the cytoplasm and cell membrane , 65% same but not as clear , and in 12% not all the tumor cells stained ) ; conversely , none of the normal breast tissue showed significant staining in the cytoplasm and cell membrane just as fluorescent anti - trkc mab did ( figure 2a ) . we infer 1-f has potential for histochemistry and as a surgical marker for trkc - expressing cancers . ( a ) histochemical stains for a library of 96 breast tissue slices were performed using 1-f ( top ) and anti - trkc antibody as control ( bottom ) , and the three illustrative ones shown here illustrate staining of the malignant tumor , whereas normal tissue is not stained . no staining was observed in the tissues without the small molecule probe or mab . ( b ) cell imaging on 4t1 cells shows 1-f was internalized into lysosomes just as the natural trkc ligand nt3 is . intracellular imaging studies featuring 1-f on murine 4t1 cells ( figure 2b ) showed compound 1-f is internalized and partially colocalizes with a lysosome tracker dye . thus , 1-f localizes into the lysosome , just as the natural trkc ligand neurotrophin-3 ( nt3 ) does when it is internalized via the trkc receptor , implying 1-f also enters the cell via trkc . similar experiments were performed using ( the less fluorescent agent ) 1-pdt , and the outcome was much the same ( supporting information , figure s3 ) . moreover , import of 1-pdt at 2 h was more than 2-pdt into 4t1 cells and that uptake of 1-pdt was suppressed by pretreatment with the natural trkc ligand , nt3 . binding nt3 to trkc on the surface of living cells causes growth and survival . some common breast cell lines used for cancer research express trkc ( e.g. , human hs578 t and murine 4t1 ) but not in others ( e.g. , skbr3 , 67nr , and mcf-10a ) . figure 3a shows photocytotoxicities of 1-pdt are greater for the trkc+ breast cancer cell lines hs578 t and 4t1 than the immortalized , trkc , breast cell lines 67nr and mcf-10a . comparison of photocytotoxicities for the targeted pdt agent 1-pdt with the nontargeted form 2-pdt , and i2-bodipy on trkc+ expressing cells ( figure 3b , c ) shows the targeted agent is more photocytotoxic ( ic50 = 0.325 m in 4t1 ; ic50 = 0.285 m in hs578 t ) than the control ones , 2-pdt and i2-bodipy ( undetermined ic50 ) , which does not bind trkc . these results suggest that 1-pdt induces selective photocytotoxicity in trkc - expressing cells via trkc receptor targeting . some photocytotoxicity was observed for 2-pdt on the human hs578 t but not on the murine 4t1 cells , indicating the scrambled control 2-pdt might be binding to some other receptor on the human line . ( a ) photocytotoxicities for 1-pdt are more for the following breast cells , murine metastatic 4t1 and human metastatic , hs578 t ; compared with the following breast cell lines , murine nonmetastatic , 67nr ; human immortalized mcf-10a . ( b ) photocytotoxicities on the 4t1 and hs578 t cells were enhanced for 1-pdt compared to the scramble control 2-pdt featuring an isomer of the targeting fragment that does not adhere to trkc cells and control i2-bodipy . ( d ) photocytotoxicities for 1-pdt on 4t1 and hs578 t cells are dose dependent ( red bars ) and suppressed by fixed concentrations of competing : ( i ) natural ligand nt3 ( blue ) and ( ii ) targeting ligand without a pdt group data shown are mean sem of three independent experiments . * , p < 0.05 ; * * , p 0.01 ; * * * , p 0.001 vs control using one - way anova ( ( a ) trkc cell line , ( b ) i2-bodipy , ( d ) red bars ) . figure 3d shows the photocytotoxicities of 1-pdt on trkc+ cells can be suppressed by the natural trkc ligand ( nt3 ) or the targeting agent without any bodipy dye attached , iy - iy - teg ( structure in supporting information , figure s1 ) . when interpreting this data , it is important to note that the concentration of nt3 is constant throughout , so 1-pdt only becomes noticeably competitive with the small molecule ligand at higher concentrations ( e.g. , > ca . all the photocytotoxicity experiments described above involved adding the test compounds for 2 h incubation , washed off before illuminating the cells . effects of prolonging the incubation on the cell viability in pdt revealed that more selective cell - growth inhibition was achieved for trkc+ 4t1 compared to trkc 67nr at 2 h incubation ( supporting information , figure s2 ) , and the difference becomes less noticeable when 4 and 6 h incubation was used ; thus a shorter incubation time is optimal for selective photokilling by the trkc - seeking conjugate . for a full comparison , the same time course experiments for the untargeted 2-pdt and the parent iodinated bodipy , i2-bodipy , however , the cell viability observed between 4t1 and 67nr cells were similar across the different time points for incubation , implying no selective binding to cell surface receptors ( supporting information , figure s2 ) . 1-pdt at 20 , 30 , and 100 mg / kg was administered to mice intravenously via the tail vein , and toxicity was evaluated based on typical symptoms ( apathy , horrent fur , diarrhea , behavior changes , and loss of body weight ) . all mice receiving doses of 30 mg / kg or more experienced motility and balancing difficulties and died 13 h post drug administration . however , 20 mg / kg 1-pdt was well tolerated and gave no signs of toxicity and death up to 17 days of post - treatment ( figure 4 ) , and no sign of gross organ toxicity was found in autopsies performed at the end of study . no death or signs of toxicity were observed in mice receiving equivalent doses of 2-pdt and bodipy . these results suggest 20 mg / kg is the maximum tolerated dose ( mtd ) for 1-pdt . if , after further studies , these compounds were to be used therapeutically , then the dose should be significantly less than this ; however , modifications to the dye structures are required first ( see below ) . healthy 78 weeks old balb / c female mice were administered intravenously via tail vein respectively with 1-pdt and 2-pdt at 20 , 30 , and 100 mg / kg ( i2-bodipy content equivalent to 6.25 , 10 , and 30 mg / kg , respectively , i.e. , corrected for mw ) , and the parent i2-bodipy ( 30 mg / kg ) . data represent the average body weight ( grams ) of two mice / treatment group . the biodistribution of 1-pdt and the isomeric non - trkc targeting control 2-pdt were monitored in 4t1 tumor bearing mice ( n = 3 ) up to 72 h. significant and prolonged accumulation of 1-pdt was observed in tumor ( figure 5 ) . at 1 h post administration , the fluorescence intensity of tumors in mice treated with 1-pdt was 2.1 higher ( 897000 135800 ) than the corresponding intensities for tumors treated with the nontargeted control 2-pdt ( 416000 43000 ) ( p < 0.05 , student s t test ) . the 1-pdt dye intensity in tumor tissue remained significantly higher compared with 2-pdt for up to 6 h , but there were no significant differences at 24 h onward . trends in the 1-pdt tumor distribution were significantly different to that of 2-pdt , indicating selective accumulation of 1-pdt in the tumor . maximum accumulation of 1-pdt at 1 h post administration led us to adopt a drug - to - light interval of 1 h in determining 1-pdt s in vivo antitumor efficacies in the subsequent studies . 1-pdt demonstrated significant and prolonged accumulation in tumor tissue for up to 6 h and cleared from the body 72 h postadministration . 4t1-tumor bearing female balb / c mice were treated at 10 mg / kg via the tail vein . mice ( n = 3 ) were sacrificed at 0 , 0.25 , 1 , 3 , 6 , 24 , 48 , 72 h. ( a ) organs and tissues ( tumor , draining lymph nodes , spleen , kidney , liver , lung , skin , and eye ) were harvested , and ( b ) fluorescence intensities in each organ were imaged using an in vivo imager ( data represent mean sem of three mice at each time point ) . * p < 0.05 ; * * p < 0.01 ; for 1-pdt vs 2-pdt . a large amount of 1-pdt accumulated in the liver ( ca . 20-fold more than in the tumor ) , then in the kidney and lung within the first 3 h post administration ( figure 5 ) , but these accumulations dissipated swiftly in the subsequent monitoring period . swift clearance of both compounds in these organs is typical of small molecular weight compounds and indicates that the accumulation in these organs was random and not due to trkc receptor binding . nonselective accumulation of 1-pdt and 2-pdt was also observed in lymphoid organs such as spleen and lymph node at a much lower level ( figure 5 ) ; others have observed no significant trkc expression for these organs . interestingly , 2-pdt but not 1-pdt was found to accumulate significantly in the lymph node for a prolonged period of time . the eye has relatively impermeable blood capillaries and low trkc receptor expression , whereas murine eyes express relatively few trkc receptors , concentrated mainly at nerve bundle portions . compound targeting efficacies and effects on trkc tumors were assessed in the following experiments . aggressive trkc murine breast carcinoma ( 4t1 ) cancer cells were subcutaneously injected to the murine mammary fat pad and then treated with 1-pdt , 2-pdt , and i2-bodipy when the tumor sizes reached 80 mm . in a control , mice inoculated with trkc murine breast carcinoma 67nr cancer cells were also treated using these compounds . agent 1-pdt significantly reduced tumor growth after illumination when treated at 2 and 10 mg / kg ( equivalent to 0.6 and 3.0 mg / kg of i2-bodipy , respectively ) . inflammation and erythema surrounded the irradiated tumor region was observed in the 1-pdt treated groups at 46 days post pdt but was less pronounced or not observed in the control groups ( figure 6a ) . subsequently , tumor sizes were drastically reduced in the 1-pdt treated groups 46 d postillumination ( 61% and 96% maximum tumor reduction in mice treated with 2 and 10 mg / kg of compound compared to pretreatment tumor size , figure 6b ) . in contrast , 2-pdt and i2-bodipy treatment induced only moderate tumor size reduction within the first 6 d after illumination ( 20% reduction for mice treated with 10 mg / kg of 2-pdt , and 11% reduction for mice treated with 10 mg / kg of i2-bodipy , figure 6b ) . at day 13 , both 2-pdt and i2-bodipy treated mice showed rapid tumor regrowth at the necrotic tumor tissue periphery while tumor regrowth in 1-pdt treated mice was minimal and delayed ( figure 6a , b ) . more importantly , 1 out of 7 mice ( 14% ) , treated with 2 mg / kg , and 5 out of 7 mice ( 71% ) , treated with 10 mg / kg of 1-pdt , healed from eschar by day 1315 after illumination and showed no palpable tumor for up to 90 d post - treatment . such total tumor remission was not found in both the 2-pdt and i2-bodipy treated groups . to confirm the targeting ability of 1-pdt on trkc+ cells in vivo , the compound efficacy in the non - trkc expressing 67nr tumor cell line in mice was examined and compared to that in the 4t1 model ; as expected , neither 1-pdt nor 2-pdt at 10 mg / kg fully eradicated the 67nr tumors in mice ( figure 6c ) ; the tumor volumes were reduced at day 46 days postillumination but regrew at day 9 . 1-pdt effectively suppressed the growth of trkc+ ( 4t1 ) tumor but not in trkc ( 67nr ) . ( a ) regrowth of trkc+ 4t1 tumor ( yellow arrow ) in female balb / c mice receiving 2-pdt ( 10 mg / kg ) , i2-bodipy ( 3.0 mg / kg ) , and saline controls but not in mice receiving 1-pdt ( 10 mg / kg ) . ( b ) significant dose dependent mean tumor volume reduction and delayed tumor regrowth in trkc+ 4t1 tumor bearing mice receiving 2 and 10 mg / kg 1-pdt as compared to rapid tumor growth in mice receiving the control substances . ( c ) 1-pdt gave impermanent and nonselective antitumor effect ( resembled that with 2-pdt ) in mice bearing trkc 67nr tumor . photoactivation was conducted at 100 j / cm with a fluence rate of 0.16 w / cm 1 h after intravenous injection of the compounds . all graphs showed mean tumor volume sem ( n = 7 ) . * p < 0.05 , * * p < 0.005 , for i2-bodipy vs 1-pdt and 2-pdt group using one - way anova . ( d ) there were no tumor metastases in 1-pdt treated survivor mice post 90 d. mice treated with 10 mg / kg 1-pdt that survived up to 90 d with no palpable primary tumor found were metastases free in all the major organs assessed ( liver , lung , draining lymph node , and spleen , representative histological images ) . control ( tumor free healthy and 4t1 tumor burden mice ) results were included for comparison ( yellow arrow = 4t1 tumor metastases ) . scale bar : 100 m . all 1-pdt treated mice that showed complete tumor regression remained disease free with no palpable tumor at the primary site up to 90 d. tumors from 4t1 cancer cells are known to be aggressive and typically metastasize to lymph nodes , liver , and lung , even in the early stages of the disease . thus , at 90 d postillumination , the surviving mice that were physically active were sacrificed for major organ / tissue histopathology by a certified veterinary pathologist . h&e staining showed no 4t1 tumor metastases in all the examined organs ( liver , lung , draining lymph node , spleen , kidney , and heart ) of the 10 mg / kg 1-pdt treated survivor mice and tumor free control mouse ( figure 6d ) . however , in 4t1 tumor bearing control mice , tumor metastases were found in liver , lung , lymph nodes , and spleen , with extramedullary hematopoiesis observed in this animal ( figure 6d ) . these results show effective eradication of trkc expressing 4t1 tumor by 1-pdt post pdt treatment in the survivor mice . the assertion that agent 1-pdt targets trkc+ breast cancer cells is supported by the observation that it has a more profound effect on these tumors than the isomeric compound , 2-pdt , and the pdt agent without any appendage , i.e. , i2-bodipy . this is consistent with the significant in vivo selective accumulation of 1-pdt in trkc+ tumors 1 h after administration , as calibrated relative to levels of 2-pdt at the same time . moreover , 1-pdt , 2-pdt , and i2-bodipy have comparable effects on tumors from trkc cell lines . the data outlined above are consistent with ex vivo studies on cells stably transfected with trkc , but those experiments did not involve breast cancer cells . in this article , we report selective photocytotoxicity of 1-pdt ex vivo correlates with natural levels of trkc+ expression in breast cell lines . the cell studies established that a 46 h interval between treatment and illumination decreased the selectivity for trkc - expressing cells relative to a 2 h interval , perhaps due to relatively slower , and nonselective , interaction with cells , something that could be anticipated from the literature . consequently , we used a 1 h interval between injection and light treatment for the in vivo work . it is remarkable that 1-pdt at 10 mg / kg in vivo caused , on average , 96% tumor volume reduction in the mice bearing trkc+ tumor at day 6 post - pdt . among these mice , 71% showed full remission and were tumor - free for 90 days after therapy , and histology indicated no metastasis development in these animals . the fact that 1-pdt was ineffective for suppressing trkc ( 67nr ) tumors in mice supports the overall assertion that this compound targets trkc+ expressing tissue in vivo . long- and short - term toxicities of agents like 1-pdt must be considered in the context of experimental therapeutics . in the dark , the trkc - targeting fragments featured are capable of transducing signals similar to nt3 upon binding to trkc , so it is conceivable that in the long term 1-pdt might induce tumorigenesis just as nt3 does . however , this was not the case in the extended time course of these experiments because , in the dark , mice receiving saline control or 2-pdt ( no trkc binding ) have comparable tumor volumes , with no significant differences , to the 1-pdt group ( supporting information , figure s5 ) . intravenous administration of photosensitizers can result in accumulation in different tissues and undesirable photosensitivities . adverse photosensitivity is common in pdt , but targeting pdt agents to tumors that express trkc should alleviate some of these effects . short - term toxicities for agents that bind trkc could be anticipated because nt3 promotes neuronal cell survival , differentiation , and synapse transmission , and antagonism at the trkc receptor might have undesirable effects . short - term toxicities of agent 1-pdt are a concern because , while doses of 20 mg / kg were tolerated , 30 mg / kg was not . a claim that high doses of neurotrophins ( including nt3 ) promote relatively rapid excitotoxic necrosis of neurons might be pertinent . in any event , 1-pdt requires further structural modifications because the light wavelength to excite this is optimally around 520 nm whereas pdt agents should absorb above 700 nm if they are to be addressed at more than 1 cm tissue penetration . consequently , 1-pdt is a prototype for other compounds , currently under development in our laboratories , that involve the same targeting fragments differently disposed around other pdt active dyes . it is anticipated that the short - term toxicities of the second - generation systems will be structure dependent . overall , the data presented here demonstrate the potential of targeting trkc+ tumors with pdt agents . excellent therapeutic indices can be achieved because pdt is spatially restricted to the illumination area , and active targeting accumulates the agents in the tumors . this study features dosing with a targeted - pdt agent alone , but there is also the intriguing possibility of combination therapies featuring trk inhibitors currently in trials as chemotherapeutic agents ( e.g. , lestauritinib and plx7486 ( http://clinicaltrials.gov/show/nct01804530 ) ) . two slides of human breast cancer tissue microarrays ( brc962 ) were purchased from us biomax , inc . the arrays include 36 cases of breast cancers and 12 cases of normal , reactive , and benign tumor tissues of the breast in duplicates . the slides were transferred to a xylene bath for 10 min and then rehydrated in two changes of fresh absolute ethanol for 7 min each . excess liquid was shaken off , and the slides were incubated in fresh 90% , 70% ethanol then water for 7 min each . the slides were washed in two changed of pbs for 5 min each , then incubated with pbs containing 4% bsa for 30 min . the tissues were rinsed with pbs and incubated again in two changed of pbs for 5 min each . 1-f solution in 4% pbs / bsa and anti - trkc antibody were added to separated slide and incubated overnight at 4 c . the slides were rinsed twice in pbs , then in water ( 10 min each ) . then the slide contained 1-f was mounted in permanent mounting media with dapi ( vector ) and incubated at 4 c for 4 h. the slide contained anti - trkc antibody was incubated with rodamine - tag primary antibody for 30 min and then rinsed twice in pbs and in water ( 10 min each ) before putting in the mounting media . two slides were imaged with a zeiss stallion dual detector imaging system consisting of an axiovert 200 m inverted fluorescence microscope , coolsnap hq digital cameras , and intelligent imaging innovations ( 3i ) software . digital images of 1-f and dapi were captured with a c - apo 63x/1.2 w corr d = 0.28m27 objective with the following filter sets : exciter bp470/20 , dichroic ft 493 , emission bp 505530 for 1-f , emission bp 565615 for rodamine and exciter g 365 , dichroic ft 395 , emission bp 445/50 for dapi . sequential optical sections ( z - stacks ) from the basal - to - apical surfaces of the cell were acquired . digital image acquisition was initiated approximately 1 m below the basal surface of the cells , and optical slices were collected at 0.5 m steps through the apical surface of the cells with a high numerical objective lens ( c - apo 63x/1.2 w corr d = 0.28m27 ) . these wide - field images were subjected to deconvolution using 3i software . 4t1 , mcf-10a , and hs578 t ( atcc ) and 67nr ( barbara ann karmanos cancer institute , detroit , mi ) cells were cultured on 75 cm culture flasks in dulbecco s modified eagle medium / nutrient mixture f-12 ( dmem / f12 , sigma chemical , st . all cells were cultures in a humidified incubator at 37 c with 5% co2 and 95% air . intracellular localization of the 4t1 cells was measured using a zeiss 510 meta nlo multiphoton system containing of an axiovert 200 mot microscope . throughout 1-f was excited at 488 nm , and the emission bp used was 500530 nm ; for lysotracker red , the excitation was at 543 nm and the emission bp was 565615 nm . sequential optical sections ( z - stacks ) from the basal - to - apical surfaces of the cell were acquired , initiated approximately 1 m below the basal surface of the cells , and optical slices were collected at 0.5 m steps through their apical surface using a high numerical objective lens ( c - apo 63x/1.2 w corr d = 0.28m27 ) . these wide - field images were subjected to deconvolution using intelligent imaging innovations ( 3i ) software . 4t1 cells were incubated with 1-f , 1 m , for 12 h at 37 c . after the cells were washed with pbs , lysotracker red ( life technology , 500 nm ) approximately 5000 cells / well in culture medium containing 10% fetal bovine serum were seeded in a 96-well plate . ( 0.02 m in dmso ) were diluted with protein - free medium ( pfhm - ii ) , 1 l stock solution/1 ml pfhm - ii , to make master stock solutions . the master stock solutions were further diluted with pfhm - ii to the desired final concentrations varying from 0.02 to 10 m to test on the cells ( less than 0.001% dmso contained in the final solutions ) . after 2 h of treatments , cells were washed twice with pbs , then culture media without any additives was added ( acas ) before irradiation , irradiated with a light dose of 7.3 j / cm from a broad - spectrum halogen light source and fluence rate of 12.2 mw / cm , then further incubated for 24 h ; viabilities were assessed through mtt conversion . briefly , 20 l of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide mtt ( 5 mg / ml , in hank s balanced salt solution ) were added and the cells were incubated for an additional 3 h. the medium was then removed , and 100 l of dmso was added to dissolve the formazan crystal formed . the optical density of each well ( at 570 nm ) was measured with a biotek synergy 4 microplate reader . the viability of each cell line in response to the treatment with tested compounds was calculated as % of cell viability = ( od treated / od control ) 100 . female 810 week old , wild - type balb / c mice were purchased from monash university , malaysia campus ( sunway , malaysia ) for in vivo studies and maintained in the satellite animal facility , department of pharmacology , faculty of medicine , university of malaya . all animal experiments were performed according to protocol approved by the faculty of medicine institutional animal care and use committee , university of malaya ( fom iacuc ) ( ethics approval no . the toxicity profiles of 1-pdt and 2-pdt were determined after intravenous administration of these compounds at 20100 mg / kg to the mice via tail vein . toxicity was observed based on typical symptoms such as apathy , horrent fur , behavior changes , and loss of body weight for 2 weeks . 4t1 tumor bearing female balb / c mice with an average tumor volume of 80 mm were divided into two groups and intravenously administered with 10 mg / kg of photosensitizer 1-pdt and 2-pdt , respectively . mice ( n = 3 for each time point ) were then sacrificed at different time points ( 0 , 15 min , 1 , 3 , 6 , 24 , 48 , and 72 h post compound administration ) , and major organs such as liver , spleen , lung , kidney , lymph nodes , skin , eye , and tumor tissues were harvested . organs and tissues were imaged using an in vivo ms fx pro ( carestream molecular imaging , woodbridge , ct ) with an excitation filter at 530 nm and emission filter at 600 nm . fluorescence intensities of each organ and tissue were quantified using carestream molecular imaging software 5.0 ( woodbridge , ct ) . the fur of the balb / c mice was shaved , and murine 4t1 and 67nr cells at a density of 5 10 cells in 0.1 ml of medium was orthotopically injected into the mammary fat pad of the mice ( 1820 g , 810 weeks old ) , respectively . the mice were then randomly divided into groups for pdt at 8 days post injection , when the tumor size reached 80 mm . compounds ( 1-pdt , 2-pdt , and i2bodipy ) at 210 mg / kg body weight were dissolved in a cocktail of 2.5% ethanol and 2.5% cremophore el . the mixture was then further dissolved using saline to a volume of 0.2 ml and administered by intravenous tail vein injection into the mice . the mice were then kept in the dark for 1 h before an anesthesia cocktail of 90 mg / kg of ketamine and 10 mg / kg of xylazine cocktail was administered . thereafter , pdt was performed using lumacare lc-122a fiber optic light delivery system ( standard fiber optic probe model lum v , 400700 nm , lumacare medical group , newport beach , ca , usa , with a 500/585 nm bandpass filter from omega optical , catalogue no . a 4 mm thick glass slide ( 1.0 mm 1.2 mm , purchased from sail brand , china , catalogue no . 7101 ) was used as a barrier to avoid direct photothermal effect on tumor . the illuminating spot was positioned at the tumor and the surrounding was covered using black cloth to avoid pdt effect on nontumor parts of body . pdt was conducted at 100 j / cm with the fluence rate of 160 mw / cm . after pdt , the mice were kept in dark and tumor size was measured 3 times per week . tumor volume changes were determined by caliper measurements with tumor volume , mm = ( l w/2 ) , where l is the longest dimension and w is the shortest dimension . during the study survivor mice were sacrificed at 90 days post therapy , and major organs such as liver , kidney , spleen , draining lymph node , lung , and heart were isolated for histological analysis . the isolated specimens were fixed with 10% formalin solution for a minimum of 48 h at room temperature , following which , tissues were trimmed into representative segments and then dehydrated using an ascending series of alcohol , cleared in xylene , and embedded in paraffin . microtomy was performed using a leica rm2255 microtome ( leica microsystems , germany ) . sections were cut at 5 m thickness and placed onto appropriately labeled microscope slides . the slides were then stained with haematoxylin and eosin ( h&e ) and coverslipped , then evaluated by a board certified pathologist at the institute of molecular and cell biology , agency for science , technology and research , in singapore . in vitro and in vivo experiments were performed to compare the efficacy of the three compounds , and statistical analysis was analyzed using spss . results were analyzed using one - way anova with dunnett s multiple comparisons when comparing among the three groups of compounds . student t test was used to analyze between two groups ; differences were considered statistically significant at the p < 0.05 ( * ) , p < 0.01 ( * * ) , and p < 0.001 ( * * * ) levels .

gynecological cancers are a frequent group of malignancies in women , accounting for approximately 18% of all cancers of women worldwide.1 cervical cancer ( cc ) is the second most deadly gynecological malignancy in the world . in developing countries , 500,000 new cases are diagnosed every year , some of which are the actual cause of death among women of age 35 to 45 years . the cc risk is 1% during the life of a woman living in a developed country , whereas the corresponding value for a woman living in a country without preventive programs is 5%.2 the development of screening programs and new advances in treatment have contributed greatly to increased survival rates and , in some cases , cure of gynecological cancers . these resulted in a subset of women , known as survivors , who return to their normal functioning and live years after the initial diagnosis without symptoms of the disease . survivors may experience a wide range of side effects that do not dissipate with time and may persist for a long - term period , such as sexual problems , pain , premature menopause , fatigue , and decreased physical functioning . the recognition that qol of cc survivors is of great importance led to the emergence of a body of research that has been focusing on qol issues . knowledge about qol issues is crucial to establish follow - up care programs adjusted to the survivors needs and to provide appropriate education in prevention and early detection of survivors needs , and ultimately improve their qol.3 patients diagnosed with early stage cc are usually treated either with surgery ( surg ) ( complete removal of internal genitals ) , radiotherapy ( rad ) ( irradiation of the pelvis and occasionally the para - aortic lymph nodes at target doses of approximately 50 gy ) , or a combination of both . standard treatment for patients with advanced stages of cc is external pelvic rad combined with brachytherapy to the cervix and today , usually , combined with chem ( rad / chem).46 in our study we decided to compare the qol of cc patients who were treated with either rad or rad / chem . for the estimation of their qol we used 4 different questionnaires : eortc qlq c-30 , eortc qlq - c24 , questionnaire of post - traumatic psychological disorder , and greek symptom control questionnaire by m.d . the patients included in this study suffered from cc stages ia iiia and filled in the questionnaires 1 year after treatment end . our aim was to compare the impact of monotherapy ( rad ) and combined therapy ( rad / chem ) on the qol of cc patients . a total 105 consecutive cc patients ( 93% participation ) stages ia iiia , who met the inclusion criteria , were enrolled to the study ; 58 ( 55% ) had been treated with rad / chem ( group 1 ) and 47 ( 45% ) with rad ( group 2 ) . eligible women had squamous cell tumors < 6 cm at diagnosis and were currently disease - free . all participants were patients in theagenio cancer hospital , thessaloniki , greece and filled in the questionnaires 1 year after treatment completion at a scheduled follow - up check . women who verbally agreed to participate were read an informed consent statement that met criteria defined by the health insurance portability and accountability act , as well as by our institution . in our study the proposed method for patients suffering from squamous cervical cancer , nonresected , stages ia iiia ( by the international federation of gynecology and obstetrics ) is concurrent rad / chem . rad is applied as external treatment with pelvic fields and 4500 cgy , combined with weekly administration of cisplatin , at the radiosensitizer dose of 40 mg / m body surface ( the therapeutic dose of platinum amounts to 100 mg / m body surface ) and afterwards brachytherapy , as additional dose . the type of radiation used was photons generated by linear accelerator with 18 mev energy . the eortc qlq - c30 incorporates 9 multi - item scales : 5 functional scales ( physical , role , cognitive , emotional , and social ) , 3 symptom scales ( fatigue , pain , and nausea and vomiting ) , and a global health and qol scale . it is internationally considered as a reliable and valid measure of the qol of cancer patients in multicultural clinical research settings . an essential aspect of the modular approach to qol assessment adopted by the eortc qlg ( quality of life group ) is the development of modules specific to tumor site , treatment modality , or a qol dimension , to be administered in addition to the core questionnaire ( eortc qlq - c30 ) . the modules , like the core questionnaire , are designed for use in cancer clinical trials . specifically for cc , we used the eortc qlq - c24 , which consists of 3 multi - item scales and 5 single - item scales.7 two complementary questionnaires were used , the greek symptom control questionnaire by m.d . anderson and questionnaire of post - traumatic psychological disorder , to supplement the assessment of the 2 major questionnaires . all statistical analysis was performed using spss for windows , version 15.0 ( spss inc . , we tried to make the design , methodology , statistical analysis , presentation , and interpretation of results in accordance with international standards to ensure reliability and comparability of research results . a total 105 consecutive cc patients ( 93% participation ) stages ia iiia , who met the inclusion criteria , were enrolled to the study ; 58 ( 55% ) had been treated with rad / chem ( group 1 ) and 47 ( 45% ) with rad ( group 2 ) . eligible women had squamous cell tumors < 6 cm at diagnosis and were currently disease - free . all participants were patients in theagenio cancer hospital , thessaloniki , greece and filled in the questionnaires 1 year after treatment completion at a scheduled follow - up check . women who verbally agreed to participate were read an informed consent statement that met criteria defined by the health insurance portability and accountability act , as well as by our institution . in our study the proposed method for patients suffering from squamous cervical cancer , nonresected , stages ia iiia ( by the international federation of gynecology and obstetrics ) is concurrent rad / chem . rad is applied as external treatment with pelvic fields and 4500 cgy , combined with weekly administration of cisplatin , at the radiosensitizer dose of 40 mg / m body surface ( the therapeutic dose of platinum amounts to 100 mg / m body surface ) and afterwards brachytherapy , as additional dose . the type of radiation used was photons generated by linear accelerator with 18 mev energy . the eortc qlq - c30 incorporates 9 multi - item scales : 5 functional scales ( physical , role , cognitive , emotional , and social ) , 3 symptom scales ( fatigue , pain , and nausea and vomiting ) , and a global health and qol scale . it is internationally considered as a reliable and valid measure of the qol of cancer patients in multicultural clinical research settings . an essential aspect of the modular approach to qol assessment adopted by the eortc qlg ( quality of life group ) is the development of modules specific to tumor site , treatment modality , or a qol dimension , to be administered in addition to the core questionnaire ( eortc qlq - c30 ) . the modules , like the core questionnaire , are designed for use in cancer clinical trials . specifically for cc , we used the eortc qlq - c24 , which consists of 3 multi - item scales and 5 single - item scales.7 two complementary questionnaires were used , the greek symptom control questionnaire by m.d . anderson and questionnaire of post - traumatic psychological disorder , to supplement the assessment of the 2 major questionnaires . all statistical analysis was performed using spss for windows , version 15.0 ( spss inc . , we tried to make the design , methodology , statistical analysis , presentation , and interpretation of results in accordance with international standards to ensure reliability and comparability of research results . the results showed that in 2 patients ( 2% ) the disease was stage ia , in 19 ( 18% ) stage ib , in 34 ( 32% ) iia , in 29 ( 28% ) iib , and in 21 ( 20% ) iiia . out of 105 patients , 58 ( 55% ) underwent rad / chem ( = group 1 ) and the other 47 ( 45% ) only rad ( = group 2 ) . local recurrences occurred in 33 patients ( 31% ) and metastasis in 25 patients ( 24% ) . the results showed that the questionnaires had a high reliability during the sensitivity test ( high cronbach s alpha values , table 2 ) . groups comparisons showed statistically significant differences in descriptive data ( rad patients were older , with more children and fewer years of contraceptive use ) and early toxicity in lower digestive system and pelvis , genetic and urinary system , blood and hematological elements : white cells , platelets , hemoglobin , and hematocrit ( higher in the rad / chem group ) . no statistically significant differences between the 2 groups were observed in general status , physical activity , fatigue , nausea , vomiting , constipation , diarrhea , perception , mood , impact on life and health ( eortc qlq-30 ) , abdominal spasms , defecation , urination , limb problems , vaginal problems , sexuality , vagina condition during the last week and the last 4 weeks ( eortc qlq - c24 ) , post - traumatic stress in the last week due to disease knowledge ( questionnaire of post - traumatic psychological disorder ) , symptoms pain intensity , and impact on life in the last 24 hours ( greek symptom control questionnaire by m.d . for the main subject of our study , question 30 of eortc qlq-30 ( q30 = quality of life ) , no difference was apparent between groups 1 and 2 ( significance level p = 0.119 > 0.005 ) , ie , there is no difference in qol related to the treatment type followed . in addition , we observed no significant differences for any of the aspects of life examined ( table 2 ) . with increasing survival rates of cc patients , health - related qol of the survivors becomes an important issue . qol is , in this context , defined as a person s self - reported perception of physical , psychosocial , and sexual well - being . the different treatment programs affect the lives of surviving women to a varying degree in terms of physical , sexual , and psychosocial functioning.814 despite the trend towards more toxic , multimodal treatment regimes , little focus has been put on the long - term effects of therapy . however , it is more common , nowadays , to include qol measurements in clinical trials . in our study we compared the qol of cc patients who were treated with either combined rad / chem or rad , 1 year after treatment completion . statistically significant differences were found in descriptive characteristics ( such as age , number of children , and use of contraceptives ) and early toxicity in the lower digestive system and pelvis , genetic and urinary system blood , and hematological elements . in the main areas of physical , emotional , and sexual functioning , no significant differences were observed , indicating that the addition of chem in the treatment does not significantly affect the qol of cc patients . our results are in agreement with the conclusion of a study conducted by greimel et al , which corroborated findings from previous research , in which qol of survivors treated with adjuvant rad was compared with qol of patients treated with surg with or without adjuvant chem.1517 there were no differences between treatment groups in overall health status and qol , emotional functioning , fatigue , insomnia , and diarrhea . patients treated with rad reported more impairment in several domains of qol , such as physical functioning , role functioning , and cognitive and social functioning . also , they had more symptoms , such as nausea , vomiting , pain , appetite loss , tight vagina , leaking of urine , and frequent urination than the other 2 groups ( surg and surg / chem ) . survivors treated with adjuvant rad reported more financial difficulties because of their physical condition than women treated with the other 2 treatment modalities . compared with normative reference data , women treated by surg with or without chem had similar qol to healthy women , whereas those treated with rad had lower qol . the latest group reported a lower sexual activity rate than the other groups of survivors ; however , they had similar sexual pleasure and sexual discomfort . frumovitz et al compared the qol and sexual functioning of cc survivors treated with either radical hysterectomy and lymph node dissection or rad at least 5 years after initial treatment.18 compared with surg patients and controls using univariate analysis , rad patients had significantly poorer scores on standardized questionnaires measuring health - related qol ( physical and mental health ) , psychosocial distress , and sexual functioning . their conclusion that rad is responsible for worse qol of cc patients agrees with our results . in a study by korfage et al , surg and a combination of therapies ( surg , rad , and rad / chem ) were compared for their effect on the qol of cc survivors.19 after controlling for background characteristics ( age , education , job , marital status , children , and country of birth ) , generic qol scale scores were similar to those of the reference population , except for worse mental health in survivors . the most frequent symptoms were crampy pain in the abdomen or belly ( 17% ) , urinary leakage ( 15% ) , menopausal symptoms ( 18% ) , and problems in sexual activity . more sexual worry and worse body image were reported by the 2- to 5-year survivors than the 6- to 10-year survivors . compared with surg only , especially primary rad was associated with an increased frequency of treatment - related side effects , also after controlling for age and disease stage at diagnosis and follow - up . according to a recent systematic cochrane review of acute and late toxicity after concomitant chem and rad , late effects of treatment were not well reported and therefore the effect of chem / rad on these effects could not be determined with confidence.15 currently , the 2 successful strategies in treating cc , concomitant chem / rad and chem followed by surg , are being compared in a large phase iii study performed by the european organization for the research and treatment of cancer ( eortc-55994 ) . one of the objectives in this trial is to compare the qol of patients under these regimes . as with all studies based on surveys , selection bias occurs when patients who finally take part in the study have a better qol than those who are unreachable or refuse to participate . also , since data were collected retrospectively , we do not know whether a qol difference may have existed in the treatment groups before therapy . our data suggest that cc survivors treated either with rad / chem or rad have no significant differences in qol and physical , psychosocial , and sexual functioning . these findings suggest that chem addition in cc treatment does not affect qol of patients , at least for this particular protocol . different protocols of treatment or different patients characteristics could have varying effects on patients qol ; thus this study should be expanded to other treatment protocols and/or other cc patient groups as well as control groups . by taking into consideration well - established information on long - term qol outcomes of cc treatments , our data may assist both the patient and the physician in selecting the most appropriate treatment protocol . since the qol of cc patients is of great importance , more interest should be focused on the investigation of the effect of treatment type on the survivor s life .

for biotechnology development , some conditions are required but are not always provided at the adequate level in each country . these conditions include : the role of different actors such as scientists , educators , journalists , politicians , and economic partners;the existence of specialized structures concerned with biotechnology ( research , training , coordination , and supervision structures);the functioning of democratic mechanisms which enable civil society to act its will ( ethics committees , laws regulating biotechnology , monitoring structures , and technological means ) . the role of different actors such as scientists , educators , journalists , politicians , and economic partners ; the existence of specialized structures concerned with biotechnology ( research , training , coordination , and supervision structures ) ; the functioning of democratic mechanisms which enable civil society to act its will ( ethics committees , laws regulating biotechnology , monitoring structures , and technological means ) . our purpose is to evaluate to which extent these conditions are offered to biotechnology development in tunisia and to compare the attitudes towards and the conditions offered to biotechnology development in tunisia through three examples : organ transplant ( ot ) , assisted reproductive techniques ( art ) , and genetically modified organisms ( gmos ) , in order to identify the factors that have allowed success of the first two applications and failure of the latter . in practice , the ot , art , and gmos are the main biotechnology applications which raised public interest which concerns ethical problems in tunisia . conditions offered for ot development concern information , specialized structures , and legislation . good level of information is provided by radio and tv programs and by papers in the local press on organ donation . technological structures consist in a national center for bone marrow transplant , a bank for tissues and eyes , the blood transfusion national center , and immunology laboratories ( hla typing ) . the last creation in june 1995 is the national center for organs transplantation promotion ( ncotp ) . the aims of this centre are increasing awareness for organ donation , establishing a waiting list of patients for ot , ensuring transparency and safety of ot , training of medical and paramedical staff , training and coordination between medical teams . with regard to legislation , two laws are particularly in favour of ot : the law 91 - 22 created in march 1991 and regulating ot and the law on the donor mention registered on the national identity card of the donor . the spirit of these laws was to promote organ donation ( table 1 ) . the main points of the two laws on organ transplantation ( ot ) in tunisia . some recommendations of the tunisia 's national medical ethics committee with regard to the assisted reproductive techniques ( art ) . the main points of the law relative to the assisted reproductive techniques ( art ) in tunisia . comparison of the regulatory situation and current usage for the three technologies ot , art , and gmo . ( + + + , + + , and + are strong , medium , and weak level , resp . ) for ot issues , the high islamic council ( hic ) has published its academic resolution authorizing ot [ 3 , 4 ] . tunisia was the first african country to set up a national medical ethics committee ( nmec ) . statistics of the year 2000 , in authorized public hospitals , permit to record the realization of 43 transplantations ( kidney : 39 ; heart : 1 ; and liver : 3 ) and 626 grafts ( cornea : 578 and marrow : 50 ) ( activity report of national center for organs transplantation promotion , tunisia , 2001 ) . a percentage of 10%15% of tunisian couples are concerned , with 6000 to 8000 candidates for art per year . with regard to the success rate the available methods proposed to couples suffering from infertility are artificial insemination , in vitro fertilization , and the microinjection technique . the in vitro fertilization ( ivf ) is available since 1991 in private and in public hospitals and many hundreds of such operations are realized each year . the major brake in the use of these technologies is the cost which is very expensive ( about $ 2000 to $ 3000 ) . in a study performed in another african country , it was found that one out of thirteen patients who were referred to art centres was able to achieve her objective of motherhood . at the legislative level , there was a lack of a law regulating art until july 2001 . at this period the nmec gave some rules to conduct art that take into account the islamic viewpoint ( table 2 ) . recently , since august 2001 , the law no 01 - 93 , published in , strictly controls art ( table 3 ) . as the law was inspired by the hic and the nmec opinions on art , there is no tension between the islamic viewpoint and the secular one . considering the difficulty to obtain gametes from a donor other than the partner , some couples go abroad , often to europe , to benefit from the ivf technique or the artificial insemination ( procreative tourism ) . historical and geographical reasons made europe the first destination for north african couples while the us centres are preferred by egyptians and saudi patients ( center for surrogate parenting and egg donation inc , encino , calif , http://www.creatingfamilies.com ) . with regard to the use of embryos created by ivf , which are supernumerary , the law 01 - 93 ( table 3 ) as well as the nmec in tunisia ( table 2 ) have stated their opposition to all experimentation on the embryo which is regarded as a potential person ( national medical ethics committee , opinion no . 1 , tunisia , 12 december 1996 ) . in the french law , supernumerary embryos could be destroyed but could also be given to another couple who fulfils some conditions . swiss law also permits embryos donation and the child could have access to data related to the sperm donor . despite years of deployment of genetically modified crops ( gmcs ) , public opinion on gmos , one gene - technology - based application , varied from the condemnation or the distrust to the support , the encouragement , and even the defense [ 10 , 12 , 13 ] . finally , for others the harmlessness of gmos is real and easy to demonstrate , while the escape of genes from gmcs to native species is considered inevitable [ 14 , 15 , 16 ] . many observations and reasons suggested the importance of building public awareness and involving the public in the biotechnology development process . with regard to general opinion in tunisia the only study reporting public opinion about biotechnology was conducted under our supervision and has affected a population of 700 students ( ben ameur r. biologie et thique : attitudes des tudiants du grand tunis en matire de biothique ; unpublished data ) . the results show that 61% of students were rather against the consumption of gm food . interestingly , a clear refusal was recorded with girls ( 69% versus 53% ) and literary sections , especially philosophical and sociological sections ( 84% of refusal attitudes ) ; the mean refusal in scientific sections was about 48% . furthermore it was striking to note poor knowledge score related to gmos even among scientific students ( 17% of correct answers versus 2% in literary sections ) . in tunisia all experiences are enclosed in laboratories and research centres and there is no gmo culture in fields ( ie , field - testing ) . with regard to animal feeding , corn and soya grains are imported without certification . as a very high percentage ( about 80% ) of sailed corn in the world is transgenic , we think that corn bought in tunisia is gm . indeed , until now there was no procedure control and the country needs great quantity of corn for animal use . moreover , there is no research on gm animals in tunisia and officially there is no gm - based seed or seedling . probably by application of the famous precautionary principle the elaboration of such regulation is delicate and tricky because it must protect , at the same time , consumers , economy , environment , and scientific research . the main points of this law which have been discussed by different experts will be as follows . the law will concern gmos importation , stocking , and transport.the law will not refuse gm products but will specify and make clear the conditions of importation.some repressive measures will be taken to protect the consumer , to preserve the environment , and to control economic exchanges the law will not refuse gm products but will specify and make clear the conditions of importation . some repressive measures will be taken to protect the consumer , to preserve the environment , and to control economic exchanges . since european countries constitute the main economic partners of tunisia , one has to notice that the tunisian law will take into account the european regulation . in the same way , the law project will specify the composition and the functioning of the new national committee of biosafety and the conditions to produce gmos . the organization of african unity stressed the request of gmos approval before its importation . each gmo must be labelled such , and its producers would be responsible in the eyes of the law ( zone libre d'ogm , les moratoires travers le monde , http://www.infogm.org ) . in egypt , gmcs were found to benefit egyptian farmers by the reduction of pesticide application . gmcs authorized in this country are cotton , squash , potato , tomato , and corn . in kenya , a gm bt - maize has also been introduced and assayed against local stem borer species . kenya , south africa , and egypt are the first african countries that accepted gmos testing in fields . applications of biodigesters to generate biogas for lighting and heating are undertook in ghana . in zambia , government bans gm food aid offered by the us agency for international development via the world food program ( wfp ) . in north africa ( ie , maghreb ) , the people 's reactions are likely to be similar to tunisian ones because the three countries ( tunisia , algeria , and morocco ) have the same religious considerations and cultural context . however , political will , specialized structures , and legislation are rather different . the practice of the art today in morocco knows a fabulous fancy even in the absence of medical coverage which could help in the repayment of the medical expenses . it is interesting to note that the 15 centres of art practising in morocco are connected with private healthcare centres . with about forty transplants , morocco is far behind countries such as tunisia and kuwait . the legislation stays unsuitable and the organs donation is governed by the dahir of 1952 , an obsolete text . with regard to morocco 's position to gmos , and in application of the principle of precaution , a circular was established in august 1999 which forbids the introduction , on the national territory , of products and food preparations containing stemming products from gmo . the only products allowed are those intended for the animal diet ( eg , corn ) and the import of gmos seeds is forbidden . finally , a project of law related to the control of the gmos use and scattering , which aims to protect the human health , the animal health , and the environment of possible risks connected to the gmos , is elaborated at the level of the agricultural department and submitted for approval . in algeria , the order no 30 of october 15th , 2002 , authorizes the taking and the transplantation of organs from corpses . in this country , gmos import is banned since december 2000 and the sociocultural context is rather unfavourable to research development ( http://www.infogm.org ) . until today , there are no structures capable of detecting gmos in the maghreb countries . however , algerian specialists think that the research in reproductive health is marginalized and very few efforts are granted in this direction . the first step was realized in february 2003 , by the birth of the first baby by art in the private hospital of boussouf in constantine ( http://www.1sante.com ) . conditions offered for ot development concern information , specialized structures , and legislation . good level of information is provided by radio and tv programs and by papers in the local press on organ donation . technological structures consist in a national center for bone marrow transplant , a bank for tissues and eyes , the blood transfusion national center , and immunology laboratories ( hla typing ) . the last creation in june 1995 is the national center for organs transplantation promotion ( ncotp ) . the aims of this centre are increasing awareness for organ donation , establishing a waiting list of patients for ot , ensuring transparency and safety of ot , training of medical and paramedical staff , training and coordination between medical teams . with regard to legislation , two laws are particularly in favour of ot : the law 91 - 22 created in march 1991 and regulating ot and the law on the donor mention registered on the national identity card of the donor . the spirit of these laws was to promote organ donation ( table 1 ) . the main points of the two laws on organ transplantation ( ot ) in tunisia . some recommendations of the tunisia 's national medical ethics committee with regard to the assisted reproductive techniques ( art ) . the main points of the law relative to the assisted reproductive techniques ( art ) in tunisia . comparison of the regulatory situation and current usage for the three technologies ot , art , and gmo . ( + + + , + + , and + are strong , medium , and weak level , resp . ) for ot issues , the high islamic council ( hic ) has published its academic resolution authorizing ot [ 3 , 4 ] . tunisia was the first african country to set up a national medical ethics committee ( nmec ) . statistics of the year 2000 , in authorized public hospitals , permit to record the realization of 43 transplantations ( kidney : 39 ; heart : 1 ; and liver : 3 ) and 626 grafts ( cornea : 578 and marrow : 50 ) ( activity report of national center for organs transplantation promotion , tunisia , 2001 ) . the second technology which is much in demand is the art . a percentage of 10%15% of tunisian couples are concerned , with 6000 to 8000 candidates for art per year . with regard to the success rate the available methods proposed to couples suffering from infertility are artificial insemination , in vitro fertilization , and the microinjection technique . the in vitro fertilization ( ivf ) is available since 1991 in private and in public hospitals and many hundreds of such operations are realized each year . the major brake in the use of these technologies is the cost which is very expensive ( about $ 2000 to $ 3000 ) . in a study performed in another african country , it was found that one out of thirteen patients who were referred to art centres was able to achieve her objective of motherhood . at the legislative level , there was a lack of a law regulating art until july 2001 . at this period the nmec gave some rules to conduct art that take into account the islamic viewpoint ( table 2 ) . recently , since august 2001 , the law no 01 - 93 , published in , strictly controls art ( table 3 ) . as the law was inspired by the hic and the nmec opinions on art , there is no tension between the islamic viewpoint and the secular one . considering the difficulty to obtain gametes from a donor other than the partner , some couples go abroad , often to europe , to benefit from the ivf technique or the artificial insemination ( procreative tourism ) . historical and geographical reasons made europe the first destination for north african couples while the us centres are preferred by egyptians and saudi patients ( center for surrogate parenting and egg donation inc , encino , calif , http://www.creatingfamilies.com ) . with regard to the use of embryos created by ivf , which are supernumerary , the law 01 - 93 ( table 3 ) as well as the nmec in tunisia ( table 2 ) have stated their opposition to all experimentation on the embryo which is regarded as a potential person ( national medical ethics committee , opinion no . 1 , tunisia , 12 december 1996 ) . in the french law , supernumerary embryos could be destroyed but could also be given to another couple who fulfils some conditions . swiss law also permits embryos donation and the child could have access to data related to the sperm donor . despite years of deployment of genetically modified crops ( gmcs ) , there is no evidence of injury to people . public opinion on gmos , one gene - technology - based application , varied from the condemnation or the distrust to the support , the encouragement , and even the defense [ 10 , 12 , 13 ] . finally , for others the harmlessness of gmos is real and easy to demonstrate , while the escape of genes from gmcs to native species is considered inevitable [ 14 , 15 , 16 ] . many observations and reasons suggested the importance of building public awareness and involving the public in the biotechnology development process . with regard to general opinion in tunisia the only study reporting public opinion about biotechnology was conducted under our supervision and has affected a population of 700 students ( ben ameur r. biologie et thique : attitudes des tudiants du grand tunis en matire de biothique ; unpublished data ) . the results show that 61% of students were rather against the consumption of gm food . interestingly , a clear refusal was recorded with girls ( 69% versus 53% ) and literary sections , especially philosophical and sociological sections ( 84% of refusal attitudes ) ; the mean refusal in scientific sections was about 48% . furthermore it was striking to note poor knowledge score related to gmos even among scientific students ( 17% of correct answers versus 2% in literary sections ) . in tunisia , only microorganisms and plants are involved in the gene technology . all experiences are enclosed in laboratories and research centres and there is no gmo culture in fields ( ie , field - testing ) . with regard to animal feeding , corn and soya grains are imported without certification . as a very high percentage ( about 80% ) of sailed corn in the world is transgenic , we think that corn bought in tunisia is gm . indeed , until now there was no procedure control and the country needs great quantity of corn for animal use . moreover , there is no research on gm animals in tunisia and officially there is no gm - based seed or seedling . probably by application of the famous precautionary principle the elaboration of such regulation is delicate and tricky because it must protect , at the same time , consumers , economy , environment , and scientific research . the main points of this law which have been discussed by different experts will be as follows . the law will concern gmos importation , stocking , and transport.the law will not refuse gm products but will specify and make clear the conditions of importation.some repressive measures will be taken to protect the consumer , to preserve the environment , and to control economic exchanges . the law will concern gmos importation , stocking , and transport . the law will not refuse gm products but will specify and make clear the conditions of importation . some repressive measures will be taken to protect the consumer , to preserve the environment , and to control economic exchanges . since european countries constitute the main economic partners of tunisia , one has to notice that the tunisian law will take into account the european regulation . in the same way , the law project will specify the composition and the functioning of the new national committee of biosafety and the conditions to produce gmos . the organization of african unity stressed the request of gmos approval before its importation . each gmo must be labelled such , and its producers would be responsible in the eyes of the law ( zone libre d'ogm , les moratoires travers le monde , http://www.infogm.org ) . in egypt , gmcs were found to benefit egyptian farmers by the reduction of pesticide application . gmcs authorized in this country are cotton , squash , potato , tomato , and corn . in kenya , a gm sweet potato resistant to virus has been introduced . bt - maize has also been introduced and assayed against local stem borer species . kenya , south africa , and egypt are the first african countries that accepted gmos testing in fields . applications of biodigesters to generate biogas for lighting and heating are undertook in ghana . in zambia , government bans gm food aid offered by the us agency for international development via the world food program ( wfp ) . in north africa ( ie , maghreb ) , the people 's reactions are likely to be similar to tunisian ones because the three countries ( tunisia , algeria , and morocco ) have the same religious considerations and cultural context . however , political will , specialized structures , and legislation are rather different . the practice of the art today in morocco knows a fabulous fancy even in the absence of medical coverage which could help in the repayment of the medical expenses . it is interesting to note that the 15 centres of art practising in morocco are connected with private healthcare centres . with about forty transplants , the legislation stays unsuitable and the organs donation is governed by the dahir of 1952 , an obsolete text . with regard to morocco 's position to gmos , and in application of the principle of precaution , a circular was established in august 1999 which forbids the introduction , on the national territory , of products and food preparations containing stemming products from gmo . the only products allowed are those intended for the animal diet ( eg , corn ) and the import of gmos seeds is forbidden . finally , a project of law related to the control of the gmos use and scattering , which aims to protect the human health , the animal health , and the environment of possible risks connected to the gmos , is elaborated at the level of the agricultural department and submitted for approval . in algeria , the order no 30 of october 15th , 2002 , authorizes the taking and the transplantation of organs from corpses . in this country , gmos import is banned since december 2000 and the sociocultural context is rather unfavourable to research development ( http://www.infogm.org ) . until today , there are no structures capable of detecting gmos in the maghreb countries . however , algerian specialists think that the research in reproductive health is marginalized and very few efforts are granted in this direction . the first step was realized in february 2003 , by the birth of the first baby by art in the private hospital of boussouf in constantine ( http://www.1sante.com ) . the conditions offered in tunisia to each of the 3 biotechnology examples presented above are different . for ot , we can note that its development is ensured by wide information as a result of a legislation in its favour , the existence of specialized structures , and a clear political will as a decisive factor of success . in fact , developing ot at the national level allows the formation of skilled personnel and restricts expenditure . furthermore , ot constitutes a response to a real need . because the kidney diseases are frequent in the country , the first transplantation in tunisia was kidney transplant . in the case of the art , the strong point in favour of its development is users pressure and population need . the art is supported in tunisia by a strong demand at the population level but it is limited by religious considerations ( table 4 ) . in fact , the islamic religion , by prohibiting the child adoption , pushes the couple who wants his with regard to gmos , there is a lack of expression of a real need at the national economic level . furthermore , this biotechnology is limited by safety considerations and the conditions for its development are rather difficult because decisions were taken at the political level and not at the individual one . public debate on acceptance of biotechnology varied among countries and among technology 's main applications . generally , concerns are about the health , the food , and the environment . biotechnology contribution to the progress of medicine through the last two decades did not arouse issues . there is an evident perceived potential benefit arising from biotechnology . on the other hand , in the agricultural field , those pros and cons have arguments and the planet has never been more divided over a question than on transgenic crops . this situation leads some organizations to participate in the debate and propose their viewpoints . the united nations development programme ( undp ) , for example , judges that development perspectives offered by gmos require strong national policies with clear regulations to ensure that this technology does not destabilize development ( making new technologies work for human development . annual report , undp , usa , july 2001 ) . as for the international service for national agricultural research ( isnar ) , the government should not act hastily , but should rather approach the issue slowly and carefully to allow time to study the full implications of the new technology ( isnar annual report 1998 , april 2000 ) . finally , the african agency of biotechnology ( aab ) encourages the production , the distribution , and the commercialization of biotechnology products ( aab , http://www.aab.org.dz/francais.htm ) . if we limit the analysis to the tunisian country , it would be interesting to express the following recommendations . our study has demonstrated a lack of information among public , even the educated people . it is worthwhile to notice that in tunisia , although biotechnology is well accepted in the medical field , the actual conditions do not seem to be favourable to gmos because of precisely , a lack of information . scientists and the mass media would find the appropriate way to communicate with the public . they should inform the nonspecialists on stakes and help the society to take advantage of the biotechnology advances . political and financial measures should be taken to encourage the dissemination of scientific and technological information . concretely , scientific and medical associations , the city of sciences , and the journalists could play this role . the role of these actors is to report the biotechnology facts as well as to present the issues and the expected benefits . thus , the public will be able to judge and to separate between what is factual from what is speculative . what we need is the establishment of a balanced view of biotechnology : its risks and its beneficial potential . our analysis has shown that in tunisia , biotechnology supported by law and regulations ( ie , art and ot ) is more accepted than that waiting for a legislation ( ie , gmos ) . moreover , legislation reassures the public and the scientists and protects the country 's economical interests . it is true that since 1992 , the tunisian country has been dotted with an ethics committee but this committee is active rather in medical fields and has never published opinions related to plant biotechnology ( ie , gmos ) . we believe that for a successful policy framework , it is necessary to first set up a local national biotechnology committee of experts , government , and private sector representatives to overview biotechnology activities . in conclusion , when safety exceeds risks , intrinsic considerations are over external pressures and when the political will and the popular demand go hand in hand , then the conditions are favourable to the development of a given technology . in any case , more information , training , research , regulation , coordination , democracy , ethics , and more wisdom can be recommended .

patient adherence to glaucoma treatment regimens is essential to maintain maximum intraocular pressure ( iop ) reduction and to delay disease progression in patients with primary open - angle glaucoma.1 however , adherence with glaucoma treatment is a known problem , with multiple studies suggesting that more than half of glaucoma patients who initiate treatment fail to refill prescriptions.26 reasons for nonadherence can vary , ranging from patient characteristics , such as race , travel , or financial challenges , to poor patient - doctor communication , limited understanding of the risks of nonadherence , or not being willing to discuss adverse events with the physician.7 some of these reasons are common across medications and medication classes , but adverse events is one area in which differences between medications may have an impact on patient adherence and treatment efficacy . for example , recent estimates suggest that ocular surface disease ( osd ) is experienced by more than half of all glaucoma patients.8 limiting the use in iop - lowering medications of the preservative benzalkonium chloride ( bak ) , which is associated with increased rates of osd,8,9 may minimize these rates . minimizing preservatives such as bak can be accomplished in a number of ways and can have important longterm eye health benefits . using a bak - free product and limiting exposure by prescribing monotherapy instead of polypharmacy are ideal ways to do this , but monotherapy may not be sufficient to lower iop adequately and adjunctive therapies are required . the introduction of adjunctive therapies multiplies negative influences on adherence ( ie , complexity of regimen , likelihood of adverse events)10,11 and increases exposure to bak which has been linked to decreased success with filtration surgery.12 therefore , the simplest treatment regimen , with lowest exposure to bak and the least need for adjunctive therapy , is optimal . not only does increased medication complexity decrease adherence , 10,13 but adverse events such as osd associated with bak can affect adherence.7 in theory , these recommendations seem reasonable , but only a real - world comparison of treatments can address the issue adequately . several studies with one - year follow - up of patients treated with a bak - free prostaglandin analog ( bak- free travoprost ) compared with those treated with alternative prostaglandin analogs ( bimatoprost and latanoprost ) have been published.46 each study used the same inclusion criteria and identified patients with a prescription for a topical prostaglandin analog during a six - month period and who had no glaucoma therapy claims in the six months prior and had at least 12 months of data available after the initial prostaglandin analog claim . these studies have consistently found that during the first year of treatment , a bak - free prostaglandin analog was associated with lower rates of adjunctive therapy , a longer time until initiating adjunctive therapy , and lower first - year costs . while these studies present evidence that bak - free travoprost provides treatment advantages within a single year of follow - up , it was useful to extend this analysis to include two years and to examine glaucoma surgical procedures as a possible confounder of previous study findings , ie , whether surgical procedures were performed at different rates across groups and thus affected the need for further pharmacotherapy . the goals of this study are similar to the previous analyses , ie , to evaluate the treatment patterns and costs of patients newly initiating therapy with prostaglandin analogs as first - line therapy . however , a different health care database was used , which included glaucoma surgical procedural data . thus , this study includes an analysis of these findings , along with an evaluation of pharmaceutical treatment data over a two - year period . the patients described in the retrospective cohort analysis were receiving prescription benefits and were included in the integrated healthcare information services national managed care benchmark database , a medical claims database covering managed care plans that serve more than 30 million members across the us . all data were deidentified in accordance with protected health information standards under the health information portability and accountability act , so that no individually identifiable information was included in the study database . therefore the study cohort included patients who first initiated therapy with one of three prostaglandin analog products ( bimatoprost , latanoprost , or bak - free travoprost ) between september 1 , 2006 and march 30 , 2007 . to qualify , patients had to have at least one claim for primary open - angle glaucoma ( international classification of diseases , ninth revision , clinical modification [ icd-9-cm ] code 365.11 ) during the observation period and six months of prior claims data in which there were no glaucoma therapy claims of any class . the index date was defined as the first date on which a prostaglandin analog was filled . the study population was further defined by requiring patients to have at least 24 months of uninterrupted use of the index prostaglandin analog following the index date ( initial prescription fill ) . this was operationalized as not having prescriptions for any other prostaglandin analogs during the follow - up period and having at least one prescription for the index prostaglandin analog during months 1924 of the follow - up period . patients were required to be continuously enrolled , as per the eligibility database , during the 30-month observation period ( six months prior to the first prostaglandin claim and the 24 months of follow - up ) . patients who did not meet these requirements ( sufficient prior claims data , uninterrupted use , and repeat use of the index prostaglandin ) were excluded from the study database . we considered that patients added an adjunctive medication , defined by national drug code , if there was a sequential and subsequent refill of an adjunctive agent in the presence of continued refills for the index agent . adjunctive medications were described by type ( adrenergics , beta - blockers , carbonic anhydrase inhibitors , miotics , prostaglandin analogs , fixed combinations , others ) . the analysis identified patients by the prostaglandin prescription they first filled ( ie , the index medication ) during the study period , with one exception . patients who initiated treatment with the original formulation of travoprost but then switched to the bak - free formulation after a single prescription for the original formulation were included in the bak - free cohort . gender was compared using a chi - square test and age was compared using analysis of variance ( anova ) . median and mean number of days to initiation of adjunctive therapy were calculated for each cohort , with the distribution of number of days examined to determine which measure to use in the model . the median numbers of days until patients added adjunctive therapies were compared using a wilcoxon rank sum approach , and mean times were compared using anova . the rate of selected surgical procedures for glaucoma , as specified in the published literature,14,15 was calculated over the observation period , with rates compared across patient cohorts , controlling for age and gender . the american academy of ophthalmology preferred practice patterns for glaucoma suggest that follow - up care should be based on achievement of target iop and the amount of disease progression,16 neither of which was available in the prescription database used for this study . therefore , the base case analysis uses resource rates from a survey - based study17 while sensitivity analyses explore visits , as recommended by other studies and guidelines.16,18 because all patients in the model are assumed to undergo the same procedures and tests at their visits regardless of the prostaglandin analog they receive , the relatively small differences across studies in terms of the components of each visit have minimal impact on the study findings . at the initial visit , patients are assumed to have a level 4 ( comprehensive ) evaluation ( current procedural terminology , 92004 ) . follow - up visits are assumed to be level 2 ( intermediate ) visits ( current procedural terminology 92012 ) , with three follow - up visits during the first year ( likely but not necessarily at 30 and 90 days following initiation of the index therapy and at 12 months , and then an additional two visits in the following year ) . two additional follow - up visits are assumed to be associated with the initiation and monitoring of adjunctive medication among patients for whom it is prescribed . thus , patients who stay on monotherapy are assumed to have one comprehensive visit and five intermediate visits , while those who require adjunctive therapy had one comprehensive visit and seven intermediate visits . table 1 presents the procedures and diagnostic tests that comprised each visit , as well as the costs used for each . we used average wholesale price ( awp ) for 2009 as the basis of prescription costs.19 the range of published awps for the prostaglandin analogs was fairly narrow ( $ 80.53 to $ 82.67).19 for the prostaglandin analogs , the midpoint cost for the agents is used in the model because awp is the best publicly available estimate for the analysis . further , the number of prescriptions for prostaglandin analogs was expressed as 2.5 ml size bottle equivalents , because this is the most common size found in the claims database and represents approximately a 3045 day supply when used as per label in both eyes . the awps for adjunctive therapies were also reviewed in the red book.19 for the base case of the model , branded products were used , but sensitivity analyses explored lower costs . days of use per bottle are based on data from previously published studies.4,5 number of bottles required is rounded to the nearest tenth . medical charges are estimated using the 75th percentile of physicians fees from a published benchmark.20 all the costs used in the model are presented in table 2 . a total of 5018 patients were identified who had six months of glaucoma - free eligibility and 24 months of continuous eligibility after an initial prescription for bimatoprost , latanoprost , or bak - free travoprost ( figure 1 ) . of the 1382 bimatoprost patients , 747 had repeat prescriptions , and of the 3247 latanoprost patients , 1651 had repeat prescriptions . among the 389 patients who initiated with bak - free travoprost , 203 had repeat prescriptions ( ie , no other prostaglandins during the follow - up period plus at least one repeat of the index prescription in months 1924 ) . fifty - six of these 203 had an initial prescription with the original formulation of travoprost and were then transitioned to the bak - free formulation . persistence patterns differed only slightly across groups , with 54% of bimatoprost patients remaining on the index therapy , while 51% of latanoprost patients and figure 1 details the patient population persistence patterns . demographic characteristics by index medication are presented in table 1 . average age across all groups was 64 years , and 52% of the patients were female . gender was similar across groups , although there were significant differences in age across groups ( range was 6265 years , p < 0.01 ) . in the bimatoprost , latanoprost , and bak - free travoprost treatment groups , 51.3% , 37.3% , and 35.0% , respectively , initiated adjunctive therapy during the course of the year at significantly different proportions ( p < 0.0001 , see table 3 ) . patients on bak - free travoprost were able to continue without adjunctive therapy longer than patients treated with other prostaglandins . the median number of days until the patients added adjunctive therapies was 83 for bimatoprost , 101 for latanoprost , and 113 for patients for bak - free travoprost . the mean number of days to initiating adjunctive therapy were 221 in the bak - free travoprost group , 140 for patients receiving bimatoprost , and 181 for patients receiving latanoprost ( p < 0.0001 ) . the types of adjunctive therapies used also appeared to differ across index therapies , although comparisons are difficult to interpret because of the use of fixed combination therapies . for example , among patients treated with bak- free travoprost and latanoprost , beta - blockers were the most commonly used adjunctive medication , while for the bimatoprost- treated patients , a fixed combination therapy of beta - blocker and carbonic anhydrase inhibitor was most common . however , across therapies , the rate of use of any beta - blocker ( ie , alone or as part of a combination therapy ) was fairly consistent , ranging from 60% to 64% , and the rate of use of any carbonic anhydrase inhibitor was only slightly more variable ( 35%42% ) . since the other adjunctive therapy category includes other fixed combination therapies , such as a combination of an 2-adrenegeric receptor agonist and beta - blocker , or different doses of medications , comparisons are not useful . medical costs , ie , ophthalmic visits and procedures ( excluding surgical procedures ) , prostaglandin analogs , and adjunctive therapies , were estimated at $ 2557 for patients initiating with bak - free travoprost , $ 3147 for patients initiating with bimatoprost , and $ 2843 for patients initiating with latanoprost . sensitivity analyses that explored clinical and cost parameters did not change the direction or magnitude of findings substantially . surgical procedures were not included in the costs because of the low frequency and the similarity in rates across index prostaglandins . table 4 presents findings on the rate of surgical procedures for glaucoma among the study population . there were too few patients who received bak - free travoprost to compare rates for certain procedures , but rates for most procedures were similar across treatment groups . this analysis adds to findings from previous studies that explored the one - year treatment patterns of patients initiating on prostaglandin analogs4,5 by providing a more comprehensive and longer follow - up analysis . the extended follow - up period ( 24 versus 12 months ) and availability of procedural data provides information on long - term patterns of care for patients with glaucoma not available in previous published studies . while previous studies found rates of adjunctive therapy use to range from 12% ( bak - free travoprost6 ) to 23% ( bimatoprost6 ) over one year , we found that this increased to a range of 35% ( bak - free travoprost ) to 51% ( bimatoprost ) of patients requiring adjunctive therapy over two years . interestingly , the median time to adding adjunctive therapy was in the same range for bak - free travoprost ( 116 days in this analysis versus 109159 days in previous analyses ) while the median times to initiating adjunctive therapy with bimatoprost or latanoprost were longer in this analysis than in previous analyses ( for bimatoprost 83 days compared with 5373 days in previous studies ; for latanoprost 101 days compared with 6374 days in previous studies).4,5 a full electronic health record might provide more information explaining these differences , and a meta - analysis of previous studies suggests that there may be significant differences in efficacy across these medications.21 consistent with previous studies , the average cost per patient over the observation period was lowest in the cohort with the longest duration of monotherapy . since the difference in monotherapy rates was more than 15% ( 48.7% for bimatoprost and 65.0% for bak - free travoprost ) , the additional follow - up visits for patients who switched therapies also had an impact on the cost differences . in fact , when the rates of monotherapy were equalized across cohorts in sensitivity analyses , the magnitude of the difference in total costs was halved . previous analyses for which only prescription claims data were available left open the question about whether low persistence was associated with the use of glaucoma surgical interventions . that is , were patients able to discontinue therapies because of a successful glaucoma surgical intervention ? while these data do not show the success rates of interventions , the analysis demonstrated very low use of glaucoma surgical interventions across index medications in the first two years after initiating treatment . therefore , for those patients who are new to glaucoma therapy , surgical interventions would not be the reason for the high rate of patients stopping all glaucoma medications . as with any claims database analysis , there are certain limitations that should be acknowledged . the use of a large claims database offers an important strength in terms of sample size but shortcomings remain . for example , the patient population in any given database may not match the general population of patients with the condition in question , although in this analysis , the age and gender of the population reflect that of the general glaucoma population.22 other characteristics , such as race , educational status , or self - reported health status , among others , are unknown . along those lines , it is important to note that patients in the study database were not randomized to treatment . further , various clinical or personal characteristics that may be associated with treatment assignment can not be known from the database . some clinical characteristics can be examined , eg , there is some evidence that glaucoma patients taking antihypertensive drugs may have different adjunctive therapy rates23 but we did not undertake such an analysis . the population of prescribers may not accurately reflect national patterns , which could affect generalizability . similarly , the treatment selection process may vary based on region , past success with the treatment , and many other factors.24,25 most claims databases have little or no data on the characteristics of prescribers but , because the data were from a large managed care organization , it is likely that there is a wide distribution of prescribers and practices . by their nature , claims databases only provide information on people with some type of insurance coverage , and those with coverage may be more likely to seek out and adhere to treatment . finally , claims databases may contain coding biases or errors , although there is no reason to believe that these errors would be different across index treatments . in addition , the claims in this database were reviewed and adjudicated before the database was prepared for this analysis . the study attempted to identify only patients who were new to therapy by requiring six months of glaucoma - free claims prior to the f irst prostaglandin analog prescription . however , there is no way to account for product samples , which one study identified as being received by 20% of patients,3 and which could translate into patients who were already taking prostaglandin analogs before our study identified them . also , based on this study methodology , patients with poor adherence could have been included , because patients in the cohort analysis were required to have a minimum of two glaucoma prescriptions filled during the 24-month follow - up period , but no attempt was made to assess medication possession ratio . this study found that over two years , the rate of adjunctive therapy use was lower and time to initiating adjunctive therapy was longer with a bak - free prostaglandin analog compared with other alternatives . it can not be ruled out that differences in physicians prescribing patterns affects these findings . this difference in treatment patterns was not associated with glaucoma surgical interventions , which were at a low rate of use and similar across patient cohorts . different rates of use of adjunctive therapy led to a substantial difference in costs over a two - year period . a full medical record review could help explore the reasons for low persistence rates and the advantages to minimizing bak in the treatment of patients with primary open - angle glaucoma .

remote monitoring of cardiac implantable electronic devices ( cieds ) has been suggested as a new standard for patient follow - up and it has been accepted as an alternative to the majority of scheduled follow - up visits in the international guidelines . expected benefits include reduction of outpatient clinic workload , better patient quality of life , improved implanted system surveillance , continuous patient monitoring to early detect harmful clinical events , and to improve patient outcome . some studies have demonstrated favourable effects of remote monitoring on device and patient management with potential benefits on clinical outcome , but few data are available in daily practice . furthermore , introduction of telemedicine in outpatient clinic for cied patients needs deep changes in workflow , but to date an optimal organizational model is still to be defined . the homeguide registry is an italian multicentre study , designed to provide an organizational model for implementing remote monitoring of cieds in daily clinical practice . the aim was to estimate the effectiveness of device remote monitoring in clinical event detection and management and to analyse the associated outpatient clinical workload and impact on resource consumption . the homeguide registry is an investigator - initiated , prospective , multicentre observational study ( clinicaltrials.gov identifier nct01459874 ) , evaluating the effectiveness of an outpatient clinic workflow model implementing remote monitoring for detection and management of unselected cardiovascular events in patients with class i / ii indications for pacemaker ( pm ) or implantable cardioverter defibrillator ( icd ) , either with or without cardiac resynchronization therapy ( crt ) . the study was designed by a coordinating investigator and reviewed by a steering committee , which included the study coordinator , five investigators , one representative nurse , one statistician , and one biotronik representative . homeguide was approved by an institutional review board and conducted in 75 italian sites recruiting patients after obtaining written informed consent to study participation . the primary objective of the homeguide registry was to collect and document all major cardiovascular events ( mce ; as better defined below ) that are normally observed and managed in the participating outpatient clinics to assess the rate of the events that home monitoring ( hm ) allowed to remotely detect and treat during patient follow - up . eligible subjects were patients indicated to first implantation or upgrade of pm or icds with or without crt . remote monitoring was accomplished with the biotronik hm system ( biotronik se & co. kg ) based on ultra - low power daily or event - triggered transmissions in the mics ( medical implant communication service ) band , from the implanted device to a mobile patient unit , forwarding data via gsm ( global system for mobile communications ) with gprs ( general packet radio service ) protocol to a service centre with encrypted access . in this study , hm was routinely implemented in the outpatient clinics of the participating sites according to an organizational model setting roles , responsibilities , and workflow . this model ( the homeguide model ) derived from a previous pilot experience and has been accepted as a national standard by the italian association of arrhythmology and cardiostimulation . practical feasibility and clinical effectiveness of such model have never been assessed on a large - scale clinical trial . the homeguide model is essentially based on a cooperative interaction between the roles of an expert reference nurse and a responsible physician with an agreed list of respective tasks and responsibilities . each nurse physician pair , within one outpatient clinic , is exclusively dedicated to an assigned subgroup of remotely controlled patients . physicians and nurses tasks are detailed in the figure 1 along with action items and responsibilities . ep , electrophysiology / cardiac pacing laboratory ; icd , implantable cardioverter defibrillator ; hm , home monitoring ; pm , pacemaker ; rn , responsible nurse ; rp , responsible physician . ep , electrophysiology / cardiac pacing laboratory ; icd , implantable cardioverter defibrillator ; hm , home monitoring ; pm , pacemaker ; rn , responsible nurse ; rp , responsible physician . home monitoring transmissions were reviewed by the nurse within two working days upon hm notifications of critical events flagged for attention . in case of no alerts , custom software was implemented to measure and record the duration of all the hm sessions conducted by the nurse and the physician . nurse communications to individual patients were triggered by : hm transmission interruptions for more than 7 days , recall for an unscheduled in - clinic visit or a skipped scheduled in - clinic visit , assessment of patient compliance , and clinic status after medical actions . in - clinic visits were scheduled at post - implant discharge , at 1 month and then once in a year . unscheduled in - clinic visits were further classified in : visits required by the patients ( including visits following an emergency department admission ) or triggered by hm notifications , reports , and trend reviews . major cardiovascular events were defined as any untoward cardiovascular occurrence , disease or signs ( including abnormal device findings ) faced in an outpatient cied clinic whether or not related to the implanted device . such definition was broad enough to encompass a large class of significant events , including arrhythmias , worsening heart failure ( hf ) , device - related complications , as well as events that are not supposed to be detected by cieds or remotely transmitted via hm ( such as strokes or acute myocardial infarctions ) . from early detection to final decision when a mce was a combination of multiple events , a multiple diagnosis was allowed in the electronic case report forms . major cardiovascular events were classified by the investigators according to specific guidelines developed by the steering committee , according to the following properties : source of information ( hm session , in - person visit , or other circumstances);home monitoring - witnessed : a mce was considered as hm - witnessed if hm is technically able to detect the event;appropriateness of initial diagnosis;absence of associated symptoms;corrective action required.investigators classifications were reviewed and adjudicated by an independent five - member board , blinded to the investigational sites and investigators . source of information ( hm session , in - person visit , or other circumstances ) ; home monitoring - witnessed : a mce was considered as hm - witnessed if hm is technically able to detect the event ; appropriateness of initial diagnosis ; absence of associated symptoms ; corrective action required . the implanted study devices were equipped with several indexes for hf monitor , depending on the device type and model . the homeguide protocol did not provide reaction algorithm , programming or threshold setting recommendations which were left to investigators discretion . therefore , the results represent current standard practice at the present stage of clinical experience and hm technology development . to provide estimates of sensitivity and ppv of hm event detection , adjudicated mces were divided in : true - positive ( events detected during periodic or alert - triggered hm sessions ) ; false - negative ( events actually occurred but not detected with hm for any reason ) ; false - positive ( false events notified by automatic hm alerts and/or misinterpreted ) . home monitoring sensitivity and ppv were then estimated as usually by the ratios of the number of true - positive events over the number of true documented events and the number of events detected during hm sessions , respectively . estimates were reported as percentages with the respective 95% confidence interval ( ci ) boundaries . generalized estimating equation ( gee ) models were used to adjust sensitivity and ppv estimates for within - individual repeated mces . it is worth noting that the broad definition of mce , assumed in the homeguide registry , and the particular characteristics of hm as a diagnostic tool , may impair practical significance of sensitivity and ppv . we therefore sought to develop an index , derived from the expected utility concept normally used in markov processes and based on the rate of some properties of interest shown by mces . the latter may in fact be considered as a measure of the utility of the follow - up strategy by which the mce was first detected ( hm sessions or in - person visits / other circumstances ) , and assumed as a score . the sum of the products of the scores by the respective probabilities is normally known as an expected utility value and can be easily estimated for both monitoring strategies with experimental data . an incremental utility ( iu ) index can be defined as the difference between the respective expected utilities of both follow - up strategies returning a numerical value in the ( 1 , 1 ) range , that can be estimated with its 95% ci and tested against the hypothesis of iu = 0 ( with our assumptions , a positive iu value was in favour of a higher expected utility of the hm strategy ) . generalized estimating equation models may be used as well to adjust for within - individual repeated events correlation . in principle , calculations may be performed for each set of arbitrarily chosen properties , or according to the clinical interest of the particular aspect analysed . we considered a set of four properties : hm - witnessed ; correct initial diagnosis ; asymptomatic events ; corrective action required ( actionability ) . the homeguide registry was 90% powered to reject the null hypothesis that sensitivity of hm mce detection was 70% at an one - sided test with a significance level of p = 0.025 . simulations showed that a priori assuming a sensitivity value of 75% would have required 844 adjudicated mces . with such a collection of mces it was also estimated that there was a 97% power available to reject the null hypothesis that the hm ppv was 80% assuming a ppv of 85% . it was then predicted that 55% of enrolled patients would have experienced one valid mce during at least follow - up of 1 year . with a hypothesized patient dropout rate of 10% , the prediction of 55% mce rate per patient proved false , as it resulted remarkably higher at the end of the study . the steering committee decided to terminate patient follow - up after the target population was enrolled and the last patient in reached the 3-month follow - up . continuous variable distributions were checked for normality with the shapiro wilk test and reported as mean sd if the normality hypothesis could not be rejected . median and interquartile range ( iqr ) were used in the opposite case . proportions were reported as percentages along with the exact 95% ci . statistical significance was set at p = 0.05 level . to account for the bias deriving from repeated mces in individual patients ( = unadjusted ) , hm sensitivity , ppv , and iu were adjusted using gee models with a first - order autoregressive correlation . the primary objective of the homeguide registry was to collect and document all major cardiovascular events ( mce ; as better defined below ) that are normally observed and managed in the participating outpatient clinics to assess the rate of the events that home monitoring ( hm ) allowed to remotely detect and treat during patient follow - up . eligible subjects were patients indicated to first implantation or upgrade of pm or icds with or without crt . remote monitoring was accomplished with the biotronik hm system ( biotronik se & co. kg ) based on ultra - low power daily or event - triggered transmissions in the mics ( medical implant communication service ) band , from the implanted device to a mobile patient unit , forwarding data via gsm ( global system for mobile communications ) with gprs ( general packet radio service ) protocol to a service centre with encrypted access . in this study , hm was routinely implemented in the outpatient clinics of the participating sites according to an organizational model setting roles , responsibilities , and workflow . this model ( the homeguide model ) derived from a previous pilot experience and has been accepted as a national standard by the italian association of arrhythmology and cardiostimulation . practical feasibility and clinical effectiveness of such model have never been assessed on a large - scale clinical trial . the homeguide model is essentially based on a cooperative interaction between the roles of an expert reference nurse and a responsible physician with an agreed list of respective tasks and responsibilities . each nurse physician pair , within one outpatient clinic , is exclusively dedicated to an assigned subgroup of remotely controlled patients . physicians and nurses tasks are detailed in the figure 1 along with action items and responsibilities . ep , electrophysiology / cardiac pacing laboratory ; icd , implantable cardioverter defibrillator ; hm , home monitoring ; pm , pacemaker ; rn , responsible nurse ; rp , responsible physician . ep , electrophysiology / cardiac pacing laboratory ; icd , implantable cardioverter defibrillator ; hm , home monitoring ; pm , pacemaker ; rn , responsible nurse ; rp , responsible physician . home monitoring transmissions were reviewed by the nurse within two working days upon hm notifications of critical events flagged for attention . in case of no alerts , custom software was implemented to measure and record the duration of all the hm sessions conducted by the nurse and the physician . nurse communications to individual patients were triggered by : hm transmission interruptions for more than 7 days , recall for an unscheduled in - clinic visit or a skipped scheduled in - clinic visit , assessment of patient compliance , and clinic status after medical actions . in - clinic visits were scheduled at post - implant discharge , at 1 month and then once in a year . unscheduled in - clinic visits were further classified in : visits required by the patients ( including visits following an emergency department admission ) or triggered by hm notifications , reports , and trend reviews . major cardiovascular events were defined as any untoward cardiovascular occurrence , disease or signs ( including abnormal device findings ) faced in an outpatient cied clinic whether or not related to the implanted device . such definition was broad enough to encompass a large class of significant events , including arrhythmias , worsening heart failure ( hf ) , device - related complications , as well as events that are not supposed to be detected by cieds or remotely transmitted via hm ( such as strokes or acute myocardial infarctions ) . from early detection to final decision , mces were tracked by a custom made software . when a mce was a combination of multiple events , a multiple diagnosis was allowed in the electronic case report forms . major cardiovascular events were classified by the investigators according to specific guidelines developed by the steering committee , according to the following properties : source of information ( hm session , in - person visit , or other circumstances);home monitoring - witnessed : a mce was considered as hm - witnessed if hm is technically able to detect the event;appropriateness of initial diagnosis;absence of associated symptoms;corrective action required.investigators classifications were reviewed and adjudicated by an independent five - member board , blinded to the investigational sites and investigators . source of information ( hm session , in - person visit , or other circumstances ) ; home monitoring - witnessed : a mce was considered as hm - witnessed if hm is technically able to detect the event ; appropriateness of initial diagnosis ; absence of associated symptoms ; corrective action required . the implanted study devices were equipped with several indexes for hf monitor , depending on the device type and model . the homeguide protocol did not provide reaction algorithm , programming or threshold setting recommendations which were left to investigators discretion . therefore , the results represent current standard practice at the present stage of clinical experience and hm technology development . to provide estimates of sensitivity and ppv of hm event detection , adjudicated mces were divided in : true - positive ( events detected during periodic or alert - triggered hm sessions ) ; false - negative ( events actually occurred but not detected with hm for any reason ) ; false - positive ( false events notified by automatic hm alerts and/or misinterpreted ) . home monitoring sensitivity and ppv were then estimated as usually by the ratios of the number of true - positive events over the number of true documented events and the number of events detected during hm sessions , respectively . estimates were reported as percentages with the respective 95% confidence interval ( ci ) boundaries . generalized estimating equation ( gee ) models were used to adjust sensitivity and ppv estimates for within - individual repeated mces . it is worth noting that the broad definition of mce , assumed in the homeguide registry , and the particular characteristics of hm as a diagnostic tool , may impair practical significance of sensitivity and ppv . we therefore sought to develop an index , derived from the expected utility concept normally used in markov processes and based on the rate of some properties of interest shown by mces . the latter may in fact be considered as a measure of the utility of the follow - up strategy by which the mce was first detected ( hm sessions or in - person visits / other circumstances ) , and assumed as a score . the sum of the products of the scores by the respective probabilities is normally known as an expected utility value and can be easily estimated for both monitoring strategies with experimental data . an incremental utility ( iu ) index can be defined as the difference between the respective expected utilities of both follow - up strategies returning a numerical value in the ( 1 , 1 ) range , that can be estimated with its 95% ci and tested against the hypothesis of iu = 0 ( with our assumptions , a positive iu value was in favour of a higher expected utility of the hm strategy ) . generalized estimating equation models may be used as well to adjust for within - individual repeated events correlation . in principle , calculations may be performed for each set of arbitrarily chosen properties , or according to the clinical interest of the particular aspect analysed . we considered a set of four properties : hm - witnessed ; correct initial diagnosis ; asymptomatic events ; corrective action required ( actionability ) . the homeguide registry was 90% powered to reject the null hypothesis that sensitivity of hm mce detection was 70% at an one - sided test with a significance level of p = 0.025 . simulations showed that a priori assuming a sensitivity value of 75% would have required 844 adjudicated mces . with such a collection of mces it was also estimated that there was a 97% power available to reject the null hypothesis that the hm ppv was 80% assuming a ppv of 85% . it was then predicted that 55% of enrolled patients would have experienced one valid mce during at least follow - up of 1 year . with a hypothesized patient dropout rate of 10% , the prediction of 55% mce rate per patient proved false , as it resulted remarkably higher at the end of the study . the steering committee decided to terminate patient follow - up after the target population was enrolled and the last patient in reached the 3-month follow - up . continuous variable distributions were checked for normality with the shapiro wilk test and reported as mean sd if the normality hypothesis could not be rejected . proportions were reported as percentages along with the exact 95% ci . statistical significance was set at p = 0.05 level . to account for the bias deriving from repeated mces in individual patients ( = unadjusted ) , hm sensitivity , ppv , and iu on the whole , 1650 patients , mean age 69.5 11.4 years , 76% male , were enrolled in 75 italian centres ( 22 enrolments per site on average , range 1221 ) from march 2008 to september 2011 . almost one - fifth of the patients actually implanted in the participating sites during standard clinical practice were selected for enrolment , with similar rates among implant types . implanted devices were single - chamber icds in 444 patients ( 27% ) , dual - chamber icds in 359 ( 22% ) , cardiac resynchronization therapy defibrillators ( crt - d ) devices in 399 ( 24% ) , single - chamber pms in 4 ( 0% ) , dual - chamber pms in 417 ( 25% ) , and cardiac resynchronization therapy pacemaker devices in 27 ( 2% ) . among pm patients , sinus node dysfunction was the main indication in 45% , atrioventricular block in 40% , vasovagal syncope in 8% , and hf requiring crt in 6% . among icd patients , population baseline characteristics and pharmacological therapies at implant are reported in tables 1 and 2 , respectively . the majority of enrolled patients ( 66% ) had emergency department visits or hospitalizations because of cardiovascular events within 1 year prior to device implantation . table 1population characteristicsall patients ( 1650)pm ( 448)icd ( 803)crt - d ( 399)mean age ( years)69.5 11.473.7 10.367.0 12.269.9 9.5male1261 ( 76%)282 ( 63%)660 ( 82%)319 ( 80%)hd no hd183 ( 11%)168 ( 38%)15 ( 2%)0 ( 0% ) cardiomyopathy851 ( 53%)30 ( 7%)496 ( 62%)325 ( 81% ) ischaemic hd689 ( 43%)82 ( 18%)421 ( 52%)186 ( 47% ) valvular hd107 ( 7%)20 ( 4%)63 ( 8%)24 ( 6% ) channelopathies27 ( 2%)0 ( 0%)27 ( 3%)0 ( 0% ) congenital hd9 ( 1%)3 ( 1%)5 ( 1%)1 ( 0% ) others5 ( 0%)3 ( 1%)2 ( 0%)0 ( 0%)nyha class i309 ( 19%)189 ( 42%)112 ( 14%)8 ( 2% ) ii751 ( 46%)181 ( 41%)456 ( 57%)114 ( 29% ) iii553 ( 33%)73 ( 16%)220 ( 27%)260 ( 65% ) iv37 ( 2%)5 ( 1%)15 ( 2%)17 ( 4%)qrs width ( ms)120 ( 100142)100 ( 87120)108 ( 98120)146 ( 125160)lvef ( % ) 30.0 ( 25.042.0).60.0 ( 50.060.0)30.0 ( 26.038.0)28.0 ( 25.030.0)ventricular tachyarrhythmias447 ( 27%)4 ( 1%)355 ( 44%)88 ( 22%)supraventricular tachyarrhythmias416 ( 25%)135 ( 30%)186 ( 23%)95 ( 24% ) at93 ( 6%)34 ( 7%)37 ( 4%)22 ( 6% ) af356 ( 22%)118 ( 26%)160 ( 20%)78 ( 20% ) paroxysmal155 ( 44%)72 ( 61%)53 ( 33%)30 ( 38% ) persistent71 ( 20%)33 ( 28%)27 ( 17%)11 ( 14% ) permanent130 ( 37%)13 ( 11%)80 ( 50%)37 ( 47%)bradyarrhythmias538 ( 33%)354 ( 79%)92 ( 11%)92 ( 23%)hd , heart disease ; lvef , left ventricular ejection fraction ; at , atrial tachycardia / flutter ; af , atrial fibrillation.individual patients could have multiple supraventricular arrhythmias . table 2baseline pharmacological therapydrugall ( 1650)pm ( 448)icd ( 803)crt - d ( 399)ace - inhibitor971 ( 59%)177 ( 40%)479 ( 60%)315 ( 79%)arbs244 ( 15%)81 ( 18%)81 ( 10%)82 ( 21%)-blockers1056 ( 64%)126 ( 28%)573 ( 67%)357 ( 89%)diuretics1050 ( 64%)171 ( 38%)536 ( 67%)343 ( 86%)spironolactone237 ( 14%)17 ( 4%)101 ( 13%)119 ( 30%)calcium channel blockers161 ( 10%)82 ( 18%)53 ( 7%)26 ( 7%)vasodilator231 ( 14%)43 ( 10%)111 ( 13%)77 ( 19%)digitalis131 ( 8%)11 ( 2%)62 ( 8%)58 ( 15%)antiplatelets741 ( 45%)174 ( 39%)383 ( 48%)184 ( 46%)anticoagulants467 ( 28%)102 ( 23%)241 ( 30%)124 ( 31%)antiarrhythmics386 ( 23%)103 ( 23%)181 ( 23%)102 ( 26% ) class i49 ( 3%)40 ( 9%)8 ( 1%)1 ( 0% ) amiodarone312 ( 19%)54 ( 12%)162 ( 20%)96 ( 24% ) sotalol25 ( 2%)9 ( 2%)11 ( 1%)5 ( 1%)arbs , angiotensin ii receptor blockers ; ace , angiotensin - converting - enzyme . population characteristics hd , heart disease ; lvef , left ventricular ejection fraction ; at , atrial tachycardia / flutter ; af , atrial fibrillation . baseline pharmacological therapy arbs , angiotensin ii receptor blockers ; ace , angiotensin - converting - enzyme . during a mean follow - up of 20.4 12.6 months , 3364 hm sessions were performed ( 74% by the nurse ) , during which 15 984 patient reports were reviewed . each session had a median duration of 5.5 ( 2.011.1 ) min to review three ( 16 ) patient reports , if conducted by the nurse ; and 4.6 ( 1.810.5 ) min to review two ( 14 ) patient reports , if conducted by the physician . overall , with the homeguide model workflow , hm required a median manpower of 55.5 ( 22.0107.0 ) min health personnel per month every 100 patients . two thousand four hundred seventy - one mces were detected in 838 patients ( 51% ) , with a median of 2.0 ( 1.03.0 ) events each : 2033 events ( 82% ) were detected during hm sessions , 165 ( 7% ) during in - person visits , and 273 ( 11% ) in other circumstances . false - positives were 60 ( 57 during hm sessions , 1 during in - person visits , and 2 in other circumstances ) . the 2411 true - positive single and multiple mces generated 2848 event classifications : 134 deaths ; 5 strokes ; 6 myocardial infarctions ; 137 worsening hf episodes ; 19 syncope episodes ; 868 atrial arrhythmias ; 612 sustained or non - sustained ventricular arrhythmias ; 318 effective / ineffective / inappropriate ventricular therapies ; 433 device - related issues ; 8 pocket / device infections ; and 351 other events . table 3classifications of 2411 true - positive mcesevent descriptionallduring hm sessionsdeaths1340strokes50acute myocardial infarctions62worsening heart failures13774syncope events195atrial arrhythmias868808sustained ventricular arrhythmias434394unsustained ventricular arrhythmias178170effective / ineffective ventricular device therapies246223ineffective maximal energy shocks107inappropriate device therapies6257sensing failures193174capture failures or threshold raises134103out - of - range impedances4341suboptimal device programming5940battery depletion or device error status44pocket / device infections80others351276 classifications of 2411 true - positive mces among the 2411 true mces , 1976 events were detected during hm sessions , resulting in an unadjusted sensitivity estimate of hm in mce detection of 81.9% ( 95% ci , 80.383.5% ) ; gee - adjusted , 84.3% ( 82.586.0% ) . unadjusted hm positive predictive value ( ppv ) was 97.2% ( 96.497.9% ) ; gee - adjusted , 97.4% ( 96.598.2% ) . among the 435 true events detected during in office visits or in other circumstances , 262 ( 60% ) were not witnessed by hm , while 173 ( 40% ) could have been potentially documented by hm but they actually were not . maximum unadjusted estimated sensitivity of hm , including potentially hm - witnessed events , could reach 89.1% ( 87.890.3% ) ; gee - adjusted , 91.3% ( 90.092.6% ) . one thousand four hundred eighty - seven events detected during hm sessions ( 75% ) were asymptomatic , vs. 87 ( 20% ) asymptomatic events not detected during hm sessions . seven hundred twenty - three events detected during hm sessions ( 37% ) were actionable , vs. 262 ( 60% ) of those detected not during hm sessions . on the whole , 73% of actionable events were detected during hm sessions with a median reaction time ( defined as the period from detection to clinical decision ) of 3 days ( iqr , 114 days ) . reactions to clinical events are reported in detail in table 4 ( events could have required multiple reactions ) . oral anticoagulation introduction , antiarrhythmic therapy starting or adjustment , -blockers titration , and diuretic dosage increasing were the most commonly reported drug therapy changes . table 4clinical reactions to actionable eventsclinical reactionallduring hm sessions number ( % ) pharmacological therapy optimization381297 ( 78%)therapy compliance recommendation4844 ( 92%)device reprogramming315244 ( 77%)device replacement178 ( 47%)implantation surgical revision7347 ( 64%)device upgrading125 ( 42%)pharmacological af cardioversion64 ( 67%)electrical af cardioversion3526 ( 74%)radiofrequency ablation2115 ( 71%)further diagnostics exams2821 ( 75%)hospitalizations15169 ( 46%)others6958 ( 84%)af , atrial fibrillation . clinical reactions to actionable events af , atrial fibrillation . incremental utility of hm , calculated according to four properties ( hm witnessed , appropriate detection , asymptomatic events , and actionability ) , was 0.53 ( 95% ci 0.510.55 ) ; gee - adjusted , 0.56 ( 0.530.58 ) p < 0.0001 . in figure 2 , gee - adjusted hm sensitivity , ppv and iu , as well as home monitoring sensitivity was > 90% for atrial and ventricular arrhythmias and device - related issues , while it was < 35% for stroke , syncope , and acute coronary syndromes . an intermediate hm sensitivity value was found for early detection of hf impairment ( 59% ) , with a maximum estimated sensitivity of 87% . figure 2generalized estimating equation - adjusted overall hm sensitivity , ppv , and iu . home monitoring sensitivity estimates for subgroups of mces are also displayed.gee , generalized estimating equation ; hm , home monitoring . home monitoring sensitivity estimates for subgroups of mces are also displayed.gee , generalized estimating equation ; hm , home monitoring . the main results of our study showed that using hm within the homeguide outpatient clinic workflow model , 82% of all mces observed in normal daily practice were detected remotely , with early clinical reaction , by devoting < 1 h healthcare personnel per month every 100 patients . remote monitoring is rapidly becoming the standard of care for patients with cieds with the aim to enhance patient clinical management and to reduce healthcare source consumption . remote monitoring has been introduced in the international guidelines and , accordingly , it may replace the majority of in - person follow - up visits . the trust study has demonstrated that remote monitoring safely reduces healthcare source consumption and shortens the reaction time to clinical events . early detection of adverse events and prompt reaction to them may lead to major clinical benefits , particularly in patients with device or lead malfunction . remote monitoring has been also associated to a reduced hospitalization rate for atrial fibrillation and a potential lower risk for stroke , to less appropriate and inappropriate icd shocks and to shorter hospitalization stays for hf . the homeguide registry is the first large registry that investigated the impact of remote monitoring of cieds on patient management and clinical event detection and treatment in daily practice . a predefined organizational model , deeply involving allied professionals , was applied in all 75 participating italian centres and the data were homogeneously collected by using custom made software . the results of the registry showed a high effectiveness of remote monitoring in detecting clinical events . almost three out of four events needing clinical intervention were remotely detected and reaction time to events was short ( median 3 days ) . the most common clinical reaction was drug therapy adjustment ; oral anticoagulation introduction in patients with asymptomatic atrial fibrillation at high risk for stroke and hf therapy titration were the most clinically significant . the impact of home monitoring - guided anticoagulation on stroke risk in patients with icd and crt - d devices is being evaluated in the ongoing randomized impact trial , with a target population of more than 2700 . sensitivity and ppv concepts have been applied to hm detection of generic cardiovascular events , excluding deaths , with no further restrictions to those normally detected by an implanted device . the scope was to stress the evaluation of the event detection performance by hm when embedded in the homeguide workflow model : therefore the 82% sensitivity obtained ( 84% , gee - adjusted ) returns an estimate of the conditional probability that for any cardiovascular event a patient experienced , the outpatient clinic personnel became aware of it remotely . events detected outside hm sessions included both events not expected to be detected by an implanted device ( infections , strokes , myocardial infarctions , etc . ) and events not detected with hm due to technical or organizational reasons . including the latter class of events led to a maximum sensitivity estimate of 89% ( 91% gee - adjusted ) . these estimates appear remarkably high in the light of the special meaning of sensitivity assumed here . similarly , the 97% ppv estimate returned the conditional probability that a remote notification of an event by hm was trustworthy . due to the broad definition of mce assumed in the homeguide registry encompassing such a large variety of unpredictable events , sensitivity , and ppv concepts may however appear incomplete to assess hm detection performance , since events may have several characteristics or properties with completely different clinical and practical significance . the iu , based on the expected utility concept and derived from the game theory , may be regarded as an additional index introducing a qualitative differentiation among events . although calculations may be performed for any set of arbitrarily chosen properties of interest , the method does not introduce any difference between the clinical or practical significance of properties . with the set of four properties we chose ( hm - witnessed , correct diagnosis , asymptomatic , and actionable ) the events collected during hm sessions were associated with a significantly positive iu of + 0.56 ( unadjusted , + 0.53 ; p < 0.0001 ) as compared with the events detected during in - person visits / in other circumstances . roughly speaking , this means that for any event detected during an in - person visit showing one of the considered properties , there was an event detected during a hm session showing on average two properties more . this gives an extra value to hm ; as a matter of fact , hm allowed early reaction mainly to asymptomatic events which could have lead to serious adverse events if missed or late detected . home monitoring sensitivity was calculated also for event types . as expected , sensitivity for atrial and ventricular arrhythmias , for device therapies , and for implanted system malfunctions ( including pacing , sensing , and lead impedance issues ) was very high ( > 90% ) . on the contrary , a modest sensitivity ( < 35% ) was observed for events such as stroke or acute coronary syndromes , for which direct event detection is not currently in the system capability and it may be only indirectly suspected ( e.g. an episode of ventricular tachycardia due to myocardial ischaemia ) . in patients with syncope , hm could identify the cause ( for instance ventricular fibrillation ) , only in 26% . this result may be basically due to suboptimal efficiency of the currently available hf predictors , as well as to a still imperfect interpretation of them during normal clinical practice . continuous monitoring of markers of hf status has been demonstrated to help identifying patients at higher risk of developing acute hf . however , an algorithm combining all data in a score index capable of alerting the physician is still lacking . it should be noted that despite the homeguide organizational model may be applied to any remote monitoring system , our results can not be generalized as , unlike other systems , the hm is essentially characterized by daily device - to - server transmissions providing a patient - independent continuous data flow . finally , data on healthcare resource consumption should be taken into account . since the preliminary studies , remote monitoring has consistently shown ability to reduce patient visits ( almost 50% ) , time required for patient follow - up , physician time , costs of patient transport , and hospital incurred costs without compromising safety . the results of the homeguide registry confirmed that continuous remote monitoring of patients with cieds may be obtained in standard clinical practice with a very low manpower and resource consumption . the homeguide registry is the first large registry , providing a model for implementing cied remote monitoring in daily clinical practice , which evaluated the impact on detection and treatment of mces . the large majority of the events were detected during hm sessions and were asymptomatic and actionable . conflict of interest : r.p.r . received minor consultancy fees by medtronic and biotronik ; n.r . and are employees of biotronik italia ; and the remaining authors have no major conflicts of interest to disclose . all funds and technical and logistical support necessary for this research were provided by biotronik italia , although biotronik has not sponsored the study , did not provide grants or fees per patient , nor is formally the owner of the collected data .

eccrine porocarcinoma is a very rare sweat gland tumor that commonly occurs on the lower limb and trunk . malignant eccrine poroma , also known as eccrine porocarcinoma ( epc ) , is a tumor arising from the epidermal portion of eccrine sweat gland . this tumor may occur de novo or develop from a preexisting lesion as degenerative progression , and it can manifest clinically as a solitary lesion with ulceration or as a plaque , polypoid , or verrucous lesion . it mostly affects the elderly , and most frequent site of involvement is lower limb followed by trunk then head and neck . we report this case as malignant eccrine poroma presenting over the vulva is extremely rare and may cause diagnostic confusion . a 75-year - old female presented with two erythematous plaques over the vulval region of the left side . the lesions started 2 years back as small papules and enlarged gradually with relatively rapid growth for the past 6 months . no other systemic complaint was present . on examination , there were two ulcerated erythematous swellings on the left labia majora [ figure 1a ] . the larger one measured 3 cm 3 cm and was covered with yellowish necrotic material [ figure 1b ] . we also found multiple enlarged , nontender , inguinal lymph nodes [ figure 1c ] . ( b ) a large ulcerated lesion covered with yellowish necrotic material . ( c ) enlarged inguinal lymph node of the right side histopathology from the larger vulval lesion showed dermal tumor comprising broad anastomosing bands and trabeculae of cells having large hyperchromatic as well as vesicular irregularly shaped nuclei . overlying epidermis was atrophic and separated from the tumor [ figure 2a and b ] . magnetic resonance imaging of the pelvis revealed the neoplastic involvement of lower one - third of the anterior vaginal wall [ figure 3a ] along with encasement of bilateral femoral veins by the inguinal mass [ figure 3b ] . the patient was referred to the gynecology department where her radical vulvectomy with bilateral inguinal and femoral lymph node dissection was done , and she is now undergoing radiotherapy . ( a ) photomicrograph showing a collection of nests of tumor cells in dermis separated by anastomosing bands ( h and e , 100 ) . ( b ) photomicrograph showing collection of clear and vesicular cells with some areas of necrosis ( h and e , 400 ) magnetic resonance imaging of the pelvis showing ( a ) heterogeneously enhancing hyperintense mass lesion in the lower third of vagina in sagittal section . epc is a rare malignancy of sweat glands accounting for only 0.005% of all the epithelial cutaneous neoplasms . pinkus and mehregan reported the first case in 1963 and called it as epidermotropic eccrine carcinoma , later , in 1969 , mishima and morioka introduced the term eccrine porocarcinoma . most common site of involvement is lower limb accounting for 50% of cases . other affected sites are face , ear , eyelids , abdomen , vulva , penis , and pubis . no sexual predilection is seen , however prevalence varies in both sexes in diverse studies . this is supported clinically by long history , mean duration of 8.5 years , and recent onset of rapid growth in longstanding cases . histological reports of dysplasia and malignant change in benign variants including eccrine poroma and hidroacanthoma simplex support this theory . however , in this case , a 75-year - old female presented with de novo development of two malignant eccrine poroma lesions over the vulva with metastasis to regional lymph nodes and other local structures recently , role of p53 gene involved in tumor suppression has been implicated in epc carcinogenesis . treatment of choice for epc is total surgical excision with broad tumor margins and regional lymph node dissection if involved . there are still insufficient data on cryosurgery and electrosurgery to accurately assess indication and tumor recurrence . chemotherapy with methotrexate , cisplatin , adriamycin , and bleomycin or isotretinoin and interferon alpha has been used with partial or little response . epc must be considered in the differential diagnosis of patients older than 50 years with longstanding tumors in the limbs and head including basal cell carcinoma , paget 's disease , melanoma , and metastatic cancer . biopsy is indicated and , even with a previous diagnosis of benign eccrine poroma , one must be attentive to risk of malignancy in the future or if it is a recurrence . its occurrence over the vulval region is extremely unusual and hence diagnosis requires a very high index of suspicion . it may metastasize to regional lymph nodes and adjoining structures making early diagnosis and treatment very important for these cases . if treated in the early stage , surgery can be curative . due to the limited availability of literature and cases documented , it is important to have a basic knowledge of this disease process as a differential diagnosis when dealing with cutaneous lesions of the vulva . malignant eccrine poroma lesions on the vulva with metastasis to bilateral inguinal lymph nodes are very rare and carry a sinister prognosis . malignant eccrine poroma lesions on the vulva with metastasis to bilateral inguinal lymph nodes are very rare and carry a sinister prognosis . malignant eccrine poroma lesions on the vulva with metastasis to bilateral inguinal lymph nodes are very rare and carry a sinister prognosis .

collapsing glomerulopathy ( cg ) is being increasingly recognized as a cause of end - stage renal disease . it is currently classified as one of the pathological variants of focal segmental glomerulosclerosis ( fsgs ) . the exact cause of this lesion is still not known , but the list of associated genetic and acquired diseases has been growing . cg is characterized by focal to diffuse , segmental to global , implosive collapse of the glomerular capillary tufts associated with marked proliferation and swelling of overlying podocytes resulting in the formation of pseudo crescents on light microscopy . immunofluorescence study is usually negative or shows only focal segmental positivity of immunoglobulin m ( igm ) , c3 and occasionally c1q . electron microscopy shows collapse and wrinkling of glomerular basement membrane ( gbm ) and greatly hypertrophied overlying podocytes with diffuse foot process effacement . cg associated with certain infections such as human immunodeficiency virus ( hiv)-1 infection , parvovirus b19 , cytomegalovirus ( cmv ) infection , human t - cell lymphotropic virus-1 , hepatitis c virus ( hcv ) , leishmaniasis and febrile illness has been documented . a 40-year - old man with diabetes and hypertension for 5 years presented with prolonged fever , cough and hemoptysis for 6 weeks duration and anasarca with breathlessness and oliguria ( urine output < 200 ml / day ) of 2 weeks duration . he denied smoking , alcohol consumption and intravenous ( iv ) drug abuse . on clinical examination , he was febrile , pale , edematous , tachypneic ( respiration rate 26/min ) , tachycardic ( pulse rate 104 bpm ) and hypertensive ( blood pressure 160/100 mmhg ) with absent diabetic retinopathy . he had few crepitations and cavernous type of bronchial breath sounds in the infraclavicular and axillary region of the right lung . urine analysis showed protein 3 + , blood 3 + and pus cells 1 - 2/hpf . complete blood count revealed a total white cell count of 10400/mm ( polymorphs 58% , lymphocytes 36% , others 6% ) , hemoglobin of 8.3 g / dl and a platelet count of 3.69 lakhs / mm . serum urea was 124 mg / dl , and creatinine was 6.4 mg / dl . his fasting lipid profile was deranged with elevated total cholesterol ( 369 mg / dl ) and triglyceride ( 542 mg / dl ) . liver tests showed normal bilirubin and transaminase levels with low total protein ( 4.9 g / dl ) and albumin ( 2.0 g / dl ) levels . anti - nuclear antibodies , anti - neutrophil cytoplasm antibodies , anti - gbm antibody tests were negative . after stabilizing him with three hemodialysis sessions , renal biopsy was performed . on light microscopy , 6 out of 9 glomeruli showed the collapse of capillary tuft with florid hyperplasia of the overlying podocytes [ figure 1 ] . tubular epithelial cells showed signs of acute injury , interstitial edema with inflammatory cell infiltrate . immunofluorescence microscopy was negative for antisera to igg , igm , iga , c3 , c1q , kappa , and lambda light chains . some podocytes contain protein resorption droplets , a common finding in collapsing lesion ( pas , 400 ) occasional microcystically dilated tubules containing proteinaceous casts were seen in the edematous interstitium ( pas , 200 ) our patient presented with nephrotic proteinuria , severe renal failure , and sputum positive pulmonary tb . the absence of lambda and kappa light chains in the immunofluorescence of renal biopsy and normal serum protein electrophoretic pattern ruled out multiple myeloma . hence , after excluding all other possible causes , we surmise that the most probable cause of cg in our patient could be pulmonary tb . at present disturbance in the immune system , coupled with genetic susceptibility , likely contribute to the changes in glomerulus . coventry and shoemaker reported a case of cg in a 16-year - old girl who presented with steroid - resistant nephrotic syndrome and pulmonary tb . in the absence of the usual associations ( adult age group , african - american race , or history of iv drug abuse ) , this lends support to the hypothesis that immune dysregulation due to infection per se , rather than infection by specific viral agents , may lead to cg in susceptible individuals . rodrigues et al . reported an hiv - negative patient with tb - related cg who needed dialysis for 5 months but presented full renal recovery after tb treatment and corticotherapy . light microscopy picture of our patient showed podocyte hyperplasia with the collapse of glomerular tuft . the therapeutic approaches empirical and analogous to those used for noncollapsing fsgs , i.e. , use of steroids or immunosuppressive agents . our patient was started on category i anti - tuberculous treatment ( att ) as per directly observed treatment strategy and oral prednisolone 1 mg / kg / day . we stopped oral steroids after tapering at the end of 2 months as his blood sugars became uncontrollable with insulin . he became sputum negative for mycobacterium tb and dialysis - independent at the end of 2 months . his serum creatinine and 24 h urine protein were 3.4 mg / dl and 2 g / day during the last follow - up . long - term follow - up is needed to assess the progression of renal disease in this patient .

spontaneous pneumomediastinum and pneumothorax commonly occur in healthy young men or parturient women , in whom increased intra - alveolar pressure ( valsalva maneuver , cough , emesis ) leads to the rupture of marginal pulmonary alveoli ( 1 ) . in contrast , pneumomediastinum and pneumothorax following cervical emphysema are very rare and few case reports have been published . however , they have been reported in relation to dental surgical procedures , head and neck surgery , or orofacial trauma . in thyroid surgery , pneumothorax and pneumomediastinum are usually associated with difficult dissection toward the pleura or mediastinum ( 2 ) . we review one case of pneumomediastinum and pneumothorax following cervical emphysema after thyroid surgery without mediastinal dissection and pleural injury . a 60-year - old woman admitted for elective excision of a right thyroid papillary carcinoma . preoperative computed tomography ( ct ) detected a solid thyroid mass invading the perithyroid tissue with suspicion of right tracheoesophageal groove invasion . intraoperatively , the tumor was found to invade the right recurrent laryngeal nerve and along the right tracheoesophageal groove ( fig . the right recurrent laryngeal nerve was sacrificed and the tumor invading the tracheoesophageal groove was shaved off the trachea and esophagus . after tracheal shaving , a small perforation ( 0.50.5 cm ) was noticed in the posterior membranous portion of first tracheal ring ( fig . the trachea did not leak by underwater testing during positive anesthetic ventilation and the operation was completed . one day after surgery , the patient developed dyspnea , cervical emphysema , and ecchymosis over the neck after an episode of vigorous coughing . with the impression of a postoperative hematoma a small hematoma was found beneath the platysma and strap muscles , no obvious bleeding point was evident and the reconstructed trachea portion was relatively intact . postoperatively , while still in the recovery room , the patient showed signs of acute respiratory failure . a chest radiograph revealed bilateral pneumothorax , pneumomediastinum , and massive subcutaneous emphysema ( fig . her dyspnea and cyanosis were relieved after an emergency chest tube was inserted . over the next week , the pneumomediastinum and pneumothorax resolved gradually . pneumomediastinum and pneumothorax are defined as the presence of air in the mediastinum and thoracic cavity , respectively . air may gain access to the mediastinum and thoracic cavity through several anatomic routes : directly , through a perforation of the trachea , bronchi , or esophagus ; by dissection along the fascial planes of the neck ; from the interstitial tissue of the lung ; or from the retroperitoneal space ( 1 ) . in most cases of pneumothorax and pneumomediastinum , air ascends along the mediastinum toward the subcutaneous space of the neck after rupture of marginal pulmonary alveoli . in contrast , pneumothorax and pneumomediastinum following cervical emphysema are very rare . in thyroid surgery , pneumothorax and pneumomediastinum are usually associated with a difficult dissection toward the pleura or mediastinum . as a result the most likely scenario is that air tracks through the cervical fascial planes under low pressure or that the pleura is violated inadvertently . nevertheless , the resulting pneumothorax or pneumomediastinum are usually of little clinical importance ( 2 ) . however , they can be associated with several serious complications including cardiac tamponade , tension pneumomediastinum , air embolism , mediastinitis and neck abscess ( 3 ) . in this patient , pneumothorax , and pneumomediastinum occurred through a minor tracheal perforation around the tracheoesophageal groove after tracheal shaving , despite preservation of intact pleura of the lung and mediastinum . initially , dyspnea was considered as a consequence of airway compression due to postoperative hematoma . however , after thorough review of the patient 's hospital course , we speculate that she had already developed pneumomediastinum and pneumothorax prior to our exploration of the neck . the mechanisms of her pneumothorax may be as follows . first , postoperative hematoma and narrowed airway from medially fixed unilateral vocal fold aggravate dyspnea . second , a high - pressure air leak occurred through the perforated trachea due to deep breathing and vigorous coughing . finally , along the fascial planes of the neck , pneumomediastinum and bilateral tension pneumothorax occurred before neck exploration . consistent with this route , air leak can be aggravated by tracheal injury during intubation and by positive - pressure ventilation anesthesia during a subsequent operation . there are two possisle points of inadvertent tracheal injury exists : one is at freeing the isthmus from the trachea and the other at dissecting the lateral and posterior aspect of the trachea close to the area where the recurrent laryngeal nerve enters the tracheal cartilage ( 4 ) . although secondary complications such as subcutaneous emphysema , pneumothorax and pneumomediastinum may occur very rarely , most patients recover without any problems with primary tracheal repair . repair of tracheal perforations usually performed by re - approximating the tracheal edges with an absorble suture , and sometimes reinforced by adjacent strap muscle flaps using fibrin glue ( 5 ) .

the incidence of breast cancer is very high in karachi and the highest in asia.1 early diagnosis is the most important factor in increasing the survival rate . regular mammographic screening has shown great promise in this regard by decreasing breast cancer mortality by 15%20%.2,3 there are no government - funded screening programs for breast cancer in pakistan . this provides information for an accurate diagnosis and subsequent surgical and neoadjuvant treatment.4 in order to standardize mammographic reporting , the american college of radiology developed breast imaging reporting and data system ( bi - rads ) lexicon terms to describe breast density , lesion features , impressions , and recommendations for further patient management.5 broadly speaking , there are two final assessment categories . assessment is considered incomplete when the radiologist orders further imaging to make a definitive mammographic diagnosis . assessment is considered complete when the radiologist is sure of the diagnosis and categorizes mammograms from 1 to 6 . bi - rads categories 1 and 2 denote a benign lesion and category 3 requires short - term follow - up of the patient . categories 4 and 5 define a suspected malignancy , and biopsy or other appropriate action is advised . category 6 depicts a biopsy - proven malignancy.5 bi - rads has been used for over a decade to provide a uniform and standardized system for reporting of mammographic findings.4 it also helps in advising on subsequent management for patients , which is necessary for adequate treatment.6 previous research on bi - rads has evaluated interobserver and intraobserver variability in description of lesions.7,8 there have been studies examining the consistency between assessment categories and management recommendations.9 a study conducted in australia examined the quality and completeness of the contents of imaging reports with respect to bi - rads.4 however , anecdotal experience suggests that the quality of breast imaging reports is highly variable and information relevant to patient care may be omitted or partially reported in these reports.4 to the best of our knowledge , no such study has been conducted in pakistan . accurate reporting and appropriate recommendations for further workup are of the utmost importance , given that many patients either self - refer or are referred by primary care physicians , and mammography reports are the driving force for appropriate treatment at the outset . in pakistan , breast imaging services are provided by either private imaging centers or government - funded ( public sector ) services that operate independent of treatment centers . except for a minority of tertiary care hospitals in pakistan , no data confirm the involvement of a radiologist in multidisciplinary meetings , whether post diagnosis or during treatment planning . in our system , primary care physicians are responsible for breast imaging and referral of the patient to a breast surgeon if necessary . therefore , it is imperative that mammography reports communicate adequate and clear information to primary care physicians and treatment teams . we conducted this study to evaluate the mammography reports of representative newly diagnosed breast cancer cases from both public sector hospitals and diagnostic centers with respect to their completeness and concordance with bi - rads standards , in the hope of identifying issues for improvement in reporting , particularly in areas which have an impact on patient care . a multicenter , prospective , descriptive study was carried out in the radiology sections of five institutions in karachi , pakistan . the aga khan university hospital is a tertiary care private sector hospital.10 the pakistan naval station shifa hospital is a 600-bed tertiary care public sector hospital serving inservice and retired naval personnel , with an established radiology section.11 the advanced radiology center is a private sector diagnostic radiology facility in karachi for the general public.12 the karachi institute of radiotherapy and nuclear medicine is a public sector cancer diagnostic and treatment center in karachi.13 the civil hospital karachi is a 1900-bed tertiary care public hospital with an established radiology section.14 approval for the study was obtained from the ethical review committee of aga khan university , after seeking consent from participating radiologists . the data were collected prospectively from may to october 2010 . fifty consecutive mammograms reported as bi - rads category 4 or 5 at each center all mammograms reported as bi - rads category 0 , 1 , 2 , or 3 were excluded . fifty reports were obtained from each study site , thus making a total of 250 reports . category 4 and 5 mammography reports were collected from all the study centers , after obtaining consent from the corresponding consultant radiologists . mammogram reports were entered onto a questionnaire developed by the principal investigator in order to maintain confidentiality and privacy . this questionnaire was designed to extract data regarding the components of breast imaging reports taking into consideration the bi - rads lexicon for describing malignant lesions in terms of clinical indication , breast density , lesion location and description , calcification , and status of axillary lymph nodes . the data included patient age , marital status , date of enrollment , registration number , institution from which the report had been generated , and the name of the reporting radiologist . in addition to clinical indication , the type of imaging service and findings based on detailed description of masses , classifications , and distribution and morphology of masses were also recorded . concordance of reporting was ascertained by comparing the radiologist s description for the given diagnosis with standard american college of radiologist reports for bi - rads 4 and 5 categories . the data analysis was performed using the statistical package for the social sciences version 19.0 ( spss inc , chicago , il ) . the chi - square test was used to assess the completeness of reports for the two groups in accordance with the bi - rads lexicon . for the variables stated above , a p value of < 0.05 at a 95% confidence interval was considered to be statistically significant the data analysis was performed using the statistical package for the social sciences version 19.0 ( spss inc , chicago , il ) . the chi - square test was used to assess the completeness of reports for the two groups in accordance with the bi - rads lexicon . for the variables stated above , a p value of < 0.05 at a 95% confidence interval was considered to be statistically significant . the most frequent parameter described in the reports was location of the lesion within the breast followed by a description of the lesion . this was followed by reporting of breast parenchymal density , which was reported for 92% of mammograms ( table 1 ) . the lesions were detected in the background of 61 fatty , 126 fibroglandular , 37 heterogeneously dense , and 26 dense breast parenchymal patterns . in total , 225 were bilateral mammograms and only 25 were unilateral mammograms . one hundred and twenty - six lesions were reported in the left breast and 124 lesions in the right breast . the upper outer quadrant was most frequently involved , and the lower inner quadrant was the least frequent site of the lesions described . clinical indication , breast parenchymal density , lesion location , and presence of calcification were all more frequently mentioned by private centers , with statistically significant p values , while lymph node description parameters were more frequently stated by the government centers , again with a statistically significant p value of 0.013 . description of the masses by the two groups was not significantly different ( p = 0.446 , table 2 ) . the general description of mass was equally good both for the private sector and the government / public sector . the bi - rads system for mammographic reporting is now being used as a universal language in breast imaging centers to enable easy and understandable communication between the radiologist and the breast surgeon . in pakistan , patients are referred for breast imaging not only by breast surgeons , but also by other specialists and family practitioners . self - referral is also common , so clear reports and further workup recommendations communicated in a clear language assume even greater importance . the results of this study can be applied in our country and can be generalized to other developing countries where diagnostic and treatment services are not integrated or where radiologists do not routinely participate in multidisciplinary meetings . almost 40% of reports in this study did not mention the clinical indication for mammography . this is similar to a study conducted by houssami et al4 in which clinical indication was not mentioned in about one - fifth of reports . in our study , breast density , location of the lesion , and a lesion description were given in 92% , 96% , and 93% of reports , respectively . these are much higher percentages than those reported by houssami et al,4 who found these to be 80% , 75% , and 77% , respectively . the difference is most likely to be due to the training of radiologists regarding mammography reporting . all authors except one were either american board - certified or fellows of the college of physician and surgeons , pakistan . both of these training programs include the american college of radiology bi - rads lexicon in their mandatory core syllabus , so these radiologists were likely to be better acquainted with the lexicon . patient selection , patient age , and quality of mammograms may also affect visibility and description of the lesion . reporting of breast parenchymal density is important , because it has an impact on accuracy of diagnosis and indeed the risk of having breast cancer . a statistically significant difference was noted in the description of clinical indication , breast parenchymal density , lesion location , and presence of calcification between public and private centers . this may be due to the frequent participation of private sector radiologists in multidisciplinary meetings and/or continuing medical education events . training in bi - rads leads to improved consensus agreement between experienced breast radiologists with regard to analysis of lesion features and final assessment . this has been documented earlier by berg et al.15 in addition , most centers in the private sector get frequent feedback on their reports , either by the referring physicians or patients , because if there is inadequate or ambiguous information in the reports , the private centers are contacted for clarification . this also results in increased knowledge about what the clinician wants in the reports , but no such mechanism exists in the government sector . this may be due to the fact that mammograms reported as bi - rads category 4 or 5 were included in the study , and every report had a lesion which needed a description . this may be due to late presentation of patients with advanced disease due to low socioeconomic status and lack of awareness among less educated patients presenting in the government sector . the data were collected only from mammography reports and the actual mammograms were not seen at the time of data collection . secondly , the lead radiologist knew that the data were being collected for the study . this might have affected the final reporting quality if the lead was also the reporting radiologist . the third limitation was that there were two private institutions and the rest were government institutions , which might have affected the results . our study shows that overall mammographic reporting for bi - rads categories 4 and 5 in the private sector in karachi , pakistan is more in line with the bi - rads lexicon , as compared with the government sector . based on our findings , recommendations for the future are to arrange refresher courses for radiologists in the government sector and to improve the quality of reports . a checklist for style of breast imaging reports that includes key items

in mammals , the central nervous system ( cns ) has the second highest concentration of lipids after adipose tissue . lipids play a critical role in neuronal membrane function as well as in enzyme , receptor , and ion channel activities [ 1 , 2 ] . one of the main constituents of brain phospholipids is the omega-3 group of polyunsaturated fatty acids ( n-3 pufas ) . there are three major n-3 pufas : alpha - linolenic ( ala ) , eicosapentaenoic ( epa ) , and docosahexaenoic ( dha ) acids . dha ( 22:6 , n-3 ) , the longest and most unsaturated fatty acid , is an essential n-3 pufa for brain it is highly enriched in neural membranes , constituting 3040% of phospholipids in the cerebral cortex and retina [ 3 , 4 ] . because brain tissue is unable to make n-3 pufas , it is remarkably sensitive to adequate diet supplementation during all stages of cns development from embryonic differentiation to adulthood and aging [ 2 , 47 ] . neural trauma and neurodegeneration are associated with significant disturbances in neuronal membrane phospholipid metabolism [ 810 ] , suggesting that supplementation with n-3 pufas may be beneficial for recovery . omega-3 deficiency induces structural and functional abnormalities in the hippocampus , hypothalamus , and cortex - brain areas that mediate spatial and serial learning . omega-3 deficiency significantly reduces the level of cerebral catecholamines , brain glucose transport capacity and glucose utilization , cyclic amp level , and the capacity for phospholipid synthesis . such a deficiency also markedly affects activity of membrane - bound enzymes , receptors and ion channels ( e.g. , na , k - atpase ) , production of neurotransmitters and brain peptides , gene expression , intensity of inflammation , and synaptic plasticity [ 1 , 7 , 11 , 12 ] . conversely , diet supplementation with dha modulates gene expression , neurotransmitter release , restores synaptic activity reduced by age or trauma , and improves memory and learning abilities [ 10 , 1319 ] , while the effect of n-3 pufas on membrane fluidity remains to be a controversial . numerous studies conducted over the past decade suggest that n-3 pufa has a significant neuroprotective and proregenerative potential [ 2130 ] . particularly , acute intervention or dietary supplementation with n-3 pufas have been found to be protective in animal models of acute neurologic injury such as cerebral stroke , traumatic brain and spinal cord injuries [ 2326 , 2830 ] , and some case studies . recent study has demonstrated the improved outcome after peripheral nerve injury in transgenic mice with elevated level of endogenous n-3 pufa . the neuroprotective properties of n-3 pufas are in part attributed to their strong anti - inflammatory action , mediated partially by dha 's inhibition of aa catabolism and modulation of cytokine levels , and antioxidant potential [ 11 , 12 ] . it has been recently demonstrated that after the onset of brain injury , dha could be released from membrane phospholipids by ca - dependent phospholipase a2 and generates neuroprotective d1a compound that differentially regulates the expression of pro- and antiapoptotic proteins from bcl-2 family , known as a critical players in cell fate . despite the wide range of targets and proposed mechanisms of n-3 pufa beneficial action , the remaining question is how they ( e.g. , targets and mechanisms ) are activated in order to execute these effects . within the cell , the mitochondrial membrane is one of the primary sites for n-3 pufa incorporation along with endoplasmatic reticulum and plasma membrane [ 14 , 3235 ] . brain , cardiac and liver mitochondria fatty acids turnover requires 3 - 4 weeks and is highly regulated by diet [ 3436 ] . a growing body of evidence has established that mitochondria are a key component in the signaling pathway(s ) underlying cell death [ 16 , 3641 ] . to some extent , mitochondria serve to integrate different apoptosis - inducing stimuli ( ca , proapoptotic bcl-2 family proteins , reactive oxygen species , etc . ) and direct them into a common downstream pathway [ 36 , 37 , 39 , 41 ] . mitochondria are enlisted to initiate the downstream stages of cell death through mitochondria - permeability - transition-(mpt ) dependent and -independent mechanisms . the mpt is a multiprotein pore complex of as yet unidentified structure that is presumably localized at the contact sites between the inner and outer mitochondrial membranes . the mpt begins as a permeabilization of the inner membrane , which prevents buildup of a mitochondrial membrane potential and leads to loss of the ability to sequester calcium from the medium , progressive osmotic swelling , disruption of the outer membrane , loss of matrix and intermembrane proteins , and initiation of caspase - dependent and caspase - independent cell death pathways [ 36 , 39 ] . mitochondrial damage , occurring via the mpt , has been identified as a critical event in stroke and stroke - related injuries , secondary injury following brain trauma ( tbi ) , and chronic neurodegeneration [ 16 , 21 , 2327 , 36 , 38 , 39 , 4143 ] . in light of the aforementioned links between mitochondria and cell death , mitochondria and n-3 pufas , and n-3 pufas and neuronal function , it is noteworthy that recent evidence shows that n-3 pufas can modulate processes that contribute to the secondary degeneration of the cns [ 10 , 1719 , 44 , 45 ] . particularly , administration of n-3 pufas after spinal cord compression injury in rats significantly increased neuronal survival and improved locomotive performance for up to 6 weeks after injury . furthermore , preinjury diet supplementation with omega-3 pufas prevented some tbi - induced effects a reduction in synaptic plasticity and impaired learning ability and reduced oxidative damage . recent data suggest that eight weeks of dietary supplementation with dha delays induction of mpt mediated swelling and increases ability to retain exogenously added calcium in cardiac mitochondria . these data , coupled with the above background , suggests that diets enriched in n-3 pufas might affect mitochondria in a way that makes them more resistant to the oxidant- and calcium - mediated injury associated with both acute neurological injury and induction of the mpt . the goal of present study was , therefore , to test directly the involvement of the mpt pathway in n-3 pufa - mediated protection in brain and liver mitochondria isolated from healthy rodents . specifically , we determined whether 4 weeks dietary supplementation in rats with 1 , 3 , or 10% fo , containing essential n-3 pufas epa and dha , changes the resistance of isolated nonsynaptosomal brain and liver mitochondria to proapoptotic signals such as ca and prooxidants . tetramethylrhodamine methyl ester ( tmrm ) and ca - green-5n were purchased from invitrogen , inc . lc - ms grade acetonitrile ( acn ) , methanol ( meoh ) , and isopropanol ( ipa ) , as well as high - performance liquid chromatography ( hplc ) grade dichloromethane ( dcm ) and dimethyl sulfoxide were purchased from fisher scientific ( pittsburg , pa ) and ammonium formate was purchased from sigma - aldrich ( st . louis , mo ) . lipid standards purchased for lc - ms as well as their abbreviations and sources are in the supplemental information ( tables s1 and s2 ) from [ 48 , 49 ] . this study was carried out in strict accordance with the recommendations of the guide for the care and use of laboratory animals of the national institutes of health . the protocol was approved by the harvard medical school standing committee on animals ( protocol no . ninety - six male fisher 344 x brown norway f1 ( fbfn1 ) rats ( four groups , 24 animals in each ) , three weeks of age were housed in cages ( two rats per cage ) and maintained in an environmentally controlled room . all animal procedures were performed in accordance with the guide for the care and use of laboratory animals and were approved by the animal studies committee at harvard university . after acclimatization for 1 week on standard rat chow , the rats were randomly assigned to diets supplemented with fish oil ( fo ) containing n-3 pufas dha and epa ( final fo 1 , 3 , or 10% of total fat in a fixed 10% fat diet , i.e. , the 10% fo diet is 1% omega-3 pufas by weight ) for 4 weeks . in all diets , including in the control diet , the total fat content made up 10% of the diet by weight ; of that fat component , total saturated fats made up 30% , total monounsaturated fats made up 26% , and total polyunsaturated fats made up 44% ( table s1 ) . the diets , fed ad libitum , were provided as pellets ( research diets , inc . ) and contained a standard vitamin and mineral mix with all essential nutrients . brain and liver tissues were rapidly dissected and placed into ice - cold buffer for isolation of mitochondria . greater details of the husbandry and diets are presented in the supplementary material following the arrive criteria for reporting animal studies . nonsynaptosomal brain mitochondria were isolated from ~2 - 3-month - old rats using a discontinuous ficoll gradient according to the commonly used method of lai and clark , with slight modifications as previously described . liver mitochondria were isolated from ~2 - 3-month - old rats by the standard differential centrifugation method in sucrose - based buffers as described and as used previously in our lab . liver isolation buffer contained 0.3 m sucrose , 10 mm hepes , 1 mm egta , and 0.5% bovine serum albumin ( bsa ) . mitochondrial yield was identical in all samples from all tested dietary groups for both tissues . immediately before extraction , brain and liver mitochondria isolated from each animal had their membranes disrupted by sonication . both a brain and a liver mitochondrial pool sample were created by combining 10 l from the sonicated mitochondria of each rat in addition to a serum pool . these samples were processed for quality control ( qc ) and lipid identification studies at the same time as the dietary samples . lipids were extracted according to the method of bligh and dyer , substituting dcm for chloroform . first , 10 l of an internal standard mixture containing 5 lipids as outlined in detail previously [ 48 , 49 ] was added to each 30 l sample ( either mitochondria or serum ) , followed by 190 l of meoh , 20 seconds of vortexing , 380 l of dcm , and 120 l of water was added to induce phase separation . the samples were then vortexed for 10 seconds and allowed to equilibrate at room temperature for 10 minutes before centrifugation at 8000 g for 10 minutes at 10c . a total of 320 l of the lower lipid - rich dcm layer was then collected and the solvent evaporated to dryness under vacuum . samples were reconstituted in 300 l of acn / ipa / h2o ( 65 : 30 : 5 v / v / v ) containing pg ( 17 : 0/17 : 0 ) at a concentration of 1 g / ml before lc - ms analysis . ten l of sample was injected onto the lc - ms system . lipids were extracted from 30 l serum and mitochondria aliquots of all individual rat samples and total study pool samples , created by taking aliquots from each sample as described previously [ 48 , 49 ] . details of the lc - ms method have been described previously [ 48 , 49 ] . briefly , lipid extracts were separated on an ascentis express c18 2.1 150 mm 2.7 m column ( sigma - aldrich , st . louis , mo ) connected to a thermo fisher scientific autosampler and accela quaternary hplc pump ( thermo fisher , san jose , ca ) . a binary solvent system was used , in which mobile phase a consisted of acn : h2o ( 60 : 40 ) , 10 mm ammonium formate , 0.1% formic acid and mobile phase b of ipa : acn ( 90 : 10 ) , 10 mm ammonium formate , 0.1% formic acid . separations were done over a 30-minute - period following the conditions set by hu and colleagues . the hplc system was coupled to an exactive benchtop orbitrap mass spectrometer ( thermo fisher , san jose , ca ) equipped with a heated electrospray ionization ( hesi ) probe . for full scan only experiments , the ms was operated between m / z 1202000 in high resolution mode , corresponding to a resolution of 60 k and a 2 hz scan speed . the instrument was tuned by direct infusion of pg ( 17 : 0/17 : 0 ) in both positive and negative mode , and external mass calibration was performed according to the manufacturer 's protocol . hcd experiments were performed by alternating between full scan acquisitions and hcd scan acquisitions , both run at 2 hz . three different hcd energies , 30 , 60 , and 100 ev , were used in separate experiment as previously described . for lipid identification studies results from all lc - ms profiling experiments were analyzed using the ms label free differential analysis software package sieve v 1.3 ( thermo fisher scientific and vast scientific , cambridge , ma ) ( for details see [ 48 , 49 ] ) . mitochondrial respiration was measured with the oxygraph 2 k electrode system from oroboros instruments ( http://www.oroboros.at ) . brain mitochondria ( 0.25 mg / ml ) were incubated in buffer containing 100 mm sucrose , 65 mm kcl , 10 mm hepes , ph 7.4 , 2 mm kh2po4 , 3 m edta , and 5 mm glutamate / malate . liver mitochondria ( 0.25 mg / ml ) were incubated in buffer containing 250 mm sucrose , 10 mm hepes , ph 7.4 , 1 mm kh2po4 , and 5 mm succinate + 1 m rotenone . isolated brain or liver mitochondria were added after the addition of 5 mm glutamate / malate ( complex i substrate ) or 5 mm succinate + rotenone ( complex ii substrate ) into media . respiration in the presence of substrates corresponds only to state 2 respiration ( v2 ) . addition of 200 m adp initiates atp synthesis coupled to proton reentry across the membrane , which corresponds to state 3 ( v3 ) . adp exhaustion leads to a reduction in the respiratory rate and corresponds to state 4 ( v4 ) . addition of 0.5 m of the ionophore carbonyl cyanide - p - trifluoromethoxyphenylhydrazone ( fccp ) induced uncoupled respiration ( vunc ) . the respiratory control ratio ( rcr ) was calculated as the ratio between the rates of respiration in states 3 and 2 ( by lardy , rcr3/2 ) ; states 3 and 4 ( by chance , rcr3/4 ) ; fccp - stimulated respiration and state 3 ( vunc / vadp ) . the measurement of these parameters was performed simultaneously on a multichannel dye fluorimeter ( c&l instruments , inc . , http://www.fluorescence.com/ ) . liver mitochondria were incubated in buffer containing 250 mm sucrose , 10 mm hepes , ph 7.4 , 1 mm kh2po4 , and 5 mm glutamate / malate or succinate and were used at a concentration of 0.25 mg / ml mitochondria [ 43 , 52 ] . brain mitochondria were incubated in buffer containing 100 mm sucrose , 65 mm kcl , 10 mm hepes , ph 7.4 , 2 mm kh2po4 , 150 m atp , 150 m mgcl2 , 3 m edta , and 5 mm glutamate / malate at a concentration of 0.10 mg / ml mitochondria . the mitochondrial membrane potential changes ( ) were estimated by measuring changes in the fluorescence intensity of tmrm ( 60 nm ) ( molecular probes ) at excitation and emission wavelengths of 543 and 590 nm , respectively . mitochondrial ca fluxes were measured as changes of extramitochondrial [ ca ] , which were followed by monitoring the fluorescence intensity of ca - green-5n ( 125 nm ) ( invitrogen ) at excitation and emission wavelengths of 482 and 535 nm , respectively . mitochondria were challenged to single or multiple ca additions . for liver mitochondria , each addition was 2040 nmol ca / mg mitochondrial protein ; for brain mitochondria , each addition was 200 nmol / mg protein . mitochondrial calcium retention capacity ( crc ) was determined as amount of ca sequestered by mitochondria without incurring structural damage and expressed in nmol / mg protein . the redox state of pyridine nucleotides in the mitochondrial suspension was followed by monitoring nadh autofluorescence at excitation and emission wavelengths of 350 and 450 nm , respectively . mitochondrial swelling was measured as a function of light scattering at excitation and emission wavelengths of 587 nm or by a standard spectroscopic assay on a plate reader at 540 nm . original respiration and fluorimeter - based data were analyzed using origin v.8.0 ( originlab ) software . we used a recently developed kinetic model of ca - induced mpt to analyze the effects of the supplemented diets on liver mitochondrial function . the model describes mpt induction as a series of 2 sequential steps defined as ca - uptake ( k1 ) and formation of an intermediate step ( k2 ) followed by mitochondrial membrane permeabilization accompanied with a putative mpt pore opening ( kp ) , scheme 1 . in the framework of the model at least 3 possible mechanisms of action for different modulators of mitochondrial dysfunction numerical analysis of the kinetics of simultaneously measured ca fluxes and swelling provides information on the contribution of each of the steps into the process of mpt induction . the rate k1 is easily assigned to the ca uptake by active mitochondria [ mh-(ca)i]a via ca uniporter . the formation of an intermediate state [ mh]i , on the other hand , is a complex function of the number of ca ions absorbed by the mitochondrion and expressed as k2 = k 2 ( [ ca]m/[mh]a ) . here , [ ca]m is the concentration of ca that has been absorbed by mitochondria ; thus , the ratio [ ca]m/[mh]a is essentially an average number of ca ions absorbed per active mitochondrion . n is an apparent order of the k2 step with respect to ca , and k 2 is the reaction constant of this step . kinetic parameter k2 is related to the ability of the ca sequestered by mitochondria to facilitate the formation / assembly of the mpt pore intermediate . functionally , the intermediate represents mpt pore assembled but still closed . in our model , parameter n interpreted as an order of reaction reflects quantitative characteristics of mpt intermediate formation in respect to ca . therefore , any change in n would be expected to correlate with an ability of mitochondria to resist ca - induced mpt . data were presented as the mean + / the standard error of the median ( sem ) . group comparisons of the effects of diet modulation on mitochondrial parameters were determined by a two - sample t - test ( origin 8.0 ) . rats consumed 14.1 3.1 g of food a day and gained 4.85 1.55 g of body weight a day for 28 days . food intake and body weight gain of all the groups were not significantly different . by using lc - ms profiling epa , dha , aa and the aa , synthesis intermediate dgla ( 20:3 , n-6 ) in both the serum and mitochondrial samples . this method did not provide absolute quantitative amounts of the fatty acids in each sample ; however , it provided a means to quantitatively monitor these species across the biological samples . the study gives information regarding relative amounts of species present and allows for direct comparisons across samples to be performed [ 48 , 49 ] . the fo diets increased the level of the free fatty acid epa in serum of tested animals in a dose - dependent manner ( note that in a control diet it was almost absent ) ( figure 1(a ) ) . the sera level of dha , in contrast , reached a plateau in the 1% fo dietary group ( figure 1(b ) ) . conversely , the level of n-6 fatty acids aa and dgla were both reduced with an increase in n-3 pufas supplementation . diet - driven fatty acid changes in mitochondria ( figure 2 ) were similar to those seen in sera . the lc - ms profiling method assumes uniform starting material concentration . for lipid extraction , the mitochondria were not uniformly concentrated based on amount of protein ; therefore , the signal was standardized by comparing the ratio of n-3 to n-6 which should be consistent regardless of starting amounts . the ratio of dha to aa in brain mitochondria was particularly elevated with a maximum in 3% fo dietary group ( figure 2(a ) ) . brain mitochondria level of epa was very low and dietary - driven changes were undetectable . fo dietary supplementation induced a dose - dependent elevation of the ratios of epa to aa , dha to aa , epa to dgla , and dha to dgla ( figures 2(b)2(e ) ) . respiratory parameters of brain and liver mitochondria isolated from tested animal groups were measured in different metabolic states : ( state 2 ( v2 ) , state 3 ( v3 ) , state 4 ( v4 ) , fccp - stimulated , or uncoupled respiration ( vunc ) ) . the substrate glutamate / malate was used to stimulate respiration initiated at complex i and the substrate succinate + rotenone was used to stimulate respiration initiated at complex ii . brain mitochondria isolated from rats fed fo diet had higher rates of adp- and fccp - stimulated respiration ( vadp and vunc , resp . ) , with succinate as a substrate ( figure 3(c ) ) , but not with glutamate as a substrate ( figure 3(a ) ) . rcr3/2 , rcr3/4 , and rcrvunc / vadp , calculated as the ratios of respiration rates between state 3/state 2 , state 3/state 4 , and uncoupled state / state 3 , respectively , were also unchanged by fo diet ( figures 3(b ) and 3(d ) ) . challenging brain mitochondria with isoketals ( isoks , 2 m ) , which are prooxidative ketoaldehydes [ 47 , 52 ] , caused no statistically significant differences in the rates of respiration between mitochondria from the different dietary groups ( figure 4(a ) ) . however , brain mitochondria isolated from animals fed 10% fo diet had lower rate of fccp - stimulated respiration compared to mitochondria isolated from brain tissue of another dietary group . the rcr3/4 , rcr3/2 , and vunc / adp also were lower in the presence of isoks in all tested animal groups , but this observation was similar for all diets ( figure 4(b ) ) . respiratory parameters of liver mitochondria were measured similarly to those of brain mitochondria ( figure 5 ) . as shown , fo 110% dietary supplementation did not induce statistically significant changes in liver mitochondria respiration . figure 6(a ) shows a representative record of basic parameters ( e.g. , , ca uptake / release , redox state of pyridine nucleotides and swelling ) of nonsynaptosomal brain mitochondria isolated from control group of animals . however , mitochondria isolated from animals fed fo 1% or 3% diets showed a weak tendency to be more resistant to a ca challenge ( figure 6(b ) ) . resistance was defined as an increase in ca retention capacity ( crc ) , that is , the ability to sequester ca without incurring structural damage . cyclosporine ( cs a ) , the prototypical mpt pore inhibitor , increased the crc in all tested groups , with potentially enhanced protection in the groups fed fo 1% and 3% diets ( figure 6(c ) ) . promethazine ( pt ) and nortriptyline ( nt)drugs that possess potent mpt inhibitory properties confer less protection to brain mitochondria than cs a , but similar to all tested dietary groups ( figure 6(c ) ) . incubation of mitochondria with a low concentration of prooxidants ( 0.5 m isoks ) resulted in a significant reduction of the crc in all tested groups ( figure 6(b ) ) , as we have shown previously . animals in the fo 10% group demonstrated a tendency to be more susceptible to isoks . isoks ( 2 m ) also significantly reduced the crc in all groups ; the effect was again more pronounced in mitochondria isolated from 10% fo group . the kinetic analysis of the ca induced mpt ( figure 7 ) revealed that in all tested groups of animals , net ca - uptake rate was not changed ; that is , dietary intake did not affect ca - uniporter activity nor modulated the factors that affect electrogenic ca uptake , such as , activity of pi carrier , and activity of ca release pathways other than mpt ( data not shown ) . the analysis of the rate of the formation of an mpt intermediate ( k2-step ) revealed that fo diets had no statistically significant changes in the resistance of mitochondria to ca ( e.g. , parameter n ) ( figure 7 ) , demonstrating only the tendency to be more protective ( e.g. , 1% or 3% fo diets ) or harmful ( e.g. , 10% fo diet ) . the results from our pilot study demonstrate that four weeks of dietary supplementation with 1 , 3 , or 10% fo enriched with an essential n-3 fatty acids epa and dha induced the expected significant changes in plasma and mitochondrial membrane fatty acid composition . the observed changes in brain and liver mitochondrial membrane phospholipid composition did not , however , significantly influence their resistance to ca and prooxidative agents ( tbh and isoks ) . only slight trend towards protective effects , insufficient to underlie the reported neuroprotective effects of n-3 pufa supplementation , were ever seen with lower levels of supplementation ( e.g. , figure 6(b ) ) , and , higher levels of n-3 pufas supplementation were , if anything , detrimental . while these trends could be more systematically studied with very large experimental protocols , this would not advance our understanding of protection , and many spurious and nonrobust statistical observations would be mathematically expected to result from multiple comparison - related trends . therefore , we do not believe that higher n studies are scientifically warranted . detailed evaluation of brain and liver mitochondrial function demonstrated almost no diet - associated changes in respiration , membrane potential , ca transport , redox state of pyridine nucleotides , or swelling , despite the elevation of epa and dha and the reduction of aa content in mitochondrial membrane . we also did not find differences in the sensitivity of mitochondria to the recently discovered mpt inhibitors promethazine and nortriptyline [ 40 , 41 ] or the prototypical mpt inhibitor cs a. the most parsimonious explanation is that n-3 pufas do not exert neuroprotection by augmenting mitochondrial resistance to calcium and oxidants . the data presented contradict our proposed hypothesis , suggesting another explanation must link dietary omega-3 fatty acids with the inhibition of cell death cascades that involve mitochondria . one possibility is that other mitochondrial pathways ( e.g. , involving bcl-2 family members bcl-2 , bax , bid , bak , etc . ) although this explanation is feasible , the injury used in experiments discussed earlier [ 17 , 44 , 45 ] is most consistent with the involvement of oxidant- and calcium - mediated damage , rendering the possibility of other mitochondrial pathways being involved somewhat unlikely . alternatively , differences in the dietary protocols used in different experiments / laboratories as well as the animals ' health status ( e.g. , the presence of pathology or injury ) might underlie the different results . a review of the literature revealed at least two critical parameters that might influence the outcome : ( i ) the dosage of n-3 fatty acids dietary supplementation and ( ii ) the total fat content in the diet . the dosage of fo used in dietary studies varies up to 60% of total fats . the suggested n-3 pufa therapeutic dose for humans is 1 to 3 g / day , which is equivalent to 1% fo diet for animals in the present study . the 10% fo diet in our study corresponds to an abnormally high dose for humans ( approx . our data suggest that this dosage does not induce dramatic beneficial changes in mitochondrial functions ; in fact , it tended to worsen them ( e.g. , it caused brain mitochondria to become more sensitive to ca and prooxidants and accelerated some respiratory parameters in liver ) . the lowest dose used in the present study , that is , the 1% fo diet that corresponds to an acceptable intake in humans showed no effect on the mitochondrial parameters tested . recent study suggests that a supplementation of healthy humans with dha has dose - dependent effect : 200400 mg / day of dha had antioxidant effect on low - density lipoproteins , whereas 8001600 mg / day of dha was associated with elevated level of reactive aldehydes ( e.g. , 4-hydrohyhexenal ) in plasma . these data are broadly consistent with our own and previous evidence showing aldehyde - induced mitochondrial dysfunction [ 41 , 52 ] . with respect to total dietary fat content , we noted that brain and liver mitochondria isolated from control animals in this study exhibited less sensitivity to ca than we usually observed in our routine experiments with identical rats fed diets that have a lower percentage of total fat ( 4.5% versus 10% in the present study ) . specifically , crc for brain mitochondria isolated from animals fed with 4.5% is equal to 250300 nmol of ca / mg protein , whereas crc for tested in the present study animal groups vary from 360 to 460 nmol of ca / mg protein . kinetic analysis done on the ca induced mpt in liver mitochondria isolated from animals fed with 4.5% of fat revealed ca requirement ( kinetic parameter n ) to be about 2.27 0.05 , while the present study demonstrated considerably elevated n ( up to 3.1 ) . such an increase was found upon application of promethazine or in the medium depleted of inorganic phosphate ( unpublished data ) . we demonstrated previously that well - characterized mpt protecting agents such as mg or promethazine increased n , thereby increasing the resistance of mitochondria to ca - induced mpt . on the other hand , addition of very low ( 0.5 nm ) concentration of fccp decreased n ( not shown ) facilitating mpt induction via a decrease of the , which is in line with data published earlier . these data are consistent with an argument that an increase in dietary percent fat is associated with increased resistance of isolated mitochondria to calcium - overload injury . unfortunately , many of the critical papers in this area fail to report the percentage of total dietary fat , and/or their control and experimental fo - enriched diets have different total fat contents . also , in some studies , the diet enrichment with n-3 pufas is conducted in such a way as to increase total fat content . these observations suggest the need to investigate the effects of total dietary fat content , as well as the effects of prolonged n-3 pufas supplementation , on mitochondrial function . in conclusion , we tested and rejected one of the possible mechanisms linking n-3 pufa dietary supplementation and brain mitochondria functional parameters and injury mechanisms . the absence of a protective effect of diet in an in vitro model of ischemic injury ( caoverloading and prooxidant exposure ) , despite the observed changes in plasma and mitochondrial fatty acid content , suggests that increased resistance to ca- and oxidant - mediated mitochondrial damage is not central to the well - documented neuroprotection induced byn-3 pufas . investigation of other potential mechanisms , such as extramitochondrial targets and a potential role of total fat , will require further study .

vitiligo is a non - contagious acquired pigmentation disorder characterized by defined white patches of variable shape and dimensions , increasing in size and number with time . the prevalence range in india found to be 0.46 - 8.8% with gujarat and rajasthan states have highest prevalence of approximately 8.8% . approximately 30% acquire the disease before the age of 20 years and 14% before the age of 10 years , with decreasing incidence in later life , and fewer than 10% develop vitiligo by the age of 42 years . infants 4 - 6 months of age may develop typical vitiligo mainly in the genital or perianal area . etiology of vitiligo is not known but the loss of melanocytes in epidermis is explained by certain pathogenetic hypothesis such as genetic hypothesis , autoimmune hypothesis , neural hypothesis , autocytotoxic hypothesis , novel microenvironment - related hypothesis and convergence theory . as a depigmenting disease vitiligo also has a greater impact on patients psychosocial aspects . persons affected with vitiligo especially children and females have greater difficulty in their social relationships due to psychological depression . our study group consists of 100 vitiligo patients . the demographic features including age and genders were included in this study . patients with vitiligo skin lesions were only included in this study . vitiligo associated with systemic conditions such as thyroid disorders , juvenile diabetes mellitus , pernicious anemia , addison 's disease was not included . the frequency of the occurrence of oral mucosal vitiligo and most commonly involved mucosal site were observed in this study . the minimum age of 4 years and the maximum age of 76 years were observed in this study . among the 100 vitiligo patients 18% were up to 25 years , 14% within 25 to 35 years , 30% within 35 to 45 years , 19% within 45 to 55 years and 19% more than 55 years ( graph 1 ) . in the present study of 100 vitiligo patients , oral mucosal depigmentation were presented in lip ( 42% ) , palate ( 8% ) , buccal mucosa ( 5% ) , labial mucosa ( 5% ) and gingiva ( 2% ) [ table 1 and graph 2 ] . the association of oral vitiligo with respect to age was 10% within 25 years , 6% among age group of 26 - 35 years , 14% among age group of 36 - 45 years , 6% within 46 - 55 years and 9% of patients above 55 years [ table 2 and graph 3 ] . regarding the gender , the association of oral presentation among our study group showed to be 18% in males and 27% in females [ table 3 and graph 4 ] . pie chart showing the age distribution among the 100 vitiligo patients presence of depigmentation in various sites of oral cavity percentage of presence and absence of oral depigmentation in lips , buccal mucosa , labial mucosa , palate and gingiva among the study group presence of oral findings with respect to age presence of oral findings with respect to age presence of oral findings with respect to gender presence of oral findings with respect to gender vitiligo is a common non - infectious dermatological disorder characterized by acquired , idiopathic , progressive , circumscribed hypomelanosis of the skin and hair , with the total absence of melanocytes microscopically . out of 100 patients in this study , 44 patients were males and 56 patients were females showing higher incidence . shajil et al . , pandya et al . showed 33.8 years , 25.59 years and 21 to 30 years , respectively . our study of vitiligo patients showed a minimum age of 4 years and a maximum age of 76 years . another study by gonul et al . showed minimum and maximum age as 8 years to 72 years respectively which is closely correlating with our study . the oral presentation of vitiligo in the present study showed depigmentation of lip in 42 patients , buccal mucosa in 5 patients , labial mucosa in 5 patients , palate in 8 patients , gingiva in 2 patients . in our study out of 100 vitiligo patients , 21 patients showed intra oral mucosal depigmentation . among the intraoral mucosal sites palate was found to be the common followed by labial and buccal mucosa , gingiva and alveolar mucosa . the number of vitiligo patients in our study showing oral depigmentation was about 9 up to 25 years , 8 in the age range of 25 to 35 years , 19 among the age of 35 to 45 years , 15 in the age range of 45 to 55 years and 11 above 55 years . thus , higher incidence of oral mucosal vitiligo was noted in patients of age 35 to 45 years of age in the present study . also , females showed higher oral presentation of about 33 patients and 29 patients were males . in a report of three vitiligo cases by dummett et al . , one patient had complete clinical depigmentation of all the oral tissues and another patient with history of heavy gingival pigmentation showed pink edentulous ridges at the time of examination . mucosal involvement occurring frequently in lips , genitals , gingival , areolar and nipples have been reported in the literature . solitary presentation of palatal depigmentation in a vitiligo patient was reported by lawoyin et al . in our study 42 age range up to 25 years with lip involvement was noted in 7 patients , 25 - 35 years of age range showed 6 patients , 10 patients with lip involvement were within 35 - 45 years , 45 - 55 years of age showing lip depigmentation in 11 patients and above 55 years 8 patients presented lip depigmentation [ figure 1 ] . depigmentation in lip among 100 patients in our study depigmentation of buccal mucosa was present in five patients in which three were males and two females . with age of range up to 25 years none , within 25 - 35 years of age two patients , among patients of age 35 - 45 years two patients , within age range of 45 - 55 years one patient and above 55 years of age none of the patients showed depigmentation in buccal mucosa [ figure 2 ] . depigmentation in palate in the present study labial mucosa showed depigmentation in a male patient and four female patients . the following age ranges of up to 25 years , 25 - 35 years , 35 - 45 years , 45 - 55 years , above 55 years presented depigmentation of labial mucosa in one patient , none , three patients , one patient and none , respectively [ figure 3 ] . depigmentation in buccal mucosa in our study palatal depigmentation was found in eight patients out of them five patients were males and three were females . up to age range of 25 years one patient had palatal depigmentation , 25 - 35 years of range showed none , within age of 35 - 45 years three patients , 45 - 55 years of age range two patients and above 55 years of age two patients showed depigmentation in palate [ figure 4 ] . with respect to age of up to 25 years , 25 - 35 years , 45 - 55 years age none showed gingival depigmentation . patients within the age range of 35 - 45 years and above 55 years showed one patient each with depigmentation of gingiva [ figure 5 ] . depigmentation in gingiva among the 100 patients in our study 2 patients showed oral depigmentation as the only manifestation of vitiligo both manifestating in the lip . in relation to age up to 25 years one patient and 35 - 45 years one patient showed oral depigmentation . depigmentation of oral mucosa can be noted as an early feature in oral submucous fibrosis . in reverse these depigmentations are excluded based on the clinical history . in our study no relation of previous dental procedures to the depigmentation was noted . as far as our knowledge no literature states about the koebner phenomenon as a cause for oral vitiligo . out of 100 patients in this study , 44 patients were males and 56 patients were females showing higher incidence . , nigam et al . , pandya et al . , chanda et al . the study conducted by shameer et al . showed male predilection . shajil et al . , pandya et al . showed 33.8 years , 25.59 years and 21 to 30 years , respectively . our study of vitiligo patients showed a minimum age of 4 years and a maximum age of 76 years . another study by gonul et al . showed minimum and maximum age as 8 years to 72 years respectively which is closely correlating with our study . the oral presentation of vitiligo in the present study showed depigmentation of lip in 42 patients , buccal mucosa in 5 patients , labial mucosa in 5 patients , palate in 8 patients , gingiva in 2 patients . in our study out of 100 vitiligo patients , 21 patients showed intra oral mucosal depigmentation . among the intraoral mucosal sites palate was found to be the common followed by labial and buccal mucosa , gingiva and alveolar mucosa . the number of vitiligo patients in our study showing oral depigmentation was about 9 up to 25 years , 8 in the age range of 25 to 35 years , 19 among the age of 35 to 45 years , 15 in the age range of 45 to 55 years and 11 above 55 years . thus , higher incidence of oral mucosal vitiligo was noted in patients of age 35 to 45 years of age in the present study . also , females showed higher oral presentation of about 33 patients and 29 patients were males . in a report of three vitiligo cases by dummett et al . , one patient had complete clinical depigmentation of all the oral tissues and another patient with history of heavy gingival pigmentation showed pink edentulous ridges at the time of examination . mucosal involvement occurring frequently in lips , genitals , gingival , areolar and nipples have been reported in the literature . solitary presentation of palatal depigmentation in a vitiligo patient was reported by lawoyin et al . in our study 42 patients presented lip depigmentation out of which 20 were males and 22 females . age range up to 25 years with lip involvement was noted in 7 patients , 25 - 35 years of age range showed 6 patients , 10 patients with lip involvement were within 35 - 45 years , 45 - 55 years of age showing lip depigmentation in 11 patients and above 55 years 8 patients presented lip depigmentation [ figure 1 ] . depigmentation in lip among 100 patients in our study depigmentation of buccal mucosa was present in five patients in which three were males and two females . with age of range up to 25 years none , within 25 - 35 years of age two patients , among patients of age 35 - 45 years two patients , within age range of 45 - 55 years one patient and above 55 years of age none of the patients showed depigmentation in buccal mucosa [ figure 2 ] . depigmentation in palate in the present study labial mucosa showed depigmentation in a male patient and four female patients . the following age ranges of up to 25 years , 25 - 35 years , 35 - 45 years , 45 - 55 years , above 55 years presented depigmentation of labial mucosa in one patient , none , three patients , one patient and none , respectively [ figure 3 ] . depigmentation in buccal mucosa in our study palatal depigmentation was found in eight patients out of them five patients were males and three were females . up to age range of 25 years one patient had palatal depigmentation , 25 - 35 years of range showed none , within age of 35 - 45 years three patients , 45 - 55 years of age range two patients and above 55 years of age two patients showed depigmentation in palate [ figure 4 ] . with respect to age of up to 25 years , 25 - 35 years , 45 - 55 years age none showed gingival depigmentation . patients within the age range of 35 - 45 years and above 55 years showed one patient each with depigmentation of gingiva [ figure 5 ] . among the 100 patients in our study 2 patients showed oral depigmentation as the only manifestation of vitiligo both manifestating in the lip . in relation to age up to 25 years one patient and 35 - 45 years one patient showed oral depigmentation . depigmentation of oral mucosa can be noted as an early feature in oral submucous fibrosis . in reverse these depigmentations are excluded based on the clinical history . in our study no relation of previous dental procedures to the depigmentation was noted . as far as our knowledge no literature states about the koebner phenomenon as a cause for oral vitiligo . vitiligo lesions presenting as skin depigmentation may also show oral mucosal depigmentation with lip and palate being the most common sites . sometimes oral depigmentation may be the first indicator of vitiligo which may manifest in unnoticeable areas . so educating patients regarding oral depigmentation and possibility of occurrence in other parts of body will help in the earlier commencement of treatment , thereby battling against a socially distressing disorder could be possible . very few articles have been published regarding oral manifestations of vitiligo . these articles report depigmentation of lip , palate and gingiva . in our study we have identified the presence of depigmentation of labial mucosa and buccal mucosa as well .

it is characterized by the accumulation of extracellular amyloid ( senile ) plaques , composed mainly of a peptides , and intracellular neurofibrillary tangles , containing abnormally aggregated and hyperphosphorylated tau protein . the a peptides are generated by the sequential cleavage of amyloid precursor protein ( app ) by -secretase ( bace1-dependent ) and -secretase ( psen / presenilin - dependent ) . based on the discovery of rare familial forms of ad caused mutations in the app and psen genes , hardy and selkoe proposed the amyloid cascade hypothesis , which suggests that a overproduction alone is sufficient to trigger the molecular events leading to both tangle formation and neuronal cell death [ 1 , 2 ] . over the past two decades , several breakthroughs have been made with regard to modeling ad pathology in vivo in mice , providing important tools for various areas of basic and translational research . several ad mouse models harboring human app , psen , and/or mapt ( tau ) transgenes support to some extent the amyloid cascade hypothesis ; however , recent clinical trials in humans suggest that amyloid - dependent signaling pathways are insufficient to cause the severe neurodegeneration and dementia in ad patients [ 35 ] . it is therefore perhaps not surprising that most , if not all , ad mouse models , albeit displaying massive a deposits and/or tangles , do not recapitulate the full - blown neuropathological biochemical , cellular , and morphological changes observed in ad brain [ 68 ] . this opens the door to the identification of novel factors important for ad development , which could equally serve as potential diagnostic and therapeutic tools . the past years have witnessed an explosion of papers linking microrna ( mirna ) dysfunction to human disease , including cancer , cardiology , liver disease , viral infection , and many others . perhaps expectedly , mounting evidence also involve mirnas in neurodegenerative disorders , with potential implications in ad [ 9 , 10 ] , parkinson 's disease ( pd ) , huntington 's disease ( hd ) [ 12 , 13 ] , frontotemporal dementia ( ftd ) [ 14 , 15 ] , and amyotrophic lateral sclerosis ( als ) . because of the rapid growth of mirna research in general , it is likely other neurodegenerative disorders will soon be added to this list . recent rna deep sequencing efforts have identified more than 1400 mirna genes in the human genome ( 700 in mice ) ( mirbase.org ) . several of these are specifically expressed in the brain , where they are proposed to function in neuronal processes such as neurite outgrowth and synapse formation [ 17 , 18 ] . the biogenesis and mode of action of mirna molecules in mammals is complex and has extensively been reviewed elsewhere ( see , e.g. , [ 1922 ] ) . in short , mirna precursors ( pre - mirs ) are cleaved in the cytoplasm by the rnase dicer to produce small ( ~21 nt in length ) single - stranded nonprotein - coding rnas . these latter function as part of the rna - induced silencing complex ( risc ) , which targets specific mrna transcripts with imperfect complementarity . binding of the mirna to its target leads to translation inhibition and/or mrna degradation [ 23 , 24 ] , which ultimately leads to down - regulation of the encoded protein . it is predicted that 25 to 70% of all protein - coding genes can be regulated by mirnas , depending on the developmental , cellular , and/or physiological context . moreover , each mirna can target up to several hundred transcripts in vivo , thus potentially regulating multiple biological pathways , including those implicated in neuronal survival . it is therefore not surprising that genetic ablation of dicer in the brain , which leads to an overall reduction in mirna production , results in rapid neurodegeneration [ 2628 ] . the somewhat humble introduction of mirnas in the ad field came in 2007 when lukiw studied the expression levels of 13 brain mirnas in control and ad patients , some of which were specifically altered in disease . since then , several groups have performed global mirna expression analysis of ad brain [ 9 , 10 , 2931 ] and peripheral system [ 32 , 33 ] . these profiling experiments tend to show a trend towards the identification of ad or neurodegenerative disease - specific mirnas , including mir-29 , mir-9 , mir-15a , mir-181c , mir-101 , mir-106b , mir-146a , and mir-107 , which have been identified in two or more independent studies . interestingly , several of these mirnas may have a direct role in modulating app ( mir-106 , mir-101 ) [ 3436 ] or bace1 ( mir-29 , mir-107 ) [ 9 , 3739 ] expression , therefore potentially contributing to increase amyloid production . because ( 1 ) mirnas regulate app and bace1 expression , whose increased protein levels are linked to genetic and sporadic ad , respectively ( reviewed in [ 21 , 40 ] ) and ( 2 ) mirnas control several pathways involved in neuronal function , inflammation and survival , mirna research provides an interesting new perspective to study the underlying mechanisms involved in ad development . while depleting all mirnas remain a conceptually crude approach , it is interesting to observe several parallels between dicer - deficient brain and ad brain . for instance , neuronal dicer conditional knockout ( cko ) mice develop progressive neurodegeneration , have reduced brain size , enlarged ventricles , neuroinflammation , apoptosis ( in some cases ) , as well as impaired dendritic branching , and spine length [ 26 , 41 , 42 ] . in addition , neuronal dicer cko mice exhibit ad - like hyperphosphorylation of endogenous tau , which is not observed in nontransgenic mice . these latter results succeed previous studies in the fly linking tau toxicity to mirna dysfunction . interestingly , specific loss of dicer in oligodendrocytes results in axonal degeneration accompanied by abnormal axonal transport and endogenous app accumulation . this model also displays signs of oxidative stress , and , taken together , these results point out the importance of dicer and mirnas in maintaining neuronal function . although controversial , one study suggests that neuronal dicer deficiency promotes learning and memory , at least at stages prior to neuronal loss . how these observations translate to human disease remains speculative , but nevertheless provides proof - of - principle that loss ( and perhaps gain ) of brain mirna function can participate in several neuropathological features of ad . apart from the more obvious role of mirnas in regulating the expression of disease - related genes ( e.g. , app and bace1 ) , it is likely that a combination of more subtle ( direct or indirect ) mechanisms alter disease progression over years , possibly decades . as example , sustained mir-29 deficiency may not only increase bace1 and a levels , but also affect dna methylation and neuronal survival [ 46 , 47 ] . in addition , it remains difficult to predict whether the observed changes in mirna levels in humans are a cause or consequence of the neurodegenerative process . the study of mirna expression profiles in ad mouse models may help to address these questions . of the 37 differently expressed mirnas , several ( mir-20a , mir-29a , mir-125b , mir-128a , and mir-106b ) mirnas were significantly downregulated , while others ( mir-34a , let-7 , mir-28 , and mir-98 ) were upregulated . interestingly , some mirnas were similarly shown to be affected in ad brain in humans , including mir-29a and mir-106b [ 9 , 30 , 31 , 40 , 49 ] . it is noteworthy that mirna alterations were measured at 3 months of age prior to a plaque formation . in most cases , mirna alterations were maintained or even accentuated during amyloid plaque formation at 6 months of age , therefore supporting the cause hypothesis . the increase in mir-34a in the mutant mice is proposed to function in regulating apoptosis via bcl-2 modulation . in a follow - up study , the group showed by sensitive mirna quantitative rt - pcr that mir-106b is upregulated in 3-month - old ad mice but downregulated at 6 months . these changes correlated to some extent with transforming growth factor , beta receptor ii ( trii ) expression , and a putative mir-106b target gene . while these studies highlight the importance of microarray validation , they also suggest a possible transient effect of ad pathology ( in this case a plaque formation ) on mirna expression and vice versa . more recently , schonrock et al . studied the effects of exogenous a on mirna expression levels in mouse hippocampal neurons in culture . again , several mirnas downregulated by a treatment were previously found to be decreased in human ad brain , including mir-9 , mir-181c , mir-30c , mir-148b , mir-20b , and let-7i . of interest , certain mirnas decreased concomitantly with a pathology progression in vivo in app23 mice expressing human app751 with the k670n / m671l mutations . while these observations support the consequence hypothesis of mirna dysregulation in ad , it is noteworthy that some mirna molecules became affected prior to a plaque formation ( like mir-409 - 3p and let-7i ) similar to what is seen in the wang et al . furthermore , the expression of certain mirnas changed over time ( from up- to downregulated or vice versa ) , again supporting the transient effect on mirna expression during ad development . while studying the role of actin and the actin - binding protein cofilin in ad , yao et al . observed decreased mir-103 and mir-107 levels in 4-month old ( a plaque bearing ) tg19959 mice that express mutant app with km670/671nl and v717f fad mutations . as mentioned above , both mir-103 and mir-107 were shown to be decreased in mci and late - onset ad . the authors further showed that these mirna paralogues could effectively regulate cofilin expression in vitro , providing a mechanism for the observed increase in rod - like structures in this mouse model . loss of presenilin function is proposed to underlie memory impairment and neurodegeneration in the pathogenesis of ad . interestingly , small - scale mirna profiling from psen1 ko mice with , as a result , reduced -secretase activity and a production , showed that mir-9 down - regulation coincided with neurodegeneration . it is noteworthy that mir-9 was shown to be an important regulator of neurogenesis , both in zebrafish and mice [ 55 , 56 ] . based on these observations , it is tempting to speculate that mir-9 , which is downregulated in ad brain , participates actively in neuronal maintenance , and functions in a feedback loop with a. candidate mirna approaches have equally been performed . for instance , li et al . studied mir-146a expression in five different ad mouse models , including tg-2576 , tg - crnd8 , psapp , 3xtg - ad , and 5xfad . it turned out that mir-146a was significantly increased in age ( 4- to 12-month - old ) when compared to young ( 1- to 2-month - old ) mice , and this , independently of the model tested . notably , mir-146a has repeatedly been shown to be implicated in the regulation of the inflammatory response . moreover , neuroinflammation is thought to play a critical role in the pathogenesis of chronic neurodegenerative diseases including ad , evoking the hypothesis that mir-146a overexpression in these ad models could reflect a defense mechanism against the deleterious effects of neuroinflammation . interestingly , synthetic a was shown to induce mir-146a expression in cultured human neuronal ( and glial ) cells . taken together , the abovementioned observations suggest that mirna - regulated gene pathways , such as the mir-146a pathway , could function both upstream and downstream of ad pathology ( cause and consequence ) . one of the main challenges in the mirna field is the identification of bona fide target genes . several genome - wide methods are currently available to address this question , including microarray expression analyses following mirna transfection / inhibition , argonaute cross - linked immunoprecipitation ( clip)-seq assays , and silac - based proteomic approaches [ 6163 ] . so far , however , most mirna studies involving ad - related genes have relied on artificial 3utr reporter systems ( e.g. , luciferase - based assays ) as well as single mirna gain- or loss - of - function experiments in cells . while these methods remain indispensable in the validation of mirna : mrna targets , they rarely put both molecules in their physiological context . in addition , it is important to consider that some mirna : mrna targets are not conserved in lower organisms such as c. elegans and drosophila , making extrapolations to mammalian brain sometimes difficult ( e.g. , see ) . as of now , most studies addressing the role of mirnas in ad gene regulation have focused on app . these have lead to the identification of at least six mirnas that could regulate app in vitro and in cells , including the mir-20a family ( i.e. mir-20a , mir-17 - 5p , and mir-106a / b ) , mir-101 , and mir-16 [ 40 , 6468 ] . interestingly , liu et al . showed that mir-16 inhibition in 8-month - old samp8 mice , a model for accelerated senescence , reduced endogenous app levels by 50% . again , the effects on a production remain to be determined . while providing the first proof - of - principle of app regulation by mirnas in vivo , this study does not exclude the role of other mirnas in this process in the brain . for instance , our preliminary data indicate that app mrna levels are increased in mir-20a/17 - 5p double ko mice ( hebert , s.s . in addition , it remains uncertain whether other mir-16 homologues , that is , mir-15a , mir-15b , mir-195 , and mir-495 are involved in app regulation in vivo . interestingly , mir-16 family members have also been implicated in endogenous tau phosphorylation in neurons . while investigating the molecular mechanisms involved in endogenous bace1 overexpression in 6-month - old ( a plaque bearing ) 5xfad , o'connor et al . observed no significant changes in mir-29a , mir-29b , and mir-9 levels , previously shown to regulate bace1 in vitro , when compared to nontransgenic controls . another study suggests however , that mir-29c ( the third mir-29 family member ) overexpression is sufficient to decrease endogenous bace1 protein levels in wild - type mice . the functional effects on bace1 activity and a production were unfortunately not evaluated in this latter model . showed that bace1 protein increases in 19-month - old ( a plaque bearing ) versus 4-month - old appswe - ps1a246e mice , an effect not observed in wild - type mice . interestingly , two mirnas shown to regulate murine bace1 expression in vitro , that is , mir-298 and mir-328 were downregulated in this mouse model . whether other mirnas are affected in the 5xfad and appswe - ps1a246e mice remains to be determined . . showed that bace1 protein and bace1 antisense ( noncoding ) transcript levels were increased in 6-week - old tg19959 mice . whether decreased mir-103/107 levels observed in this mouse model ( see yao et al . thus , among at least five mirnas shown to target bace1 in vitro , that is , mir-29 , mir-9 , mir-107 , mir-298 , and mir-328 , no clear evidence can be drawn from these mouse models with regard to the physiological or pathological regulation of bace1 by mirnas in vivo . while the abovementioned results are interesting , the overall contribution of mirnas in bace1 and app expression regulation remains unclear . the use of gene knockout mice will be indispensible to make definitive conclusions with regard to the role of mirnas or mirna gene families in bace1 , app , and other ad - related gene expression regulation . more transient approaches , for instance using mirna mimics or inhibitors , will be necessary to address the cause - consequence relationship between mirna dysregulation and ad progression in the brain . thus , both classical genetics and mirna functional tools will be necessary to fully investigate the role of mirnas in ad mouse models . much advancements have been made in the cancer field , for instance , with regard to mirnas as potential diagnostic tools , some of which are currently available in the clinic . the interest in mirnas in this field comes from the fact that they are readably detected in human body fluids , making them attractive biological markers . in addition , mirnas are in general quite stable when compared to protein and/or other rnas , providing reliable markers of cell or tissue state . while a few groups have explored the role of mirnas in blood and csf [ 32 , 33 ] , most ad - based studies have focused mainly on soluble a ( and its derivatives ) and tau [ 72 , 73 ] . in the future , it will be interesting to combine and/or compare these species with circulating mirnas , which can be found in body fluids including exosomes . it is noteworthy that groups have already shown this applicability in mouse models for the duchenne muscular dystrophy and liver injury [ 75 , 76 ] . as summarized above , most studies addressing the role of mirnas in ad pathology remain correlative in nature , and little or no definitive proof with regard to mirna target gene regulation in vivo is currently unavailable . addressing these issues is crucial in order to advance our knowledge of the contribution of mirnas , if any , in ad neuropathology . because mice are genetically homogenous in nature when compared to humans , they provide unique tools to study mirna - regulated gene pathways in ad development . human profiling studies clearly indicate that mirna expression profiles are altered in ad brain . whether these changes are specific to ad or reflect an overall loss ( or gain ) of mirna function in neurodegenerative disease remains to be determined using adequate comparative analyses . the current profiling data from ad mice suggest that mirna changes equally occur in disease models , yet some discrepancies still exist . given our current state of knowledge , the role of mirnas in ad development , and their applicability as diagnostic and perhaps therapeutic tools into clinic , will require extensive follow - up studies in both in vitro and animal models . specific mirna gene knockout and/or transgenic mouse is required to address these and other fascinating questions .

the use of genome - wide association studies ( gwas ) has successfully linked genetic variants with susceptibility to a wide range of common polygenic diseases . such gwas , however , have almost exclusively focused on single nucleotide polymorphisms ( snps ) . additional studies targeted to specific copy number variant loci , as well as the alternative approach consisting of using snps to tag copy number variations ( cnvs ) , have suggested that cnvs may contribute significantly to genetic predisposition to several common diseases . the tagging of cnvs using snps is often imperfect , and therefore motivates the development of association studies directly targeting cnvs . importantly , association tests directly targeting cnvs rely on the development of maps of common cnv polymorphism in the human genome [ 5 - 7 ] . the process of discovering common cnvs is related but distinct from the association testing procedure . software reviewed here assume that the user is considering known cnv regions , with properly mapped boundaries . programs designed to identify previously unknown cnvs ( eg wang et al . ) are not discussed here . moreover , association tests outlined in this review typically consider the common variant / small effect situation . various assays can estimate cnv status directly --in particular , real - time polymerase chain reaction ( rtpcr ) analysis , comparative genomic hybridisation ( cgh ) arrays and snp genotyping arrays . the throughput of these assays is sufficient to analyse the large cohorts required to detect subtle effects previously reported by snp - based association studies . these cnv assays typically generate a one - dimensional continuous measure per dna sample and per cnv probe . provided that the quality of the cnv assay is sufficiently clear to convert these raw continuous measures into discrete calls , cnv - based association testing is analogous to the snp situation , with a few minor differences , which are outlined in this review . when uncertainty in the calling makes this discretisation difficult , however , a different statistical approach is necessary . cnv association testing must then be based on the raw continuous data instead of potentially inaccurate discrete calls . here , these alternative approaches are outlined , both for case control and family - based association studies . even though the underlying cnv state is a discrete integer , cnv assays typically only provide a continuous measure for each cnv and individual , and this continuous trait is a surrogate for the actual , discrete cnv state . therefore , when testing for association at a cnv locus , the first choice is to decide whether to analyse the cnv as a continuous or a discrete measurement . in an ideal scenario , the distribution of this surrogate measure can be properly separated in discrete clusters , which can then be linked to discrete numbers of dna copies . in this case , it is appropriate to discretise the data and base the association test on these discrete calls . unfortunately , and because of technical difficulties associated with the assessment of cnv status , this continuous measure can not always unequivocally be converted into discrete numbers of dna copies . erroneous calls can inflate the rate of false - positive associations and limit the statistical power to detect true associations . therefore , when discretisation is difficult , it is preferable to base the association test on the raw continuous measurement . if the cnv status is summarised using a discrete measure , the association test is analogous to the snp situation , and traditional statistical software such as r http://www.r-project.org , or more specialised tools such as plink , are appropriate ; however , additional factors complicate the analysis of cnvs compared with snps . first , association tests require assumptions on the model linking cnv status to disease risk . for example , a traditional cochran - armitage test assumes that , on the log - scale , the risk is proportional to the number of copies . such assumptions may not be appropriate for some cnvs ; for example , for some diseases only extreme numbers of copies may have a causal role in disease aetiology . especially for multi - allelic cnvs , for which a wide range of models can be investigated , the choice of model must be driven by prior belief on disease aetiology . in the absence of prior knowledge , it is advisable to constrain the analysis to a small number of simple models to avoid misleading p - values generated by multiple testing . secondly , cnv assays do not always provide information about the exact number of copies , but often only on the ' relative ' numbers . for example , one can not always distinguish cnvs with zero , one or two copies from cnvs with two , three or four copies . thirdly , the phase is often unknown and only the total number of copies can be measured ; for example , most cnv genotyping assays can not distinguish two haplotypes with one copy on each from one haplotype with two copies combined with a complete deletion . phasing cnvs is difficult , but if sufficient marker density is available it can be achieved using software such as fastphase . lastly , it is common when using snp genotyping arrays to assess cnv status to obtain information jointly on snp genotypes and dna copy number . rather than a single number , each individual is therefore summarised by a pair ; the first element indicates the number of dna copies and the second element indicates the associated snp genotype . this added information can potentially provide additional power when testing for association , and a recent study has proposed a new approach to integrating snp and cnv information in a unique association testing procedure . this joint snp / cnv association is implemented as part of the analysis software plink . cnv genotyping arrays , in particular high - density cgh arrays , typically use multiple probes to interrogate a single cnv . this is particularly useful when we expect variable probe performance or , more generally , when measurements are noisy and a combination of probes can provide more accurate measurements . some association testing procedures require the combination of information from all probes into a single measurement for each individual , while others deal directly with multi - marker data . when the genomic location of the cnv is well defined , and there is strong evidence that all probes are located within the copy number variable region , simply averaging the probe intensity across all probes for each individual is sensible . in many situations , however , the boundaries of the cnv are not well defined , and some probes may lie outside of the region of interest . when this is the case , it is advantageous to identify and down - weight , or even simply remove , these non - informative probes to lower the measurement noise . this down - weighting procedure can be done using a principal component analysis that should down - weight the non - informative probes provided that the signal generated by the probes in the cnv region is sufficiently strong . this down - weighting procedure is also relevant when dealing with complex cnv regions , where several distinct cnvs may be overlapping . a principal component analysis can help separate a set of probes in genetically relevant discrete groups . when the uncertainty in the calling procedure is too large to call the data confidently , it is advisable to base the association test on the raw , continuous data summary . in the context of family - based association studies , pbat implements a broad class of association tests and has been extended to deal with raw measurements from cnv assays . in its simplest form , this test compares the cnv measure for an affected offspring with the average cnv measure of both parents in parent - offspring trios . a consistently elevated number in affected offspring indicates an association between high copy number and elevated disease risk . a commercial version of the pbat statistical tools , including a graphical user interface , is now part of the golden helix genetic association software http://www.goldenhelix.com . a previous study has highlighted the potential for different dna sourcing , handling , extraction or storage to affect the measures obtained from genotyping assays , potentially creating biases between sample collections that do not reflect actual genotype differences . therefore , even when cases and controls are analysed using the same genotyping assay and at the same time , differences in the distribution of the raw cnv measures can not always be interpreted directly as actual genotype differences . to circumvent these issues , barnes et al . this approach provides calls by clustering the data using a gaussian mixture , but the association test statistic is designed to account for the uncertainty in these calls . in addition , the calling procedure implements a hierarchical clustering model that lets the parameters of the gaussian mixture vary across cohorts in order to account for potential biases correlated with the origin and handling of dna samples . this method is , in fact , general and can be used to combine data generated by different cnv genotyping assays at the same cnv locus . a drawback of the clustering approach implemented by cnvtools is the fact that the clustering procedure relies on the data quality being sufficient to fit a gaussian mixture with a limited number of components . for example , when analysing highly polymorphic short tandem repeats , it is unreasonable to expect that the cnv assay can properly separate samples that differ by one or very few repeats . it makes little sense , in this situation , to cluster the cnv measurement , and cnvtools is not an appropriate tool for that type of data . the pbat testing procedure , however , is still usable , provided that one is analysing family data . while a wide range of options is available for analysing the cnv status as a discrete trait , software tools to analyse directly raw continuous measurements from cnv assays are currently limited to pbat for family and cnvtools for case control association studies . both pbat and cnvtools share a frequentist statistical approach ; however , a bayesian approach to this problem appears well suited to average over the uncertainty in the calling procedure efficiently , which is the main hurdle for cnv association tests . as gwas targeting cnvs become more common , we expect that several bayesian approaches will be developed to complement currently available software .

the basic principles of root canal treatment are the eradication of root canal irritant , obturation of the root canal system and preservation of the natural dentition . traditionally root canal treatment was performed in multiple visits , with medication between root canal preparation and obturation , which mainly aims to reduce or eliminate microorganisms and their by - products from the root canal system before obturation . however , with various advances in endodontics , single visit root canal treatment is commonly performed . however , despite advances in endodontic therapy , studies have shown that pain frequency and intensity vary after root canal treatment . there is no consistency in the literature , the frequency of postoperative pain is mentioned to be lower in single visit or multiple visits or to have similar results . post - operative pain after endodontic procedures is an undesirable occurrence for both patients and clinicians . patients might consider post - operative pain as a benchmark against which the clinician 's skills are measured . it might undermine patient 's confidence in their dentist or patient satisfaction with their treatment . the occurrence of mild pain is relatively common even when the treatment has followed the highest standards and should be expected and anticipated by patients . numerous studies with post - operative pain after root canal treatment have been published , with the incidence of post - operative pain ranging from 1.9% to 48% , and they showed conflicting findings . pain associated with root canal therapy is a poor indicator of pathosis and an even more unreliable predictor of long - term success . prospective , randomized studies are generally believed to provide the highest level of support for evidence - based clinical practice . thus , a prospective , randomized study was carried out to evaluate the incidence and severity of post - operative pain following single sitting root canal therapy in indian population . the relationship of certain factors ( age , gender , pulpal status , arch , and presence of pre - operative pain ) with post - operative pain was also analyzed . one thousand three hundred and twenty eight patients were included in the study . of the 1328 patients , 593 ( 44.7% ) were women and 735 ( 55.3% ) were men . of all the teeth 1097 ( 82.6% ) were vital and 231 ( 17.4% ) teeth were non - vital . 606 ( 45.6% ) treated teeth were in the maxilla and 722 ( 54.4% ) teeth were in the mandible . all the teeth were molars , either first molar ( 783 ) or second molar ( 545 ) . single sitting root canal therapy was explained to the patient and oral and written informed consent was obtained . patients who were pregnant , need retreatment , having complications during treatment ( calcification , impossibility to achieve apical patency ) , taking antibiotics or steroids at the time of treatment , immuno - compromised or under 15-year - old were excluded from this study . the working length of each canal was determined by an electronic apex locator and 2 or more angled radiographs . canals were prepared with a combination of hand files ( k file , mani , japan ) and rotary protaper system ( dentsply maillefer , ballalgues , switzerland ) by using rc prep ( premier dental product co. king of prussia , pa , usa ) . the canals were obturated with protaper gutta percha points ( dentsply maillefer ) using ah plus sealer ( dentsply , konstanz , germany ) . then the post - obturation restoration was done with amalgam to maintain a good coronal seal . post - operative pain was measured by using a visual analogue scale ( vas ) of 0 ( no symptom ) to 3 ( severe pain and/or swelling ) . vas was taught to the participants as well as reporting the post - operative pain after 12 h , 24 h , and 48 h. patients were contacted by telephone if they did not return the vas form . the post - operative evaluation was recorded as 0 ( no pain ) , 1 ( slight pain / discomfort ) , 2 ( moderate pain relived by analgesics ) and 3 ( severe pain / swelling not relieved by analgesics and required unscheduled visit ) . all patients were given a prescription for 50 mg of diclofenac potassium , with the dose of 1 tablet every 8 h taken only if they experienced moderate pain . the working length of each canal was determined by an electronic apex locator and 2 or more angled radiographs . canals were prepared with a combination of hand files ( k file , mani , japan ) and rotary protaper system ( dentsply maillefer , ballalgues , switzerland ) by using rc prep ( premier dental product co. king of prussia , pa , usa ) . the canals were obturated with protaper gutta percha points ( dentsply maillefer ) using ah plus sealer ( dentsply , konstanz , germany ) . then the post - obturation restoration was done with amalgam to maintain a good coronal seal . post - operative pain was measured by using a visual analogue scale ( vas ) of 0 ( no symptom ) to 3 ( severe pain and/or swelling ) . vas was taught to the participants as well as reporting the post - operative pain after 12 h , 24 h , and 48 h. patients were contacted by telephone if they did not return the vas form . the post - operative evaluation was recorded as 0 ( no pain ) , 1 ( slight pain / discomfort ) , 2 ( moderate pain relived by analgesics ) and 3 ( severe pain / swelling not relieved by analgesics and required unscheduled visit ) . all patients were given a prescription for 50 mg of diclofenac potassium , with the dose of 1 tablet every 8 h taken only if they experienced moderate pain . results are shown in tables 1 - 6 : the overall incidence of post - operative pain after rct during the follow - up period of 12 h , 24 h , and 48 h was assessed according to patient 's record in the vas [ table 1 ] . 1190 ( 89.6% ) patients had no pain , 14 ( 1.1% ) patients had mild pain , 5 ( 0.4% ) patients had moderate pain , and 119 ( 9.0% ) patients experienced severe pain after 12 h. after 48 h the number of patients with severe pain was drastically reduced to 54 ( 4% ) , and there was no patient with mild pain , and only 1 ( 0.1% ) patient with moderate pain . overall incidence of post - operative pain after 12 h , 24 h , and 48 h post - operative pain related to the age : differences were statistically significant ( p = 0.0 ) between both the groups . incidence of post - operative pain is high in group 2 ( 41 - 65 years ) at all the three - time intervals [ table 2 ] . incidence of post - operative pain in relation to age at 12 h , 24 h and 48 h post - operative pain related to the gender : statistically significant ( p = 0.0 ) difference was present between both the genders . more number of women experienced severe pain as compared to men , at all the three - time intervals [ table 3 ] . incidence of post - operative pain in relation to gender at 12 h , 24 h , and 48 h post - operative pain related to the arch : in lower teeth post - operative pain was significantly higher ( p = 0.001 ) at all the three time intervals [ table 4 ] . incidence of post - operative pain in relation to arch at 12 h , 24 h , and 48 h post - operative pain related to the vital status of the tooth : there was no statistically significant difference ( p = 0.338 ) in post - operative pain , between both the groups at all the three time intervals [ table 5 ] . incidence of post - operative pain in relation to the vitality at 12 h , 24 h , and 48 h post - operative pain related to the pre - operative pain : in cases with reported presence of pre - operative pain , the presence of post - operative pain was significantly more ( p = 0.0 ) [ table 6 ] . incidence of post - operative pain in relation to the pre - operative pain at 12 h , 24 h , and 48 h one of the main problems in studying pain is the patient 's subjective evaluation and its measurement . for this reason , design of the questionnaire is critical and must ensure that it will be fully understood by patients and easily interpreted by researchers . in this report , a simple verbal categorization was used in the feedback form with 4 categories : 0 - no pain , 1 - mild pain , 2 - moderate pain , and 3 - severe pain . it is well known that pain perception is a highly subjective and variable experience modulated by multiple physical and psychological factors . postobturation pain is considered to be related with several factors , including pre - operative pain , infection , retreatment , intracanal medications , and physical and chemical damage to periapical tissue . the lower incidence of post - operative pain in single visit root canal treatment might be attributed to immediate obturation , thereby to avoid passage of medications , repeated instrumentation and irrigation . the reported prevalence of post obturation pain ranges widely from 0% ( at 30 days ) to 65% ( at 1 day ) , generally declines over time and should therefore be qualified by duration after last treatment episode . in this investigation , overall incidence of post - operative ( severe ) pain in rct was 9% after 12 h , which was reduced to 8.6% after 24 h and drastically to 4% after 48 h. thus , under sound biological principles and by using contemporary scientifically based techniques , a low incidence of post - operative pain can be expected . the pain was more frequent during the first 24 h of the observation period and quickly decreased thereafter . these findings were in agreement with those of some authors.[2711 ] in this study , five variables ( age , gender , arch , vitality , and presence of pre - operative pain ) were considered separately to explore the potential influence on the prevalence of post - obturation pain . except for one variable ( i.e. , vital status of the tooth ) , the remaining four variables were significantly associated with presence of post - obturation pain . prevalence of post - operative pain is significantly higher ( p = 0.0 ) in old patients ( 7.8% ) as compared to young patients ( 0.8% ) , at all the three time intervals . this may be because of less pain tolerance , less blood flow and delayed healing . the prevalence of post - operative pain is very high in women ( 7.4% ) as compared to men ( 1.8% ) ( p0 = 0.0 ) . this finding is in agreement with ryan et al . , who presented gender ( women ) as a factor that significantly influenced post - operative pain . a more legitimate explanation is based on emerging evidence that biological differences between genders may explain increased pain prevalence in women . there are two possible explanations : differences in pelvic and reproductive organs may provide an additional portal of entry of infection in females leading to possible local and distant hyperalgesia.fluctuating female hormonal levels may be associated with changing levels of serotonin and noradrenaline leading to increased pain prevalence during the menstrual period and in women receiving hormonal replacement therapy or oral contraceptives . differences in pelvic and reproductive organs may provide an additional portal of entry of infection in females leading to possible local and distant hyperalgesia . fluctuating female hormonal levels may be associated with changing levels of serotonin and noradrenaline leading to increased pain prevalence during the menstrual period and in women receiving hormonal replacement therapy or oral contraceptives . in mandibular molars post - obturation pain is significantly higher ( 6% ) as compared to maxillary molars ( 2.2% ) ( p = 0.001 ) . this might be because mandible has a dense trabeculae pattern , thus there is reduced blood flow and more localization of infection and inflammation , which might lead to delay healing . analysis of post - obturation pain shows no statistically significant difference between vital ( 3.7% ) and non - vital teeth ( 5.6% ) ( p = 0.338 ) . in cases with presence of pre - operative pain , frequency , and degree of postoperative pain were significantly more ( p = 0.0 ) ( 10.5% ) as compared to patients with absence of pre - operative pain ( 0.9% ) . this finding could be explained by the presence of infection of the root canal system and periapical region pre - operatively . thus , within the limitation of this study , it is concluded that post - obturation pain is likely to occur in first 24 h which further reduces as time passes . thus , it is a strong indication that clinician should not overreact to early post - obturation pain by immediately initiating endodontic retreatment procedures or extraction of the involved tooth . analysis of the influence of patient 's age , gender , arch , vitality of the tooth , and presence of pre - operative pain , on prevalence of the post - operative pain showed that it is higher in the old age group ( group 2 : 41 - 65 years ) , women , mandibular teeth , and with presence of pre - operative pain . the vital condition of the tooth does not affect the intensity and frequency of post - obturation pain . other factors reported to be significantly related to post - obturation pain such as apical periodontitis , retreatment , irrigating solution , intracanal medicament , tooth positions were not analyzed in this study .

the recent world health organization report on the worldwide incidence of tuberculosis ( tb ) indicates that in 2012 there were an estimated 8.6 million incident cases of tb and 1.3 million deaths . in addition , it is estimated that one third of the world 's population is latently infected with mycobacterium tuberculosis ( mtb ) and is at a 5 - 10% lifetime risk of reactivation , thereby fueling the cycle of transmission . clearly , tb remains a global emergency . a significant impediment to tb treatment and global eradication of the disease is a 6-month multi - drug treatment regimen . prolonging treatment reduces rates of relapse , but the extended period leads to non - compliance and the emergence of drug - resistant mutants additionally , despite successful treatment , relapses still occur in 5% of the patients because of bacterial persistence . furthermore , tb - hiv co - infected patients receiving anti - retroviral treatment and tb chemotherapy are at risk of developing immune reconstitution inflammatory syndrome . other major challenges to tb treatment and significant threats to global health are the worldwide increase in multi - drug - resistant ( mdr ) tb and the reported presence of extensively drug resistant tb in 92 countries . in the last decade , therefore , a great deal of research effort in tb was invested in the discovery and development of new tb drugs that target the bacteria and in conducting clinical trials to evaluate their efficacy in shortening tb treatment regimens . however , there is high likelihood of also developing resistance against these newly discovered drugs . though still incomplete , our understanding of how mtb evades the host response has advanced significantly . in addition , through the use of in vivo animal models , immune factors that restrict or promote mtb growth have been identified . this commentary will focus on lipid mediators , since converging findings from several groups point to their significant role in regulating multiple pathways of host control of mtb . we integrate these studies to highlight how lipid mediators form the central nexus influencing the various macrophage outcomes , including cell apoptosis , necrosis , and tumor necrosis factor ( tnf ) production , thereby positioning them as strong targets for host - directed therapy ( hdt ) . hdts have the potential to shorten tb treatment regimens with first - line tb drugs and , importantly , to improve treatment outcomes when used in conjunction with second - line drugs for mdr patients . another potential advantage of hdt is the ability to restrain inflammatory responses in the lung and thereby ameliorate pulmonary pathology . in this regard , treatment of mtb - infected mice with ibuprofen alone was found to be sufficient to reduce the number and size of lung lesions , and a meta - analysis study of 41 clinical trials found that corticosteroids were effective in reducing mortality in patients with tb . eicosanoids are a family of bioactive lipid mediators derived from arachidonic acid ( aa ) , which is released from membrane phospholipids by phospholipases . there are three major pathways that are involved in the production of eicosanoids : ( i ) the cyclooxygenase pathway ( cox-1 and cox-2 ) , which produces prostaglandins and thromboxanes ; ( ii ) the lipooxygenase ( lox ) pathway ( 5-lox , 12-lox , and 15-lox ) , which catalyzes the production of leukotrienes and lipoxins ; and ( iii ) the cytochrome p40 pathway , which generates hydroxyeicosatetraenoic acids and epoxyeicosatrienoic acids . in addition , omega-3 essential eicosapentaenoic acid ( epa ) and docosahexaenoic acid are substrates for the production of resolvins , protectins , and maresins that are termed as specialized pro - resolving mediators ( spms ) [ 710 ] . prostaglandins and leukotrienes are the initial mediators of the inflammatory response and stimulate recruitment of neutrophils and monocytes . lipoxins and spms have anti - inflammatory pro - resolving activities and contribute to the resolution phase of an inflammatory response . prostaglandin e2 ( pge2 ) and prostaglandin d2 ( pgd2 ) function as pro - inflammatory mediators during the initial phase of an inflammatory response . however , later , through a process termed lipid mediator class switching , these mediators reprogram exudate neutrophils to a pro - resolution phenotype by inducing 15-lox and lipoxin generation . lipoxins are also double - edged mediators with anti - inflammatory and pro - resolving activities . ligation of the lipoxin a4 ( lxa4 ) receptor on neutrophils arrests their migration but in monocytes signals for migration and non - phlogistic responses . thus , lipid mediators participate in the initiation and resolution of inflammation and aid in the return to homeostasis . in this commentary , we will first present evidence that eicosanoids regulate host defense against mtb . based on fundamental findings , it is well established that tnf is critical to restricting mtb growth but that inhibition of apoptosis is a virulence strategy that mtb employs to evade innate defense mechanisms of the host . furthermore , type i interferons ( ifns ) have been shown to impinge on host - protective responses against tb . we will then review the growing body of literature that mechanistically links these three host responses to regulation by lipid mediators . we conclude that these advances are paving the way for lipid mediator manipulation as promising targets for hdt in tb . however , one needs to be mindful of the possibility that these host - targeted therapeutic interventions for tb may have a deleterious effect on the control of coincident viral infections . there is rapidly accumulating evidence that the eicosanoids leukotrienes , prostaglandins , and lipoxins are critical to determining the outcome of mtb infection in the host . the first evidence that lipid mediators may serve as druggable host targets came from mtb infection studies in mice deficient in 5-lox ( alox5 ) , the enzyme required for both leukotriene and lipoxin biosynthesis . these mice had elevated expression of interleukin-12 ( il-12 ) and ifn , and exhibited significantly lower mtb burdens in their lungs at 5 weeks following infection , compared with wild - type ( wt ) mice . the further finding that administration of a lipoxin analog to mtb - infected alox5 mice reversed the protection indicates that lipoxins , and not leukotrienes , negatively regulate host control of mtb . in contrast , other studies have reported that inhibition of 5-lox with the pharmacological inhibitor mk886 abrogated host control of experimental pulmonary tb and that this was associated with a reduction in leukotrienes . despite the discrepant outcomes between alox-5 and mk886-treated mice , the finding that alox-5 variants are associated with susceptibility to tb supports that 5-lox products , lipoxin and leukotrienes , regulate host - protective immune response against mtb . findings from a forward genetic screen in zebrafish larvae found that mutations in the ita4h locus encoding leukotriene a4 hydrolase ( lta4h ) conferred hypersusceptibility to mycobacterium marinum infection . lta4h catalyzes the conversion of leukotriene a4 to the pro - inflammatory eicosanoid , leukotriene b4 ( ltb4 ) . the study found that the phenotype of the mutant was not due to the lack of ltb4 but rather to the deviation of the eicosanoid substrates toward the production of anti - inflammatory lipoxins . the increased levels of lipoxin inhibited tnf production and resulted in increased growth of mycobacteria . contrary to expectation , modulation of lta4h expression with anti - sense rna and morpholinos showed that not only lta4h - low but also lta4h - high zebrafish exhibited increased bacterial growth . lta4h - high animals , on the other hand , expressed enhanced levels of ltb4 , increased tnf , and an overall heightened inflammatory state . additional experimental manipulations of tnf levels in the fish revealed tnf to be the key executioner of increased necrosis and bacterial growth in both states of lta4h expression . together , these studies indicate that a fine balance of the eicosanoid mediators is essential to generate the right level of tnf since either too little or too much of the cytokine can be detrimental to host control of mtb . the finding that optimal tnf levels and control of mycobacterial growth can be achieved by modulation of lipoxin levels with pharmacologic modulators or via overexpression of an enzyme that inactivates ltb4 underscores the potential of targeting lipid mediators for the development of hdts . subsequent work on the lta4h promoter showed that a functional single - nucleotide polymorphism influenced the level of transcription in macrophages , thereby providing an explanation for why adjunctive therapy with dexamethasone benefitted only a subset of patients with tb meningitis . reanalysis of study patients for lta4h polymorphism showed that patients who benefitted from dexamethasone adjunctive treatment had a high lta4h - expressing genotype but that those who did not benefit had the low - lta4h - expressing genotype . this finding is paradigm - shifting and indicates that tb treatment with hdt may have to take into consideration the individual 's genotype . modulation of the macrophage cell death pathway is exploited by mtb as a virulence strategy to survive in the host . early studies clearly delineated two distinct outcomes following infection of alveolar macrophages with virulent and avirulent mtb strains . infection of murine bone marrow - derived macrophages or human monocyte - derived macrophages with avirulent mtb strains resulted in tnf - dependent apoptosis of the infected cell while virulent mtb evaded this innate defense mechanism and instead induced macrophage necrosis as the major mode of cell death . mechanistically , virulent mtb , via annexin-1 blockade , prevents the complete formation of the apoptotic envelope and inhibits apoptosis induction . apoptosis is host - protective since it restricts mtb growth , enhances antigen presentation by dendritic cells , and induces an early mtb - specific adaptive th1 response . on the other hand , necrosis results from an irreversible increase in the mitochondrial permeability transition , resulting in mitochondrial damage and enhanced release of cytochrome c . together , these studies underscore the pivotal role for the necrotic form of macrophage cell death in creating a safe niche for virulent mtb . in subsequent studies , the mechanistic basis for the induction of disparate cell death pathways by avirulent and virulent strains of mtb avirulent mtb induced pge2 production which protected against cellular necrosis by preventing mitochondrial inner membrane damage and by promoting rapid plasma membrane repair . on the other hand , lxa4 was the dominant lipid mediator produced by macrophages infected with virulent mtb and was responsible for induction of necrosis . lxa4 suppressed cox-2 expression and synthesis of pge2 , thereby deviating the infected macrophage toward a necrotic fate . the ensuing findings that prostaglandin e synthase ( ptges)-deficient mice and mice lacking the prostaglandin receptor ep2 have increased susceptibility to mtb infection provide strong evidence that pge2 and the apoptotic death of macrophages are critical to regulating mtb growth in vivo . in this commentary , the role of apoptosis in mtb infection is discussed under the rubric of lipid mediators and in this context the literature supports apoptosis as being host - protective . however , we would like to emphasize that the role of apoptosis in mtb infection is contentious and that several studies have reported that the induction of apoptosis is indeed a virulence strategy of mtb [ 3741 ] . clearly , additional extensive studies with mtb clinical isolates are necessary to determine how the two forms of cell death regulate mtb growth and pathogenesis in vivo . , clinical isolates of the beijing family induce a strong type i ifn response , and in vivo studies have shown that type i ifns exacerbate disease in mtb - infected mice . enhancing type i ifn expression in the lungs of wt mice through intranasal poly - ic administration or exposure to influenza a virus also resulted in increased pathology and bacterial load in the lungs in comparison with control mice . consistent with ifn 's deleterious effect on the host , ifn receptor knockout mice ( ifnarko ) mice were found to be more resistant to mtb infection compared with their wt counterparts , and poly - ic treatment had no effect on ifnarko mice . removal of tpl2 , a negative regulator of type i ifn , also led to excess type 1 ifn and exacerbated disease in mtb - infected mice . type i ifn downregulates the host immune response by inhibiting the production of il-1 , a cytokine critical to protection against mtb infection [ 5154 ] . findings from a seminal study performed in a cohort of tb patients from london and south africa showed that active disease was associated with a type i ifn gene signature and that the intensity of the signature correlated with severity of disease . subsequently , a similar disease - related type i ifn gene signature was reported in south african and gambian cohorts and in an indonesian cohort . despite the growing evidence of the cross - talk between il-1 and type i ifn and the importance of il-1 to host protection , the mechanism of in a recent study , mayer - barber and colleagues probed this question by first examining the typically studied mediators of protection , including ifn , nitric oxide synthase 2 ( nos2 ) , il-12p40 , and tnf , and found that these were not abrogated in the susceptible il-1r1ko mice . instead , the authors discovered differential expression levels of eicosanoid mediators between wt and il-1rko mice . in comparison with bronchoalveolar lavage fluid of wt mice , that of il-1r1ko mice had significant reductions in pge2 and pgf2a and concomitant increases in the levels of lxa4 and ltb4 . using a number of elegant in vitro and in vivo approaches to ablate cox-2 and pge2 expression , the authors were able to effectively demonstrate that , during mtb infection , il-1 induces cox-2 expression which in turn upregulates pge2 and subsequent mtb growth restriction . ifnarko mice that are resistant to mtb express significantly high levels of il-1 and pge2 . furthermore , removal of ifnar1 in il-1r1ko mice reduced their susceptibility compared with il-1r1ko alone , clearly indicating that type i ifn abrogates host protection . this study definitely shows that the suppression of type 1 ifn by il-1-induced pge2 is a key step in conferring resistance to mtb . importantly , the authors were able to confirm their observations in two human cohorts where they found that patients with a lower pge2-to - lxa4 ratio had worse sputum grade . treatment of these mice with zileuton , a us food and drug administration - approved 5-lox inhibitor , together with pge2 led to decreased lung pathology and bacterial burden , indicating that lipid mediators are attractive host targets for therapeutic intervention in tb . in figure 1 , we summarize the findings that demonstrate the contribution of the three major eicosanoid pathways to the ability of the host to restrict mtb . aa metabolism leads to the induction of pge2 , lipoxin , and leukotrienes ( i ) . pge2 promotes apoptosis and prevents necrosis by inhibiting mitochondrial damage ( ii ) and inducing rapid plasma membrane repair ( iii ) . however , lipoxin can inhibit pge2 via inhibition of cox-2 expression ( iv ) and ablate the protective effect of pge2 in the macrophage , leading to necrosis . il-1 enhances pge2 expression ( v ) , but il-1 expression is inhibited by type i ifn ( vi ) . thus , in the presence of type i ifn , il-1 expression is abrogated and consequently pge2 levels decrease . this results in an overall increased ratio of lipoxin to pge2 in the host , promoting mtb growth ( vii ) . . low expression of lt4ah leads to reduced tnf ( viii ) and inability to control bacteria and subsequent necrosis of macrophages . the fact that removal of lta4h deviates eicosanoid expression toward enhanced lipoxin levels ( ix ) indicates that an increased ratio of lipoxin to leukotrienes leads to macrophage necrosis ( x ) . high expression of lta4h is also detrimental since it tips the balance toward high leukotriene expression , excess tnf and macrophage necrosis ( xi ) , and enhanced growth of mtb . this indicates that the levels of lipoxin and leukotriene should be in equilibrium for optimal macrophage response to mtb . abbreviations : cox , cyclooxygenase ; ifn , interferon ; il-1 , interleukin-1-beta ; lox , lipooxygenase ; lta4 , leukotriene a4 ; lta4h , leukotriene a4 hydrolase ; ltb4 , leukotriene b4 ; mtb , mycobacterium tuberculosis ; pge2 , prostaglandin e2 ; pm , plasma membrane ; tnf , tumor necrosis factor . together , the findings demonstrate that striking the proper balance between the levels of lipoxin , pge2 , and leukotrienes is critical for successful control of mtb . thus , several drug targets in the eicosanoid pathway can be exploited to modulate the expression of the individual eicosanoids to the right level . the paradigm that pge2-mediated acquisition of the apoptotic phenotype by macrophages is host - beneficial is not without caveats . for example , lipid bodies contained in foamy macrophages promote the intracellular survival of mycobacteria but are also intracellular sites for pge2 generation . also , contrary to expectation , mice treated with ibuprofen , a drug that suppresses pge2 production , exhibit reduced bacterial load . of note , this study used the c3heb / fej strain of mice that develop necrotic lung pathology similar to active tb . clearly , further mechanistic studies are required to determine how ibuprofen treatment and decreased pge2 can be beneficial in the context of extensive lung pathology . also , in vivo studies need to examine whether ibuprofen and other non - steroidal anti - inflammatory drugs protect the host against tb by mechanisms that are independent of host lipid mediators . to understand the role of lipid mediators during influenza infection , lipidomic profiling was performed in mice infected with a low - pathogenicity influenza strain ( x31/h3n2 ) and a high - pathogenicity strain ( pr8/h1n1 ) . comparison of lipid class composition showed that 12/15-lox mediators were associated with the resolution phase of the x31 infection but that a high percentage of pro - inflammatory 5-lox mediators correlated with the h1n1 strain . these data suggest that a similar analysis of the composition of lipid mediators induced by different clinical strains of mtb may yield important data regarding the temporal role of lipid mediators in regulating pulmonary pathogenesis and inform whether the infecting strain of mtb may have to be considered in hdts . as clinical trials with hdts are implemented , it is important to consider the duality in function of several of the lipid mediators . however , lipoxin also reduces airway inflammation and therefore it would be important to investigate whether hdt targeting the lipoxin pathway may have adverse effects of enhanced immunopathology . in this regard , it is worth investigating whether pro - resolving mediators that have no anti - inflammatory effects will provide a new therapeutic opportunity for complete resolution of lung pathology in patients with tb . it may also be important to study the effect of lipoxin on non - hematopoietic cells since it induces bactericidal / permeability - increasing protein in human mucosal epithelia . a recent study reported that influenza virus a uses the pge2 pathway to enhance its replication in the host . the virus limits the induction of type i ifn and also inhibits cell apoptosis by upregulating the expression of pge2 . inhibition of pge2 via genetic or pharmacological means enhanced the ability of the host to control viral replication . this finding suggests that pge2 that is protective in mtb infection has an opposing role in influenza virus infection and raises a note of caution regarding lipid mediator - directed hdt against mtb . conversely , it is equally important to consider whether inhibition of pge2 as an hdt against influenza virus infection may lead to a burst in type i ifn and be detrimental if a co - infection with mtb is present . despite these caveats , the exciting findings on how the balance of lipid mediators dictates the ability of macrophages to control mtb growth will open up novel pathways for developing hdts . targeted delivery of the hdts

keratoconjunctivitis sicca ( kcs ) , or dry eye , is an eye disease characterized by dryness of the cornea and conjunctiva.1,2 dry eye is usually a chronic disease , affecting more than 10% of the population worldwide . in particular , it is more common among women and people > 65 years old.2,3 dry eye is a multifactorial disease and is accompanied by increased osmolarity of the tear film and inflammation of ocular surface.1 symptoms of dry eye vary among patients , and most commonly they include itching , grittiness , burning , sensitivity to bright light , foreign - body sensation , irritation , pain , blurred vision , and contact lens intolerance . in severe cases , dry eye disease can also lead to permanent visual impairment.4,5 clinical signs of dry eye also vary among patients depending on the specific cause of the disease and include decreased tear film stability as measured by tear break - up time ( tbut).5 patients with severe kcs may lose the ability to tear in response to neural stimulation,68 and are prone to sight - threatening corneal infection and ulceration.2 the symptoms of kcs can be managed and complications can be prevented by the use of artificial tears and lubricating gels that restore normal tear film.9,10 in general , artificial tears moisturize the eye surface by simply increasing the water content of the tear film or by preventing tear evaporation . osmoprotection , however , is a new approach to treating dry eye by providing protection against hypertonicity below the corneal surface via hydrating the epithelial surface . evidence suggests that the combination of sodium carboxymethylcellulose ( 0.5% ) and glycerol ( 0.9% ) , optive ( allergan inc . , irvine , ca , usa ) , not only treats the tear film but also offers osmoprotection.11 the effects of optive are long - lasting , benefiting patients with dry eye symptoms . the purpose of the present study was to evaluate efficacy and tolerability of optive in a broad population of patients with kcs in a real life setting . this study was a multicenter , non - interventional , observational , open - label study conducted in germany . eight hundred thirty - five participating ophthalmologists provided anonymous patient data using a standardized data - collection instrument . german law does not require the collection of informed consent from patients in non - interventional studies . all patients included in the analysis were diagnosed with dry eye symptoms ranging from mild to severe . these patients were either nave to treatment with any dry eye medication , or were using only 1 artificial tear product at the time of initial assessment . at the initial assessment , patients received or were switched to optive treatment , depending on if they were nave to treatment or had been using an artificial tear , respectively . patient demographics , baseline characteristics ( tbut and severity of dry eye ) , type of prior treatments , and the reason for changing the kcs treatment were recorded . adverse events and efficacy outcomes were measured and recorded by physicians at the final exam ( 24 weeks after the initial treatment ) . efficacy endpoints were changes in clinical signs and symptoms , local comfort , tbut , and the assessment of satisfaction with optive . local comfort was measured by a 5-point scale ranging from comfort was markedly improved to comfort was worse . the rating was determined both by physician and patient and was described as the optive experience versus previous therapy . tbut was measured using a sterile fluorescein paper diluted with a nonpreserved , balanced salt solution . statistical analyses of changes in tbut from baseline to final exam across 3 disease severity categories were done via t - tests for paired samples . this study included analyses of patients who were using the most widely used artificial tears in germany prior to study entry : systane ( alcon pharma gmbh , freiburg / breisgau , germany ) , hylo - comod ( ursapharm arzneimittel gmbh , saarbrucken , germany ) , lacophtal ( dr winzer pharma gmbh , berlin , germany ) . the major active ingredients in systane , hylo - comod , and lacophtal were hydroxypropylguar , hyaluronic acid ; and povidone , respectively . an additional group included patients who were nave to dry eye treatment ( nave group ) . this study was a multicenter , non - interventional , observational , open - label study conducted in germany . eight hundred thirty - five participating ophthalmologists provided anonymous patient data using a standardized data - collection instrument . german law does not require the collection of informed consent from patients in non - interventional studies . all patients included in the analysis were diagnosed with dry eye symptoms ranging from mild to severe . these patients were either nave to treatment with any dry eye medication , or were using only 1 artificial tear product at the time of initial assessment . at the initial assessment , patients received or were switched to optive treatment , depending on if they were nave to treatment or had been using an artificial tear , respectively . patient demographics , baseline characteristics ( tbut and severity of dry eye ) , type of prior treatments , and the reason for changing the kcs treatment were recorded . adverse events and efficacy outcomes were measured and recorded by physicians at the final exam ( 24 weeks after the initial treatment ) . efficacy endpoints were changes in clinical signs and symptoms , local comfort , tbut , and the assessment of satisfaction with optive . local comfort was measured by a 5-point scale ranging from comfort was markedly improved to comfort was worse . the rating was determined both by physician and patient and was described as the optive experience versus previous therapy . tbut was measured using a sterile fluorescein paper diluted with a nonpreserved , balanced salt solution . statistical analyses of changes in tbut from baseline to final exam across 3 disease severity categories were done via t - tests for paired samples . this study included analyses of patients who were using the most widely used artificial tears in germany prior to study entry : systane ( alcon pharma gmbh , freiburg / breisgau , germany ) , hylo - comod ( ursapharm arzneimittel gmbh , saarbrucken , germany ) , lacophtal ( dr winzer pharma gmbh , berlin , germany ) . the major active ingredients in systane , hylo - comod , and lacophtal were hydroxypropylguar , hyaluronic acid ; and povidone , respectively . an additional group included patients who were nave to dry eye treatment ( nave group ) . of the initial dataset collected ( n = 14,012 patients with dry eye ) 5,277 patients fulfilled the selection criteria . as shown in table 1 , the majority of the patients were female and over 60 years old . the lack of efficacy of the prior treatments or the expectancy for a higher efficacy with the new treatment in relieving dry eye symptoms was the most commonly reported reason for switching to optive ( 68.6% ) . the severity of kcs was moderate in 3,162 ( 59.9% ) patients ( table 1 ) . prior to switching to optive , patients with kcs used a variety of other artificial tear products ( n = 66 ) or were nave to dry eye treatments . among the most common treatment choices were systane ( n = 396 ; 7.5% ) , hylo - comod ( n = 367 ; 7.0% ) , and lacophtal ( n = 305 ; 5.8% ) . four and a half percent of patients ( n = 237 ) were nave to treatment . n = 3972 ) used 1 of the other 63 different artificial tears prior to study entry . at baseline , disease severity varied in patients using different artificial tear products prior to study entry . in the hylo - comod group , severe dry eye was more frequent ( 29.2% ) , and mild dry eye was less frequent ( 12.8% ) compared with all the other treatment groups , including the nave group . on the other hand , in the lacophtal treated group , mild dry eye was more frequent ( 25.9% ) and severe dry eye was less frequent ( 12.5% ) compared with other groups that used artificial tears prior to study enrollment . in the nave group , mild dry eye was much more frequent ( 42.6% ) and severe dry eye was much less frequent ( 8.0% ) than in any other groups . at the final visit , 3963 ( 75.1% ) patients of 5,277 patients reported improvement in clinical signs and symptoms ( figure 1 ) . in particular , 68.9% ( n = 253 ) , 78.5% ( n = 311 ) , 83.0% ( n = 253 ) , 75.1% ( n = 2,983 ) , and 69.6% ( n = 165 ) of patients showed improvement in the hylo - comod , systane , lacophtal , other , and nave group , respectively . only a few patients ( n = 136 , 2.6% ) reported worsening of the symptoms . figure 2 illustrates that the majority of all 5,277 patients ( 85.4% ) rated local comfort as an improvement after using optive for 2 to 4 weeks . specifically , 80.1% ( n = 294 ) , 84.6% ( n = 335 ) , 90.8% ( n = 277 ) , 85.1% ( n = 3,381 ) , and 92.0% ( n = 218 ) of patients reported local comfort improvement in the hylo - comod , systane , lacophtal , other , and nave group , respectively . figure 3 ( top panel ) illustrates the tbut across 7 time categories for all patients . at baseline , the highest number of patients had a tbut of 6 to < 8 seconds , which increased to > 10 to < 12 seconds after 2 to 4 weeks of using optive . in particular , as shown in figure 3 ( bottom panel ) , tbut significantly increased from baseline to final assessment in all 3 disease severity categories ( p < 0.001 ) . table 2 summarizes the change in the mean tbut from baseline to final assessment across all prior treatments used before study entry . within 2 to 4 weeks , tbut increased in 29.9% of 4,338 patients , indicating a robust improvement in the clinical sign of kcs . as shown in figure 4 , most patients ( n = 4,797 , 90.9% ) were somewhat to very satisfied with using optive . in detail , 86.9% ( n = 319 ) , 90.4% ( n = 358 ) , 94.1% ( n = 287 ) , 90.7% ( n = 3,603 ) , and 97.0% ( n = 230 ) of patients were satisfied with the new therapy in the hylo - comod , systane , lacophtal , other , and nave groups , respectively . of 5,277 patients , 107 ( 2.0% ) reported adverse events ( aes ) , none of which was considered serious . only 20 of the aes ( 0.4% of all patients ) were identified as treatment - related . the most commonly reported adverse events were eye irritation ( n = 53 ; 1% ) , ocular hyperaemia ( n = 12 ; 0.2% ) , sensation of foreign body ( n = 8 ; 0.2% ) , blurred vision ( n = 7 ; 0.1% ) , and drug intolerance ( n = 6 ; 0.1% ) . of the initial dataset collected ( n = 14,012 patients with dry eye ) 5,277 patients fulfilled the selection criteria . as shown in table 1 , the majority of the patients were female and over 60 years old . the lack of efficacy of the prior treatments or the expectancy for a higher efficacy with the new treatment in relieving dry eye symptoms was the most commonly reported reason for switching to optive ( 68.6% ) . the severity of kcs was moderate in 3,162 ( 59.9% ) patients ( table 1 ) . prior to switching to optive , patients with kcs used a variety of other artificial tear products ( n = 66 ) or were nave to dry eye treatments . among the most common treatment choices were systane ( n = 396 ; 7.5% ) , hylo - comod ( n = 367 ; 7.0% ) , and lacophtal ( n = 305 ; 5.8% ) . four and a half percent of patients ( n = 237 ) were nave to treatment . n = 3972 ) used 1 of the other 63 different artificial tears prior to study entry . at baseline , disease severity varied in patients using different artificial tear products prior to study entry . in the hylo - comod group , severe dry eye was more frequent ( 29.2% ) , and mild dry eye was less frequent ( 12.8% ) compared with all the other treatment groups , including the nave group . on the other hand , in the lacophtal treated group , mild dry eye was more frequent ( 25.9% ) and severe dry eye was less frequent ( 12.5% ) compared with other groups that used artificial tears prior to study enrollment . in the nave group , mild dry eye was much more frequent ( 42.6% ) and severe dry eye was much less frequent ( 8.0% ) than in any other groups . at the final visit , 3963 ( 75.1% ) patients of 5,277 patients reported improvement in clinical signs and symptoms ( figure 1 ) . in particular , 68.9% ( n = 253 ) , 78.5% ( n = 311 ) , 83.0% ( n = 253 ) , 75.1% ( n = 2,983 ) , and 69.6% ( n = 165 ) of patients showed improvement in the hylo - comod , systane , lacophtal , other , and nave group , respectively . only a few patients ( n = 136 , 2.6% ) reported worsening of the symptoms . figure 2 illustrates that the majority of all 5,277 patients ( 85.4% ) rated local comfort as an improvement after using optive for 2 to 4 weeks . specifically , 80.1% ( n = 294 ) , 84.6% ( n = 335 ) , 90.8% ( n = 277 ) , 85.1% ( n = 3,381 ) , and 92.0% ( n = 218 ) of patients reported local comfort improvement in the hylo - comod , systane , lacophtal , other , and nave group , respectively . figure 3 ( top panel ) illustrates the tbut across 7 time categories for all patients . at baseline , the highest number of patients had a tbut of 6 to < 8 seconds , which increased to > 10 to < 12 seconds after 2 to 4 weeks of using optive . in particular , as shown in figure 3 ( bottom panel ) , tbut significantly increased from baseline to final assessment in all 3 disease severity categories ( p < 0.001 ) . table 2 summarizes the change in the mean tbut from baseline to final assessment across all prior treatments used before study entry . within 2 to 4 weeks , tbut increased in 29.9% of 4,338 patients , indicating a robust improvement in the clinical sign of kcs . as shown in figure 4 , most patients ( n = 4,797 , 90.9% ) were somewhat to very satisfied with using optive . in detail , 86.9% ( n = 319 ) , 90.4% ( n = 358 ) , 94.1% ( n = 287 ) , 90.7% ( n = 3,603 ) , and 97.0% ( n = 230 ) of patients were satisfied with the new therapy in the hylo - comod , systane , lacophtal , other , and nave groups , respectively . of 5,277 patients , 107 ( 2.0% ) reported adverse events ( aes ) , none of which was considered serious . only 20 of the aes ( 0.4% of all patients ) were identified as treatment - related . the most commonly reported adverse events were eye irritation ( n = 53 ; 1% ) , ocular hyperaemia ( n = 12 ; 0.2% ) , sensation of foreign body ( n = 8 ; 0.2% ) , blurred vision ( n = 7 ; 0.1% ) , and drug intolerance ( n = 6 ; 0.1% ) . in the present study , therapy with optive greatly improved the signs and symptoms of kcs in the vast majority of patients within 4 weeks . the dual action of optive is likely the reason that optive improved signs and symptoms of kcs in our study . the vast majority of patients were very satisfied or at least somewhat satisfied with the new product , and the local comfort it provided was rated excellent . patients valued the comfort of optive as much better or at least somewhat better compared to their previous dry eye therapy . in accordance with these observations , the clinical sign of kcs ( tbut ) was also improved significantly in all stages of the disease ( mild , moderate , and severe ) . optive contains sodium carboxymethylcellulose and glycerol , which lubricate the tear film at the eye surface and also promote the growth of epithelial cells to provide osmoprotection.11 the tear film may become damaged under hypertonic stress . restoring physiologic osmolarity is an important goal in kcs treatment to avoid serious complication , such as sight - threatening corneal infection.12 a number of different products are available for dry eye treatment , including various polymers and numerous products with diverse viscosities and with or without preservative formulations.13 however , these products are not known to possess osmoprotective properties , and therefore likely provide only lubrication and do not protect epithelial cells against hypertonic stress . the formulations of the artificial tear products play key roles in their efficacy in treating kcs . in particular , in patients who used hylo - comod , which is a preservative - free formulation , severe dry eye was more frequent at baseline compared with other treatment groups . in the lacophtal group , severe dry eye was less frequent at baseline compared to other groups that had prior artificial tear therapy , which , in part , might be due to the low viscosity of this product . the active ingredients in optive are sodium carboxymethylcellulose , which is used in several artificial tears and is an effective lubricant , and glycerol , which is a non - blurring and long - lasting moisturizer . sodium carboxymethylcellulose is an anionic water soluble polymer , which binds to the cell surface and aids in reducing water loss.11 glycerol restores the lost cellular volume14 and protects cells , but does not increase viscosity that could contribute to blur . optive also includes a preservative , purite , which is gentle to the ocular surface and has an excellent safety record.15 the safety and tolerability of purite has been also established in a study of 62 patients with mild to moderate dry eye who were treated with 0.5% carboxymethylcellulose that was preserved with purite ( refresh tears ) 4 to 8 times a day for 4 weeks.16 the characteristics of optive formulation suggest that it would be efficient in restoring physiologic osmolarity of the tear film with minimal side - effects . as anticipated , optive was well tolerated and efficient in this clinical study . given the excellent efficacy and tolerability of optive in the present study , a longer - term treatment with this formulation may result in further improvements . in this observational study , the use of optive was effective and well tolerated in patients with dry eye .

epilepsy has a prevalence of approximately 1% ( sander and shorvon , 1996 ) . over half have focal epilepsy and around one - third are refractory to medical treatment ( semah and ryvlin , 2005 ) . magnetic resonance imaging ( mri ) is the imaging modality of choice to detect possible structural lesions underlying the epilepsy , to assess comorbidities and to evaluate individuals with medically refractory epilepsy for potential surgery . uk guidelines recommend mri in all patients with epilepsy , except those children and young persons with idiopathic generalised epilepsy who respond to drug treatment ( national institute for health and clinical excellence , 2012 ) . identification of a lesion is crucial for the consideration of surgical treatment and is associated with a greater chance of seizure freedom following surgery ( kuzniecky et al . , 1993 ; mosewich et al . , 2000 ) . in hippocampal sclerosis , co - existent abnormalities ( dual pathology ) are common , affecting 15% of adults and up to 67% of children ( bocti et al . , correct recognition of abnormalities influences management and prognosis . despite advances in imaging technology , around 2030% of patients with refractory focal epilepsy have normal mri scans ( mri - negative ) ( duncan , 2010 ) . it is widely held that an increase in field strength from 1.5 t to 3 t is associated with greater sensitivity for the detection of lesions . however , comparisons of imaging in patients with epilepsy at different field strengths are limited to small series of patients being considered for surgery and are not unanimous in their conclusions . in a series of 40 patients undergoing surgical evaluation , 3 t mri identified a lesion in 65% of the patients considered mri negative at 1.5 t ( knake et al . , 2005 ) . in contrast another study of 37 patients undergoing surgical evaluation failed to demonstrate any superiority of 3 t mri over 1.5 t mri ( zijlmans et al . , 2009 ) . other technological developments , in particular changes in the design of head coils and gradients , may contribute but have not been assessed in patients with epilepsy . the present study presents a retrospective review of a large cohort of patients attending a tertiary epilepsy referral centre who had undergone mri at 1.5 t and later at 3 t with improved head coils . the aims were to determine the range of underlying pathologies and to identify that proportion in whom modern scanner technology yielded additional findings and how these affected clinical management . the national hospital for neurology and neurosurgery in conjunction with the epilepsy society at chalfont runs a large tertiary referral epilepsy service with 1300 new referrals for epilepsy and 50 undergoing surgery per year . the epilepsy society mri unit opened in 1995 with a 1.5 t general electric horizon echospeed scanner ( ge medical systems , milwaukee , usa ) employing quadrature coils . this was replaced by a 3 t general electric signa excite hdx scanner ( ge medical systems , milwaukee , usa ) using an 8-channel phased array receive coil in 2004 . a dedicated epilepsy protocol mri acquisition was used throughout ( supplementary tables 1 and 2 ) . during the period 19952010 , two neuroradiologists reported the scans and since november 2010 , a single neuroradiologist has reported the scans . the mri database was queried for the periods may 1995june 2004 ( 16,692 bookings for the 1.5 t scanner ) and july 2004july 2011 ( 8823 bookings for the 3 t scanner ) to identify patients who had undergone cranial mri on the 1.5 t scanner and subsequently on the 3 t scanner . scan reports were reviewed to determine the diagnosis and in the case of multiple scans on one scanner the most recent diagnosis was used . only patients in whom the indication for scanning was epilepsy and with reports available on both scanners were included . the medical notes were reviewed to determine the treating clinician 's classification of the epilepsy ( focal , generalised or unclassified ) . printed films of the 1.5 t scans were reviewed and the most recent 3 t scans were reviewed electronically to identify illustrative cases . the 1.5 t electronic data are no longer available and the printed films represent only a subset of the sequences acquired . this retrospective evaluation of clinically acquired data was considered by the joint research ethics committee of the national hospital for neurology and neurosurgery and institute of neurology to be audit / service evaluation so individual patient consent was not required . the majority suffered focal epilepsy ( 697 , 87% ) , with the remainder either generalised ( 66 , 8% ) or unclassified epilepsies ( 41 , 5% ) . 37% of the mostly recently performed 3 t scans were normal ( 37% ) and 20% had only incidental findings ( table 1 ) . the remainder showed abnormalities ( 13% hippocampal sclerosis with or without other pathology , 8% malformations of cortical development , 4% other ) or previous surgery ( 18% , predominantly temporal ) . a total of 97 ( 12% ) new diagnoses were found with the 3 t scans that were not made on the previous 1.5 t scans ( table 2 ) . whilst many were incidental , they were of direct relevance in 37 patients ( 5% , figs . 13 ) , particularly hippocampal sclerosis , focal cortical dysplasia and dysembryoplastic neuroepithelial tumour ( dnet ) . several patients underwent surgical procedures , including anterior temporal lobe resection , removal of an epileptogenic tuber , gamma knife surgery for hypothalamic hamartoma and insertion of a shunt for hydrocephalus ( table 2 ) . of the 13 patients newly diagnosed with focal cortical dysplasia , 3 underwent surgery , 1 underwent intracranial recording but was unsuitable for resection , 2 declined intracranial recording , 2 are pursuing anti - epileptic drugs currently , 1 was considered unsuitable and 4 are currently undergoing further presurgical investigations . progression of the underlying abnormality ( e.g. atrophy , ischaemia ) was observed in a further 9 patients . a change in diagnosis was made in 18 patients and 91 patients ( 11% ) had undergone surgery , predominantly temporal ( 79 patients ) . the changes in diagnosis were mostly reinterpretation of the imaging data ( 12 patients ) , for example from dnet to glioma . these changes were histologically confirmed in the two patients proceeding to surgery . in the remaining 6 patients a previously reported abnormality magnetic resonance imaging plays a key role in the assessment of patients with epilepsy and in this study a relevant finding was detected in 30% of patients who had not already undergone surgery . this exceeds a previously reported yield for relevant abnormalities of 18% at 3 t mri in focal epilepsy ( craven et al . , 2011 ) significant improvements in imaging technology have occurred over recent years and a number of factors may influence the diagnostic yield . the key finding in this study was that 3 t scans with modern head coils and gradients yielded a relevant new diagnosis in 5% of those patients who had previous 1.5 t scans in the same centre and which had been reported as normal , and a further , smaller proportion had diagnoses clarified that were not due to disease progression between the two scans . high resolution fast sequences at 1.5 t , including 3d volumetric t1-weighted and t2-weighted fast spin echo ( fse ) sequences ( barkovich et al . , 1995 ) and fluid - attenuated inversion recovery ( flair ) imaging improves the detection of relevant pathology ( bergin et al . , 1995 ) so it is important to implement a specialist epilepsy imaging protocol ( recommendations for neuroimaging of patients with epilepsy , 1997 ; guidelines for neuroimaging , 1998 ) . , 1998 ; von oertzen et al . , 2002 ) of patients with focal epilepsy who were mri - negative with conventional sequences at 1.5 t. two major technical developments are the increase in field strength from 1.5 t to 3 t ( alvarez - linera , 2007 ) and the change in coil design from quadrature to phased array coils . the increased signal to noise ratio ( snr ) from the latter can increase diagnostic yield at 1.5 t ( grant et al . 2004 ) and in combination with doubling the field strength is estimated to increase snr by a factor of 68 ( knake et al . , 2005 ) . this large increase in snr allows faster imaging for a given resolution , a higher resolution for a given scan time or a combination of the two ( alvarez - linera , 2007 ) . of particular importance is the ability to acquire thinner slices and our protocol has recently been extended by the addition of 3.5 mm thick axial flair and 3 mm thick coronal fse t2 imaging . diagnostic yield can be further improved by scans being read by expert neuroradiologists working in concert with neurologists with expertise in epilepsy . detection of abnormalities on conventional 1.5 t mri images had a sensitivity of 39% with non - experts compared to 50% in experts ( von oertzen et al . , 2002 ) . however , some abnormalities escape visual detection even by experienced observers and the introduction of computer - based image analysis techniques such as voxel - based flair ( focke et al . , 2008 ) or surface - based morphological analysis for focal cortical dysplasia ( besson et al . , 2008 ; thesen et al . , the increased detection of hippocampal sclerosis at 3 t is likely a combination of improved sensitivity and the development over time of this pathology between the scans , which can be further studied by t2 relaxometry and hippocampal volumetry ( woermann et al . , 1998 ) . however , malformations of cortical development are likely to have been present throughout and the increased detection of focal cortical dysplasia ( fcd ) represents a significant advance which deserves special mention . the correct identification of fcd is challenging . in a retrospective series of pathologically proven fcd , standard imaging fails to identify fcd in up to 80% of patients when the abnormality lies in the depth of a sulcus ( besson et al . , 2008 ) . the improved snr at 3 t improves the detection of focal abnormalities such as fcd through better definition of the grey / white matter junction ( which becomes blurred in fcd ) and a more uniform signal intensity in normal cortex ( in contrast to hyperintense signal within the dysplastic region ) . two studies have compared 1.5 t and 3 t scans in patients with epilepsy and reached contradictory conclusions . in the first a series of 40 patients undergoing presurgical evaluation underwent scans on a siemens 3 t scanner at a specialist centre with phased array coils ( knake et al . , 2005 ) . in comparison to prior standard 1.5 t mri performed and reported elsewhere , lesions were detected in 65% of previously mri negative patients and clinical management was affected in 38% . however , four variables were different field strength , availability of a phased array coil , use of a specialist epilepsy protocol and reporting by an experienced observer . given these numerous differences and the inclusion of only patients being considered for surgery , it is not surprising that there was a higher yield of new diagnoses in previously mri - negative patients . the second study included 37 patients being considered for surgery who were scanned on 1.5 t and 3 t scanners both using phased array coils and reported by the same observers ( zijlmans et al . , 2009 ) . this in conjunction with the selection of only patients considered ineligible for surgery following 1.5 t scans might explain the failure to demonstrate increased yield at 3 t. whilst dysplasia was detected more frequently , hippocampal sclerosis was reported less often . in the context of limited and contradictory previous literature , the present study represents the largest comparison of repeat imaging in patients with epilepsy at a tertiary referral centre over a period of 16 years . it is not possible to disentangle the relative contributions to diagnostic yield of an increase in field strength from the introduction of phased array coils ( used on the 3 t scanner , but not the 1.5 t scanner ) . however , it is not feasible to apply a prospective study to such a large cohort with the previous studies only assessing 37 ( zijlmans et al . , 2009 ) or 40 patients ( knake et al . , 2005 ) the particular strengths of the current study are the size of the cohort , the use of dedicated epilepsy protocols throughout and the scan reporting by experienced neuroradiologists , with the majority of scans at both field strengths being reported by the same two observers . in the current study , the scans were clinically reported with knowledge of the clinical details , rather than blind , and only the scan reports rather than the imaging itself were reviewed for this study . in some cases it may be possible in hindsight to identify a lesion only identified with the 3 t scan on the earlier 1.5 t scan . however , this does not represent clinical practice and the full 1.5 t imaging data from the 1990s are not available for review . this chance is minimised by the use of the same two experienced neuroradiologists for the majority of the reporting period and many of the new diagnoses obtained were those that theoretically benefit from the improved scan technology . finally , some selection bias may have been introduced as those having repeated scans may be more likely to have been previously mri negative and to have epilepsy that had not gone into remission , and thus be biased to those with a higher likelihood of structural pathology than those who remitted and who would thus be less likely to be rescanned . magnetic resonance imaging plays a key role in the assessment of patients with epilepsy and in this study a relevant finding was detected in 30% of patients who had not already undergone surgery . this exceeds a previously reported yield for relevant abnormalities of 18% at 3 t mri in focal epilepsy ( craven et al . , 2011 ) significant improvements in imaging technology have occurred over recent years and a number of factors may influence the diagnostic yield . the key finding in this study was that 3 t scans with modern head coils and gradients yielded a relevant new diagnosis in 5% of those patients who had previous 1.5 t scans in the same centre and which had been reported as normal , and a further , smaller proportion had diagnoses clarified that were not due to disease progression between the two scans . high resolution fast sequences at 1.5 t , including 3d volumetric t1-weighted and t2-weighted fast spin echo ( fse ) sequences ( barkovich et al . , 1995 ) and fluid - attenuated inversion recovery ( flair ) imaging improves the detection of relevant pathology ( bergin et al . , 1995 ) so it is important to implement a specialist epilepsy imaging protocol ( recommendations for neuroimaging of patients with epilepsy , 1997 ; guidelines for neuroimaging , 1998 ) . von oertzen et al . , 2002 ) of patients with focal epilepsy who were mri - negative with conventional sequences at 1.5 t. two major technical developments are the increase in field strength from 1.5 t to 3 t ( alvarez - linera , 2007 ) and the change in coil design from quadrature to phased array coils . the increased signal to noise ratio ( snr ) from the latter can increase diagnostic yield at 1.5 t ( grant et al . 2004 ) and in combination with doubling the field strength is estimated to increase snr by a factor of 68 ( knake et al . , 2005 ) . this large increase in snr allows faster imaging for a given resolution , a higher resolution for a given scan time or a combination of the two ( alvarez - linera , 2007 ) . of particular importance is the ability to acquire thinner slices and our protocol has recently been extended by the addition of 3.5 mm thick axial flair and 3 mm thick coronal fse t2 imaging . diagnostic yield can be further improved by scans being read by expert neuroradiologists working in concert with neurologists with expertise in epilepsy . detection of abnormalities on conventional 1.5 t mri images had a sensitivity of 39% with non - experts compared to 50% in experts ( von oertzen et al . , 2002 ) . however , some abnormalities escape visual detection even by experienced observers and the introduction of computer - based image analysis techniques such as voxel - based flair ( focke et al . , 2008 ) or surface - based morphological analysis for focal cortical dysplasia ( besson et al . , 2008 ; thesen et al . , the increased detection of hippocampal sclerosis at 3 t is likely a combination of improved sensitivity and the development over time of this pathology between the scans , which can be further studied by t2 relaxometry and hippocampal volumetry ( woermann et al . , 1998 ) . however , malformations of cortical development are likely to have been present throughout and the increased detection of focal cortical dysplasia ( fcd ) represents a significant advance which deserves special mention . the correct identification of fcd is challenging . in a retrospective series of pathologically proven fcd , the mri was reported as normal in 34% ( tassi et al . , 2002 ) . standard imaging fails to identify fcd in up to 80% of patients when the abnormality lies in the depth of a sulcus ( besson et al . , 2008 ) . the improved snr at 3 t improves the detection of focal abnormalities such as fcd through better definition of the grey / white matter junction ( which becomes blurred in fcd ) and a more uniform signal intensity in normal cortex ( in contrast to hyperintense signal within the dysplastic region ) . two studies have compared 1.5 t and 3 t scans in patients with epilepsy and reached contradictory conclusions . in the first a series of 40 patients undergoing presurgical evaluation underwent scans on a siemens 3 t scanner at a specialist centre with phased array coils ( knake et al . , 2005 ) . in comparison to prior standard 1.5 t mri performed and reported elsewhere , lesions were detected in 65% of previously mri negative patients and clinical management was affected in 38% . however , four variables were different field strength , availability of a phased array coil , use of a specialist epilepsy protocol and reporting by an experienced observer . given these numerous differences and the inclusion of only patients being considered for surgery , it is not surprising that there was a higher yield of new diagnoses in previously mri - negative patients . the second study included 37 patients being considered for surgery who were scanned on 1.5 t and 3 t scanners both using phased array coils and reported by the same observers ( zijlmans et al . , 2009 ) . this in conjunction with the selection of only patients considered ineligible for surgery following 1.5 t scans might explain the failure to demonstrate increased yield at 3 t. whilst dysplasia was detected more frequently , hippocampal sclerosis was reported less often . in the context of limited and contradictory previous literature , the present study represents the largest comparison of repeat imaging in patients with epilepsy at a tertiary referral centre over a period of 16 years . it is not possible to disentangle the relative contributions to diagnostic yield of an increase in field strength from the introduction of phased array coils ( used on the 3 t scanner , but not the 1.5 t scanner ) . however , it is not feasible to apply a prospective study to such a large cohort with the previous studies only assessing 37 ( zijlmans et al . , 2009 ) or 40 patients ( knake et al . , 2005 ) the particular strengths of the current study are the size of the cohort , the use of dedicated epilepsy protocols throughout and the scan reporting by experienced neuroradiologists , with the majority of scans at both field strengths being reported by the same two observers . in the current study , the scans were clinically reported with knowledge of the clinical details , rather than blind , and only the scan reports rather than the imaging itself were reviewed for this study . in some cases it may be possible in hindsight to identify a lesion only identified with the 3 t scan on the earlier 1.5 t scan . however , this does not represent clinical practice and the full 1.5 t imaging data from the 1990s are not available for review . this chance is minimised by the use of the same two experienced neuroradiologists for the majority of the reporting period and many of the new diagnoses obtained were those that theoretically benefit from the improved scan technology . finally , some selection bias may have been introduced as those having repeated scans may be more likely to have been previously mri negative and to have epilepsy that had not gone into remission , and thus be biased to those with a higher likelihood of structural pathology than those who remitted and who would thus be less likely to be rescanned . in conclusion , this is the largest study of a cohort of patients with epilepsy undergoing mri at 1.5 t and subsequently at 3 t with improved head coils with both employing a specialist epilepsy protocol at a tertiary referral centre . 3 t mri identified a relevant abnormality in around 30% patients who had not already undergone surgery . in 5% of patients , a relevant diagnosis was reached with the 3 t scan which had not been reported on the previous 1.5 t scan . the new information contributed to clinical management , particularly in the detection of focal cortical dysplasia . the yield of diagnostic imaging is dependent upon many factors , including high resolution sequences , magnetic field strength , phased array coils , gradient performance , a specialist epilepsy protocol and dedicated neuroradiologists and may be increased further by the introduction of computer - based analysis techniques . the point of practical importance is to rescan individuals with previously unclear mri using optimal technology . john duncan has served as a consultant for ge healthcare on the development of pet tracers .

the prevalence of autism has been increasing in the us over the last few decades , but its causes are not well understood . several ecological analyses of autism have appeared in the last five years , reporting correlations with reported mercury emissions [ 2 , 3 ] , with precipitation , and with race . these study findings have been cited extensively by other peer - reviewed publications and widely circulated by the popular press [ 7 , 8 ] . although it is well known that ecological studies may be affected by aggregation bias when used to estimate individual level effects , ecological studies of medical conditions such as autism are typically susceptible to an additional hazard that is seldom recognized : censoring of low disease counts . autism is not a reportable disease , but states do track and report the total number of students receiving special education services for conditions such as autism to the us department of education under the 1990 individuals with disabilities education act ( p.l . statewide totals are routinely published by the us department of education , but prevalence by school district or county must be obtained from each state 's department of education . countywide environmental releases of mercury are also available publicly through the us environmental protection agency toxic release inventory , through a mandatory reporting system that includes any facility emitting 10 lbs . or more of mercury per year . educators have legal and moral obligations to protect the privacy of students and their educational records . some states impose limits upon enumeration of small numbers of students receiving particular special education services in order to ensure privacy for these students . for example , the texas educational agency provided us with exact autism counts for districts with 5 or more students with autism and for districts with no students with autism , but it used a special code ( 999 ) for districts with 14 students with autism . 35% of the districts fell into the latter category requiring the special code ( table 1 ) . similarly , waldman et al . reported that oregon censored the autism counts ( i.e. , substituted a special code for the actual counts ) for counties with fewer than 10 students with autism at any one year of age . these autism counts likelihood - based statistical methods for administratively censored outcomes have long been available for classical parametric fixed effects models and are widely implemented for well - known distributions in major statistical packages . however , previous ecological studies of autism have cited statistical methods such as mixed effects poisson models and ordinary least - squares regression that are not designed to handle censored observations [ 25 ] . the absence of any explicit description of methods for handling censored observations in these previous publications suggests that censored values were deleted or substituted with fixed values such as zeros . indeed , one might substitute zeros after incorrectly assuming that a missing autism count indicates that a school district had no children with autism to report . palmer et al . [ 2 , 3 ] fit random effects poisson models associating 2000 - 2001 school district autism counts and 2001 countywide total mercury releases , adjusting for percent white , district wealth , percent economically disadvantaged , and urbanicity , but they did not discuss censoring . we were able to compile these variables for 1029 of the school districts , and previously reanalyzed the data for these districts in various years using the model described by palmer et al . . we previously reported similar results to palmer 's for 2000 - 2001 when using the zero substitution approach , with rrs of 1.29 to 2.03 per 1000 lbs of total mercury releases , depending on the estimation algorithm , versus palmer 's originally reported rr of 1.61 . however , when we substituted larger fixed values ( threes ) for the censored counts , our rr estimates decreased to values near unity . statistically significant associations between mercury emissions and autism did not persist in later years . substantial differences between the results of ad hoc fixed value substitution methods indicate that the method of handling censored counts is critical in this setting , motivating the following analysis . in general , the contribution of each interval censored observation to the likelihood is pr ( l < yi u ) , where yi is a random variable describing the count in district i and ( l , u ] is the interval in which the censored value lies . the following statements comprise a censored poisson random effects model for the ecological association between mercury releases and autism using the texas data : ( 1 ) pr(yi = yi ) = eiiyiyi ! for yi=0 or yi>4pr(0<yi4 ) = yi=14eiiyiyi ! , for censored counts ( yi coded as 999),ln ( i)=ln ( districtsizei)++0j+1mercuryi + 2whitei+3taxbasei+4econi+5urbani + 6suburbani+7other urbanicityi , 0j~n(0,02 ) , where yi is the observed autism count for school district i , i is the poisson rate parameter modeled as an exponentiated linear combination of seven covariates and an offset for the log of the number of students in the district , and 0j is a random effect of county j ( many counties have more than one school district ) . , 0 , and the s are unknown parameters , and 1 is the parameter of interest : the log rr for autism associated with each 1000 lbs increase in total mercury emissions . n(0 , 0 ) denotes a normal distribution with mean 0 and variance 0 . although censored poisson fixed effects regression is widely implemented by major statistical software packages , interval - censored poisson random effects regression is a newer area of statistical research . moreover , frequentist poisson random effects regression algorithms rely on differing likelihood approximations that are known to produce disparate estimates for these particular data and for rare outcomes in general . we use a bayesian approach for parameter estimation instead , requiring the specification of prior distributions for the unknown parameters . we chose relatively uninformative priors ( 2)~n( 6 , 10),k~n(0,100 ) for all k,02~uniform ( 0 , 50 ) . we implemented the model using winbugs 1.4.3 , a generalized environment for conducting bayesian analyses using markov chain monte carlo methods , as well as the r2winbugs library in r. winbugs code is provided ( see algorithm 1 ) . at least , 100,000 iterations were used in each analysis for each of 3 chains ; 50% of the iterations were discarded for burn - in . convergence of the sampling chains was assessed using plots and the r - hat statistic , which was less than 1.01 for each posterior rr for mercury and less than 1.05 for all other parameters . for comparison purposes , naive approaches relying on either exclusion of censored observations or fixed - value substitution for censored observations were conducted using mixed effects poisson regression in r as described previously but with a more recent version of the lmer library ( version 0.999375 - 31 ) . we also repeated all analyses for air mercury emissions in place of total mercury emissions . air emissions are arguably a better proxy for concurrent exposure than total mercury emissions for reasons described in our previous analysis and later in this paper . the resulting bayesian posterior mean rr estimate per 1000 lbs of reported total mercury releases using the 2000 - 2001 data is 1.18 ( 95% confidence interval ( ci ) : 1.07 , 1.32 ) . in contrast , the nave zero - substitution method yields an rr estimate of 2.02 ( 95% ci : 1.96 , 2.09 ) per 1000 lbs . table 2 shows the complete set of parameter estimates for the bayesian censored likelihood analysis and for the nave substitution approach using various fixed values and for the exclusion approach . although some of the nave approaches produce reasonable central estimates , they produce artificially narrow confidence intervals . for air mercury emissions , the posterior mean rr estimate is 1.42 ( 95% ci : 1.09 , 1.78 ) per 1000 lbs . of mercury . in contrast , the zero - substitution approach yields an rr estimate of 4.44 ( 95% ci : 4.16 , 4.74 ) per 1000 lbs . of mercury . we checked for sensitivity to the choice of the prior by repeating the air mercury analysis with the prior for 1 centered on log(4.44 ) . this analysis yielded nearly identical results ( rr = 1.42 ; 95% ci : 1.10 , 1.80 ) to the primary analysis , suggesting that our choice of prior distribution for the effect of mercury is not overly influential . our results using the full censored likelihood indicate that the ecological association between total mercury emissions and 2000 - 2001 autism in texas school districts may be much smaller than previously reported . published associations between precipitation and autism are also dependent on censored data and could be affected by similar biases . rr estimates for the ecological association using the censored likelihood were fairly similar to the results using the ad hoc substitution approach when values near the middle of the censoring interval were chosen . however , confidence intervals were ( appropriately ) wider for the bayesian likelihood - based approach than for the substitution approaches . exclusion of all censored values produced similar central rr estimates to the bayesian approach but with artificially narrow confidence intervals . however , all results should also be viewed as ecologic effect estimates that may not be representative of individual level effects due to aggregation bias . another consequence of the group - level analysis is that uncertainty regarding individual - level associations between mercury exposure and autism in texas is much greater than suggested by the group - level confidence intervals reported here and in previous publications [ 3 , 5 , 12 ] . these results were surprising to us given that the cutoff for censoring was so low ( 5 students ) , but they may be explained by an apparent association between mercury emissions and the presence of censoring , whereby those districts with small autism counts tended to have lower mercury emissions . this effect may be better understood through inspection of figure 1 , which shows the data and model fit using a variety of fixed value substitution approaches . because many of the censored values occur in counties with low mercury emissions , the overall intercept from the mixed effects poisson regression is highly sensitive to the choice of fixed value substituted for the censored observations . in contrast , there are relatively few censored observations in counties with higher mercury releases , leading to a more stable position of the right - hand side of the prediction curve . the result appears to be a forcing of the slope ( the log relative risk ) to higher values when the intercept is lower . although both total mercury releases and air mercury emissions are poor surrogates for individual mercury exposures , air mercury emissions may be a better proxy for concurrent exposures than total mercury releases . total mercury releases include landfill disposals and other releases that may not lead to widespread or immediate public exposure , especially if compliant with the 1976 resource conservation recovery act ( p.l . in contrast , air mercury emissions are more widely and rapidly dispersed with wind and rainfall and are , therefore , of the two , more plausible sources of human exposure in the short term . surface water mercury emissions may also be quickly dispersed , but these emissions were relatively small in texas in 2001 : only 25% of texas counties reported any surface water mercury emissions , and the largest annual release was 16 lbs . air mercury releases occurred in much larger quantities , with reported releases in most texas counties , a mean annual release of 288 lbs . , and a maximum annual release of 1579 lbs . although these analyses are limited by the ecological nature of the data and the crude measure of exposure , we believe that the air mercury emissions are the more relevant measure within these limitations . researchers considering similar analyses should also consider time - lagged comparisons of autism counts with emissions / exposure estimates from previous years to allow enough time for environmental transport , autism development , and diagnosis to have occurred . the three substitution and exclusion approaches happen to provide a rough approximation to the central rr estimate for these particular data , but likelihood - based approaches for handling censored data produce more realistic confidence intervals and have a stronger theoretical basis . all of the previous and current approaches , however , depend on common model assumptions such as poisson distributed counts , log linearity of the predictors , and adequate control of confounding . the third assumption is of most concern in the present analysis , given the limited understanding of autism risk factors and a lack of any individual - level data on mercury exposure or confounders . ecological studies of autism are not the only studies where censored disease counts are a threat to validity . all special education category counts reported by texas are administratively censored , and the same practice appears to be followed by some other states . moreover , efforts to protect privacy under the health insurance portability and accountability act of 1996 ( p.l.104191 ) may lead to administrative censoring of medical surveillance data when counts are extremely low . with the widespread computerization of large data sets , ecological analyses are becoming ever easier to conduct and will likely appear more frequently in the scientific literature despite their limitations . censoring also arises in chemical concentration measurement , another common issue in environmental health , through reporting of low concentrations as below the limit of detection if censored values are excluded , calculated means will generally be biased upwards in this setting . environmental health researchers have a long tradition of substituting zeros , lod/2 , lod/2 , or lod for these censored values , treating the substituted values as if they were actually observed . the substitution approach is easy to implement but is inferior to formal likelihood - based censored data analysis in that it may also produce biased estimates and always fails to capture the uncertainty associated with measurements below the lod , such as in calculating confidence intervals . however , the adverse effects of substitution or exclusion may be negligible when both of the following conditions are met : ( 1 ) few samples are below the cutoff for censoring and ( 2 ) the cutoff for censoring is small relative to most of the measurements . our analyses indicate that previously reported ecological associations between mercury and autism in texas are likely to have been overestimated due to inadequate statistical analysis of censored autism counts . researchers should be aware of the issue of administrative censoring of disease counts and how ad hoc substitution methods can introduce bias and underestimate uncertainty in effect estimates . bayesian methods offer a potential solution to the problem , do not rely on likelihood approximations , and are not difficult to implement .

this research is a case study on the medical mycology and parasitology course at the school of medicine of shiraz university of medical sciences ( sums ) . the medical mycology and parasitology course is a sophomore - level course on the pathogenesis and treatment of mycotic and parasitic infections for medical students . the class included 106 medical students who attended two 90-min lectures and one 2-hours laboratory session each week for 25 weeks . the lectures were presented using powerpoint and included some case studies . to encourage active learning , the students were informed that they may be randomly called on to answer questions . moreover , the students were free to ask questions during the lectures . the course was presented in a large - sized lecture hall . as shown in the architectural plan of the hall ( fig . 1 ) , a 5 m wide screen was placed on the stage in the front of the class 10 m from the first row . there were 14 columns and 15 rows of seats arranged on a gentle slope and subdivided by aisles into six areas , as shown in fig . the position of each seat was coded in two dimensions : x ( horizontal axis ) and y ( vertical axis ) . x in each seat corresponded to the position of that seat in a row ( or number of the column ) and y in each seat corresponded to the position of that seat in a column ( or the number of the row ) y in each seat corresponded to the position of that seat in a column ( or the number of the row ) . as the distance of the seats in a column from each other is about 63.5 cm , the distance of each seat from the lecturer and the board can be estimated by its corresponding y. the architectural plan of the classroom . random sampling was performed and each student 's position was recorded using digital photos taken during 15 of the 26 sessions of the course . following identification of each student in the photos , the positions were coded by superposing ( placing ) the positions within the architectural plan . the students were told that the photographs were used to check their attendance throughout the course . to determine students preferred seating position in medical school courses , students were asked to record their preferred seating position by marking it on an architectural plan of the classroom distributed during the last session . first , as described by mccroske and mcvetta ( 7 ) , we divided the classroom into three student - teacher interactional zones : high , medium , and low . 1 , the students sitting within the triangle in the front of the class next to the teacher had a high interactional level ( h ) . the remaining students sitting in the front half of the classroom had a medium interactional level ( m ) . the students sitting at the back of the classroom had the lowest interaction level ( l ) . second , since previous studies ( 1 , 2 , 5 ) showed that the distance from the lecturer influenced students educational achievement , the classroom was divided in the y orientation into three areas named i , ii , and iii . third , the seats were divided into two groups based on the students access to the aisles : direct access ( were shown in bold ) and indirect access . the exams were composed of multiple choice questions , short essays , and true / false questions . only the multiple choice questions were used for the both mid - term and final exams and the scores of each of them were calculated out of 20 . reliability of the tests was measured by the split - half and kuder richardson methods and indicated respectively a reliability of 0.81 and 0.84 for the final exam . the final exam has been validated by measuring the correlation coefficient between midterm and final exams ( r = 0.77 , p = 0.01 ) . the students presence in the classroom was recorded by digital camera and students were divided into two groups based on the number of absences ( having more than 10 absence sessions or not ) . the study was approved by the institutional review board and ethical committee of shiraz university of medical sciences . all the students willingly signed an informed consent that was attached to the distributed architectural plan of the classroom . in addition , in the last session , the students were informed about the details of the study and gave permission to issue the results . the mantel - haenszel chi - square test was used to test for associations between categorical variables in the presence of confounding factors . one - way analysis of variance ( anova ) , the independent sample t - test , and the nonparametric mann - whitney u test were applied to test for differences between group . additionally , the chi - square test was used to test for differences between categorical variables . for the statistical analysis , the mantel - haenszel chi - square test was used to test for associations between categorical variables in the presence of confounding factors . one - way analysis of variance ( anova ) , the independent sample t - test , and the nonparametric mann - whitney u test were applied to test for differences between group . additionally , the chi - square test was used to test for differences between categorical variables . for the statistical analysis , the students mean seating positions were classified in three manners as described in the materials and methods section . under the interactional zones classification , 27 ( 25.5% ) students had high interactions with the teacher , 60 ( 56.6% ) had medium interactions , and 19 ( 17.9 ) had low interactions . based on the area classification , 39 ( 36.8% ) students sat in area i , 49 ( 46.2% ) sat in area ii , and 18 ( 17% ) sat in area iii . under the classification based on student access to the aisles , 48 ( 45.3% ) of the 106 students had direct access and 58 ( 54.7% ) had indirect access to the aisles . in addition , the students seat position changes were measured by calculating the standard deviations from their mean positions in the x ( sd - x ) and y ( sd - y ) orientations . of the studied students , 56 ( 54.9% ) had an sd - x2 , 24 ( 23.5% ) had 2<sd - x3 , and 22 ( 21.6% ) had an sd - x>3 . in addition , the majority of the students ( 71 students , 69.6% ) had an sd - y2 , 18 ( 17.7% ) had 2<sd - y3 , and 13 ( 12.7% ) had an sd - y>3 . there was a significant correlation between sd - x and sd - y ( r=0.31 , p=0.001 ) . the students preferred positions were determined from the questionnaire , and there was a significant correlation between the students seating positions and their preferred positions in both the x ( r=0.62 , p<0.001 ) and y ( r=0.72 , p<0.001 ) orientations as shown in fig . the students preferred seating positions and mean seating positions in the x ( a ) and y ( b ) orientations . the seating distribution of the female and male students in the three classifications is shown in table 1 . statistically , the proportion of females in areas i and ii was significantly higher than the number of males . however , in area iii , the proportion of males was significantly higher than the proportion of females ( odds ratio=8.3 , p<0.01 ) . moreover , independent sample t - tests showed that there were no significant differences in sd - y and sd - x between males and females . by focusing on the 10% of the students with the highest level of changes in seat position ( sd - y5 ) , there were significant differences between the sexes ( eight males vs. two females , p=0.041 ) . distribution of female and male students in the three seating classifications the students educational performance was assayed by their final exams scores . our results showed that there were statistically significant correlations between the final ( r=0.2 , p=0.031 ) exam scores and the students seating positions in the y orientation ( fig . 3a ) . of the nine students with final exam grades of a ( grades 17 ) , six students ( 66.6% ) were seated in the h zone and three were seated in the m zone . none of the a - level students were seated in the l zone . as shown in fig . 3b , a statistically significant correlation was found between the students final exam scores and sd - y ( r=0.29 , p=0.003 ) . of the students , 97 ( 90.7% ) had a sd - y4 , and interestingly all nine students with final exam grades of a ( scores17 ) had a sd - y2 . moreover , the students with a sd - y5 had average final exam scores of 12.20.9 , while the other students had average final exam scores of 13.92.2 . distribution of the students based on their final exam scores and either their mean seating positions ( a ) or the standard deviations of their seating positions ( b ) in the y orientation . 4a , there was a significant positive correlation between the number of absences and the distance from the board ( r=0.41 , p<0.001 ) . in other words , the number of absences increased with increasing distance from the board . there were significant negative associations between the number of absences and the students final exam scores ( r=0.36 , p<0.001 ) as shown in fig . although there was no significant correlation between the number of absences and sex , when we divided the students into two groups based on the number of absences , there were significant differences between the sexes ( p<0.01 ) . of the 21 ( 20.4% ) students who were absent from more than 10 class sessions , 16 ( 76.2% ) were male and 5 ( 23.8% ) were female . distribution of the students based on their number of absences and either their mean seating positions in the y orientation ( a ) or their final exam scores ( b ) . traditional classrooms are the most common type of educational setting in universities , especially for medical students ( 13 ) . in this study , we examined the mutual interactions among the chosen seating of medical students , exam scores , and the students attendance . we observed that only five ( 4.6% ) of the students sat in the same seat , all of whom , interestingly , had no movement in the y orientation during the semester . we compared the students educational achievements and attendance based on three types of spatial classification : distance from the lecturer in the y orientation , interactional zones , and access to the aisles . the majority of the students ( n=88 , 82.2% ) chose seats in specific areas . the remaining students changed their positions frequently in both the x and y orientations ( sd - x>3 , sd - y>3 ) . previous studies distributed the students in the classroom by assigning their positions randomly or by assigning the students their favorite seating positions . in our study , students had the opportunity to choose their seat since the number of seats was nearly double the number of students . moreover , the students actual seating positions and their preferred seats were correlated , indicating that the association between seating position and educational performance was not a random event as the students intentionally chose the places where they sat . a previous report by cinar ( 13 ) showed that the distance to the stage is negatively associated with feeling secure in large spaces . in the present study , the number of males in area iii was five times greater than the number of females . several authors ( 1 , 2 , 4 , 5 ) have shown that there is an association between better academic performance and the option of sitting close to the board , while other researchers did not observe this correlation even when the students sat a considerable distance away from the board in very large classrooms ( 11 , 12 ) . although the data presented in this report demonstrated no significant correlation between the students exam performance and their seating in different areas ( i , ii and iii ) of the classroom , a significant association was found between the students performance and seating positions in the high and medium interactional zones . the differences between the results of this study on student performance in different interactional zones or area classifications and previous reports ( 1 , 2 , 4 , 5 ) may be due to differences in the types and sizes of the classrooms . in the present study , the comparison of area i with zone i showed that those students seated in the corners of the area i did not have a high level of interaction with the lecturer despite their close proximity to the stage . moreover , of all the a - level students , almost two thirds were seated in the h zone , and the rest were in the m zone , which suggests that eye - contact and interaction with the lecturer contributed to better performance . these data supported the previous hypotheses ( 10 , 13 , 14 ) that proximity to the lecturer enhances student motivation and improves their scholastic performance . another possible explanation for these results was provided by a recent study , which noted the possible relationship between seat location and test scores was mainly due to the motivation of the students who sat in the front of the class rather than their seat position ( 11 ) . in large classrooms with assigned seating , the researchers observed no effect or a negative correlation between the seat location and exam scores . meanwhile , when the students were free to choose their seats , those in the front received higher grades than those in the back , suggesting that more motivated and engaged students chose seat nearer to the lecturer ( 11 ) . on the other hand , we observed a negative significant association between student performance and changes in seat location , with a - level students rarely changing their seating locations ( sd - y2 ) . likewise , the students who changed their seating locations frequently received significantly lower grades than the rest of the class . however , another variable like motivation may exist that affects both seat location and educational performance . these students might have lower motivation than the fixed seat students , which may in turn affect their educational performance . typically in classes , those who are frequently late also perform less well than those who arrive on time . tardy students also have to take whatever empty seats are left and easy to get to , rather than the seats they would prefer if they had more choice . our results demonstrate that the students number of absences increased with increasing distance from the board . as in previous studies ( 4 , 10 , 15 ) although no significant correlation was found between the number of absences and sex , when we divided the students into two groups based on the number of absences , we observed there were significantly more males in the group of students that was absent from more than 10 sessions . on the other hand , the low number of students attending the educational classes might be natural consequence of modern educational techniques used . in some medical schools such as ours , all the lectures are presently recorded , typed and accessible for all students and these together with the online atlases and slides , has resulted in the students feeling no necessity to attend the class physically . by monitoring the students participation through taking unannounced quizzes , we are making an attempt to obligate the students to participate in the class . ( 16 ) reported that there was a relationship between educational achievement in sophomore students and sex . we conclude that two factors may impact educational achievement : student seating in the high interactional zone and minimal changes in seating location . our results showed that the students in the high interactional zone had higher educational performance and attendance than the rest of the class . this observation is consistent with the view that students in the front of the classroom are likely more motivated and interact with the lecturer more than their classmates . these results suggest that engaging students at the back of the class ( area iii ) or persuading them to sit closer might positively influence their academic performance and participation . however , further studies in large size lecture halls is still needed to verify if assigning poorer performing students and those who change their seat frequently to the seats , would improve their performances and participation . the authors have not received any funding or benefits from industry or elsewhere to conduct this study .

constructs were cloned from full length mouse jmy4 , fly spire2 , and rat n - wasp using standard techniques . jmy fragments were expressed as gst - fusions in e. coli and purified using a combination of glutathione and cation chromatography . except for individual wh2 domains ( supplementary fig . s2 ) , the gst was removed to prevent dimerization of the recombinant protein , which results in a marked increase in the rate of both intrinsic nucleation and activation of arp2/3 ( data not shown ) . to improve reproducibility of fluorimetry reactions jmy concentrations were calculated using predicted molar extinction coefficients for jmy peptides that contained tryptophan residues ( protparam ) , or by quantitative sds - page with sypro - red staining ( invitrogen ) . actin was purified from acanthamoeba castellani as described21 , labelled with pyrene iodoacetamide as described22 , and stored on ice . for all assays , arp2/3 was thawed daily and diluted with 1 mg / ml bsa in buffer a ( 0.2 mm atp , 0.5 mm tcep , 0.1 mm cacl2 , 0.02% w / v sodium azide , 2 mm tris , ph 8.0 at 4c ) . actin polymerization assays were performed in 1x kmei ( 50 mm kcl , 1 mm mgcl2 , 1 mm egta , 10 mm imidazole , ph 7.0 ) . ca - actin was converted into mg - actin by incubation of actin in me ( 50 mm mgcl2 , 0.2 mm egta ) for two minutes prior to adding 10x kmei and test components . pyrene fluorescence was measured with an iss pci / k2 fluorimeter . unless otherwise noted , polymerization reactions contained 2 m actin ( 5% pyrene labelled ) , 2.5 nm arp2/3 and 167 nm jmy or n - wasp . jmy proteins were diluted with 10 mg / ml bsa in buffer a to prevent loss of activity . to normalize fluorimetry data , we subtracted the offset from zero then divided by the plateau value of actin alone , so as to not mask the effects of sequestration by jmy . for half - time calculations , reactions were normalized by dividing by their own plateau and solving for time at half - maximal fluorescence ( 0.5 a.u . ) . to visualize actin filaments we polymerized 2 m actin under the same conditions used for fluorimetry , then arrested reactions at the time indicated with alexa fluor 488 phalloidin and latrunculin b. this technique preserves the ratio of monomeric to filamentous actin at the moment of quenching24 . to keep the concentration of f - actin constant we arrested reactions at their individual ts:1 m 40 s for jmy+arp2/3 ; 6 m 20 s for scar+arp2/3 ; 37 m 0 s for actin alone . filaments were diluted to low nanomolar concentrations and spotted on poly - l - lysine ( sigma ) coverslips , using wide - bore pipette tips to minimize shearing . u2os ( atcc ) and hek 293 cells were cultured in dulbecco s modified eagle s medium supplemented with 10% fbs , 2 mm l - glutamine , non - essential amino acids , and penicillin - streptomycin ( ucsf cell culture facility ) . for transfection , cells were seeded onto glass coverslips and transfected with ha - jmy4 using genejuice ( merck ) , or gfp - jmy and gfp - rnai constructs using lipofectamine ltx ( invitrogen ) , according to manufacturer s protocol . hl-60 cells were cultured as described25 , in rpmi-1640 with 25 mm hepes , 2.0 g / l nahco3 , 10% fbs , 1% antibiotic - antimycotic ( fisher ) , and were passaged every 34 days to a density of 0.2 10 cells / ml . immunofluorescence of hl-60 cells was performed as described25 . briefly , flamed coverslips were treated with 200 g / ml bovine plasma fibronectin ( sigma ) in pbs , washed with pbs , and blocked with 1.8% low endotoxin bsa ( sigma ) in modified hanks buffered saline solution ( mhbss : 150 mm nacl , 4 mm kcl , 1 mm mgcl2 , 10 mm glucose , 20 mm hepes , ph 7.4 at 22 c ) for 5 minutes prior to adhering cells . cells were pelleted and resuspended in bsa / mhbss , and adhered to coverslips for 3060 minutes at 37 c , then washed to remove unbound cells . stimulation or mock stimulation was in bsa / mhbss with 100 nm ( hl-60s ) or 20 nm ( human neutrophils ) fmlp , or dmso ( carrier ) , for 5 minutes at 22 c. cells were fixed in 3.2% formaldehyde in cytoskeletal buffer ( 138 mm kcl , 3 mm mgcl2 , 2 mm egta , 320 mm sucrose , 10 mm hepes , ph 7.2 at 22 c ) . for immunofluorescence of u2os cells , cells were plated on flamed , fibronectin - coated coverslips , and fixed for 30 minutes in 3.2% formaldehyde in pbs . triton in pbs with 1.4 u / ml alexa fluor 568 phalloidin ( invitrogen ) to stabilize and visualize filaments . for jmy immunolocalization , rabbit polyclonal jmy antibody 12895 or anti - ha antibody ha11 ( babco ) was used at a 1:500 dilution . alexa fluor 488-labeled goat - anti - rabbit secondary ( invitrogen ) was used at a 1:500 dilution . dapi ( sigma ) was used at 0.5 g / ml . samples were mounted with fluorescent mounting medium ( dakocytomation ) . epifluorescence and wide field images were acquired on a nikon te300 inverted microscope equipped with a hamamatsu c4742 - 98 cooled ccd camera , with simple pci software ( compix ) , using 100x and 60x 1.4 na plan apo objectives ( nikon ) , or with a 10x 0.6 na phase objective , using micromanager software27 . for quantification of f - actin levels in cells expressing gfp - pwwwca or gfp , micrographs were acquired with an ix micro automated microscope , using identical illumination conditions . cells were identified and outlined using imagej software , background was subtracted , and the average intensity in the red and green channels were measured . we used imagej ( national institutes of health ) and adobe photoshop ( adobe ) for image analysis and contrast adjustment . for wound healing experiments , cells were plated on marked coverslips at the same density , scratch wounded with a micropipette tip , washed to removed detached cells , and then given fresh medium and kept at 37c during image acquisition . stable lines of u2os cells stably expressing gfp - jmy and variants were selected in 500 g / ml g418 for at least 3 weeks , then enriched by facs ( ucsf flow cytometry core ) . sirna was used at a final concentration of 25 nm ( hjmy sirna from santa cruz and control non - targeting 2 from dharmacon ) and transfected into cells with oligofectamine or dharmafect 1 ( both according to manufacturer s protocols ) , and cells were grown for 72 hours prior to experiments . to discriminate between rnaied and non - rnaied cells , pl - ugih plasmid ( atcc ) was modified to contain a human jmy - specific hairpin based on the sequence of jmy - specific sirna-3 by the method of weiner et al . hek 293 cells transiently transfected with this construct were grown for 7 days prior to fixation or immunoblotting . standard methods were used for western blotting , using 1:500 1289 or 1:1000 l-16 ( santa cruz biotechnology ) jmy primary antibodies and 1:5000 hrp - anti - rabbit secondary ( jackson immunoresearch ) . p300-ct primary antibody ( millipore ) was used at 1:500 , goat - anti human gapdh ( santa cruz ) was used at 1:10,000 , and mouse anti - human actin ( sigma ) was used at 1:20,000 . hrp secondaries ( dako ) were used at 1:10,000 , and ecl reagent ( supersignal west pico , pierce ) was used according to the manufacturer s instructions . we used two - tailed unpaired t - tests , assuming unequal variance , to calculate p - values ( microsoft excel ) . constructs were cloned from full length mouse jmy4 , fly spire2 , and rat n - wasp using standard techniques . jmy fragments were expressed as gst - fusions in e. coli and purified using a combination of glutathione and cation chromatography . except for individual wh2 domains ( supplementary fig . s2 ) , the gst was removed to prevent dimerization of the recombinant protein , which results in a marked increase in the rate of both intrinsic nucleation and activation of arp2/3 ( data not shown ) . to improve reproducibility of fluorimetry reactions jmy concentrations were calculated using predicted molar extinction coefficients for jmy peptides that contained tryptophan residues ( protparam ) , or by quantitative sds - page with sypro - red staining ( invitrogen ) . actin was purified from acanthamoeba castellani as described21 , labelled with pyrene iodoacetamide as described22 , and stored on ice . for all assays , arp2/3 was thawed daily and diluted with 1 mg / ml bsa in buffer a ( 0.2 mm atp , 0.5 mm tcep , 0.1 mm cacl2 , 0.02% w / v sodium azide , 2 mm tris , ph 8.0 at 4c ) . actin polymerization assays were performed in 1x kmei ( 50 mm kcl , 1 mm mgcl2 , 1 mm egta , 10 mm imidazole , ph 7.0 ) . ca - actin was converted into mg - actin by incubation of actin in me ( 50 mm mgcl2 , 0.2 mm egta ) for two minutes prior to adding 10x kmei and test components . pyrene fluorescence was measured with an iss pci / k2 fluorimeter . unless otherwise noted , polymerization reactions contained 2 m actin ( 5% pyrene labelled ) , 2.5 nm arp2/3 and 167 nm jmy or n - wasp . jmy proteins were diluted with 10 mg / ml bsa in buffer a to prevent loss of activity . to normalize fluorimetry data , we subtracted the offset from zero then divided by the plateau value of actin alone , so as to not mask the effects of sequestration by jmy . for half - time calculations , reactions were normalized by dividing by their own plateau and solving for time at half - maximal fluorescence ( 0.5 a.u . ) . to visualize actin filaments we polymerized 2 m actin under the same conditions used for fluorimetry , then arrested reactions at the time indicated with alexa fluor 488 phalloidin and latrunculin b. this technique preserves the ratio of monomeric to filamentous actin at the moment of quenching24 . to keep the concentration of f - actin constant we arrested reactions at their individual ts:1 m 40 s for jmy+arp2/3 ; 6 m 20 s for scar+arp2/3 ; 37 m 0 s for actin alone . filaments were diluted to low nanomolar concentrations and spotted on poly - l - lysine ( sigma ) coverslips , using wide - bore pipette tips to minimize shearing . u2os ( atcc ) and hek 293 cells were cultured in dulbecco s modified eagle s medium supplemented with 10% fbs , 2 mm l - glutamine , non - essential amino acids , and penicillin - streptomycin ( ucsf cell culture facility ) . for transfection , cells were seeded onto glass coverslips and transfected with ha - jmy4 using genejuice ( merck ) , or gfp - jmy and gfp - rnai constructs using lipofectamine ltx ( invitrogen ) , according to manufacturer s protocol . hl-60 cells were cultured as described25 , in rpmi-1640 with 25 mm hepes , 2.0 g / l nahco3 , 10% fbs , 1% antibiotic - antimycotic ( fisher ) , and were passaged every 34 days to a density of 0.2 10 cells / ml . briefly , flamed coverslips were treated with 200 g / ml bovine plasma fibronectin ( sigma ) in pbs , washed with pbs , and blocked with 1.8% low endotoxin bsa ( sigma ) in modified hanks buffered saline solution ( mhbss : 150 mm nacl , 4 mm kcl , 1 mm mgcl2 , 10 mm glucose , 20 mm hepes , ph 7.4 at 22 c ) for 5 minutes prior to adhering cells . cells were pelleted and resuspended in bsa / mhbss , and adhered to coverslips for 3060 minutes at 37 c , then washed to remove unbound cells . stimulation or mock stimulation was in bsa / mhbss with 100 nm ( hl-60s ) or 20 nm ( human neutrophils ) fmlp , or dmso ( carrier ) , for 5 minutes at 22 c. cells were fixed in 3.2% formaldehyde in cytoskeletal buffer ( 138 mm kcl , 3 mm mgcl2 , 2 mm egta , 320 mm sucrose , 10 mm hepes , ph 7.2 at 22 c ) . for immunofluorescence of u2os cells , cells were plated on flamed , fibronectin - coated coverslips , and fixed for 30 minutes in 3.2% formaldehyde in pbs . triton in pbs with 1.4 u / ml alexa fluor 568 phalloidin ( invitrogen ) to stabilize and visualize filaments . for jmy immunolocalization , rabbit polyclonal jmy antibody 12895 or anti - ha antibody ha11 ( babco ) was used at a 1:500 dilution . alexa fluor 488-labeled goat - anti - rabbit secondary ( invitrogen ) was used at a 1:500 dilution . dapi ( sigma ) was used at 0.5 g / ml . samples were mounted with fluorescent mounting medium ( dakocytomation ) . epifluorescence and wide field images were acquired on a nikon te300 inverted microscope equipped with a hamamatsu c4742 - 98 cooled ccd camera , with simple pci software ( compix ) , using 100x and 60x 1.4 na plan apo objectives ( nikon ) , or with a 10x 0.6 na phase objective , using micromanager software27 . for quantification of f - actin levels in cells expressing gfp - pwwwca or gfp , micrographs were acquired with an ix micro automated microscope , using identical illumination conditions . cells were identified and outlined using imagej software , background was subtracted , and the average intensity in the red and green channels were measured . we used imagej ( national institutes of health ) and adobe photoshop ( adobe ) for image analysis and contrast adjustment . for wound healing experiments , cells were plated on marked coverslips at the same density , scratch wounded with a micropipette tip , washed to removed detached cells , and then given fresh medium and kept at 37c during image acquisition . stable lines of u2os cells stably expressing gfp - jmy and variants were selected in 500 g / ml g418 for at least 3 weeks , then enriched by facs ( ucsf flow cytometry core ) . sirna was used at a final concentration of 25 nm ( hjmy sirna from santa cruz and control non - targeting 2 from dharmacon ) and transfected into cells with oligofectamine or dharmafect 1 ( both according to manufacturer s protocols ) , and cells were grown for 72 hours prior to experiments . to discriminate between rnaied and non - rnaied cells , pl - ugih plasmid ( atcc ) was modified to contain a human jmy - specific hairpin based on the sequence of jmy - specific sirna-3 by the method of weiner et al . hek 293 cells transiently transfected with this construct were grown for 7 days prior to fixation or immunoblotting . standard methods were used for western blotting , using 1:500 1289 or 1:1000 l-16 ( santa cruz biotechnology ) jmy primary antibodies and 1:5000 hrp - anti - rabbit secondary ( jackson immunoresearch ) . p300-ct primary antibody ( millipore ) was used at 1:500 , goat - anti human gapdh ( santa cruz ) was used at 1:10,000 , and mouse anti - human actin ( sigma ) was used at 1:20,000 . hrp secondaries ( dako ) were used at 1:10,000 , and ecl reagent ( supersignal west pico , pierce ) was used according to the manufacturer s instructions . we used two - tailed unpaired t - tests , assuming unequal variance , to calculate p - values ( microsoft excel ) .

diabetes mellitus has become a public health problem for the past couple of decades , and the exploration for therapies to combat this continues unabated . bariatric surgery is already recognized as an effective therapeutic option for producing weight loss in morbidly obese patients , and it not only leads to subsequent weight loss but also ameliorates or resolves various comorbidities including type 2 diabetes mellitus , and vast research has been undergone regarding the mechanism underlying this in an attempt to understand it hoping that it would pave a way for new and more efficient therapeutic strategies . it has also been considered beneficial in nonobese patients with diabetes , but scant literature is available regarding low bmi patients with diabetes in context to bariatric surgery [ 24 ] . adiponectin is an idiosyncratic collagen - like cytokine secreted by mature fat cells , and it directly regulates glucose metabolism and insulin sensitivity in vitro and in vivo through the activation of amp - activated protein kinase ( ampk ) . it also increases fatty acid oxidation in skeletal muscle cells by sequential activation of amp - activated protein kinase , p38 mitogen - activated protein kinase , and peroxisome proliferator - activated receptor - alpha , thereby contributing to enhanced insulin sensitivity . visfatin exerts insulin - mimetic effects in cultured cells and lowers plasma glucose levels in mice , and it binds to and activates the insulin receptor . visfatin has a role in inhibiting apoptosis of -cells through the activation of mapk - dependent and phosphoinositide 3-kinase ( pi3k)-dependent signaling pathway . visfatin is involved in inflammatory process , and sicam-1 plays an important role in diabetic vascular disease . so , we investigated the endocrine and metabolic changes post - lrygb in chinese patients with t2 dm , whose body mass indices were between 22 and 30 kgm , focusing on adiponectin , visfatin , and sicam-1 and explored their relationship with postsurgical insulin sensitivity . thirty - three patients of t2 dm ( bmi 26.71 0.69 kgm ) ( aged 49.51 1.33 years ) , who underwent lrygb performed in the third xiangya hospital of central south university from june 2009 to july 2011 , were randomly included in this case study . among them , twenty - four were male , and nine were females . inclusion criteria included ( 1 ) diagnosis of t2 dm based on the guidelines set by the ada in 2007 , ( 2 ) the ratio of peak to the basic value of cprt > 2 ( the basic value of cprt by ogtt > 0.33 g / l ) , ( 3 ) bmi : 2230 kgm , ( 4 ) age 65 years , and ( 5 ) duration of diabetes < 10 years . exclusion criteria included patients with a history of open abdominal surgery , unstable psychiatric illness , inability or reluctance to cooperate in follow - up , alcohol or drug dependence , and relatively high surgical risks such as active ulcer or helicobacter pylori infection . fasting blood samples were collected prior to the surgery and at 3 months after surgery . the study was conducted in compliance with the protocol approved by the ethics committee of the third xiangya hospital , central south university , and all patients provided written informed consents for participation in the study . all the operations were performed in the department of surgery of the third xiangya hospital of central south university . the proximal jejunum was transected 40 cm distal to the ligament of treitz , the alimentary limb was measured at 100 cm , and a side - to - side jejunostomy was performed using 60 mm linear cutter and 3.5 mm stapler . the jejunostomy and the mesenteric defects were subsequently closed with manually placed polyglactin ( 20 vicryl , ethicon , usa ) sutures , and with nylon ( 20 ethibond , ethicon , uk ) sutures respectively . a 25 ml proximal gastric pouch was created with 60 mm linear cutter and 3.5 mm stapler . then , a 2.5 cm long gastrojejunostomy was formed with 30 mm linear cutter and 2.5 mm stapler at the antecolic position . the gastrojejunostomy was closed with manual sutures ( 20 vicryl , ethicon , usa ) , and a drain was placed near gastrojejunostomy around the right - upper quadrant . standard methods were used to measure waist and hip circumferences , height , and weight . the biochemical measurement for visfatin plasma glucose was measured with glucose analyzer ( ekf biosen c - line glucose lactate analyzer , germany ) and glycated hemoglobin by high performance liquid chromatography . homeostatic model assessment of insulin resistance was calculated as a product of fasting plasma glucose and insulin divided by a constant ( homa - ir = ( fpg ( mmol / l ) fins)/22.5 ) . adiponectin , sicam-1 , and visfatin were measured by elisa ( rd company , usa ) . data are expressed as mean sd . comparisons were made using the t - test for data following the normal distribution or rank sum test for data not following the normal distribution . analysis of the preoperative and postoperative differences was done using the pearson correlation for data following the normal distribution or the spearman analysis for data not following the normal distribution . multivariate analysis was done by multiple regression analysis . the level of significance is taken at p < 0.05 . three - month postoperatively , statistically significant decrease in both bmi ( p < 0.001 ) and whr ( p < 0.01 ) was noted . three months after rygb , a significant decrease in the levels of fpg , 2hpg , hba1c , and tg was noted . three - month postoperatively , a statistically significant increase ( p < 0.001 ) in the concentration of adiponectin and significant reductions ( p < 0.001 ) in the concentration of visfatin and sicam-1 were observed ( figure 1 ) . concentrations of adipocyte hormones were further correlated with homa - ir and fins . both at baseline and at 3 months after surgery , adiponectin negatively correlated with homa - ir and fins , whereas plasma visfatin positively correlated with homa - ir and fins . a significant difference was found between the mean bmi of postoperative participants whose adiponectin values were below and above the median of adiponectin [ t(29 ) = 2.39 , p < 0.05 ] . the mean bmi of the postoperative participants whose adiponectin levels were below the median was significantly higher ( 25.81 4.08 kgm ) than of those whose adiponectin levels were above the median ( 22.96 2.38 kgm ) . however , there was no significant correlation of these adipocyte hormones with bmi , whr , fpg , 2hpg , hba1c , tg , chol , hdl - ch , ldl - c , or sicam-1 . serum sicam-1 levels and changes in bmi , whr , fpg , 2hpg , hba1c , tg , chol , hdl - ch , ldl - c , homa - ir , fins , adiponectin , and visfatin were not correlated . global cutoff points for overweight and obesity as set by the world health organization are bmi of 25.0 kgm and 30.0 kgm , respectively . there has been a lot of controversy regarding these cutoff points in asian populations for more than a decade . few studies in china [ 11 , 12 ] , japan , taiwan , and hong kong have reported an association between a bmi of 22.3 kgm and increased atherogenic risk factors . a survey by kim et al . reported that the prevalence of diabetes , hypertension , and dyslipidemia has doubled at a bmi of 23.0 to 24.0 kgm and tripled at a bmi of 26.0 kgm in the korean adult population and that body fat and fat distribution at a bmi of 23.0 kgm in asians may be similar to those in the whites at a bmi of 25.0 kgm . the who , the international obesity task force , and the international association for the study of obesity have proposed lower cutoff points for overweight ( bmi 23.0 kgm ) and obesity ( bmi 25.0 kgm ) in asian and pacific island populations . a study by huang et al . has showed that roux - en - y gastric bypass could be used in chinese t2 dm patients with bmi of 2535 kgm , and 63.6% patients showed t2 dm remission , 27.3% showed glycemic control , and 9.1% showed improvement at 12-month duration in their study . the bmi cutoff for overweight in our study was 23 kgm and that for obesity was 28 kgm . in our study , 20 patients were overweight and 9 patients were obese . our study investigated the changes of adipocyte hormones and systemic marker of inflammation following lrygb in low bmi chinese subjects with t2 dm . the mean bmi in our study was ( 26.71 0.69 kgm ) , and the mean age of the patients was ( 49.51 1.33 years ) . the hba1c level of all patients exceeded 7% despite being on maximum tolerated dose for a duration of at least six months . the major limitations of our study were that the sample size was limited to 33 individuals only , and as the sample was randomly selected , the patients could not be equally weighted for sex to allow for between sex differences . expanding the sample size and the duration of study while previous studies have reported the endocrine and metabolic changes post - rygb in obese patients with t2 dm , we believe this to be the first study to report these findings in case of low bmi patients with t2 dm . the patients were on oral hypoglycemics or insulin or both prior to the surgery . among the 33 patients , 19 ( 45.4% ) were on oral hypoglycemic agents prior to surgery , and 8 ( 21.2% ) were on insulin . after the surgery , 8 ( 24.2% ) patients totally came off these medications , whereas 4 ( 12.1% ) remained on insulin . the other 17 ( 51.5% ) three months after the surgery , statistically significant amelioration in body mass index and waist - hip circumference could be observed in our study . in addition , the indicators of glucose metabolism , namely , hba1c , fpg , and 2hpg were significantly reduced in comparison to the preoperative values . likewise , statistically significant reduction in tg decrease in homa - ir and fins levels was also noted , suggesting that lrygb could be beneficial even for low bmi individuals of t2 dm ( table 1 ) . similar findings have been reported by several studies conducted on obese individuals with diabetes [ 1923 ] . the results of our study reveal that there was an increase in the adiponectin levels ( p < 0.001 ) three months after lrygb ( table 2 ) . few other studies have come up with similar results albeit on different time frames after - surgery ( at 6 and 12 months ) and on morbidly obese subjects [ 2426 ] . three - month postoperatively , a statistically significant reduction in the concentration of visfatin ( p < 0.001 ) was noted ( table 2 ) . there has been conflicting results regarding the study of visfatin post - rygb . some studies have reported decreased levels of visfatin post - rygb [ 23 , 27 ] in consistent with the findings of our study , whereas a study reported an increase . the results for disparate visfatin levels are unknown but may be attributed to the variation in the gastrointestinal surgery , the study population , the duration of postoperative observation , the degree of inflammatory status , and the surgical approach . our study found that the serum adiponectin negatively correlated with homa - ir and fins both preoperatively and at 3 months after surgery , and visfatin positively correlated with homa - ir and fins both preoperatively and postoperatively ( table 3 ) . in addition , multivariate analysis revealed that the changes in insulin sensitivity were predicted by weight loss . no significant correlation of these adipocyte hormones with bmi , whr , fpg , 2hpg , hba1c , tg , chol , hdl - ch , ldl - c , or sicam-1 was found . reported that the proportional improvement in homa - ir over 12 months in their study was related to change in adiponectin . another prospective study on 31 morbidly obese patients followed up for a duration of 6 months showed that insulin sensitivity as estimated by the homa - ir was unchanged , but individual changes of insulin resistance and visfatin were significantly associated . rygb in individuals with t2 dm not only leads to reductions in blood sugar , blood lipids , blood pressure , and other metabolic disorders but also improves the inflammatory status of patients with t2 dm . our study shows that the level of sicam-1 was lower at 3-month post - lrygb than that before surgery ( p < 0.001 ) ( table 2 ) . other studies reported similar results on morbidly obese subjects at 3 , 6 , and 12 months [ 23 , 31 ] . changes in adipokines are related to an improvement in postsurgical insulin sensitivity even in low bmi patients with t2 dm , and the major predictor was weight loss . unraveling the pathogenesis and attaining in - depth exploration would require large - scale clinical studies and long - term followup that would further elucidate the effect of lrygb in such patients .

clinical details of our study group have been reported previously but are reproduced in table 1 for ease of reference . among the hemodialysis patients , no changes were observed in weight , kt / v , energy , and protein and mineral intakes at the beginning and end of the study ( p>0.05 ) . median ( range ) serum total alp activity in all of the samples included in the study was 76 ( 30289 ) u / l in our original report , confirming that there had been no loss of total alp activity during frozen storage . cochran 's test for outliers among duplicates identified two bone alp measurements , both from the hemodialysis group ( sample 5 from subject 9 and sample 11 from subject 19 ) ; these samples were excluded from statistical analysis . cochran 's test identified two samples that needed to be excluded from within - subject variance estimations ( sample 6 from subject 7 and sample 3 from subject 25 ) . sample 11 from subject 13 and sample 2 from subject 26 were excluded because of suspected contamination ( undetectable total alp result ) . a number of individuals ' observations displayed a significant trend over the length of the investigation ( e.g. , a consistent rise or fall in analyte concentration over time ; supplementary table s1 and figure s1 online ) . analysis of variance performed both including and excluding the subjects showing trends yielded essentially the same estimates of cva and cvi ( supplementary table s2 online ) : consequently , the data presented in this paper include all subjects ( i.e. , those who both did and did not show trends in concentration over time ) . figure 1 shows the individual median and range concentrations for bone alp in all the study subjects . the cvi for bone alp was significantly higher for the hemodialysis patients compared with the healthy volunteers ( p<0.05 ) , resulting in a greater number of specimens being required to estimate an individual 's homeostatic set point and a higher critical difference ( table 2 ) . no significant difference ( p>0.05 ) in the cvi for bone alp was observed between those patients who underwent dialysis in the morning ( 12.9% ) and those who underwent dialysis in the afternoon ( 12.2% ) . bone alp concentrations were correlated with total alp activity irrespective of whether all subjects were considered together ( rs 0.86 ) or whether the healthy control group ( rs 0.94 ) and hemodialysis patients ( rs 0.86 ) were considered separately ( p<0.0001 in all three cases ) . there were no significant correlations ( p>0.05 ) between either bone alp or total alp and pth irrespective of whether controls and patients were considered separately or together . its concentration generally reflects the rate of bone formation in skeletal tissue . as bone formation is coupled to bone resorption , bone alp measurements provide an indication of overall bone metabolism . bone alp can be used as a marker of bone turnover in ckd - mbd patients . in this study , we have described for the first time the biological variation of bone alp in hemodiaylsis patients and compared it against that we previously described for pth . the cvi for bone alp that we observed among healthy subjects ( 7.7% ) is similar to that reported by others ( 3.4 ( ref . differences between reports could , for example , reflect other studies that only included exclusively premenopausal or postmenopausal women . biological variation for bone alp was higher among hemodialysis patients than healthy volunteers , as we previously observed for total alp , calcium , phosphate , and pth . we can not exclude the possibility that this reflects the older age of our hemodialysis patients compared with the healthy volunteers . however , ckd - mbd is almost universal among patients with kidney failure , and it is perhaps not unexpected that these markers will show a significantly higher cvi in hemodialysis patients . similar observations have been made for total alp when comparing healthy controls ( cvi 4.5% ) with pagetic patients ( cvi 12.4% ) . the higher cvi in disease compared with health clearly affects the critical difference and number of specimens required to estimate an individual 's homeostatic set point . as noted by others , variance components derived from healthy subjects can not necessarily be extrapolated to pathological situations . however , the cvi of bone alp in hemodialysis patients ( 12.5% ) was less than half of that previously described for pth ( 25.6% ) , resulting in a lower number of specimens required to estimate an individual 's homeostatic set point ( 7 vs. 26 ) and a lower critical difference ( 36% compared with 72% table 2 ) . in addition , the biological variation of bone alp in dialysis patients appeared to be consistent throughout the day , whereas we previously found suggestive evidence that the cvi of pth differed between the morning and afternoon . the lower biological variation of bone alp compared with pth represents a potentially important advantage of this marker in the monitoring and treatment of ckd - mbd . in addition to higher biological variation , pth is an imperfect marker of ckd - mbd for a variety of other reasons including the following : analyte instability ; significant phlebotomy sample site variation ; standardization - related assay variability ; nonspecificity of assays , with which 50% of measured pth is of a biologically inactive form ; racial variation in the pth histology relationship ; a strong relationship between pth and body mass index and nutritional / inflammatory status ; poor histological correlation to target ranges ; and skeletal resistance . furthermore , attainment of the recommended pth target range has not been shown to improve the risk of death or cardiovascular events within the ckd population . these issues raise significant concerns over the validity of specific target concentrations of pth and the use of pth in monitoring response to treatment . there are many potential biomarkers of skeletal health and integrity , some of which are difficult to use and to interpret in kidney failure on account of renal function affecting their plasma concentrations . one of the best known , and best established , of these is the plasma activity of alp , which is increased in osteitis fibrosa . high plasma alp activity among hemodialysis patients is associated with increased risks of all - cause and cardiovascular mortality , and it has been shown to be more sensitive to the effects of vitamin d replacement than pth . however , the use of total alp in this context has always been considered limited by its nonspecificity for bone disease , with only 50% of blood activity being attributable to bone alp . specific immunoassays for bone alp have been available for nearly 20 years , but their use has not become widespread in nephrology , probably reflecting perceived difficulties of measurement and the primary role assigned to pth in the management of ckd - mbd . bone alp has a relatively long half - life of 12 days , and its plasma concentration depends only on its rate of release from the osteoblasts and on its hepatic degradation . unlike pth , bone alp does not accumulate with progressive loss of gfr . further , bone alp has a good relationship with bone histology and bone mineral density and is also a good predictor , with dexa - derived bone mineral density , of future fracture risk in dialysis patients . in addition , high concentrations of bone alp are associated with increased cardiovascular and all - cause mortality in kidney failure patients . mechanistically , there is evidence implicating bone alp as a proponent of vascular calcification in ckd - mbd . this is supported by data showing a significant association between serum alp activity and coronary artery calcification in hemodialysis patients , with serum alp > 120 u / l being a robust predictor of calcification . hence , our data , together with other published literature , advocate the use of bone alp as a potential alternative marker of ckd - mbd , providing a living biopsy of bone activity with additional potential value in the risk stratification of hemodialysis patients . however , no loss in serum total alp activity was seen during this time . as bone alp represents a significant component of total alp , it is a reasonable assumption that bone alp was also stable under these storage conditions . the cvi , and consequently the critical difference , for total alp was lower than that for bone alp ; there was a strong correlation between bone alp and total alp ; as noted above , total alp has strong relationships with outcome in patients with kidney failure ; further , total alp is easier and cheaper to measure in laboratories than bone alp . it is possible that the value of total alp as a marker of ckd - mbd should be reassessed . however , we feel that its nonspecificity for bone tissue will always remain an impediment to clinical acceptance in this situation . further , bone alp shows stronger relationships with histological changes than total alp , and better sensitivity and specificity for both low- and high - turnover bone disease . although bone alp requires far fewer samples than pth to establish a patient 's homeostatic set point , it is still clear that a single measurement is insufficient . the significance of the reduced critical difference for bone alp compared with pth is best appreciated by application to a clinical example . in the study of fletcher et al . , who used the forerunner of our assay , mean bone alp concentrations of 44 g / l were observed in dialysis patients with severe hyperparathyroidism ( mean pth 780 g / l among patients with adynamic bone disease ( mean pth 140 ng / l ) . applying critical differences ( table 2 ) to the values lying exactly intermediate between these two groups ( bone alp 27 g / l and pth 460 ng / l ) , it can be seen that a 72% increase in pth ( just significant ) will result in a concentration of 790 ng / l , in excess of the mean of the group with severe hyperparathyroidism , whereas a 72% decrease would result in a concentration below the mean of the group with adynamic bone disease ( 129 ng / l ) . this , in part , may explain the poor histological correlation of bone biopsy specimens to pth target ranges . for bone alp , the same manipulation would result in concentrations of 17 g / l and 37 g / l , above and below the adynamic and severely hyperparathyroid means , respectively . this illustrates how the better signal - to - noise ratio of bone alp compared with pth would enable clinicians to better interpret changes in concentrations in their patients . g / l among patients with osteitis fibrosa , and fletcher et al . observed mean bone alp concentrations of 44 g / l in dialysis patients with severe hyperparathyroidism ( mean pth 780 ng / l ) . two of our patients ( subjects 17 and 19 ) had markedly increased , and one ( subject 4 ) marginally increased , median bone alp concentrations : these subjects also had high pth concentrations ( > 400 overall , this would be consistent with an interpretation that the ckd - mbd among our cohort was at the milder end of the spectrum , possibly reflecting a shift in the phenotype of ckd - mbd away from high - turnover bone disease , as noted by others , compared with these earlier studies . however , the majority of the patients had pth concentrations in excess of the upper limit of normal . in the absence of bone biopsy data , one can not draw strong conclusions in this regard , but it is tempting to speculate that the high pth concentrations may not be reflecting increased bone turnover in all of these individuals , because of some of the problems alluded to above . as we have noted previously , our cohort is fairly typical of hemodialysis cohorts in the uk at the present time , and we therefore feel our conclusions are generally applicable to this population . in conclusion , we propose that the relatively low biological variability of bone alp provides further suggestive evidence that it could be a better alternative to pth measurement in the management of ckd - mbd in the adult hemodialysis population . whichever marker is used , it is clear that clinicians can not rely on single measurements assessed against rigid and somewhat arbitrary management targets , but instead must look serially for trends in patient results . twenty - two patients receiving three - times - weekly hemodialysis for renal failure at the kent kidney care centre were recruited in 2008 , with their informed consent . ethical approval for this study complete details of the patients included in the study and of the exclusion criteria applied have been published previously . briefly , all patients had been on hemodialysis for a minimum of 3 months , and their pharmacologic prescription and dietary intake remained consistent throughout the study period . most were receiving 1-hydroxycholecalciferol ( alfacalcidol ) and calcium carbonate ( calcichew ) . to standardize preanalytical variables , only patients who underwent dialysis on monday , wednesday , and friday twelve volunteer staff members who had a negative proteinuria screening test ( multistix 10 sg , siemens healthcare , surrey , uk ) and estimated gfr 60 ml / min per 1.73 m ( ref . non - fasting blood samples were collected before dialysis from the vascular access ( arteriovenous fistula ) of the patients before their dialysis sessions on wednesday and friday for 6 weeks ( i.e. , 12 samples per patient ) . blood was collected from patients who underwent dialysis in the morning ( n=13 ) between 0800 and 1000 hours , and from patients who underwent dialysis in the afternoon ( n=9 ) between 1300 and 1500 hours . concurrently , samples were collected from the 12 healthy volunteers on wednesday and friday ( between 0930 and 1130 hours ) . blood was collected in serum separator tubes ( becton - dickinson vacutainer systems , oxford , uk ) , and serum was separated within 4 h of venepuncture and stored at 80c for 22 months until assayed . before analysis , samples were thawed at room temperature for 48 h to enable reactivation of alps and then mixed by inversion . for analyte measurement , each sample serum total alp was measured on an abbott architect c8000 automated system ( abbott laboratories , abbott park , il ) at 37c using essentially the method described by the international federation of clinical chemistry . serum concentrations of bone alp were determined using a solid - phase , monoclonal antibody immunoenzymetric assay ( ostase bap , immunodiagnostic systems , tyne & wear , uk ) . the upper limit of the reference range for this assay is 22.4 g / l ( manufacturer 's product information ) . all calculations and statistical analyses were carried out with minitab ( coventry , uk ) and analyse - it ( analyse - it software , leeds , uk ) . comparisons between dietary intakes , weight , and kt / v at the start and end of the study were made using paired t - test or wilcoxon match - pairs signed ranks test as appropriate . comparisons between total alp , bone alp , and pth concentrations at the start of the study between healthy controls and patients were made using the mann cochran 's criterion test was used to identify outliers among duplicate measurements and among within - subject variances , and the reed test to eliminate mean outlying values . wilk test : approximately one - third of the data sets were not normally distributed . all variance estimates were therefore derived using natural logarithmic transformed data after exclusion of outliers as recommended , and then back - transforming the data . analytical ( sda ) and intraindividual variances ( sdi ) were calculated by nested analysis of variance according to the methods of fraser and harris . coefficients of variation ( cva and cvi ) were calculated as cv%=sd / mean analyte concentration 100 . the f - test was used to compare : ( i ) cvi obtained for the healthy volunteers and the hemodialysis patients and ( ii ) cvi among patients who underwent dialysis in the morning with those who underwent dialysis in the afternoon as the aim of the study was to establish biological variation under steady - state conditions , exploratory analysis was conducted using linear regression analysis in order to discover whether the natural logarithmic data for bone alp concentrations followed a trend over the 6-week period ( e.g. , a consistent rise or fall in concentrations ) . analysis of variance analysis to determine cvi was then performed both including and excluding the subjects showing trends . subjects with t values greater than + 2 or less than 2 had measurements that showed a significant trend . the critical difference , which is the difference that must be exceeded between two sequential results for a significant change to have occurred in either direction at p<0.05 , was derived by applying the standard equation [ 2.77 ( sda+sdi ) ] . the number of specimens required to estimate the homeostatic set point of an individual to within 10% with 95% confidence was also calculated , and this analysis was adjusted to consider 20 and 30% limits . spearman 's correlation was used to assess relationships between bone alp and both total alp and pth : in all cases the mean value for each subject was used . all calculations and statistical analyses were carried out with minitab ( coventry , uk ) and analyse - it ( analyse - it software , leeds , uk ) . comparisons between dietary intakes , weight , and kt / v at the start and end of the study were made using paired t - test or wilcoxon match - pairs signed ranks test as appropriate . comparisons between total alp , bone alp , and pth concentrations at the start of the study between healthy controls and patients were made using the mann cochran 's criterion test was used to identify outliers among duplicate measurements and among within - subject variances , and the reed test to eliminate mean outlying values . we tested individuals ' data for normality using the shapiro wilk test : approximately one - third of the data sets were not normally distributed . all variance estimates were therefore derived using natural logarithmic transformed data after exclusion of outliers as recommended , and then back - transforming the data . analytical ( sda ) and intraindividual variances ( sdi ) were calculated by nested analysis of variance according to the methods of fraser and harris . coefficients of variation ( cva and cvi ) were calculated as cv%=sd / mean analyte concentration 100 . the f - test was used to compare : ( i ) cvi obtained for the healthy volunteers and the hemodialysis patients and ( ii ) cvi among patients who underwent dialysis in the morning with those who underwent dialysis in the afternoon . a p - value of < 0.05 was set for statistical significance . as the aim of the study was to establish biological variation under steady - state conditions , exploratory analysis was conducted using linear regression analysis in order to discover whether the natural logarithmic data for bone alp concentrations followed a trend over the 6-week period ( e.g. , a consistent rise or fall in concentrations ) . analysis of variance analysis to determine cvi was then performed both including and excluding the subjects showing trends . subjects with t values greater than + 2 or less than 2 had measurements that showed a significant trend . the critical difference , which is the difference that must be exceeded between two sequential results for a significant change to have occurred in either direction at p<0.05 , was derived by applying the standard equation [ 2.77 ( sda+sdi ) ] . the number of specimens required to estimate the homeostatic set point of an individual to within 10% with 95% confidence was also calculated , and this analysis was adjusted to consider 20 and 30% limits . spearman 's correlation was used to assess relationships between bone alp and both total alp and pth : in all cases the mean value for each subject was used .

the decreases in the fertile and pregnant populations and birth rate have accelerated the aging of korean society , resulting in an increase in the elderly population . with the increase in the elderly population , increased attention should be given to the middle - aged population , which includes people in the stage prior to the old age1 . physical , psychological , and mental factors of middle age directly or indirectly affect the quality of life in old age . therefore , a healthy old age life can stem from a well - managed and balanced middle age life . moreover , health in middle age is being emphasized in south korea aging society . middle - aged women experience gradual weakening of physical functions with aging and show decreased physical activities . this decrease increases the obesity rate , causing middle - aged women to be susceptible to various musculoskeletal problems such as osteoporosis and other degenerative diseases2 . moreover , postmenopausal middle - aged women have reduced ovary functions due to estrogen deficiency , and the level of low - density lipoprotein cholesterol ( ldl - c ) in the blood increases due to reduced estrogen stimulation and low - density lipoprotein ( ldl ) receptor activities . in addition , they are susceptible to adult diseases such as coronary diseases because of a rapid increase in the lipid concentration in the blood and subsequent decrease in the high - density lipoprotein - cholesterol ( hdl - c ) concentration3 . regular aerobic exercises are suggested for middle - aged and older women to help them overcome their physical and physiological changes and stabilize their mental and psychological status through participation in society activities4 . among the aerobic exercises , walking is the most popular physical activity , and it can be done easily without the use of any devices . physical and physiological improvements through walking exercise have been shown in a number of studies , and positive influences are observed in most cases . hence , walking can be an effective exercise from the perspective of preventive medicine . yet studies on the effects of walking on blood and physical factors in obese middle - aged women have not been conducted sufficiently5 . this study was conducted to identify the effects of walking on the body composition , health - related physical fitness , and serum lipids as part of efforts to encourage middle - aged people to participate in walking as a regular and sustainable exercise . the study period was for 12 weeks from january to march 2010 . the participants were 43 middle - aged women ( age range : 4055 years ) residing in seoul , korea , with body fat rates over 30% . subjects in the experiment group participated in the walking exercise ( n = 38 ) , whereas the control group did not participate in the exercise ( n = 23 ) . the participants had not undergone any exercise regimen within the previous 1 year . subjects who could not undergo the exercise tests because of cardiac infarction and pulmonary embolism , confirmed using a self - answered questionnaire , were excluded . this study was approved by the seodaemun district office and the institution in which it was performed and complied with the ethical standards of the declaration of helsinki . all participants provided their written informed consent to participate in this study prior to enrollment . general characteristics of the participantsexercise group ( n=38)control group ( n=23)age ( year)46.83.146.72.8height ( cm)159.13.7157.44.6weight ( kg)58.68.756.49.0blood pressuresystolic ( mmhg)124.014.2117.79.5diastolic ( mmhg)78.011.473.409.1*p<0.05 ; * * p<0.01 ; * * * p<0.001 . * p<0.05 ; * * p<0.01 ; * * * p<0.001 this study evaluated the body composition , physical fitness , and serum lipid level . body composition was evaluated by measuring body weight , muscle mass , and body mass index ( bmi ) . physical fitness was evaluated by measuring muscle strength , flexibility , muscle endurance , and cardiopulmonary endurance . muscle strength was determined by measuring the maximum grip strength by using the dominant hand ( pf / kg ) . flexibility was measured by a sit - and reach , in which the subject sat with the knees completely straight and reached forward slowly using both the hands together ( unit : cm ) . muscle endurance was measured through and exercise test using sit - ups , and the maximum number was recorded for 30 s after a beep ( unit : number ) . cardiopulmonary endurance was measured using a treadmill and a gas analyzer ( qmc 5000 , usa ) . the bruce protocol was used for the exercise test , and the maximum exercise point of the participant was set at the maximum heart rate and a respiratory exchange rate of over 1.15 for each age range . the indicator for cardiopulmonary endurance was the point where oxygen intake did not increase even with an increase in exercise intensity . for serum lipid test , the participants fasted from 10:00 ( am ) of the previous day , and 5 ml of blood was collected from the brachial vein the following day between 08:00 and 10:00 am . after blood collection , the vials were centrifuged , and tc , tg , hdl - c , and ldl - c levels were analyzed in the hemanalysis department . the walking program was designed such that the number of calories burned was gradually increased to 150 kcal / hr by reducing the weight and increasing the walking speed . the initial exercise duration was 20 min , which was gradually increased to 4055 min ; the initial frequency was 3 times a week , which was increased to 5 times a week . details of the walking durations and frequencies are shown in table 2table 2 . twelve - week walking exercise programperiodintensityduration ( break)frequency03 weeks100 kcal / h20 min/23 times ( 5 min)3 times / week49 weeks120 kcal / h30 min/23 times ( 5 min)4 times / week1012 weeks150 kcal / h40 min/2 times ( 5 min)5 times / week . two - way repeated anova was used to observe the differences in each measurement factor by group ( exercise and control groups ) and measurement period ( before and after the 24-week walking exercise program participation ) . a paired t test was conducted to confirm the significance of the measured categories of before and after intervention in each group , and all statistical significance ( ) was set at p < 0.05 . regarding the changes in body composition , weight significantly decreased in the exercise group ( p < 0.05 ) . no main effect was observed by group ( exercise and control groups ) ; however , main and interaction effects ( p < 0.05 ) were observed by period ( before and after 12 weeks ) and period and group ( period*group ) . muscle mass significantly decreased in the exercise group ( p < 0.05).no main effect was observed by group ( exercise and control groups ) ; however , main and interaction effects ( p < 0.05 ) were observed by period ( before and after 12 weeks ) and period and group ( period*group ) . further , main and interaction effects were observed by group ( exercise and control groups ) , period , and period*group ( table 3table 3 . changes in body compositionitemgrouppretestposttestweight ( kg)exercise58.58.657.27.3***control56.49.056.69.1muscle mass ( kg)exercise37.34.038.03.8control36.74.436.74.3body fat ( % ) exercise32.53.625.54.5***control31.23.731.03.6p value of paired t - test : * p<0.05 ; * * p<0.01 ; * * * p<0.001 . p value of two - way anova test ( period*group ) : p<0.05 ; p<0.01 ; p<0.001 ) . p value of paired t - test : * p<0.05 ; * * p<0.01 ; * * * p<0.001 . p value of two - way anova test ( period*group ) : p<0.05 ; p<0.01 ; p<0.001 reagarding changes in physical fitness , no significant change was observed in grip strength ( dominant hand ) in the exercise and control groups ( p > 0.05 ) . further , no main and interaction effects ( p > 0.05 ) were observed by group , period , and period*group . no significant change was observed with regard to performance of sit - ups in the exercise and control groups ( p > 0.05 ) ; however , partial improvements were observed in the exercise group . further , no main and interaction effects were observed by group , period , and period*group ( p > 0.05 ) , ( table 4table 4 . changes in physical fitnessitemgrouppretestposttestgrip strengthexercise27.73.727.14.8control27.954.127.75.9sit - and- reachexercise15.46.616.85.6**control15.64.615.55.6sit - upexercise8.64.39.13.9control8.54.48.84.3vo2maxexercise24.43.526.94.6***control24.94.624.73.1p value of paired t - test : * p<0.05 ; * * p<0.01 ; * * * p<0.001 . p value of two - way anova test ( period*group ) : p<0.05 ; p<0.01 ; p<0.001 ) . p value of paired t - test : * p<0.05 ; * * p<0.01 ; * * * p<0.001 . p value of two - way anova test ( period*group ) : p<0.05 ; p<0.01 ; p<0.001 regarding changes in serum lipids , tc showed a significant decrease in the exercise group ( p < 0.05 ) , and main and interaction effects ( p < 0.05 ) were observed by group , period , and period*group . tg showed a significant decrease in the exercise group ( p < 0.05 ) , and main and interaction effects were observed by group , period , and period*group ( p < 0.05 ) . hdl - c showed a significant change in the exercise and control groups ( p > 0.05 ) , but main and interaction effects were not observed by group , period , and period*group ( p > 0.05 ) . ldl - c showed a significant decrease in the exercise group ( p < 0.05 ) , and main and interaction effects were observed by group , period , and period*group ( p < 0.05 ) ( table 5table 5 . changes in serum lipiditemgrouppretestposttesttotal cholesterol ( mg / dl)exercise212.232.2176.729.1control209.734.4209.032.7triglyceride ( mg / dl)exercise183.258.8138.840.0control185.345.8187.546.1hdl - c ( mg / dl)exercise56.517.455.616.1control60.816.461.116.6ldl - c ( mg / dl)exercise128.016.9111.312.0control130.120.0129.816.6p value of paired t - test : * p<0.05 ; * * p<0.01 ; * * * p<0.001 . p value of two - way anova test ( period*group ) : p<0.05 ; p<0.01 ; p<0.001 ) . p value of paired t - test : * p<0.05 ; * * p<0.01 ; * * * p<0.001 . p value of two - way anova test ( period*group ) : p<0.05 ; p<0.01 ; p<0.001 regular aerobic exercise in middle age increases the metabolic rate and improves risk factors such as obesity , hypertension , and hyperlipidemia . in addition , the functioning of the heart and blood vessels improves so cardiovascular diseases such as coronary diseases are prevented or delayed , thereby preventing various health problems in middle - aged women6 , 7 . the study results showed a significant decrease in weight and body fat in the exercise group that participated in the 12-weeks of walking exercise . in the study by nindel et al . ( 2000 ) , weight and body fat were significantly decreased as a result of 24 weeks of aerobic exercise 5 times a week8 . jang ( 2009 ) reported that the weight , body fat , and muscle mass decreased significantly in middle - aged women who participated in boxing aerobics9 . in the studies of park ( 2009 ) and park ( 2011 ) on body composition , significant decreases in weight and body fat were shown , which implies that aerobic exercise and walking affects the body composition of participants5 , 10 . this result is considered to be due to the high breakdown rate of lipids through exercise ; lipid usage is increased through an oxidation process , resulting in the reduction of weight and body fat11 , 12 . cholesterol ( tc , hdl - c , and ldl - c ) is a serum lipid found in the blood along with neutral fat and phospholipids . an increase in serum lipids expedites arteriosclerosis and is one of the main reasons for cardiovascular diseases13 . in particular , this lipid is a risk factor for adult diseases in middle - aged women having reduced female hormone levels and physical fitness because of decreased physical activities caused by aging . significant decreases in tc , tg , and ldl - c levels an increase in hdl - c level , although not significant , were observed after the 12-week walking program in this study . in the study of ju ( 2010 ) on the influence of a 12-week - walking pilates program in middle - aged women , the serum lipid , tc , tg , and ldl - c levels reduced significantly , and the hdl - c showed no significant increase14 . on the other hand , in the study kim ( 2009 ) , significant decreases in tc , tg and lds - c and significant increases in hdl - c levels were observed in obese women after a 24-week walking with running exercise program15 . we assume that a significant increase in hdl - c was not observed in this study because of a difference in the exercise period and intensity . both this study and the study of ju ( 2010 ) were conducted for 12 weeks , whereas the study of kim ( 2009 ) was for 24 weeks and was a combination program of walking and running exercises15 . generally , significant changes in serum lipid levels are observed because aerobic exercise blocks the ldl - c from accumulating on the arterial walls and facilitates the tc removal mechanism in various cells , including smooth muscles in arteries , through increased hdl - c levels , anti - arteriosclerosis , and catabolism during the transport of cholesterol to the liver .

red cell transfusion ( rbc ) has been shown to be associated with increased patient morbidity and mortality . rbc transfusion has been linked with increased duration of hospital stay , length of stay in the intensive care unit , duration on mechanical ventilation , risk of bacterial postoperative infection , and risk of multiple organ failure . observational studies have shown this association to be dose - dependent [ 14 ] . in 2012 , the joint commission and the american medical association - convened physician consortium for performance improvement determined rbc transfusion to be one of the top five overuses in us medicine . it is important to note that the us transfuses more blood than other countries for comparable procedures without improved outcomes . in 2012 , aabb ( formerly american association of blood banks ) published clinical guidelines for rbc transfusion based upon results and recommendations from 31 randomized clinical trials involving over 12 500 patients , comparing restrictive transfusion thresholds ( 7 - 8 g / dl ) with liberal thresholds ( 9 - 10 g / dl ) . although aabb could make no specific recommendation regarding liberal or restrictive transfusion strategy for patients with acute coronary syndrome , aabb recommends restrictive transfusion strategy in other stable , hospitalized patients . transfusion is likely to be indicated in a patient with hemoglobin ( hgb ) level less than 7 g / dl . aabb recommends a transfusion trigger where hgb is less than 8 g / dl in patients with symptomatic preexisting cardiovascular disease . transfusion is not likely to be indicated when hgb level is greater than 10 g / dl . the concept of liberal and restrictive transfusion strategy has been in the literature for some time . the transfusion requirements in critical care ( tricc ) trial in 1999 randomized 838 critical care patients into liberal ( transfusion trigger of hgb less than 10 g / dl with posttransfusion target hgb of 1012 g / dl ) or restrictive ( transfusion trigger of hgb less than 7 g / dl with posttransfusion target hgb of 7 to 9 g / dl ) strategy . this trial reported that restrictive transfusion strategy is at least equivalent to liberal transfusion strategy in all groups , except for in patients with severe ischemic heart disease . the trial also reported that a restrictive transfusion strategy is potentially better in less ill ( apache score less than 20 ) and younger ( less than 55 years old ) patients . this has been supported in other reports , including a systematic review with meta - analyses and trial sequential analysis of 31 trails totaling 9813 randomized patients concluding restrictive transfusion strategies to be safe in most clinical situations . the authors also concluded that liberal transfusion strategies have not been shown to convey any benefit to patients . education of ordering providers and adherence to restrictive strategy could reduce patient exposure to blood products and reduce potential risks associated with transfusion . our hospital system is a university - based teaching hospital with a level i trauma center and neonatal intensive care unit . from 2014 through mid-2016 , the computerized provider order entry ( cpoe ) system at our institution did not include orders related to massive transfusion protocol , intraoperative transfusion , outpatient transfusion , or orders from the neonatal intensive care unit . since the aabb 2012 guidelines for rbc transfusion are for stable inpatients , we selected cpoe generated rbc transfusion orders as our mechanism of capturing our target patient population . three main metrics were developed to align with aabb rbc transfusion guidelines and a restrictive transfusion strategy , including orders with a pretransfusion hgb level greater than 8 g / dl , a posttransfusion hgb level of greater than 10 g / dl , and nonemergent two - unit rbc orders . nonemergent orders were defined as orders entered into cpoe , but orders that selected active bleeding or acute cardiovascular ( cv ) insufficiency as the transfusion indication were excluded . in april 2015 , an official patient blood management program was established at our institution and a patient blood management safety coordinator was hired . an electronic database was established to obtain rbc orders entered into cpoe electronically on a daily basis and were reviewed in real time . the hospitalist group , internal medicine resident staff , and family medicine resident staff were determined to have the highest order frequencies at our institution , respectively . individual data for each of these departments and individual provider ordering information were obtained , graphed , and presented to each department . each individual provider was given their personal transfusion practice relative to the established metrics , as well as the data for their own practice group . in addition , information was provided containing aabb rbc transfusion guidelines including verbal presentation and laminated pocket cards . following this , the rbc transfusion orders were reviewed and email communications sent to the ordering provider when outside of metric indicator . if the provider was a resident physician , the email was also sent to the patient 's attending physician of record . copy of the aabb guidelines and review article regarding restrictive transfusion strategy was attached to all email communications . after six months , the data comparison before and after program metrics was represented to each individual group . in april 2016 , cpoe rbc transfusion alerts were activated . the alert is actionable , thereby allowing the ordering provider to continue the original order , cancel the order , or change the number of rbc units ordered for transfusion . the alert notifies the ordering provider that the order would likely be outside of aabb rbc transfusion guidelines and a restrictive transfusion strategy . alert firing is based upon the order indication and pretransfusion hgb , defined as most recent hgb level within 24 hours of rbc order . possible choices for rbc indication include active bleeding with hemodynamic instability , acute cv insufficiency , an hgb level less than 7 g / dl in stable patients , an hgb level less than 7.5 g / dl in stable icu patients , an hgb level less than 8 g / dl in non - icu patients with symptomatic anemia , an hgb level less than 8 g / dl in oncology patients with symptomatic anemia , and renal failure with symptomatic anemia . alerts were developed in an attempt to target transfusion scenarios mirroring aabb 2012 guidelines for stable inpatients and limit provider alert fatigue . no alert will fire if the pretransfusion hgb level is less than 6 g / dl . since chronic renal failure patients and those undergoing chemotherapy are often transfused based upon clinical findings , no alert will fire if hgb level was less than 8 g / dl with oncology or renal failure diagnosis . if the provider selects active bleeding or acute cv insufficiency , then no alerts will fire . when patients have active bleeding , the pretransfusion hemoglobin available in cpoe may not be reflective of the current clinical state or hemodynamic status . available patient electronic medical records of orders that list active bleeding as the indication are reviewed retrospectively and email communications are sent to ordering providers in which the indication does not seem to fit the available information . in addition , aabb could make no transfusion recommendations for patients with acute coronary syndrome . otherwise , the alert will fire under the following conditions : one or more rbc units ordered for transfusion and an hgb level greater than 7 g / dl without selected indication of active bleeding , acute cv insufficiency , oncology diagnosis , or renal failuretwo or more rbc units ordered for transfusion and an hgb level between 6 and 7 g / dl without selected indication of active bleeding , acute cv insufficiency , oncology diagnosis , or renal failureone or more units ordered and a pretransfusion hgb level greater than 10 g / dl with selected indication of oncology diagnosis or renal failuretwo or more units ordered and a pretransfusion hgb level between 8 and 10 g / dl with selected indication of oncology diagnosis or renal failure one or more rbc units ordered for transfusion and an hgb level greater than 7 g / dl without selected indication of active bleeding , acute cv insufficiency , oncology diagnosis , or renal failure two or more rbc units ordered for transfusion and an hgb level between 6 and 7 g / dl without selected indication of active bleeding , acute cv insufficiency , oncology diagnosis , or renal failure one or more units ordered and a pretransfusion hgb level greater than 10 g / dl with selected indication of oncology diagnosis or renal failure two or more units ordered and a pretransfusion hgb level between 8 and 10 g / dl with selected indication of oncology diagnosis or renal failure prior to the provider education program , the average rate in 2014 ( 12-month period ) for rbc orders with a pretransfusion hgb level greater than 8 g / dl was 16.64% and a posttransfusion hgb level greater than 10 g / dl was 14.03% and for nonemergent two - unit rbc orders was 45.26% . from january through march of 2015 , efforts were made to hire a patient blood management safety coordinator . after the initial 12 months of the program ( april 2015 through march 2016 ) , provider education resulted in the following metrics : the pretransfusion hgb level greater than 8 g / dl was reduced to 6.36% ( 62% reduction , p value < 0.001 ) , the posttransfusion hgb level greater than 10 g / dl was reduced to 3.78% ( 73% reduction , p value < 0.001 ) , and number of nonemergent two - unit red blood cell orders was reduced to 22.66% ( 50% reduction , p value < 0.001 ) . compared to 2014 , the total patient volume increased by 11% during the first 12-month period of the program ( 26430 annual adult inpatient admissions increased to 29404 ) . total percentage of inpatients transfused per 1000 patient days over the 12-month period decreased from 12.4% average in 2014 to 10.84% by march 2016 ( 13% reduction ) . the number of patients transfused per 1000 patient days decreased from 24.23% in 2014 to 21.26% ( 12% reduction ) . during this 12-month time period , the estimated rbc product acquisition cost savings are $ 130,000 . during the first 6 months of the program ( april through september ) , there were 2925 rbc orders in cpoe , 784 rbc transfusion orders reviewed , and 117 email communications sent ( 15% of orders reviewed received email communications ) . during the second 6-month period ( october through march ) , there were 2953 rbc orders in cpoe , 709 orders reviewed , and 81 e - mail communications sent ( 11% of orders reviewed ) . the percentage during months 10 through 12 ( january through march ) , email communications were sent to 8% of orders reviewed . in april 2016 , cpoe rbc alerts were activated . during the first 2-month period after implementation of cpoe rbc transfusion alerts , a flaw in the computer algorithm was in place and later corrected for june 2016 . during the first 4-month period after corrected implementation of cpoe alerts ( june through september 2016 ) the order continued as a two - unit order 39 times ( 22% ) , the transfusion order was changed to one rbc unit 119 times ( 68% ) , and the order changed to no rbc units 14 times ( 8% ) ( figure 1 ) . following implementation of corrected cpoe alerts for four months , rate for a pretransfusion hgb level greater than 8 g / dl was 6.1% , a posttransfusion hgb level greater than 10 g / dl was 4.47% , and nonemergent two - unit orders was 16.67% . there was no significant additional reduction in nonemergent two - unit rbc orders with implementation of cpoe alerts ( figure 2 ) . however , of the 119 patients who had an order changed from two units to one unit , 84 patients ( 71% ) did not receive any additional rbc units within 48 hours . the other 35 patients ( 29% ) , on average , received one additional rbc unit within 48 hours of the original order alert . the cpoe alerts prevented at least 98 rbc units ( 84 times two - unit order changed to one unit with no additional units ordered with 48 hours and 14 times when the rbc order was cancelled ) from being transfused within a 4-month time period . this alone is an acquisition cost savings of $ 16,600 . during this same time period , 490 rbc transfusion orders were reviewed with 36 email communications sent to ordering providers ( 7% of orders reviewed , 1.7% of total cpoe rbc orders ) . the case mix by patient location of the prestudy group ( figure 3 ) and that of study group ( figure 4 ) patient populations are similar . the prestudy group included 5963 cpoe rbc transfusion orders : 3264 ( 54.7% ) from acute care locations , 2011 ( 33.7% ) from critical care locations , and 688 ( 11.5% ) from other locations . the study group included 9331 cpoe rbc transfusion orders : 4897 ( 52.5% ) from acute care locations , 3135 ( 33.6% ) from critical care locations , and 1299 ( 13.9% ) from other locations . in addition , the distribution of male and female patients among locations within the study groups is similar ( table 1 ) . previous studies have shown a reduction in inappropriate transfusion by various intervention techniques [ 914 ] . prior to the aabb 2012 rbc transfusion guidelines , in addition , many studies do not report case mix of prestudy and poststudy patient groups . in 2002 , wilson et al . reviewed nine published articles reporting effectiveness of interventions to reduce inappropriate transfusion . while the definitions for inappropriate transfusion varied and the authors used a variety of intervention techniques , wilson et al . concluded that there is adequate evidence to support behavioral interventions to decrease inappropriate transfusion practice among health care providers . in 2005 , tinmouth et al . reported similar conclusions after reviewing 19 published studies on behavioral interventions and physicians ' transfusion practices . in our program , provider education utilizing aabb 2012 rbc transfusion guidelines was implemented as a component of the overall strategy to decrease inappropriate rbc transfusion . in order to accomplish this goal , a program coordinator was needed to help implement the process and for data retrieval and review . while hiring personnel during the current economic climate was challenging , preliminary data demonstrating the proposed metrics and current state in 2014 were presented , along with financial data , to hospital senior management . only a 5% reduction in inpatient rbc utilization would be cost neutral in hiring the coordinator . during the first 12-month period of the program , a 13% reduction in the target population was achieved , far exceeding the cost of the program coordinator . the institution also greatly benefited from the additional activities of the coordinator who was also actively involved in patient safety , review of reported potential adverse transfusion reactions , review of transfusion policies / procedures , review of training modules for nursing staff , performing direct observations of blood administration , providing educational lectures / materials to ordering providers / nursing staff , assisting with implementation of clinical pathways involving transfusion , and serving as transfusion resource throughout the institution . we implemented real - time provider communication and feedback via email , immediately following educational lectures . handouts containing aabb 2012 rbc transfusion guidelines and literature regarding the restrictive rbc transfusion strategy were distributed throughout the hospital system . following one year , we implemented cpoe alerts in attempt to further decrease inappropriate use and to help sustain the educational efforts . however , they did report that , with prospective physician notification for potential modification , they achieved an approximate 33% decrease in rbc usage from baseline over a 3-year period without evidence of change in mortality . yerrabothala et al . in 2014 reported that the use of prospective computerized order auditing ( cpoe ) alone significantly reduced number of rbc units transfused per 1000 patient - days ( 60.8 to 44.2 , p < 0.0001 ) . they also noted that the proportion of two - unit rbc orders decreased from 47% to 15% . zuckerberg et al . in 2015 reported a provider education program which was followed by cpoe algorithms ( clinical decision support ) . they noted that , with education alone for all surgical services combined , rbc utilization decreased by 16.4% . however , with cpoe algorithms the overall decrease was 14.3% . they report that adding cpoe and clinical decision support did not further reduce rbc utilization and suggested education as an effective measure to reduce rbc utilization . rbc transfusions that occurred during emergent transfusion , active bleeding , or during surgery were excluded , aligning with aabb 2012 rbc transfusion guidelines for stable adult inpatients . we initially changed the cpoe order to default to one unit and required multiple additional steps to complete an rbc order for more than one unit . the educational efforts showed the most dramatic reductions in inappropriate rbc transfusion ; however , the cpoe alerts were implemented following one year of educational efforts . with the recent implementation of cpoe within our system and concerns of alert fatigue , the educational program was initiated prior to the activation of alerts . with this strategy , the alerts are likely now more meaningful to the ordering providers and more directed at providers resistant to change following the educational program . in addition , the education program and cpoe alerts decreased the number of orders triggering manual review for appropriateness . while the implementation of cpoe alerts did not show additional reduction in our nonemergent two - unit rbc orders , we feel they are important to help encourage and maintain a restrictive transfusion strategy following the education efforts . this is supported by the fact that over 70% of the patients who had an order changed from two units to one unit following a cpoe alert did not receive any additional rbc units within 48 hours of the order alert . , aabb proposes an expanded two - tier recommendation for most inpatients , but states evidence supports transfusion decision to be within the clinical context and to also consider patient preferences and available alternatives to transfusion . the updated guidelines recommend restrictive transfusion strategy with transfusion not indicated until an hgb level less than 7 g / dl in hospitalized adult patients who are hemodynamically stable , including those who are critically ill . aabb recommends a restrictive threshold of 8 g / dl for patients undergoing orthopedic surgery and cardiac surgery and those with preexisting cv disease . they do state that restrictive transfusion threshold is likely comparable between 7 and 8 g / dl . again , aabb states that the recommendations do not apply to acute coronary syndrome patients , as well as to patients with severe thrombocytopenia and chronic transfusion - dependent anemia due to insufficient available evidence . aabb states that rbc units can be selected at any point of their licensed dating period and does not recommend limiting selected patient populations to only fresh ( less than 10 days ) rbc units . as with all areas of medicine , the field of transfusion is evolving . with the updated 2016 aabb rbc guidelines , the cpoe alerts can be further fine - tuned to incorporate the most recent evidence - based recommendations . while cpoe alerts were activated based upon 2012 aabb guidelines , the updated guidelines state that the clinical context is important in the decision to transfusion . active review of the cpoe alert orders supports that the ordering provider 's judgment within the clinical context still plays an important role in the care of patients . therefore , future studies and recommendations may include additional parameters to better access oxygenation and allow for more streamlined transfusion decision - support algorithms . we have a relatively large sample size of prestudy and study group rbc transfusion orders . our overall case mix of acute care / critical care patients and male / female ratios are very similar in both groups . we were able to define a specific target group of stable adult inpatients which directly mirror the patient population of the 2012 aabb guidelines . our cpoe alert definitions not only were developed consistently with 2012 aabb guidelines , but also demonstrate consideration of ordering provider fatigue that could potentially decrease the effectiveness of the alerts . we also developed a metric of a posttransfusion hgb level greater than 10 g / dl in order to not only monitor the pretransfusion trigger but also support restrictive posttransfusion target strategy . however , we could not define the patient population case mix stratified by comorbidities which potentially could have an impact on transfusion practice . while aabb guidelines for platelet transfusion are available , our study only addresses rbc transfusion practice and our patient population only includes stable adult hospitalized patients . provider education is an effective and low - cost method and should be considered as a first - line method to reduce inappropriate red blood cell transfusion rate in hospitalized adult patients . following an effective education program , alerts in cpoe may not further decrease inappropriate transfusion significantly but may help to encourage and maintain restrictive transfusion strategy following provider education .

vanadium is actually known as a trace element , essential for higher organisms although deficiency symptoms in humans have not yet been clearly identified ( guevara 1996 ) . trapping free oxygen radicals that might cause cardiac problems , it acts as a biological antioxidant and shows antitumoral activity ( scior et al 2005 ) . bis - maltolato - oxovanadium(iv ) ( bmov ) is under investigation in other pathological processes induced by uric acid , such as vascular endothelial dysfunction ( jindal et al 2007 ) . the development of innovative vanadium compounds against diabetes mellitus ( dm ) has attracted much pharmaceutical interest during the last decades ( saatchi et al 2005 ) . in the light of desired clinical advantages of a novel orally administered drug with selective insulin - like effects , a considerable impact on the patients health , their insulin injection - related long - term complications , or reduced side effects compared to other oral antidiabetics , can be expected ( zhang et al 2008 ) . besides our review with critical details on the protein tyrosine phosphatase ( ptpase ) inhibition , the pharmacodynamical behavior of organic vanadium compounds , their complex stabilities and inconclusive role as prodrugs for simpler inorganic vanadium ions in combination with toxicity data , it is noteworthy here to add newer reports on pharmacological findings ( scior et al 2005 ) . in 2007 the action mechanism on a molecular level was studied in bmov - treated cell cultures . it was confirmed that bmov enhanced the tyrosine - phosphorylation of upstream insulin - signaling proteins including the insulin receptor ( bose et al 2007 ) . heinemann and colleagues ( 2003 ) examined the pharmacokinetics of insulin mimetic inorganic vanadium ions : mainly vanadate ( + v , 80% ) and other oxidation states . their study in healthy humans after administration of a single intravenous dose revealed that the apparent volume of distribution was of the order of 0.6 l / kg . it is responsible for the typical heavy metal accumulation of vanadium in tissues such as liver , kidneys , bone , and spleen ( heinemann et al 2003 ) . most recently , another pharmacokinetics study in rats evaluated positively the therapeutical potential of bmov as an oral glucose - lowering drug candidate . bmov was rapidly absorbed , widely distributed in various tissues but only slowly eliminated from plasma due to accumulation to a greater extent in bone tissue . significant decrease of glucose concentrations in plasma was observed in hyperglycemic diabetic rats ( zhang et al 2008 ) . among the protein - tyrosine phosphatases , ptp1b has received much attention due to its role as a negative regulator of insulin signaling ( elchebly 1999 ; cheng et al 2002 ) . on one side the protein tyrosine phosphatase-1b ( ptp-1b ) is highly expressed in insulin - resistant persons . the absence of ptp-1b in genetically modified mice , on the other side , immunizes them against obesity and diabetes , even with rich and fatty diets . clearly , a selective control of the biological function of ptp1b is a challenging task , and an inhibitor must not only efficiently bind to the specific target enzyme but also do so without impeding the catalytic behavior of closely related enzymes . in figure 4 of our review we have already reported on the alignment studies to elucidate the regiospecificity of ptp1b ( scior et al 2005 ) . the design of small molecule ptp1b inhibitors to treat type ii diabetes has received considerable attention ( groves et al 1998 ; wang et al 2000 ; shen et al 2001 ; doman et al 2002 ; patankar and jurs 2003 ; sun et al 2003 ; szczepankiewicz et al 2003 ) . vanadium compounds have been known to mimic the effects of insulin by inhibition of tyrosine phosphatases . based on such observations that vanadate ( oxo complexes of vanadium with oxidation state + v ) electronically and structurally imitates phosphate , and phosphorylated proteins constitute the substrates of ptp-1b , vanadium - coordinated compounds should be capable of blocking ptp-1b upon competitive replacement of the natural substrates . this is one of the possible hypotheses of the reaction mechanisms for vanadium compounds with insulin - like activities ( scior et al 2005 ) . in addition , more recent papers explore these mechanisms in further detail ( zborowski et al 2005 ; adachi et al 2006 ) . the discovery of the insulin - like activity ( insulin - mimetics ) of vanadate ( [ vo4 ] or [ h2vo4 ] ) ( shechter and karlish 1980 ) , has ushered in a new era of antidiabetes research with new oxovanadium coordination complexes with inhibitory activities against protein - tyrosine phosphatases ( ptp-1b ) ( huyer et al 1997 ) , albeit not selectively . the next generation compounds have been based on organic vanadium complexes in search of higher target selectivity or less toxicity . however , some vanadium compounds have a great plethora of intrinsic enzyme affinities because of their phosphate - like physicochemical behavior : bio - isosterism ( scior et al 2005 ) . hence , it is of paramount importance whether the insulin - mimetic effect can be dissociated from the unwanted side effects based on phosphate imitation ( scior et al 2005 ) . the insulin - mimetic activity of certain vanadium coordination compounds has been well documented ( thompson and orvig 2000 ) , in particular , the improved glucose uptake and biosynthesis of glycogen as well as the inhibition of gluconeogenesis or lipolysis ( shechter et al 1998 ) . in addition , specific biomolecular targets regarding the aforementioned effects have been identified as protein phosphatases ( posner et al 1994 ; crans and tracey 1998 ; cuncic et al 1999 ; tracey 2000 ) , whereas neither insulin receptor nor glut2/glut4 pathways are directly concerned as targets ( goldwaser et al 2000 ) . in particular , bis - maltolato - oxovanadium(iv ) ( bmov or [ vo(ma)2 ] with central atom = v4 + ; and ma = monoanion maltolate ) has one of the highest antidiabetic activities among the known insulin - mimetics with vanadium coordination . syntheses in nonaqueous solvents always yield trans - bmov , while in water trans - and cis - aquo forms of bmov are present due to interconversion ( figure 1 ) . hence , the exact molecular mechanism of bmov still remains unknown ( liboiron 2005 ) . the work also aims at studying the rotational barrier with its transition state because so far , no crystal structure of bmov bound to ptp-1b has been reported ( pdb last visited august 8th , 2008 ) . intriguingly , in 2003 a report presents the binding of a trigonal - bipyramidal vanadium oxide to the active site of ptp-1b although bmov was incubated with the enzyme at the beginning of the crystallization ( peters et al 2003 ) . thus , whether bmov would constitute a sort of prodrug ( with an inorganic oxovanadium complex as its bioactive form ) or bmov itself might reach the binding cleft ( katherine et al 2006 ) , constitutes the scientific motivation for the present in silico simulations combining geometry prediction of the ligand and its docking into the suggested target . in this context we devised ab initio calculations to elucidate bmov geometries and related structures . the overall goal was to gain more insight about its possible bioactive structure by modeling an amenable binding mode of bmov and related oxovanadium complexes applying adapted force field techniques from the literature . the energy minimization and unbiased , manual docking of vanadium complex into the enzyme ptp-1b was conducted in five steps on a sgi 02 work station with 126 mb ram ( silikon 2001 ) . in general , cundari and colleagues had already demonstrated good modeling accuracy using semiempirical quantum chemistry and even molecular mechanics mm2 force fields ( cundari et al 1997 , 1998 ) . other authors applied quantum chemical methods ( zborowski et al 2005 ) . since both have been used successfully , we combined them in our joint study . we adapted the available tripos force field ( ff ) ( sybyl ; tripos inc . , st . louis , mo , usa ) and validated it against cundari s test series , which also contains cis - and trans - bmov ( table 1 ) . the brookhaven pdb database ( berman et al 2000 ) was searched for crystal structures of ptp-1b in active conformation ( the open state ) with docked ligands reflecting some structural patterns : tetraoxo - complexes or aromatic wings ( groves et al 1998 ) . the atoms of the target proteins were loaded with kollman_united pre - calculated charges ( tripos inc . ) while the charges of vanadium ligands were derived from our ab initio calculations ( gaussian 1998 ) . the geometry optimization was tailored to model the aqueous medium in a simplified approach by simulating the dumping effects of solvent molecules on electrostatic interaction in the binding groove : distance - dependent dielectric constant ( = 40 ) ; nonbonded cut off at 8 ; conjugate gradient algorithm : powell , and terminal criteria : energy change : of 0.2 kj / mol ( tripos inc . ) . the final docked poses were compared to tungstate ( wo4 sodium salt ) bound to human ptp-1b ( barford et al 1994 ) . calculations of the cis - trans conversion were carried out on two dell - computers ( dimension 2400 , with pentium iv 2.8 ghz processors , 256 mb ram , and 40 gb hard discs ) , using the gaussian 98 electronic structure software ( gaussian , inc . , wallingford ct , usa ) , and the chemoffice 2000 program ( cambridgesoft , cambridge , ma , usa ) to visualize the results . in order to draw electronic - density and electrostatic - potential graphics , the arguslab software has been used ( arguslab 2005 ) . together with the chemoffice program , both bmov structures have been generated at the b3lyp ( becke 1993 ) dft level , choosing a quadratic convergence criterion ( scf = qc ) . with these initial geometries , sto-3 g single - point energy calculations have been performed , yielding some reference parameters of both species . subsequently , a stepwise optimization of both chemical structures has been carried out by using the following basis sets : sto-3 g , 3 - 21 g , 3 - 21 g * , and 6 - 31 g . having terminated the optimization process , frequency calculations for each structure characterized the corresponding energy minimum type ( ie , a global , local or intermediate minimum ) . considering the biological activity of bmov in addition , dft calculations using the b3lyp/6 - 311 g * approach ( gaussian 03 program system ; gaussian inc . ) were carried out for trans- and cis - bmov as well as for the transition state connecting the both isomers , in order to determine the relative stabilities of these structures . to postulate a viable mechanism one has to cope with a principle obstacle : while cis - aquo - bmov is the observed active species , the isomeric trans - bmov is its administered solid form . the first part of our study simulates the blocking of ptp-1b by either cis - or trans - bmov . the calculated active configuration ( cis or trans ) is thought to suppress the intracellular insulin effects , therein assisting the cells in need of an adequate insulin response . to avoid blind docking , we conducted a pdb search for the most related crystal structure to bmov and found a tetraoxo analogue to vanadate , called tungstate . it binds to the active site of the protein tyrosine phosphatase1b ( pdb code : 2hnq ) ( figure 2 ) . applying the adapted tripos ff ( tripos inc . ) , the geometric structures of the bmov and of some related complexes ( cundari et al 1997 , 1998 ) were predicted correctly ( table 1 ) . prior to the unbiased docking of bmov into the ptp-1b targets ( pdb codes : 1bzc , 1bzh ) ( groves et al 1998 ) , the ligands were removed . in good agreement with reported data , trans - bmov meets steric hindrance whatever the docked poses around the central atom position . but for the central atom of cis - bmov , the computed pose is very similar to that described for tungstate . the final coordinates of the central atom on bmov and of inorganic vanadate resulted in practically identical coordinates as compared to the crystallographic position of tungstate ( pdb code : 2hnq ) ( barford et al 1994 ) : w in [ wo4 ] : x = 1.1596 , y = 0.0977 , z = + 9.2017 cf . v in [ vo4 ] : x = 1.5147 , y = 0.8434 , z = + 10.3426 . for validation purpose , tungstate was successfully docked back into its crystallographic position using the same force field conditions ( cf . this successful validation of the test system evidences that steric and electrostatic forces mainly control this anion binding . it is noteworthy that hydrophobic and solution effects ( entropy ) are not quantified during manual docking in the tripos ff . notwithstanding , the nonbonded electrostatic and van der waals terms were combined in a qualitative manner with the insight obtained from other docking tools ( ludi , flexx , autodock ) ( coupez and lewis 2006 ) . for instance , in autodock with it s a(romatic ) atom type and solvation descriptors for hydrophobic effects , - and cation- interactions of neutral bmov were recognized even without a set of vanadium parameters at hand ( castellano et al 2003 ) . the calculated energy ( interpreted with precaution for its limited parameterization for bonded and nonbonded electrostatic / steric terms ) for docked cis - bmov was 136 kj / mol , which lies in good proximity to the peptide ligands found in 1bzc and 1bzh after crystal strain was released by full geometry optimization ( groves et al 1998 ) : 179 kj / mol and 203 kj / mol , respectively . the bioactivity pattern of bmov is a hybrid : on the one side , it has aromatic contacts in common with the peptide ligands , on the other side it establishes the typical hydrogen bon network of other nonpeptide small ligand complexes through the many oxo groups ( figure 3 ) . figure 8 is a schematic drawing of the binding - relevant amino acids ( pharmacophore ) : tyr46 , phe182 , ser216 , ala217 , gly218 , ile219 , gly220 , and arg221 . particularly , the residues ser216 , ala217 , gly218 , ile 219 , gly220 , and cationic arg221 participate in binding of [ wo4 ] as an inorganic tetraoxo - monoanion whereas some of them ( ser216 , ala217 , gly220 , arg221 ) and others ( anionic asp181 , aromatic phe182 and tyr46 ) interact with formally uncharged , aromatic cis - bmov . moreover , a - interaction can be observed between one maltolate ring on bmov with tyr46 ( figure 3 and 8) . in addition , positively charged arg221 attracts the other maltolate anion in a typical cation- interaction ( castellano et al 2003 ) . since both identical maltolate rings on docked bmov enhance binding strength , but in two different ways this wing-symmetry can be broken up in future drug profiling in search of higher binding selectivity . in this respect , we had already aligned the ptp-1b against other phosphatase family members and have reported the results of conserved and unique amino acids at the binding site elsewhere ( scior et al 2005 ) . as a final step in docking to confirm our working hypothesis , cis- and trans - bmov there is computed evidence that cis - aquo - bmov can be favorably placed into the active site of the target enzyme , while trans - bmov can not enter there due to energetically high barriers ( figure 3 ) . due to the absence of imaginary frequencies , the ab initio optimization process for trans - bmov yielded a global minimum ( figure 4 ) on the potential energy surface . moreover , comparing both optimization results with and without solvent effects , respectively , we observed only marginal deviations . thus , we consider this structure to be stable . with respect to the cis - aquo - bmov geometry , generated by stepwise single - point energy optimization , we obtained two structures : the first one , termed closed - type cis - aquo - bmov theoretically , this structure represents a local minimum , due to its three imaginary frequencies , which might stem from a geometrical distortion generated by the water ligand ; taking into consideration the solvent effect , this structure remains stable , too . the second structure ( the stepwise optimization result using the 6 - 31 g basis set ) is the so - called open - type cis - aquo - bmov ( figure 6 ) . showing the same energy as closed - type cis - aquo - bmov , this structure represents a global minimum , since no imaginary frequencies occurred . the solvent interaction option confirms that open - type cis - aquo - bmov can be considered being stable throughout , with a slight tendency towards the aqueous phase . energy values obtained for the three species indicate , that in aqueous solution and room temperature ( ie , under physiological conditions ) the conversion from trans - bmov to cis - aquo - bmov is thermodynamically possible . hereby seems to be the stable intermediate in kinetic equilibrium with the closed - type cis - aquo - bmov form . to find out which of the two species interacts with the enzyme we determined both corresponding electrostatic potential surfaces by means of the arguslab program ( arguslab 2005 ) . the result confirms that closed - type cis - aquo - bmov shows a negative electrostatic potential of higher absolute value at the vanadium position and its close environment ( figure 7 ) . open structure , with higher affinity to the catalytic center of the enzyme ptp-1b . finally , the geometries of trans- and cis - aquo - bmov ( open - type ) were optimized at the b3lyp/6 - 311 g * level of theory . subsequent frequency calculations demonstrated , that both forms represent minima , ie , stable structures , on the energy hypersurface . the cis isomer is lower in energy by 5.6 kj / mol as compared with the trans - bmov , thus being the global minimum . the search for a transition state connecting the both forms along the reaction coordinate resulted in a structure possessing one imaginary frequency , ie , a first order saddle point , being higher in energy by 21.1 kj / mol with respect to the global minimum . to avoid blind docking , we conducted a pdb search for the most related crystal structure to bmov and found a tetraoxo analogue to vanadate , called tungstate . it binds to the active site of the protein tyrosine phosphatase1b ( pdb code : 2hnq ) ( figure 2 ) . applying the adapted tripos ff ( tripos inc . ) , the geometric structures of the bmov and of some related complexes ( cundari et al 1997 , 1998 ) were predicted correctly ( table 1 ) . prior to the unbiased docking of bmov into the ptp-1b targets ( pdb codes : 1bzc , 1bzh ) ( groves et al 1998 ) , the ligands were removed . in good agreement with reported data , trans - bmov meets steric hindrance whatever the docked poses around the central atom position . but for the central atom of cis - bmov , the computed pose is very similar to that described for tungstate . the final coordinates of the central atom on bmov and of inorganic vanadate resulted in practically identical coordinates as compared to the crystallographic position of tungstate ( pdb code : 2hnq ) ( barford et al 1994 ) : w in [ wo4 ] : x = 1.1596 , y = 0.0977 , z = + 9.2017 cf . v in [ vo4 ] : x = 1.5147 , y = 0.8434 , z = + 10.3426 . for validation purpose , tungstate was successfully docked back into its crystallographic position using the same force field conditions ( cf . this successful validation of the test system evidences that steric and electrostatic forces mainly control this anion binding . it is noteworthy that hydrophobic and solution effects ( entropy ) are not quantified during manual docking in the tripos ff . notwithstanding , the nonbonded electrostatic and van der waals terms were combined in a qualitative manner with the insight obtained from other docking tools ( ludi , flexx , autodock ) ( coupez and lewis 2006 ) . for instance , in autodock with it s a(romatic ) atom type and solvation descriptors for hydrophobic effects , - and cation- interactions of neutral bmov were recognized even without a set of vanadium parameters at hand ( castellano et al 2003 ) . the calculated energy ( interpreted with precaution for its limited parameterization for bonded and nonbonded electrostatic / steric terms ) for docked cis - bmov was 136 kj / mol , which lies in good proximity to the peptide ligands found in 1bzc and 1bzh after crystal strain was released by full geometry optimization ( groves et al 1998 ) : 179 kj / mol and 203 kj / mol , respectively . the bioactivity pattern of bmov is a hybrid : on the one side , it has aromatic contacts in common with the peptide ligands , on the other side it establishes the typical hydrogen bon network of other nonpeptide small ligand complexes through the many oxo groups ( figure 3 ) . figure 8 is a schematic drawing of the binding - relevant amino acids ( pharmacophore ) : tyr46 , phe182 , ser216 , ala217 , gly218 , ile219 , gly220 , and arg221 . particularly , the residues ser216 , ala217 , gly218 , ile 219 , gly220 , and cationic arg221 participate in binding of [ wo4 ] as an inorganic tetraoxo - monoanion whereas some of them ( ser216 , ala217 , gly220 , arg221 ) and others ( anionic asp181 , aromatic phe182 and tyr46 ) interact with formally uncharged , aromatic cis - bmov . moreover , a - interaction can be observed between one maltolate ring on bmov with tyr46 ( figure 3 and 8) . in addition , positively charged arg221 attracts the other maltolate anion in a typical cation- interaction ( castellano et al 2003 ) . since both identical maltolate rings on docked bmov enhance binding strength , but in two different ways this wing-symmetry can be broken up in future drug profiling in search of higher binding selectivity . in this respect , we had already aligned the ptp-1b against other phosphatase family members and have reported the results of conserved and unique amino acids at the binding site elsewhere ( scior et al 2005 ) . as a final step in docking to confirm our working hypothesis , cis- and trans - bmov were docked into ptp-1b . there is computed evidence that cis - aquo - bmov can be favorably placed into the active site of the target enzyme , while trans - bmov can not enter there due to energetically high barriers ( figure 3 ) . due to the absence of imaginary frequencies , the ab initio optimization process for trans - bmov yielded a global minimum ( figure 4 ) on the potential energy surface . moreover , comparing both optimization results with and without solvent effects , respectively , we observed only marginal deviations . thus , we consider this structure to be stable . with respect to the cis - aquo - bmov geometry , generated by stepwise single - point energy optimization , we obtained two structures : the first one , termed closed - type cis - aquo - bmov theoretically , this structure represents a local minimum , due to its three imaginary frequencies , which might stem from a geometrical distortion generated by the water ligand ; taking into consideration the solvent effect , this structure remains stable , too . the vacuum structure , however , seems to be slightly preferred . the second structure ( the stepwise optimization result using the 6 - 31 g basis set ) is the so - called open - type cis - aquo - bmov ( figure 6 ) . showing the same energy as closed - type cis - aquo - bmov , this structure represents a global minimum , since no imaginary frequencies occurred . the solvent interaction option confirms that open - type cis - aquo - bmov can be considered being stable throughout , with a slight tendency towards the aqueous phase . energy values obtained for the three species indicate , that in aqueous solution and room temperature ( ie , under physiological conditions ) the conversion from trans - bmov to cis - aquo - bmov is thermodynamically possible . hereby seems to be the stable intermediate in kinetic equilibrium with the closed - type cis - aquo - bmov form . to find out which of the two species interacts with the enzyme we determined both corresponding electrostatic potential surfaces by means of the arguslab program ( arguslab 2005 ) . the result confirms that closed - type cis - aquo - bmov shows a negative electrostatic potential of higher absolute value at the vanadium position and its close environment ( figure 7 ) . open structure , with higher affinity to the catalytic center of the enzyme ptp-1b . finally , the geometries of trans- and cis - aquo - bmov ( open - type ) were optimized at the b3lyp/6 - 311 g * level of theory . subsequent frequency calculations demonstrated , that both forms represent minima , ie , stable structures , on the energy hypersurface . the cis isomer is lower in energy by 5.6 kj / mol as compared with the trans - bmov , thus being the global minimum . the search for a transition state connecting the both forms along the reaction coordinate resulted in a structure possessing one imaginary frequency , ie , a first order saddle point , being higher in energy by 21.1 kj / mol with respect to the global minimum . the goal of the present in silico study is to theoretically address the missing explanation of a trans - cis conversion of bmov . to our best knowledge , it generates the first molecular binding mode model concerning the insulin - mimetic activity of bmov . it also elucidates the stereochemical requirements of bmov as an inhibitor to the biomolecular target site . in aqueous solution open - type cis - aquo species of bmov , which itself is in equilibrium with another closed - type form of cis - aquo - bmov . the postulated binding mode for bmov , ie,closed - type cis - aquo - bmov complex , is fully consistent with in vivo studies on bmov activity with respect to enzyme activity of ptp-1b : the group of mcneill reported that bmov treatment reduced the activity of ptp i b by 25% in fatty treated rats ( mohammad et al 2002 ) . our present work may lead to the detection of the activated complex upon forming a complex of cis - bmov with a h2o moiety . our report may help other researchers to devise new calculations concerning this activated complex . to this end our modeling input and output data can be obtained by email . in conclusion , we contribute with computed solutions to better understand the molecular recognition process and selectivity of nonpeptide insulin - mimetic vanadium complexes . to complete the picture , on a theoretical level it can not be discarded that vanadium coordinated organic compounds would also be prodrugs after re - arrangements with stronger chelators and following redox - reactions . thus , whether bmov or a simple ( inorganic ) tetraoxo compound is the final bioactive species acting on target structure remains an open question and disserves further investigation

ellis - van creveld syndrome ( evc , omim # 225500 ) , also known as chondroectodermal dysplasia , was first described by ellis and van creveld in 1940 . the syndrome is caused by a mutation in the two genes evc1 and evc2 , mapping both on chromosome 4p16 , in a head to head configuration . the disorder is commonly seen among the old order amish community of pennsylvania ( approximately five of 1,000 live births ) , but , today , the syndrome is known to affect all races . in the general population ellis - van creveld syndrome is a rare autosomal - recessive disorder affecting bone growth , for which minimally diagnostic criteria include short limb dwarfism , postaxial polydactyly of hands and feet , malformation of the wrist bones , hypoplastic fingernails , teeth dystrophy and congenital heart defect ( particularly atrioventricular canal with common atrium ) . the clinical report outlines the classical and unusual oral and dental manifestations , which will assist the oral health care providers in diagnosing the syndrome and timely referrals to appropriate health care professionals to prevent the patient from further complications of heart defect and bony deformity . an 11-year - old female patient reported to the department with the chief complaint of pain and mobility in the mandibular right posterior region since 1 month . family history revealed that the girl was the first child of consanguineously married and normally developed parents . she has a sister ( 9 years ) and a brother ( 7 years ) with no congenital abnormality . medical history revealed congenital heart defect ( single atrium ) , which was corrected at the age of 2 years . developmental history revealed that the patient started walking at the age of 3 years and the teeth started erupting at the same age . the patient was short statured ( 97 cm ) , which was relatively short for her age , and had a waddling gait . intraoral examination revealed carious primary mandibular right second molar , edentulous mandibular incisor region , absence of mandibular anterior mucobuccal fold , mandibular hyperplastic frenula and morphological abnormalities of maxillary and mandibular anterior teeth [ figures 2 and 3 ] . slight serrations of the alveolar ridge were observed distally to the maxillary central incisors [ figure 3 ] . panoramic radiograph showed missing permanent maxillary right lateral incisor and permanent mandibular central incisors , delayed formation of tooth buds of permanent maxillary right and left canines , first and second premolars and permanent mandibular left canines , first and second premolars [ table 1 ] . primary first molars and maxillary primary second molars were single rooted ; roots of primary mandibular second molars were conical and convergent . taurodontisum were present with all the first permanent molars [ figure 4 ] [ table 1 ] . intraoral periapical radiograph showed multiple small alveolar notches on the crest of the mandibular anterior alveolar ridge , giving a serrated appearance [ figure 5 ] . radiograph of the upper and lower limb showed shortening of the extremities , genu valgum , post - axial polydactyly , fusion of hamate and capitate bones and fusion of the 5 and 6 metacarpal bones [ figure 6 ] . based on the clinical and radiographic findings , we strongly suspected ellis - van creveld syndrome . thereafter , the patient was referred to the pediatrician for thorough medical examination to reveal any evidence of systemic involvement , such as congenital heart malformation , lung , liver , kidney and central nervous system disorders . her mental development was within the normal range and no systemic involvement was reported ; thus , confirming the diagnosis . hypoplastic fingernails of the hands intraoral photograph showing edentulous mandibular central incisor region , absence of mandibular anterior mucobuccal fold and hyperplastic frenula morphological abnormalities of maxillary and mandibular anterior teeth and serrations of the alveolar ridge comparison of oral manifestation of ellis - van creveld syndrome in the literature with the patient from this report panoramic radiograph showing missing teeth , delayed formation of tooth buds , single - rooted primary molars and taurodontisum radiograph showing multiple small alveolar notches giving a serrated appearance radiograph of the upper and lower limb showed shortening of the extremities , genu valgum , post - axial polydactyly , fusion of hamate and capitate bones and fusion of 5 and 6 metacarpals clinically , the mandibular right primary second molar was unrestorable . therefore , mandibular right primary second molar was extracted under local anesthesia using lignocaine hydrochloride ( dentocaine 2% pharma health care product , mumbai , mumbai , india ) and antibiotic coverage . to increase the mandibular anterior mucobuccal fold , the presentation of medically compromised and syndromic children in the dental office is a great challenge to the oral health care providers . ellis - van creveld syndrome is one of these syndromes , which is characterized by ectodermal disturbances and chondrodysplasia . ellis - van creveld syndrome presents phenotypic diverse oral manifestations of the soft tissues and teeth . these include hyperplastic frenula , absence of mucobuccal fold , serrations of the alveolar ridge , multiple small alveolar notches , partial cleft lip , neonatal teeth , peg - shaped laterals , partial anodontia , conical and microdontic teeth , enamel hypoplasia and delayed eruption of teeth [ table 1 ] . but , similar oral manifestations are also observed in other syndromes like jeune syndrome ( js ) , orofaciodigital syndrome ( ofds ) , mckusick - kaufman syndrome ( mkk ) and weyers acrofacial dysostosis ( wad ) . thus , we consider these syndromes in the differential diagnosis of ellis - van creveld . js is an inherited form of dwarfism that produces short limbs similar to ellis - van creveld . other symptoms the individuals of js may have and are absent in ellis - van creveld are small chest , renal dysplasia , retinal degeneration , liver dysplasia and intestinal malformation . in addition , individuals with ellis - van creveld have a finger nail hypoplasia , a finding generally absent in js . mkk syndrome , a recessively inherited disorder , can be distinguished from ellis - van creveld by the presence of hydrometrocolpos in the former , whereas was , an autosomal - dominant condition has ectodermal disturbances similar to ellis - van creveld but absence of other features like dwarfism , thoracic - dysplasia and congenital heart diseases . as all the clinical findings in the reported patient were favoring ellis - van creveld syndrome , the diagnosis was thus made . many new cases reported present a rare aberration in shape and structure of the teeth . in the present case , we noticed some unusual findings such as single - rooted and funnel - shaped primary first molars , single conical roots of primary second molars and taurodontisum [ table 1 ] . investigations have shown that dental anomalies like taurodontisum and single conical root forms of primary molars may develop in the presence of any one of a large number of different genetic alterations . these findings suggest that the chromosomal abnormalities may disrupt the development of the tooth 's form and that these anomalies are not the result of a specific genetic abnormality . thus , the additional findings of single conical root forms of primary molars and taurodontisum in the present case could possibly serve as diagnostic criteria for ellis - van creveld , but more often may lead to misdiagnosis if the more relevant oral and general findings are inconclusive . taurodontisum is a noted observation in ofds and so are the other oral features of ellis - van creveld . however , ofds can be differentiated from ellis - van creveld by the presence of cleft palate , trifurcation of soft palate , ocular hypertelorism , hypoplasia of alae nasi , polycystic kidney diseases and mild intellectual deformity ( 50% ) in the patients of ofds . single conical roots in relation to primary molars and taurodontisum were also reported by vinay and hunter , respectively . malocclusions are common in ellis - van creveld but are not of any specific type . the literature described hypoplasia of the anterior maxilla , prognathism of the mandible and increased height of the lower third of the face . in the present case report thus , the dental age of our patient does not correspond to the chronological age . unfortunately , 50% of the children with ellis - van creveld syndrome die in early infancy due to cardiorespiratory problems , and those who survive require a multidisciplinary approach for treatment , i.e. orthopedic correction of genuvalgum , amputation of extra digit , surgical repair of cardiac malformation and dental intervention . as there are many oral manifestations , there is no standard formula for dental treatment . treatment needs may include preventive measures against caries , restorations of decayed and malformed teeth , partial dentures with frequent adjustment and replacement and correction of malocclusion . children with ellis - van creveld syndrome have a high risk for caries due to malformed hypoplastic teeth and molars with deeps pits and fissures . therefore , the patient and the patient 's parents should be made aware of the importance of home - based preventive measures such as diet control , oral hygiene maintenance , daily fluoride mouth rinses and use of fluoridated denitrifies . simultaneously , preventive professional care such as pit and fissure sealants , oral prophylaxis and professional topical fluoride application must be provided . congenitally missing teeth , hypoplastic teeth and malocclusion can affect the speech , appearance and mastication of patients with ellis - van creveld syndrome . to maintain space and to improve speech , hypoplastic teeth and decayed teeth can be restored with composite restorations for better esthetics and preservation of tooth structure . parental and child counseling is often required to treat psychological trauma due to compromised oral manifestations and medical health . antibiotics prophylaxis is recommended for surgical patients with prosthetic cardiac valves , previous bacterial endocarditis , mitral valve prolapse with valvular regurgitation or thickened leaflets . for these patients , anti - staphylococcal antibiotics ( e.g. , cephalosporins ) should be given before surgery only , when the procedure involves significant risk of bacteremia ( e.g. , incision into infected tissues ) or as soon as the risk is recognized . the dental treatment must be performed under prophylactic antibiotic coverage with consideration for the high incidence of cardiac defects in ellis - van creveld patients . but , as in the present case , the defect was corrected at the age of 2 years , there was no previous history of bacterial endocarditis and the present cardiac examination was normal ( as reported by the pediatrician ) , there was no indication for antibiotic prophylaxis . however , to control the spread of local infection ( as indicated by the 2/3 pathologic root resorption of the carious primary mandibular right second molar ) , antibiotic coverage was provided . diagnosis and management of ellis - van creveld syndrome is a great challenge to the oral health care provider . because of the genetic and environmental phenotype - modifying factors in syndromes , the oral health care providers may come across some unusual oral and dental manifestations therefore , they should get updated with the latest knowledge to minimize the risk of misdiagnosis . to achieve satisfactory functional and aesthetic results , multidisciplinary treatment planning is required both in terms of preventing oral disease and in providing restorative care .

the common causes of gastric outlet obstruction include peptic ulcer disease , stomach cancer and gastrointestinal stromal tumor . impacted gallstone is a rare cause of intestinal obstruction and accounts for 1% to 3% of all intestinal obstructions.12 bouveret 's syndrome is a gastric outlet obstruction caused by an impacted gallstone that passes through a cholecysto - gastric or cholecysto - duodenal fistula.34 we present a case of bouveret 's syndrome which was successfully treated with endoscopic stone removal , followed by cholecystectomy , resection of the duodenal bulb and the distal stomach and gastro - jejunostomy . a 76-year - old woman visited a local clinic with a history of nausea and abdominal pain . a clinician found gastritis on endoscopy and a stone in the gallbladder on ultrasonography . to evaluate the gallstone , laboratory tests , including blood cell counts , liver enzymes , electrolytes and creatinine levels were within normal limits . abdominal computed tomography ( ct ) revealed a distended stomach , an irregular wall thickening at the first and second portions of the duodenum and a gallbladder with an enhancing mass in the cystic duct ( fig . 1 ) . there was a cholecysto - duodenal fistula with a 3-cm calcified stone in the duodenal lumen . magnetic resonance cholangiopancreatography revealed a mild intrahepatic duct dilatation and a mild narrowing of the confluence . positron emission tomography ( pet ) showed a slight increase in fludeoxyglucose uptake ( suv=3.9 ) in the cystic duct , which made it impossible to rule out the possibility of gallbladder cancer ( fig . 2 ) . esophagogastroduodenoscopy ( egd ) showed a 2.32.8-cm pigment stone causing gastric outlet obstruction . however , a cholecysto - duodenal fistula was not observed on egd due to the duodenal deformity . the gallbladder showed severe inflammation and adhesions to the duodenum , which had resulted in a cholecysto - duodenal fistula . after cholecystectomy , we resected the duodenal bulb along with the distal stomach and also performed gastro - jejunostomy . a percutaneous drainage tube was inserted into the anterior aspect of the stomach for removal of fluid accumulation on post - operative day 6 . in 1896 , bouveret reported 2 patients with gastric outlet obstruction due to an impacted gallstone.1 recurrent inflammation of the gallbladder frequently causes erosion and necrosis of the gallbladder wall and forms biliary - enteric fistulas . there are many types of fistulas according to the site of fistula formation , such as cholecysto - gastic , cholecysto - duodenal or cholecysto - colic fistula.34 fistulas are known to occur twice more frequently in women than in men.5 the symptoms of bouveret 's syndrome are not typical . nausea / vomiting is the most common symptom , followed by abdominal pain , abdominal distension , hematemesis , weight loss and melena . abdominal tenderness is commonly noted on physical examination.4 pneumobilia , a calcified mass or a distended stomach is frequently seen in plain abdominal radiographs . however , if a fistula tract is filled with fluid or air , it may be confused with the common bile duct . magnetic resonance cholangiopancreatography is a diagnostic tool that provides definitive imaging of cholecysto - duodenal fistulas . t-2 weighted magnetic resonance imaging shows intraluminal duodenal filling defects and cholecysto - duodenal fistulas.678 egd has diagnostic and therapeutic values . intracorporeal laser lithotripsy , electrohydraulic lithotripsy and extracorporeal shock wave lithotripsy as well as a manual endoscopic lithotripsy have recently been performed to treat patients with gallstones.910 even after successful endoscopic stone removal , surgery must be considered when there is biliary obstruction , remnant gallstone , cholecystitis or recurrent cholangitis . an one - stage operation is conducted to perform enterolithotomy , cholecystectomy and fistula repair simultaneously . a two - stage operation is considered when patients ' vital signs are unstable and consists of an enterolithotomy first and then delayed cholecystectomy with fistula repair.11121314151617 the mortality rate is reported to be higher in the one - stage operation than in the two - stage operation ( 20 - 30% vs. 12%).13 however , biliary complications are more frequent after enterolithotomy alone.15 therefore , therapeutic plans should be made based on the patient 's general condition . otherwise , gastric or duodenal resection and gastro - enteric anastomosis should be performed.111213 chronic inflammation of the gallbladder caused by cholecysto - enteric fistulas may induce malignant transformation . singer et al.18 and sanada et al.19 reported some cases of malignant transformation of bouveret 's syndrome . day and marks20 reported 2 cases of biliary - enteric fistulas associated with gallbladder cancer in 34 patients with gallstone ileus . in our case , malignancy was suspected because of the findings of an enhanced mass with increased fludeoxyglucose uptake in the cystic duct . however , there was no evidence of malignancy on histopathological examination .

multiple studies have shown that expression of tras by mtecs is critical for establishing self - tolerance to these antigens in the periphery ( 1 ) . in mice that express transgenes controlled by peripheral tissue - specific promoters , a scattered subset of mtecs express the transgenic antigen , and this expression is sufficient to tolerize the developing t cell repertoire to the antigen . the known mechanism of tolerance induction involves deletion of antigen - specific thymocytes ( 24 ) ; the effect of tra expression on other tolerance mechanisms , such as regulatory t cell development , remains to be determined . these studies also showed that the patterns of endogenous antigen expression in mtecs ( insulin , somatostatin ; c - reactive protein [ crp ] , serum amyloid p component [ sap ] ) is similar to the expression of transgenic antigens under the corresponding tissue - specific promoters ( rip - tag ; human crp ) . this shows clearly that tra expression by mtecs is a physiological phenomenon that is under endogenous transcriptional control . ( 5 ) showed that tra expression by mtecs encompasses a large set of antigens characteristic of a broad range of tissues , leading to the formulation of the notion of promiscuous gene expression as both a phenomenon and a mechanism for generating tolerance . when it was found that mutations in the gene encoding the transcriptional regulator aire ( autoimmune regulator ) were responsible for the development of a multiorgan autoimmune syndrome ( aps-1 or apeced ) in humans , it was suggested that aire might mediate tra expression in mtecs ( for review see reference 6 ) . indeed , analysis of aire mice , which have autoimmune activity targeted to multiple endocrine tissues , has shown that expression of some tras is reduced or absent in aire - deficient mtecs and that negative selection of thymocytes is impaired ( 7 ) . however , aire can not account for all promiscuous gene expression , as expression of many tras is unaffected in aire mice and some of the thymocyte selection defects observed in these mice are independent of tra expression levels ( 8) . the mechanism of aire activity has been attributed to direct transcriptional regulation , ubiquitin ligase activity , or unspecified derepression of chromatin ( 6 ) . however , a definitive molecular function for aire has not been determined yet , and therefore , the manner in which it controls thymic expression of a subset of tras remains speculative . the mechanism(s ) that may account for promiscuous gene expression by mtecs is difficult to define because mtecs themselves are difficult to define ( 9 ) . regarding mtec development , it can be said conclusively that mtecs are clonally derived , as shown by rodewald and colleagues ( 10 ) ; that development and maintenance of the highly heterogeneous medullary compartment of the thymus is dependent on reciprocal signals between mtecs and developing thymocytes ( referred to as cross - talk [ 9 , 11 ] ) ; and that the lymphotoxin receptor nf-b inducing kinase relb pathway is involved in both the induction of aire and the generation and/or maintenance of mtec heterogeneity , although whether this pathway regulates maturation , proliferation , or survival of mtecs is not clear ( 1214 ) . the identity and relationship of mtec subsets is also poorly defined ( 9 ) . although some molecules are broadly expressed by most mtecs ( such as cd80 , mhc class ii , ep - cam ) , others ( recognized by antibodies and lectins such as 8.1.1 , uea-1 , a2b5 , etc . ) are differentially expressed by mtec subsets and can be used to subdivide the broader mtec populations ( 5 , 9 ) . there has been no demonstration of precursor / progeny relationships among the various mtec subsets , and thus the maturation state , developmental potential , and homogeneity of these subsets is undetermined and open to speculation . ( see page 33 ) provide important clues to the regulation of tra expression by mtecs ( 15 ) . they describe a hierarchy of tra expression by subsets of mtecs that are defined by levels of expression of the costimulatory molecule cd80 , with the level of cd80 expression increasing as a function of mtec maturity . this correlation was noted in both aire - deficient and wild - type mtecs , emphasizing that aire - independent mechanisms play an important role in regulating tra expression . the results pertaining to the role of aire in this process will be addressed in more detail later . they also provide evidence that some of the genes encoding tras expressed by the cd80 mtec population are clustered together on chromosomes , suggesting potential epigenetic regulation of this process . whereas expression of tras in peripheral tissues is regulated by tissue - specific transcription factors , the authors propose that mature mtecs open regions of chromatin and express the clusters of genes in these open regions ( fig . the authors propose a modified version of the promiscuous gene expression model termed the terminal differentiation model in which promiscuous gene expression by mtecs is a specialized property that is attained upon terminal differentiation . models of the mechanisms resulting in tra expression by mtecs . ( a ) the terminal differentiation model . as a committed mtec progenitor ( p ) cell differentiates , it progressively expresses an increasing number of tras , starting at an immature ( i m ) stage and culminating in a terminally differentiated ( mature [ m ] ) cell that is characterized by high level expression of mhc class ii , cd80 , aire , and a broad spectrum of tras ( represented by colored circles ) . the role of aire in this model is undefined but is progressively manifested during mtec maturation . progenitors have access to a wide variety of lineage - specific transcriptional programs ( and the tras regulated by those programs ) before differentiation . as part of the differentiation process , each of these mature cells might express a peripheral lineage program ( i and iii ) , or might extinguish all peripheral regulators and express an undefined terminal mtec program ( ii ) . in the developmental model , mhc class ii and costimulatory molecules like cd80 would be inducibly expressed on mtecs throughout differentiation in response to cell cell or other extrinsic signals . we speculate that aire acts at early stages of mtec differentiation , perhaps by regulating lineage decisions or by controlling temporal patterns of the differentiation process . however , because of the large gaps in our understanding of thymic epithelial differentiation , the model proposed by derbinski et al . is based on a number of assumptions ( 15 ) . we wish to point out that these data can be interpreted differently when alternative but equally valid assumptions are made regarding thymic epithelial differentiation . for instance , by attributing tra expression to mature mtecs this model implies that thymic epithelial progenitor cells have a restricted transcriptional profile and would express mtec - specific molecules before activation of tra expression . at this time , we are not aware of many genes , structural or regulatory , that can be used to define a unique thymic epithelial identity , nor is there evidence that these progenitor cells are committed to the mtec lineage . as will be discussed in an alternative to section , there are grounds to consider the potential of epithelial progenitor cells or immature thymic epithelial cells to express tras . some have assumed that the program of mtec differentiation resembles that of dendritic cells , where high levels of cd80 , cd86 , and mhc class ii expression are considered to be markers of maturation . however , several observations suggest that expression of these molecules by nonhematopoietic cells may be inducible and not constitutive , and independent of maturation state ( 16 , 17 ) . for example , mtecs and mtec cell lines can be induced to express mhc class ii at high levels by exposure to ifn- ( 18 ) . in mice lacking promoter iv ( piv ) of the class ii transactivator ( ciita ) , hematopoietic antigen - presenting cells constitutively express mhc class ii , whereas nonhematopoietic cells do not express mhc class ii constitutively ( cortical thymic epithelial cells ) or in response to systemic ifn- treatment ( multiple cell types ; reference 17 ) . therefore , the cd80 subset of mtec may reflect a state of epithelial activation in response to local stimuli rather than a subset of thymic epithelial cells at a particular stage of maturation . further assume that the cd80 subset of mtecs represents a homogenous and mature population , even though several molecules characteristic of embryonic stem cells ( dppa3 , utf-1 ) and stem cell populations ( sca-1 ) as well as early regulators of endodermal tissue development ( cdx1 , foxa1 ) are enriched in this mature we suggest an alternative interpretation of the data presented in the study by derbinski et al . ( 15 ) that is based on several different underlying assumptions regarding the development and character of the medullary thymic epithelium . we propose that conserved developmental mechanisms regulate tra expression by mtecs , possibly in one or several ways . first , the absence of a well - defined population of thymic epithelial progenitor cells or well - defined markers that are unique for thymic epithelium suggests that the earliest thymic epithelial progenitor cells could be multipotent and not initially restricted to a thymic lineage . according to this scenario , multiple conserved transcriptional programs that reflect the multipotential nature of these progenitor cells would result in the display of a broad spectrum of tras ( fig . as cells differentiate ( defined here as a progressive restriction of developmental potential or a narrowing of developmental fate choices ) , there would be sequential silencing or inactivation of some of these transcriptional programs , leading to terminally differentiated cells . although the duration of the expression profile for peripheral transcriptional networks may vary , the products specified by these networks would contribute to the set of tissue - restricted structural and regulatory genes that are expressed by mtecs . this process might increasingly restrict the expression of discrete sets of tissue - restricted genes as the cells develop ( 1921 ) , and allow individual cells to terminally differentiate into either a peripheral lineage fate ( 2 , 22 ) ( fig . 1 b , i and iii ) or lead to the silencing of peripheral transcription networks after commitment to a terminal as yet undefined mtec fate ( depicted in fig we suggest that expression of these peripheral transcriptional programs in various individual cells might account for the molecular mosaic of peripheral self - antigens that are represented within medullary thymic epithelium . if progressive silencing of gene expression is associated with differentiation , individual immature mtecs should express a wider array of tras than mature mtecs , regardless of the terminal fate of the mature cell . in addition , the mechanisms that regulate expression of specific tras by mtecs should be similar to those active in native tissues . the terminal differentiation model proposed by derbinski and colleagues predicts the opposite , with terminally differentiated mtec expressing the highest levels of tras as a result of epigenetic derepression or other regulatory mechanisms that are not recapitulated in the periphery ( 15 ) . although the progressive restriction of transcriptional programs as a mechanism of differentiation has received little attention in epithelial cells , such mechanisms have been described in hematopoietic cells . studies using either isolated early hematopoietic progenitors ( 19 , 23 ) or more mature hematopoietic cells ( 24 ) have shown that immature cells can express genes characteristic of lineages that are distinct from their terminally differentiated fates . for example , pro b cells deficient in the transcription factor pax5 , which are incapable of commitment to the b cell lineage , can differentiate into mature t cells ( 24 ) . by examining lineage - specific gene expression patterns in early hematopoietic populations , miyamoto et al . ( 23 ) concluded that multiple lineage - affiliated differentiation programs are activated at the transcriptional level before commitment . in this context , what has been described in the past as promiscuity of gene expression or lineage commitment could simply reflect the ability of immature cells to utilize multiple developmental programs . this interpretation differs significantly from the promiscuous gene expression of the terminal differentiation model , whereby genes are expressed nonspecifically as long as their chromosomal neighborhood is accessible . the components necessary for a developmental , progressive restriction model are all present in the thymus : a cycling population of epithelial cells ( 1 ) in an environment replete with the mechanisms and signaling pathways that are involved in the development of peripheral epithelial ( and nonepithelial ) tissues . these include the notch , hedgehog , bone morphogenic protein ( bmp ) , fibroblast growth factor ( fgf ) , and wnt signaling pathways ( 9 ) . a recent study demonstrated that transplanted fetal pharyngeal pouch endoderm can generate thymus and other endodermally derived structures , such as pharyngeal and gut tissues , and that transplanted embryonic day 9 fetal pharyngeal arches ( which normally give rise to the thymus ) can generate a wide range of tissue types , including skin , hair , cartilage , bone , muscle , and adipose tissue ( 25 ) . therefore , it seems reasonable that the ability of mtecs to express a remarkably wide range of tras may reflect a general developmental process that occurs in a tissue with high levels of cellular turnover , broad developmental potential , and complex environmental cues that can direct individual mtec precursors into various developmental pathways . although the plasticity of the medullary compartment has been shown in multiple experimental contexts ( 9 ) , the identity and differentiation potential of mtec progenitors remains to be determined . the tra expression data ( 3 , 4 , 5 , 26 ) and chromosomal clustering analyses presented in previous studies ( 27 ) are consistent with either model . the clustered pattern of tra expression noted by derbinski et al . ( 15 ) was interpreted to reflect derepressed loci in a homogeneous , terminally differentiated epithelial population that is capable of promiscuous gene expression . but this clustering could also result from immature populations of precursor cells that have retained a broad transcriptional profile , or could represent the summed expression by a heterogeneous mixture of cells , each of which expresses a specific set of peripheral lineage genes . the same can be said for the data showing biallelic expression of the insulin - like growth factor 2 ( igf2 ) gene by mtecs , which derbinski et al . this biallelic expression pattern might instead reflect a lack of imprinting in immature progenitors , or a heterogeneous mixture of cells that individually express igf2 either mono- or biallelically in a developmental or lineage - specific manner ( 28 ) . the data presented in the current study indicate that aire does not regulate tra expression by random derepression of loci or chromatin remodeling mechanisms as previously proposed ( 1 , 5 , 27 , 29 ) . for example , within the casein locus the authors show that casein is not expressed by cd80 aire mtecs . however , this can not be attributed to a locus that is closed by the absence of aire , as the ability of cd80 aire mtecs to express the casein and genes ( which flank casein ; reference 30 ) demonstrates that the locus must be open in some cells . therefore , the mechanism by which aire affects casein expression appears to be independent of wholesale chromatin remodeling , run - on transcription of open loci , or direct transcriptional regulation of specific genes . by extension , that aire is required for casein expression in wild - type mtecs ( which have an accessible locus ) suggests that for both aire - dependent and aire - independent antigens epigenetic mechanisms alone are not sufficient for tra expression . the data and model put forth by derbinski et al . highlight the complex and ill - defined nature of mtec differentiation and advocate an intriguing model to account for the observed tra expression ( 15 ) . however , it remains to be determined whether tra expression reflects a novel derepression mechanism that is unique to mtecs , as they have proposed , or whether it results from developmentally conserved mechanisms that are active in a permissive environment . in either case , the results presented delineate important functional parameters of this phenomenon and help define key issues that need to be resolved before the mechanisms underlying tra expression can be understood . these include the identity , developmental potential , and plasticity of resident mtec progenitors , the precursor progeny relationships between the various subsets of mtecs , and the means to isolate defined , homogeneous subsets of mtecs to test these models directly . it seems likely that the mechanisms controlling thymic epithelial cell differentiation and those that control the expression of tras will be found to be highly convergent .

tumour size is a commonly used predictor of survival in breast cancer and correlates strongly with lymph node involvement [ 15 ] . tumour size is included in the american joint committee on cancer / union for international cancer control ( ajcc / uicc ) cancer staging manual and is represented by the maximum linear extent of disease . here we describe a novel methodology for measuring tumour volume in lumpectomies , referred to as the 3d pathology volumetric technique . for proof of concept , we demonstrate the technique using two lumpectomy specimens , comparing volumes as measured from serial whole - mount sections versus simulated conventional , sampling - based pathology . conformational distortion is minimized by first encapsulating the fresh tissue sample in a buoyant , density - matching gel . quantitative analysis is performed on the large image dataset ( 3070 gb ) using custom software tools to create volume renderings and estimate volumes of both in situ and invasive disease . tumour volume may provide a more accurate representation of size , because tumour is a 3d entity . a simple ellipsoid model has been used to approximate volume using maximum linear extent and has been shown to provide a more accurate assessment of the volume of breast tumours compared to modelling the tumour as a sphere ; in a retrospective study of 165 tumours measuring 2.5 cm or less , the largest diameters in anterior - posterior ( ap ) , medial - lateral ( ml ) , and superior - inferior ( si ) dimensions were distinct in 96.4% of the cases . biologically , tumour volume is proportional to the number of cells , and theoretical models have shown that metastatic potential depends on the total number of cells and the probability of each to disseminate . to date there are no clear data supporting the superiority of tumour volume over maximum linear dimension as a predictor of outcome in breast cancer , although current pathology guidelines are beginning to incorporate 3d parameters ( e.g. , eccentricity factor ) [ 1012 ] . some studies ( limited to unifocal tumours ) fail to demonstrate stronger prediction with tumour volume , when estimated using the ellipsoid approximation , compared to maximum linear extent . however , for lung cancer , a significant association between tumour volume and both overall survival and disease - free survival has been shown [ 14 , 15 ] . in staging of prostate cancer , typically , in conventional work for these sites volume is estimated from linear measurements assuming ellipsoid geometry . one of the impediments to establishing the prognostic value of tumour volume in breast cancer staging stems from inconsistencies in measurement technique , especially for more complex tumour patterns ( e.g. , diffusely infiltrating or multifocal ) . diffusely infiltrating tumours exhibit a morphology in which the cancer cells are interspersed with normal epithelial and stromal elements making precise measurement of the volume of tissue occupied by tumour cells difficult . multifocality occurs in about 30% of breast cancers and is associated with local recurrence and decreased survival [ 19 , 20 ] . ajcc / uicc guidelines are based on the size of largest focus while some studies demonstrate prognostic value for the aggregate diameter instead or show that volume must be controlled to demonstrate the association between multifocality and lymph node involvement . for multifocal prostate cancer , significant overestimation of mean volume has been shown and at least one measurable tumour is missed in approximately 17% of cases , when the ellipsoidal method is used , assuming a gold standard based on serial sectioning . similar observations have been noted for lung carcinoma , when estimating volume using an ellipsoidal approximation along with maximum extent measured from serial standard - format histological sections . whole - mount sections have been proposed as a gold standard for evaluating multifocality ( defined here as two or more foci of either invasive or in situ carcinoma where the foci are separated by intervening normal breast tissue ) and may permit more accurate assessment of tumour burden where conventional sampling is difficult ( e.g. , due to lack of desmoplastic reaction or infiltrative growth pattern ) . studies using whole - mount or large - section pathology techniques confirm accepted prevalence rates for multifocality ( observed in 31.9% of 114 mm invasive breast carcinomas , in a study of 301 consecutive cases ) and confirm that multifocality is an independent prognostic factor for survival at 10 years [ 26 , 27 ] . furthermore , multifocality is associated with a more than twofold increased risk of vascular invasion and lymph node metastasis compared to unifocal cancer . however , the ajcc / uicc guidelines require only measurement of the largest tumour focus . whole - mount sections would enable the entire tumour burden comprising any secondary foci to be more fully assessed . in this work , we extend the principle of increasing coverage by incorporating serial sectioning , while supporting the flaccid specimen to reduce conformational distortion , to create a 3d representation of tumour histology . technique which utilizes a set of whole - specimen , whole - mount serial section images to create volume renderings and calculate invasive and in situ tumour volumes . the volumetric analysis is demonstrated using two lumpectomies with features that might be associated with underestimation of tumour burden when conventional histological sampling is used . calculated volumes are compared with those obtained from simulated conventional histological sampling , and also with estimated volumes obtained from linear measurements assuming ellipsoid tumour geometry . two lumpectomy cases were selected retrospectively from the whole - mount tumour bank at the biomarker imaging research laboratory at sunnybrook health sciences centre . the specimens were initially obtained from the department of pathology at sunnybrook with the approval of the institutional research ethics board , excluding lumpectomies that would be submitted in toto when conventional sampling - based techniques would be used . the diagnosis in both cases one case ( case a ) is an invasive tumour that infiltrates diffusely , mostly without a desmoplastic reaction or destroying intervening benign breast elements , such that a typical tumour section shows invasive carcinoma admixed with benign breast elements . the other case ( case b ) is a localized invasive tumour with foci of in situ carcinoma away from the invasive tumour and separated from it by intervening benign tissue . case b is , therefore , representative of the challenge inherent in measuring tumour volume for multifocal disease , using conventional techniques . both specimens were prepared and processed using techniques collectively referred to as each fresh , unfixed specimen was first suspended in a buoyant gel ( 3.5% agar ) , and after setting , each tissue - gel block was serially sliced ( in the ml dimension ) into uniform , 4 mm thick slices using a rotary slicer ( berkel products co. , ltd . ; itw ; glen lake , il , usa ) . the tissue - gel slices were fixed overnight in 10% neutral buffered formalin and then processed using a 16-hour program developed for whole - mount breast tissues in an automatic tissue processor that uses microwave assistance ( pathos classic ; milestone medical srl ; sorisole , italy ) . the processed slices were embedded in custom moulds and one 4 m thick tissue section was obtained from the top of each block using a sliding microtome ( sm2500 ; leica microsystems , germany ) . the sections were stained with hematoxylin and eosin ( h&e ) using manual techniques . the two sets of whole - mount sections , mounted on 7.62 cm 10.16 cm glass microscope slides , were produced comprising 14 slides from case a , and 23 from case b. the set was digitized ( tissue scope ; huron technologies inc . ; waterloo , on , canada ) using a pixel spacing of 2 m as previously determined to optimize tumour detectability and computational feasibility . the size of the total image dataset was 28 gb and 69 gb for case a and b , respectively ( average 0.43 gb per section ) . the images were interpreted by a pathologist ( kl ) using sedeen viewing program ( selective decoding and encoding engine ; developed by dr . this software tool was developed to enable interactive display and quantitative work for large image datasets using conventional workstations . features include panning and zooming , tools for contouring , and annotating and measuring tumours or multiple regions of interest ( e.g. , invasive tumour and in situ tumour ) ( figure 1 ) . using this software , manual , digital contouring of tumour in all of the images was performed and coordinates were stored to file in extensible markup language ( .xml ) format . using a virtual sampling technique described elsewhere , a set of images simulating the conventional , sampling - based pathology evaluation ( i.e. , standard sized slides ) was created . for volume calculations and visualization , the .xml files were imported into matlab ( matlab7.11.0.584 ( r2010b ) ; math works inc . ; natick , ma , usa ) , to generate a volume rendering for each case , and to calculate tumour volumes , for in situ and invasive disease separately . for each whole - mount image , three binary image arrays were generated to represent the following three features as defined by the digital contouring : in situ disease , invasive disease , and normal tissue , with pixels enclosed in a contour set to intensity values r , g , b = 1 , and 0 otherwise . the three binary arrays were stacked and then extruded in the medial - lateral dimensions , by layering duplicate copies of the arrays at every 2 m , corresponding to the lateral resolution , to fill the 4 mm gap between whole - mount sections . in this way , tumour volumes were calculated by riemann summation of all the positive pixels ( rgb intensity = 1 ) in the volume rendering , for in situ and invasive disease separately . for the set of images simulating conventional pathology technique , volume was calculated similarly by multiplying the sampled tumour area by the thickness of 4 mm . finally , tumour volume was estimated from 2d measurements of maximum linear extent in the ml , ap , and si dimensions using the following ellipsoidal approximation : volumeellipsoid = 1/6 mlapsi . the maximum linear extent in ml , for case a , the set of serial whole - mount sections which contain tumour is shown in figure 2 . these images also depict the locations where conventional samples would be taken as determined by the pathology assistant . figures 4 and 5 present the serial whole - mount sections and volume rendering for case b. tumour volume , calculated from the full renderings as well as from the locations corresponding to conventional sampling , are compared in table 1 , along with measurements of maximum linear extent and the corresponding ellipsoidal volume . the 3d pathology volumetric technique facilitates visualization of spatial relationships within lumpectomies and may provide a more accurate surrogate for tumour burden . it is seen from case a that when the tumour infiltrates without eliciting a desmoplastic reaction and without destroying the normal benign breast elements , the conventional 2d measurements using maximum extent in the three dimensions ( ap , ml , and si ) may overestimate tumour burden . if the tumour is approximated by an ellipsoid , then using these three measurements the tumour volume is overestimated by a factor of approximately 7 ( table 1 ) . however , when the tumour is localized ( case b ) , the estimated volume based on measurements of maximum extent assuming ellipsoid tumour distribution more closely approximates the calculated volume , overestimating by a factor of 1.5 . thus , the 3d volumetric technique may remove bias introduced by assumptions of tumour geometry when less precise approximations are used . from the volume renderings ( figures 3 and 5 ) , the relationship of invasive tumour to the in situ component that is present away from the tumour can be readily appreciated . taking the close or involved margins seen in the serial whole - mount section images ( figures 2 and 4 ) as a tissue landmark , corresponding areas are seen in the volume renderings ( figures 3 and 5 ) within the context of the 3d specimen . for example , tumour at the inferior margin in the section 10 mm from the lateral aspect in case a ( figure 2 ) is seen in the volume rendering ( figure 3 ) , relative to another close margin near the inferior - posterior midpoint . even within the limitations of a 2d presentation , whole - mount serial sections can enable more accurate estimates of tumour burden ( case a , figures 2 and 4 ) compared to conventional sampling . case a indicates the power of the 3d volumetric technique to identify and measure the extent of tumour in those lesions in which the tumour cells extensively infiltrate the normal tissue . comparing tumour volume to the subset which would be sampled , consequently in this case , only 70% of the total invasive tumour volume would have been captured in the conventional approach . for the localized mass in case b , however , the representation increases to 97% . case b illustrates the ability of the 3d pathology volumetric technique to enhance visualization and resolve tumour foci which are separated by normal tissue . at least two distinct foci of in situ disease appear as follows : one at 2028 mm from the medial aspect and one at 3644 mm from the medial aspect . in this case whole - mount serial sections may also help to capture dcis , which might otherwise be difficult to sample adequately . moreover , the focality of the in situ component in the ml dimension is better appreciated from the volume rendering ( figure 5 ) compared to the serial section presentation ( figure 4 ) in which continuity between serial sections is more difficult to synthesize . studies comparing methodologies for measuring tumour volume in other cancer sites support our observations . for prostatectomies , where the majority of tumours assume a multifocal distribution , these include the ellipsoidal approximation and riemann summation for serial sections 3 mm and 6 mm apart . taking the calculation for serial sections 3 mm apart as the gold standard , using the sections 6 mm apart instead would cause the mean tumour volume to be overestimated by 29.5% . using the ellipsoidal approximation , similarly for lung tumours , it was shown that riemann summation ( using sections 10 mm apart ) is most accurate compared to the ellipsoid approximation and especially compared to a spherical approximation , with the less accurate methods overestimating volume . the 3d pathology volumetric technique is well suited to validation of noninvasive imaging modalities ( e.g. , magnetic resonance imaging ) which are used to estimate 3d tumour descriptors including volume and surface area . surface area might also serve as a useful reporter for tumour burden in conjunction with volume . our methodology lends itself to exploration of other quantitative measures including surface area as a simple extension of the volumetric principle . using the techniques we describe here , we can obtain precise , volumetric measurements of challenging features ( e.g. , separate foci of disease , infiltrating pattern ) and in future studies the prognostic significance of these presentations studies on the prognostic significance of tumour volume with relation to biology are scarce . it has been suggested that tumour volume is inversely related with microvessel density , and that metastasis may , therefore , be an early event . the 3d volumetric technique can provide a platform for precise , volumetric measurements of both morphological and biological patterns , which can be correlated to validate prognostically significant relationships . using the 3d pathology volumetric technique , tumours are visualized in 3d and spatial relationships between key features ( e.g. , close or involved margins ) are readily appreciated . the technique also enables calculation of tumour volume , which may be a more accurate representation of tumour burden or size compared to 2d linear measurements . this technique also reduces the errors associated with undersampling in conventional histopathology , which can result in underestimation of tumour burden . the volumetric technique may be particularly useful in cases where accurate representation using conventional tissue samples may be difficult ( e.g. , infiltration without desmoplastic reaction and tumour admixed with normal epithelium , or localized invasive mass with multifocal in situ carcinoma away from the tumour ) . we have shown that in such cases tumour burden can be significantly underestimated using conventional methods . conversely , tumour volume can be markedly overestimated , especially for more complex tumour distribution ( e.g. , infiltrative ) , when simple approximations such as an ellipsoid model are used instead of the volumetric approach . additional studies to evaluate the predictive value of tumour volume on patient outcome are underway .

chronic actinic dermatitis ( cad ) is a severe photosensitivity disease induced by uvb , uva and occasionally visible light . clinical manifestations include eczematous lesions , infiltrated plaques and pruritic erythematous papules , which develop on light - exposed areas , particularly the face , neck and upper chest . the severity of the disease in each individual depends on the degree of photosensitivity , ranging from chronic eczematous lesions to erythroderma . the diagnosis of cad is based on the following 3 criteria : persistent photodermatitis without history of exposure to known photosensitizers , abnormal delayed erythemal responses to uvb and/or uva and/or visible light , and a histological finding consistent with photodermatitis . the pathophysiology of cad remains unclear , but current evidence suggests a delayed - type hypersensitivity reaction to endogenous photo - induced allergens . several treatment modalities have been proposed to induce long - term remission , such as systemic corticosteroid , azathioprine , and puva ; however , the use of these treatment options has limited efficacy and is associated with severe side effects . a 37-year - old male farmer presented to our hospital with a 22-year history of itchy indurated facial erythematous plaques . he has been treated with oral corticosteroids ( prednisolone 3060 mg / day ) with only temporary improvement . he had no history of exposure to any chemicals , plants , or perfumes . additionally , he had no known underlying disease and there was no family history of similar skin conditions . physical examination showed huge indurated lichenified erythematous infiltrative plaques on his forehead , nose , and both cheeks ( fig . 1 ) , and there were discrete erythematous papules on both forearms . a skin biopsy from a lesion of the left cheek was obtained and stained with he . the biopsy specimen consisted of mounds of parakeratosis , spongiosis and epidermal hyperplasia associated with dense superficial inflammatory cell infiltrates , mainly lymphocytes , in the thickened papillary dermis . routine laboratory findings were normal , except for a very low morning serum cortisol level ( < 1 g / dl ) . patch and photopatch testing ( including analysis of common photoallergens such as sunscreen , fragrance , and sesquiterpene lactone ) revealed no contact sensitivity ( table 1 ) . the patient 's minimal erythema dose to both uva and uvb demonstrated a significantly reduced threshold for uva ( 3 j / cm ; reference value : 30 j / cm ) and uvb - induced erythema ( 12 mj / cm ; reference value : 4560 mj / cm ) for his skin phototype iv ( fig . avoidance by using sunscreen and wearing opaque clothes and a broad - brimmed hat , there was a significant improvement of his facial lesions ( fig . remission of cad could be maintained by topical tacrolimus , and the secondary adrenal insufficiency induced by exogenous corticosteroid therapy recovered after discontinuation of oral steroids . in this case , cad with extensive leonine facies was successfully treated with 0.1% tacrolimus ointment twice daily . there was no adverse effect except for a mild transient burning sensation during the first few treatment days . tacrolimus is an immunosuppressive macrolide antibiotic , which inhibits calcineurin and suppresses the activation of antigen - specific t cells and the release of inflammatory cytokines , such as interleukin 2 and tumor necrosis factor . this hypothesis is supported by both the fact that the infiltrating cells are predominantly t cells and the immunological effects of tacrolimus . this advocates the use of topical tacrolimus as maintenance therapy and to prevent recurrence . after a follow - up period of 1 year with strict sun protection and maintenance therapy with topical tacrolimus once daily , the patient eventually achieved remission . although there have been several reports of cad cases successfully treated with topical tacrolimus [ 5 , 6 , 7 , 8 ] , this is the first case with severe and extensive leonine facies , which were uncontrolled despite 20 years of oral corticosteroid use , which in contrary induced secondary adrenal insufficiency as adverse effect . after remission , the oral corticosteroid was successfully tapered and discontinued within 6 months ; thereafter , the morning serum cortisol level returned to a normal range . in conclusion , this case demonstrates for the first time that topical tacrolimus might be an appropriate topical treatment with minimal side effects to induce and maintain remission in severe cad cases .

multiple myeloma ( mm ) mainly affects men , with those aged 65 years on the average affected . as for its systemic symptoms , anemia , hypercalcemia , renal failure , increased risk of infection , are the main clinical characteristics ; for clinical symptoms at the head and neck area , pain on the affected bones , paresthesia , edema , tooth mobility , pathologic fracture of bones , etc . , are observed . we have experienced two cases of mm , which are not frequently observed at the head and neck area , and we report what we have investigated and analyzed in two mm patients who visited the department of oral and maxillofacial surgery at samsung medical center ( seoul , korea ) to find out the diagnostic and therapeutic methodologies for mm . a 44-year - old female patient who had been diagnosed with mm at the bilateral humerus bones , bilateral pelvic bones , and bilateral femur bones as observed while receiving chemotherapy using vincristine doxorubicin , dexamethasone , hematopoietic stem cell transplantation ( hsct ) , etc . , was referred to our department after masses were observed over the buccal gingiva on the left side of the maxillary anterior in may 2008.(fig . a ) no specific features were observed from the panoramic view , but osteolytic features were noted at the relevant site based on a computed tomography ( ct ) scan in may 2008.(fig . b ) m - protein was 1.07 g / dl , and there was an increase in the immunoglobulin g ( igg ) count from the serum electrophoresis ( 2,235 mg / dl , normal range=700 - 1,600 mg / dl ) . reddish swellings were observed from the intraoral environment of the patient , and there were edemas - including teeth # 22 and # 23 - with smooth surfaces . thus , based on the fact that the patient had been observed by the department of hematology and oncology due to mm and an increase of m - protein , our department temporarily diagnosed the patient with an expression of mm at the head and neck area and carried out a biopsy of the relevant lesions under local anesthesia . the sample was sent to the department of pathology at samsung medical center , and the tissue was reported to have aspects of monotonous plasmacytoid cell infiltration . consequently , our department confirmed the expression of mm at the head and neck area . though the aspects of osteolysis were observed to increase based on a follow - up ct scan in july 2008 , healing of the gingival wounds at the sites of biopsy were shown , but the m - protein level was 1.6 g / dl , whereas the igg level was 2,176 mg / dl . the department of hematology and oncology then performed chemotherapy using thalidomide , dexamethasone , velcade , etc . note , however , that pancytopenia occurred in january 2009 , and other malignant aspects had been observed , including plasma cell rate of more than 35% based on the hematological test performed in april 2009 . the patient expired in july 2009 . a 61-year - old female patient who had been diagnosed with mm at the skull and rib areas by the department of hematology and oncology in may 2009 received hsct in january 2010 . she was observed to be in a systemically stable state . in december 2010 , she complained of edemas and pain in the left mandibular buccal gingiva and the buccal mucosa ; thus , she was referred to our department . she had a history of extraction of the left lower first molar tooth due to pain and tooth mobility at the relevant sites 2 months before her visit to the department of oral and maxillofacial surgery . an edema in the left facial area was found during the first examination , and clinical symptoms such as spontaneous hemorrhage of blue - reddish color tones were observed.(fig . there were no abnormal results except decrease of hemoglobin and hematocrit from the complete blood cell count test.(table 1 ) monoclonal gammapathy was observed with 5.29 g / dl when the m - protein level increased since june 2010 , with the iga level increasing considerably in the serum immunoglobulin counts ( 2,314 mg / dl , normal range=70 - 400 mg / dl ) . since the patient had a history of existing mm , the test results seemed to be related to hematological diseases , and no changes in bones were observed from the ct scan ; our department judged the existing mm to have been manifested into the oral soft tissue area and temporarily diagnosed the patient in detail with extramedullary plasmacytoma . strong standardized uptake value ( suv ) uptake ( suvmax=4.3 ) was found at the relevant site from a positron emission tomography - ct ( pet - ct).(fig . b ) thus , our department carried out a biopsy , and tissue specimens were sent to the department of pathology at samsung medical center . after the results of the biopsy determined the disease to be plasmacytoma , chemotherapy using lenalidomide , dexamethasone , and radiotherapy ( 25 gy ) were performed . a ) ; some aspects of improvement , including decrease of iga level , were found from the blood test carried out in march 2011 ( 740 mg / dl ) ; the m - protein level also shifted to a constant decrease ( 1.9 g / dl in march 2011 ) , and there was no uptake observed from a follow - up pet - ct scan.(fig . b ) during the follow - up in august 2011 , pain and edemas at the buccal gingiva of the left maxillary posterior teeth and palatal gingiva and pain , edemas , and spontaneous hemorrhage at the left mandibular lingual and buccal sites were found.(fig . in addition , osteolytic lesion with diameter of up to 1 cm and sclerotic rim was found in plain film and ct scans . consequently , biopsy and radiotherapy were performed again , with the patient hospitalized after consulting the department of hematology and oncology for observation with chemotherapy ( dexamethasone , thalidomide , cyclo - pho sphamide , doxorubicin , cisplatin , and etoposide ) . note , however , that cytopenia , azotemia , and pleural effusion of the lungs were observed due to development of the lesions ; the patient was discharged in september 2011 due to the aggravation of general condition owing to the lesions . a 44-year - old female patient who had been diagnosed with mm at the bilateral humerus bones , bilateral pelvic bones , and bilateral femur bones as observed while receiving chemotherapy using vincristine doxorubicin , dexamethasone , hematopoietic stem cell transplantation ( hsct ) , etc . , was referred to our department after masses were observed over the buccal gingiva on the left side of the maxillary anterior in may 2008.(fig . a ) no specific features were observed from the panoramic view , but osteolytic features were noted at the relevant site based on a computed tomography ( ct ) scan in may 2008.(fig . b ) m - protein was 1.07 g / dl , and there was an increase in the immunoglobulin g ( igg ) count from the serum electrophoresis ( 2,235 mg / dl , normal range=700 - 1,600 mg / dl ) . reddish swellings were observed from the intraoral environment of the patient , and there were edemas - including teeth # 22 and # 23 - with smooth surfaces . thus , based on the fact that the patient had been observed by the department of hematology and oncology due to mm and an increase of m - protein , our department temporarily diagnosed the patient with an expression of mm at the head and neck area and carried out a biopsy of the relevant lesions under local anesthesia . the sample was sent to the department of pathology at samsung medical center , and the tissue was reported to have aspects of monotonous plasmacytoid cell infiltration . consequently , our department confirmed the expression of mm at the head and neck area . though the aspects of osteolysis were observed to increase based on a follow - up ct scan in july 2008 , healing of the gingival wounds at the sites of biopsy were shown , but the m - protein level was 1.6 g / dl , whereas the igg level was 2,176 mg / dl . the department of hematology and oncology then performed chemotherapy using thalidomide , dexamethasone , velcade , etc . note , however , that pancytopenia occurred in january 2009 , and other malignant aspects had been observed , including plasma cell rate of more than 35% based on the hematological test performed in april 2009 . a 61-year - old female patient who had been diagnosed with mm at the skull and rib areas by the department of hematology and oncology in may 2009 received hsct in january 2010 . she was observed to be in a systemically stable state . in december 2010 , she complained of edemas and pain in the left mandibular buccal gingiva and the buccal mucosa ; thus , she was referred to our department . she had a history of extraction of the left lower first molar tooth due to pain and tooth mobility at the relevant sites 2 months before her visit to the department of oral and maxillofacial surgery . an edema in the left facial area was found during the first examination , and clinical symptoms such as spontaneous hemorrhage of blue - reddish color tones were observed.(fig . there were no abnormal results except decrease of hemoglobin and hematocrit from the complete blood cell count test.(table 1 ) monoclonal gammapathy was observed with 5.29 g / dl when the m - protein level increased since june 2010 , with the iga level increasing considerably in the serum immunoglobulin counts ( 2,314 mg / dl , normal range=70 - 400 mg / dl ) . since the patient had a history of existing mm , the test results seemed to be related to hematological diseases , and no changes in bones were observed from the ct scan ; our department judged the existing mm to have been manifested into the oral soft tissue area and temporarily diagnosed the patient in detail with extramedullary plasmacytoma . strong standardized uptake value ( suv ) uptake ( suvmax=4.3 ) was found at the relevant site from a positron emission tomography - ct ( pet - ct).(fig . b ) thus , our department carried out a biopsy , and tissue specimens were sent to the department of pathology at samsung medical center . after the results of the biopsy determined the disease to be plasmacytoma , chemotherapy using lenalidomide , dexamethasone , and radiotherapy ( 25 gy ) were performed . a ) ; some aspects of improvement , including decrease of iga level , were found from the blood test carried out in march 2011 ( 740 mg / dl ) ; the m - protein level also shifted to a constant decrease ( 1.9 g / dl in march 2011 ) , and there was no uptake observed from a follow - up pet - ct scan.(fig . b ) during the follow - up in august 2011 , pain and edemas at the buccal gingiva of the left maxillary posterior teeth and palatal gingiva and pain , edemas , and spontaneous hemorrhage at the left mandibular lingual and buccal sites were found.(fig . in addition , osteolytic lesion with diameter of up to 1 cm and sclerotic rim was found in plain film and ct scans . consequently , biopsy and radiotherapy were performed again , with the patient hospitalized after consulting the department of hematology and oncology for observation with chemotherapy ( dexamethasone , thalidomide , cyclo - pho sphamide , doxorubicin , cisplatin , and etoposide ) . note , however , that cytopenia , azotemia , and pleural effusion of the lungs were observed due to development of the lesions ; the patient was discharged in september 2011 due to the aggravation of general condition owing to the lesions . as a disease showing plasma cell hyperplasia , mm can be classified into three stages based on the strengths of the activation of lesions . the first stage is monoclonal gammopathy of undetermined significance wherein m - protein is detected but no increase of plasma cells is observed in marrows and no clinical symptoms exist in relation to mm1 . in the second stage , which is a more developed stage , smoldering multiple myeloma with increased m - protein and plasma cells is observed in the marrows , but no clinical symptoms exist . the third stage is classified as symptomatic multiple myeloma wherein the contractions are clinically identified and the spread of lesions is found over numerous organs2.(table 2 ) likewise , depending on the onset sites , mm can be divided into extramedullary plasmacytoma as a single lesion limited to soft tissues , solitary bone plasmacytoma occurring as a single lesion at the bone tissue , and the kind of mm occurring systemically over numerous organs . in general , solitary bone plasmacytoma occurring in a single bone is highly likely to develop into mm . furthermore , canger et al.3 reported in 2007 that such shifts occurred over several months to several years . in contrast , in the case of the plasmacytoma occurring in soft tissues , control of disease is reported to be possible with local excision and appropriate amount of irradiation . with respect to this , tournier - rangeard et al.4 reported that the amount of irradiation necessary for treatment was 45 gy . mm is a disease showing features of hyperplasia of plasma cells along with osteolytic lesions , anemia , hypercalcemia , renal failure , etc . the most affected sites include the vertebrae , skull , pelvic bone , ribs , humerus , and femur5 . in the head and neck area , it occurs at the maxilla and mandible rather than skull sites but more frequently at the mandible than at the maxilla . in the mandible , it occurs more frequently at the posterior teeth including the body , angle , ascending ramus , etc6 . in general , chemotherapy and hsct recently , however , prognosis has improved remarkably with the use of thalidomide , bortezomib , and lenalidomide . the reported mean residual life is 5 years or longer5 ; it is not the case in our hospital , and studies report that discussion is open with respect to the prognosis . it is difficult for an oral and maxillofacial surgeon to make a diagnosis from the first examination because many internal medicine approaches are necessary for the diagnosis of mm . there have only been a few cases wherein it occurs in the head and neck area , and we have rare cases of the first onset of this disease at the head and neck area7 . table 2 shows the points that serve as references for the diagnosis of mm8 . though m - protein in serum is a clear criterion for the diagnosis , attention should be paid since m - protein is not detected in about 2% of all cases9 . the current two cases tended to be easy to diagnose because the oral symptom was expressed in patients with mm as the existing systemic disease ; thus , the patients had existing internal medicine history . note , however , that it is difficult for an oral surgeon to confirm its diagnosis if the first symptom of mm occurs in the head and neck area . in the first case , a rare case was presented wherein the disease occurred at the maxilla anterior area . after the excision of the lesions , decrease of edemas and tooth mobility note , however , that progressive development of osteolysis at the mandible was seen from a follow - up ct scan . in the second case , this case was also one wherein the lesions rapidly worsened one year after expression outside the bone marrow cavity . in the case of extra - marrow plasmacytoma , the first treatment is not surgery but radiotherapy , and the recommended dose in such case is 40 - 50 gy11 . a healing aspect was observed in this patient after 25 gy irradiation to the relevant sites . usually , bone marrow biopsy is needed to evaluate the percentage of bone marrow occupied by plasma cells , and this percentage is used in the diagnostic criteria for myeloma . immunohistochemistry can detect plasma cells that express immunoglobulin in the cytoplasm and occasionally on the cell surface ; myeloma cells are typically cd56 , cd38 , and cd138 positive as well as cd19 and cd45 negative8 . in two cases , the histopathologic samples showed fragments of soft tissue with marked plasma cell infiltrate . in an immunohistochemical staining study , the cells showed lambda light chain positive in the plasma cells . this phenotype showed a monoclonal population , indicative of a diagnosis of plasma cell tumor . surgery is not recommended when mm is found at the head and neck area since it is a systemic disease . it seems reasonable to treat patients systemically in terms of existing therapy after consulting the department of internal medicine . in the second case , we found the symptoms to have been mitigated by irradiation . though it rarely occurs in the head and neck area , mm is a disease that should be suspected for old patients who show a lot of osteoporotic lesions in the cortical bones and hematological abnormalities . in cases wherein mm is suspected clinically , m - protein tests including serum electrophoresis , serum immunofixation , and serum free light chain should be performed . though plain x - ray scan can be helpful in its diagnosis , pet - ct and magnetic resonance imaging scans enable more precise diagnosis . at the same time , clinical systemic symptoms ( osteoclastic lesions , anemia , and renal failure ) are observed . thus , one should make a plan for systemic examination and treatment as well as treatment for the head and neck areas .

as craniopharyngioma is a congenital brain tumor and benign in nature , total surgical removal is the ideal treatment . however , total removal is often difficult without deterioration of qol of the patients because the tumor originates in the hypothalamic - pituitary axis . as the second choice , the residual tumor after surgery or the recurrent tumor had been treated by fractionated - radiotherapy , adjuvantly or additionally . however , not only complications to optic nerves , pituitary gland and hypothalamus , but also control of the tumor has not been satisfied . recent progress of stereotactic irradiation such as gamma knife radiosurgery ( gkr ) and stereotactic radiotherapy ( srt ) have become accurate , and stereotactic treatments are now good indication for craniopharyngioma . one hundred and twenty eight cases of residual or recurrent craniopharyngioma have been successively treated by gkr & surgery since may 1991 at komaki city hospital . in this paper , recent 30 cases treated at nagoya kyoritsu hospital during 10 years since july 2004 have been studied and discussed by comparison with those of previous papers . male to female ratio was 19:11 , adult to child ratio was 26:4 , the ratio of post surgical residual to recurrent tumor was 23:7 . the location of tumors were suprasellar and/or intraventricular in 18 , intrasellar in 9 , and chiasm & anterior part in 3 . the nature of tumors was solid in 11 , cystic in 7 , and mixed in 12 . mean size of tumors was 2.64 ( 0.39.3 ) ml in volume and 16.3 ( 8.3123.5 ) mm in diameter . the prescribed dose of gkr was mean maximum of 23.6 ( 2028 ) gy and mean marginal of 11.7 ( 8.223 ) gy . the follow - up period was 6 months to 10 years after gkr , in which mean of 79.9 and median of 91 months were obtained . as the previous surgical treatments , intracranial and transshpenoidal removals of tumor were made 37 and 13 times , respectively . v - p shunt in two and ommaya reservoir setting in three cases were performed . the evaluation of the effects was made by repeated mris , neuro - endocrine changes , hypothalamic signs & symptoms , complications , and qol assessed by kps every 3 to 6 months . twenty - nine out of 30 patients were evaluable because one case was lost to follow - up . the change of tumors was complete remission ( cr ) in 8 , partial remission(pr ) in 12 , no change ( nc ) in 6 and progression(pg ) in 3 patients , in which 2 died by progression of hypothalamic involvement and one by infirmity . the tumor response rate was 68.9% ( 20/29 ) and the control rate was 89.7 % ( 26/29 ) . regarding signs and symptoms , ten cases showed visual disturbance , in which 3 of visual acuity , 2 of the visual field and 5 of both disturbances were found . sixteen cases showed endocrine disturbances with panhypopituitarism in 10 , gh deficiency in 4 , hypo - corticoid , hypo - gonad , hypo - tsh and hypo - igf-1 in 1 before gkr . after gkr , there was no new appearance nor progression of neuro - endocrinological symptoms , and even an improvement of visual disturbance was found in 2 cases . there were 5 cases with hypothalamic signs and symptoms in which di was found in all but other conditions were found in 3 cases such as electrolyte abnormality in 1 , temperature disturbance in 1 , appetite abnormality and obesity in 2 and hypersomnia in 2 cases . hypothalamic signs and symptoms progressed in 2 cases , from which the patients died at 8.5 and 14 years after the first treatment . the scores were excellent ( kps=100 ) in 14 , good ( 90 ) in 9 , fair ( 7080 ) in 2 and poor ( 5060 ) in a case and unknown in another . actuarial survival by kaplan - meier method revealed 96% at 5 years and 86% at 10 years . pfs was 76% at 5 years and 76% at 10 years , respectively ( figure 1 ) . survivals after surgery and gamma knife radiosurgery os= over all survival , pfs= progression free survival case 1 . a 6-year - old boy showed visual disturbance and hydrocephalus in september , 1996 . before visiting nrc at nagoya kyoritsu hospital , he had two craniotomies for subtotal removal of the tumor , and subsequent gkr in 1996 and 1999 , where the marginal doses were 11 gy and 11.5 gy , respectively . the third craniotomy was made in december 2004 because the suprasellar tumor recurred , and was followed by a third gkr with the marginal dose of 10.5 gy . fourth surgical removal was made by transsphenoidal surgery for the recurrent intrasellar tumor , followed by a 4 gkr using the marginal dose of 14 gy . thereafter , bilateral visual field defects and a tendency of obesity appeared and progressed . in june 2006 , obesity ( body mass index ; bmi>30% ) and poikilothermia developed . after the tumor recurrences , three surgical removals and a 5 gkr the patient died in august 2010 at the age of 19 . in total , the patient had nine surgeries , five gkrs and one course of chemotherapy during the 14 years from diagnosis . mri findings of hypothalamic involvement of case 1 a : mid - sagittal mri showes supra- and intrasellar tumor invading the floor of anterior iiird ventricle , where patient showed obesity and poikilothermia . b : aggressive progression of the tumor into iiird ventricle and skull base is found after multiple surgeries , gkr and chemotherapy . a 42-year - old man complained of visual disturbance of the right eye and was diagnosed as suprasellar tumor in june 1999 . the tumor regrew and a second craniotomy was made , followed by 50 gy of focal fractionated radiotherapy . after cyst evacuation , gkr was made to the tumor of 6.4 ml with a marginal dose of 9.5 gy in march 2005 ( figure 3a ) . the tumor enlarged after intratumoral bleeding and patient showed hydrocephalus in october 2006 ( figure 3b ) . although a vp shunt was made , the patient became disoriented and hypersomnic from february 2007 . the patient showed attacks of loss of consciousness , high fever , and electrolyte imbalance since july . because his family rejected further treatment , the patient died in february 2008 . sagittal and coronal mri findings of hypothalamic involement in case 2 recurrent cystic tumor is found under anterior iiird ventricle after surgeries ( a , b ) . after fractionated radiotherapy , ommaya setting and gkr , the patient showed di and visual disturbances . patient showed hypothalamic signs and symptoms such as hypersomnia , high fever and electrolyte disturbances , and died within seven months . one of the important factor which makes the prognosis of craniopharyngioma worsen will be the regrowth or recurrence of tumors after treatments . many prognostic factors have been documented for the recurrence , but none of them had been verified as significant factor . recently , we have statistically analyzed nine factors which appeared in the past using our database . they are age , sex , child or adult , residual or recurrent tumor , natures of tumor , size of tumor , pathological diagnosis , numbers of previous treatments and radiation dose . by univariate and multivariate analysis , tumor size and radiation dose are the significant factors for recurrence of tumor after partial removal followed by gkr . furthermore , roc analysis * revealed that a mean diameter less than 19 mm and a marginal dose more than 13.2 gy are favorable prognostic factors . regarding two factors , results of present study are compaired with those of previous studies ( table 1 ) . initial study presented 33 cases , in which a tumor volume of 4.4 ml was treated with the marginal dose of 12.8 gy . the tumor control rate was 84.8% , but the optico - endocrine complications occurred relatively high as 15.2% ( 5 in 33 cases ) . while the next report with 98 cases that the tumor volume of 3.5 ml was treated with a marginal dose of 11.5 gy revealed an actuarial survival of 94 % and 91% in 5 and 10 years , but pfs were 60% and 53% , respectively . however , the rate of neuro - endocrine complications decreased to 6.5% ( 6 in 98 cases ) . finally , the present study of 30 cases in which the tumor volume of 2.6 ml was treated with a marginal dose of 11.7 gy revealed a higher survival and pfs for 5 and 10 years without complications . regarding the effective dose for craniopharyngioma , it was suggested that the dose should be higher than 11.5 gy but less than 12.8 gy and the marginal dose of 11.7gy showed higher response and control rates . massive hypothalamic invasion was found in two of 30 patients ( 6.6% ) in this study in which the incidence was similar to that of previous study . the tumor was originally cystic in case 2 and mixed type in case 1 which initially located at the anterior floor of third ventricle ( figure 2a , 3a ) , and grew huge to occupy whole hypothalamus until patients death at 8.5 and 14 years . it was again suggested that the dose of each treatment seemed too low to control tumor because of oars , especially optic nerve nearby . however , the control of tumor and radiation injury to oar are the both sides . and , recent developement of stereotactic irradiation technology for these tumors has become tumor control maximum but of radiation injury minimum . chiou et al . had treated tumors using higher marginal dose of 16.4 gy . control and disappearance of tumor showed 80% and 40% respectively , however cyst enlargement in 30% and visual disturbance in 20% were found as complications . comparison of results with present and previous studies * complication rate of the group 1 ) to 3 ) and group 2 ) to 3 ) are statistically significant ( p<0.0371 ) by fischer exact test . * * indicates percentage of mean survival time ( m ) of this group abbreviations : pps = progression free survival , m = months , y = years , fu = follow - up , gy = grey while ulfarsson et al . had used a lowest marginal dose of 5 gy to mean volume of 8.0 ml , lower tumor control of 36% and response rate of 23% were obtained , along with a high recurrence rate of 63% and optic nerve injury of 42.1% . present study has confirmed that radiation injury was not found by applying relatively smaller marginal dose of 11.7 gy to a small tumor volume of 2.6 ml . also actuarial survivals were 96% and 86% for 5 and 10 years , and the pfss at same times were 76% , respectively . this implies that the effective marginal dose could be around 11.7gy to control the tumor without radiation injury . however , surgical reduction of tumor volume is another important issue to increase the effective dose as shown by present study and others . although so - called an optimal or effective dose for craniopharyngioma has not been defined , the marginal dose could be increased safely when a tumor is small . both of present authors ( t.k and y.m ) had successively engaged in gkr of craniopharyngioma since 1991 and treated consecutive 128 cases at two gk sites using common strategy of " volume reduction and setting of effective dose " . recently , many authors of gkr for craniopharyngioma have advocated the similar strategy to treat smaller tumor with higher radiation dose . it has recently been proposed by european investigators that for the treatment of pediatric craniopharyngiomas , the initial treatment should be less invasive and conservative to prevent crucial damages to the developing brain . in case of larger tumors or inoperable conditions , stereotactic fractionated to improve the survival and qol of patients with craniopharyngioma , residual or recurrent tumors should be treated by stereotactic irradiation using an effective dose to alleviate regrowth . the effective marginal dose will be around 11.7 gy when the tumor size is small ( 2.64ml in volume or 16.3 mm in diameter ) . the appearance and progression of hypothalamic symptoms other than diabetes insipidus will be signs of poor prognosis , implying hypothalamic invasion of the tumor . early diagnosis and establishment of effective treatments are crucial . * roc : a receiver operating characteristic ( roc ) from wikipedia , is a graphical plot of the sensitivity , or true positive rate , vs false positive rate this paper was presented at the 21th annual meeting of the japanese society for stereotactic radiosurgery at maebashi on june 1 2012 and the 4 annual meeting of the japan radiosurgery society on january 19 , 2013 . the authors have no personal financial or institutional interest in any of the drugs , materials , or devices in this article .

a common way to treat arterial stenoses due to atherosclerosis is percutaneous transluminal angioplasty ( pta ) , intended to improve the hemodynamic conditions . this therapeutic procedure itself involves an injury to the vessel wall and the repair of the injury starts immediately post - pta . sometimes the repair process causes a lumen narrowing that affects the flow unfavourably . to evaluate medication aimed at diminishing the vessel wall reactions there is a need for quantitative , non - invasive methods to follow the repair process in the vessel wall post - pta . magnetic resonance imaging ( mri ) is a method that is , in general , non - invasive and relatively independent of the operator . we have previously proposed a quantitative method based on a combination of mri and image processing techniques ( thresholding and 3d morphology ) , that was tested for examining severely atherosclerotic animals . we have also proposed a modified method combining mri with an alternative image processing technique , the gradient vector flow snake algorithm . this image processing procedure is less operator - dependent and less time - consuming than thresholding and 3d morphology . the results from the combined mri - snakes method were promising when animals with moderate atherosclerosis were examined . the aim of this study was to apply the mri - snakes method in a rabbit model in order to quantify vessel wall changes after angioplasty . a double - injury , cholesterol - fed new zealand white rabbit model ( 3.13.6 kg , lidkping , farms , lidkping , sweden ) was used . six rabbits were fed with a diet , rabbit chow enriched with 0.25% cholesterol ( analycen ab , lidkping , sweden ) , known to cause atherosclerosis , for 2 weeks before the entire aorta was deendothelialized with repeated passage of a 3f embolectomy catheter . the cholesterol - enriched diet was continued for four more weeks after the deendothelialization injury . angioplasty was performed in a 3 cm long segment of the infrarenal aorta ( 30 3 mm balloon , viva angioplasty catheter , boston scientific ) , and after totally 6 weeks the cholesterol - enriched diet was stopped , and the animals were fed with regular diet . the location of the expanded balloon in the aorta was documented with x - ray angiograms and was evaluated in relation to the lumbar spine and the aortic bifurcation ( figures 1(a ) and 1(b ) ) . the initial mri was performed 2 - 3 days after pta , and four weeks later all animals were subjected to a second mri examination . three of the rabbits were followed for 6 more weeks ( i.e. , 12 weeks after deendothelialization and 10 weeks after pta ) . at that time an additional mri analysis was performed and the rabbits were sacrificed by an overdose as described below . the first half ( n = 3 ) of study group was euthanatized after 6 weeks after deendothelialization , the vasculature were perfused at 120 mm hg with ice - chilled phosphate - buffered saline followed by a fixative ( 4% paraformaldehyde in phosphate buffer ) and the aorta processed further for histological analysis . the second half ( n = 3 ) of the study group were continued on the diet for 6 more weeks ( i.e. , 10 weeks after angioplasty ) . all animal interventions were performed at the department of diagnostic radiology animal laboratory , uppsala , sweden and the study protocol was approved by the local ethics committee for animal experiments . for deendothelialization and angioplasty procedures , the animals were anesthetized with 0.33 ml / kg subcutaneous hypnorm ( 0.315 mg / ml fentanyl and 10 mg / ml fluanosine , janssen pharmaceutica ) and 0.33 ml / kg intramuscular dormicum ( midazolam , 5 mg / ml , roche ) , and the anesthesia was maintained with intermittent intravenous bolus doses . preoperative metronidazole ( 125 mg , zinacef , glaxo wellcome ) was administered and the animals were heparinized ( 1000 iu ) before catheterization . all mri procedures were performed under slight fluanisone / fentanyl and midazolam sedation . at the study end - point , full surgical anesthesia was induced as above and the animals were euthanatized with an overdose of fluanisone / fentanyl and midazolam - anesthesia . the animals were examined with a philips gyroscan acs nt - ii imager with field strength of 1.5 t using a knee coil . they were anesthetized with a mixture fluanisone / fentanyl and midazolam sedation of ketamine administered by repeated i.v . the same mri technique as described in our previous studies was used , and the examination covered a 10 cm segment of the aorta with its caudal border located at the aortic bifurcation [ 1 , 2 ] . to define the region of interest , three different survey sequences were used which facilitated correct positioning of the inflow angio sequence ( m2di ) and the proton density weighted sequence ( pdw ) . both of these sequences were used to evaluate the aortic wall . for the comparison between mri - examinations , a 2 cm section , 6 axial slices , located centrally within the pta - affected part of the vessel was selected and followed over time with repeated measurements . for each rabbit a 2 cm long segment of the aorta cranial to the pta - affected section , containing 6 axial slices from each of the imaging series , was selected as control . for each individual animal 6 the m2di sequence showed the flowing blood in the vessel with high signal intensity and the pdw visualised the surroundings with different intermediate signal intensities . the pdw sequence , a turbo spin echo sequence , was applied in two consecutive acquisitions , the second filling the gaps in the first acquisition . image processing was accomplished with a gradient vector flow ( gvf ) snake algorithm ( cf . the objective of the image processing was to identify the inner and outer boundaries of the vessel wall . the inflow angio sequence shows stationary tissue ( both vessel wall and surrounding tissue ) with low signal and flowing blood in the aorta and the inferior vena cava , with high signal intensity ( figure 3 ) . the snake is a dynamic contour that is attracted to regions in the image , which are rich in edge information . when the snake is applied to the mri images , it will seek the borders between tissues that differ in signal , such as the inner and outer border of the vessel wall , in the inflow angioimages . in the pdw images the surroundings of the lumen are presented by various intermediate signal intensities . the snake will seek the areas in the image which have most edge information , thereby identifying the outer border of the vessel wall , since this seems to be the area where the major edge information is located ( figures 4(a)4(e ) ) . a complete program for dealing with and segmenting the images after the vasculature was perfused with the fixative , the length of the infrarenal aorta was measured in situ in each animal from the bifurcation to the left renal artery . thereafter this vessel segment was divided every 10 mm beginning from the distal end . specimens were then embedded in paraffin , sectioned in 4 - 5 m slices , and stained with hematoxylin and eosin . in the histopathology images wall intima - media area and thickness was measured by delineating the contours . histopathology measurement corresponding to a single mri - slice were combined by calculating the mean value . the development of mri - measured wall area and wall thickness was evaluated with analysis of variance , allowing for variation between individuals as well as between the measured slices within the pta - affected , 6 mri - slices , and proximal segments , 6 mri - slices , and change over time ( repeated measures ) . wall area measured with mri was correlated to wall intima - media area measured with histopathology by calculating a correlation coefficient ( pearson r ) , based on all the mri slices with corresponding histopathology . at baseline , there was no significant difference in wall thickness between the lesion and the proximal piece . wall thickness within the lesion , measured with mri 4 weeks after pta ( in all 6 animals , 36 mri - slices from each region ) , was significantly larger compared to the measurement from the lesion at baseline and to proximal vessel wall thickness at simultaneous examination ( figure 5 ) . wall thickness measured at 10 weeks ( in 3 animals , 18 slices from each region ) was not significantly different from the measurement at 4 weeks but significantly larger than thickness in the segment proximal to the lesion , 36 and 18 slices , respectively ( figures 6 and 7 ) . wall thickness measured proximal to the lesion did not change significantly between different points in time ( figure 6 ) . the wall area in the lesion did not change significantly from baseline to 4 weeks ( n = 6 ) , but decreased significantly from 4 to 10 weeks ( n = 3 ) ( figure 8) . the correlation between histological intima - media thickness and the last mri - snakes - measured wall area was r = 0.89 ( p < 0.0001 ) . when technically possible , pta is often chosen as the first intervention for a patient with symptomatic arteriosclerosis . restenosis after pta is a well - known complication for which several different types of treatments are available for the patients [ 69 ] . the onset of atherosclerosis can be silent and the early stages of restenosis can also elapse silently . the purpose of our study was to find out whether it is possible to follow vessel wall changes post - pta over time , during the first 10 weeks after pta , in an animal model . the complexity of histopathology in atherosclerosis is known and has been studied by several groups [ 1 , 1015 ] . the invasive in vivo methods available for animal models include angiography , intravascular mri and ultrasound ( us ) [ 1620 ] . angiography is regarded as the gold standard when hemodynamic conditions are evaluated [ 21 , 22 ] . although angiography can diagnose calcifications in the vessel wall information about other compounds in the vessel wall is very limited . electron - beam computed tomography and multi - slice helical computerized tomography [ 2325 ] are methods that could be used noninvasively for measuring the amount of calcifications in the vessel wall , but these methods also do not produce information about vessel wall thickness and/or area . risk factors associated with the method such as the x - ray exposure and the use of iodinated contrast media must also be considered . intravascular us and mri are promising modalities when assessing the amount of different components in the vessel wall . the location of probe position has to be described in relation to the surroundings or distance from closest situated vessel branches . also determining the vessel wall measurement in relation to different layers in the wall can be difficult , since the ultrasound is completely reflected if calcifications are present in the wall . artefacts from calcifications could create loss of information from adjacent tissues and consequently a loss of information about the wall composition . us is also operator - dependent to a considerable degree [ 26 , 31 ] . percutaneous ultrasound with doppler is by far the method most used in clinical practice when hemodynamic conditions are evaluated [ 27 , 28 , 32 ] . percutaneous ultrasound can be applied in animal models , but because of the inter - observer variability it is not commonly used in animal studies . intravascular mri can be used for measuring and diagnosing vessel wall morphology [ 18 , 19 ] . although the modality is less operator dependent , its invasive approach limits its use . during the last decades several groups have presented interesting results with mri used in a non - invasive way to examine vessel wall morphology [ 3441 ] . in our study we decided to use a non - invasive quantitative method applied to an animal model to assess vessel wall changes at different points in time . the pta - affected site was located in the distal part of the abdominal aorta and a 2 cm long section of the abdominal aorta cranial to the pta location was considered as a control region . the double injury , deendothelialization during cholesterol - enriched diet followed by pta , initiates different vessel wall changes at different locations [ 42 , 43 ] . in the control region , cranial to the lesion , a major change in the intimal layer could be expected , since the deendotheliazation causes neointima development . the vessel wall reaction at 4 weeks control could be a sign of intimal thickening and instigation of change in the medial layer . the thickness and area were somewhat smaller at 10 weeks than both the initial and the second measurement . a potential source of error in this study is the location of the pta - balloon . by using x - ray films from the pta session and the images from the mri survey sequences , it was possible to correlate the position of the balloon to the vertebral bodies in the lumbar spine , figures 1(a ) and 1(b ) . the balloon , 30 3 mm , would affect the vessel wall in at least 10 mri slices . by selecting six of the slices located in the middle of the part of the aorta corresponding to the balloon location , we assume that the studied slices represent pta - affected vessel and may be compared with six slices surely proximal to the lesion . it is not clear to what extent motion artifacts from respiration affect the results and whether there are ways of reducing this . a limitation of our method is that it can not distinguish between the intima and media in the arterial wall , which can be done by ultrasound [ 27 , 29 ] . however , when only information about the effect on total wall thickness is required , the proposed method appears to be an attractive alternative to histopathological studies , as the same individual may be followed over time , resulting in better statistical power for a given sample size and requiring smaller number of subjects . in conclusion , the proposed in vivo method may detect the amount of initial vessel wall changes post - pta . in future studies , it would be interesting to follow a larger number of animals , thus a higher total of measurements , for a longer period of time and to perform corresponding histopathology analyses . the ultimate goal is to develop an in vivo method that could be used clinically to measure wall thickness / area and also provide morphological information of the vessel wall .

systemic sclerosis ( ssc ) is a connective tissue disorder characterized by internal organ involvement , dermal thickening and fibrosis . ssc is categorized into two groups : limited cutaneous ( lcssc ) and diffuse cutaneous ( dcssc ) forms . the former ( the crest syndrome ) is characterized by calcinosis , raynaud 's phenomenon , sclerodactyly , esophageal dysmotility , and telangiectasia . although joint involvement is not so common in lcssc , arthritis is reported to occur at a rate of 15% . hand joints are usually affected , followed by wrist , elbow , and knee joints . however , the sternoclavicular joint ( scj ) is rarely involved in limited cutaneous ssc , and there is insufficient information about this involvement . the primary treatment of such involvement is conservative , involving nonsteroidal anti - inflammatory drugs , rest , and cold application . in patients unresponsive to conservative treatment although ultrasonography ( us ) has been increasingly used for interventional pain management in recent years , other imaging techniques are also used for intra - articular injection . one of these techniques , fluoroscopy - guided scj injection has been scarcely covered in the literature . in this report , we present the technique of fluoroscopy - guided intra - articular steroid injection and its outcomes in scj arthritis resistant to conservative management in a patient with limited cutaneous ssc . a 50-year - old woman diagnosed with limited cutaneous ssc two years earlier presented to the rheumatology outpatient clinic with swelling and pain on the right side of her neck and the ventral surface of her chest for 3 months . she had been using hydroxychloroquine 200 mg bid , nifedipine 30 mg / day , and acetylsalicylic acid 100 mg / day . laboratory tests revealed antinuclear antibody 1/1000 + , anticentromere antibody + + + , and crp : 9,49 mg / dl , while other parameters were normal . mri revealed signs of inflammation consistent with right - sided sternoclavicular joint arthritis ( fig . 1 ) . diagnosed with scleroderma - associated sternoclavicular joint arthritis , the patient was started on prednisolone 10 mg / day . having no satisfactory relief of her pain despite a 10-day trial of medical therapy , the patient was referred to our pain medicine outpatient clinic . on physical examination she had tenderness , swelling , and minimally increased temperature on her right scj upon palpation . her pain was rated 8 on the nrs ( numeric rating scale ) . a fluoroscopy - guided intra - scj steroid injection was planned . after informed written consent was given for the scj injection , the patient was placed in a supine position on the fluoroscopy table and monitored . skin overlying the area of interest was cleansed with povidine iodine and covered with a sterile drape . the right scj was palpated and then imaged with the aid of fluoroscopy in the anteroposterior view . under fluoroscopic guidance , a 5-cm , 21 g needle was entered into the right scj . the contrast material was injected to verify an intra - articular location of the needle ( fig . 2 ) . then , a mixture of 20 mg methylprednisolone and 0.5 cc bupivacaine 5% was injected . the nrs level was 0 and there was no tenderness in joint palpation at control examinations at the 1st hour , 1st month , and 3rd month . in this case , steroid injection into scj was done under fluoroscopy guidance for scj arthritis secondary to limited cutaneous scleroderma . pain level dropped to a level of nrs 0 at the control examinations at the 1st hour , 1st month , and 3rd month from a preprocedural level of nrs 8 , indicating 100% pain relief and improvement in examination findings . there are only a few reports about steroid injection into the scj under fluoroscopy guidance . in which 40 mg methyl prednisolone and 1 ml bupivacaine were injected into the scj under fluoroscopy guidance in a patient with osteitis condensans of the clavicle . that patient experienced an immediate relief of pain following the procedure , and the outcomes of the procedure were consistent with that of our patient , whose pain had regressed to nrs 0 at the 1st hour . that case had some differences from ours , including the lack of reporting of long - term outcomes ; using 40 mg methylprednisolone ; and applying an scj injection for osteitis condensans of the clavicle . in a retrospective analysis , peterson et al . explored the 20 - 30 minute results of the ct - guided injection of triamcinolone 40 mg into a total of 83 scjs of 50 patients with osteoarthritis or scj subluxation . pain relief was achieved in a short period in a majority of patients , as was the case in our study . that study , however , differs from our study with regard to reporting a ct - guided procedure ; including patients with different etiological causes ; and providing long - term follow - up data . our technique has the significant advantage of using a lower radiation dose with fluoroscopy - guided technique compared to the ct - guided technique . furthermore , taking into account the small size of scj and the possible risks of steroid injection , we preferred using a lower steroid dose compared to the cases of galla et al . and peterson et al . this case is an important contribution to the literature due to its reporting the rare scj scj involvement in a patient with limited cutaneous scleroderma , obtaining an easy access to the target area under fluoroscopy - guidance , showing a lower complication risk , and providing long - term treatment outcomes . however , it also has some limitations , including the lack of reporting long - term treatment outcomes , exposing the patient to radiation unlike the us - guided procedure , and requiring special hardware and equipment unlike the blind procedure . ultrasound provides the advantages of injection accuracy , reliability , and the ability to perform real - time image - guided procedures . in this case , fluoroscopy - guided injection was preferred , as we had much more experience with fluoroscopy - guided injections than with ultrasound - guided injections . in conclusion , scj arthritis should be considered among the etiological causes of chest pain in patients with limited cutaneous systemic sclerosis . for these patients , intra - scj steroid injection is an effective treatment option when pain does not respond to conservative treatment methods . among injection options , fluoroscopy - guided intra - sternoclavicular joint steroid injection should be regarded as a safe alternative method .

renal tubular acidosis ( rta ) is a disorder of renal acidification out of proportion to the reduction in glomerular filtration rate characterized by normal anion gap ( ag ) metabolic acidosis . type 1 rta can cause hypokalemia ; however , initial presentation of type1 rta with hypokalemic quadriparesis with bulbar weakness and cardiac arrhythmia is extremely rare . only 14 cases of distal rta with hypokalemic muscle paralysis have been previously reported in association with sjogren syndrome . a 54-year - old postmenopausal female patient presented with history of intermittent low - grade fever 8 days back followed by weakness of lower limbs ( difficulty in getting up from squatting position associated with dull aching pain in both buttocks and thighs ) 2 days prior to admission . the weakness rapidly worsened by next day to involve the trunk , upper extremities , and neck muscles such that the patient was barely able to move in bed by herself or lift her head . on the day of admission , the patient also complained of difficulty in swallowing solid food and inability to speak loudly . there was no history of visual blurring , diplopia , facial asymmetry , sensory , bladder , or bowel complains . she had a clear sensorium with no history of seizures , involuntary movements , root pains , or band - like sensation over the trunk . she did not give history of prior drug intake , colicky abdominal pain , recent vaccination , or similar episodic weakness in the past . her past medical and family histories were uneventful . on examination , patient was conscious , cooperative , and oriented . power was reduced in all limbs , proximal muscles ( grade 1/5 ) being more severely affected than distal ones ( grade 3/5 ) . muscle stretch reflexes were normal at all joints except that bilateral ankle jerks were depressed . ecg was obtained , which showed intermittent atrial fibrillation with multiple multifocal ventricular ectopics with conduction abnormalities . investigations revealed that serum sodium was 130meq / l , potassium 2.0meq / l , chloride 110meq / l , creatinine 1.5mg / dl , magnesium 1.95mg / dl , mild leucocytosis with esr of 40 mm at 1 h. random blood sugar was 89mg / dl , thyroid function tests being within normal limits . serum protein electrophoresis showed total protein of 8.9g / dl , serum albumin 3.36g / dl , serum globulin 5.54g / dl , a / g ratio 0.61 , gamma globulin 4.27g / dl , and diffuse band in gamma region suggesting polyclonal hypergammaglobulinemia . arterial blood gas ( abg ) analysis showed ph=7.22 , pco2 = 18.4 kpa , po2 = 76.3 kpa , hco3 = 7.4mmol / l , so2 = 90.7% . the urinary ph was 7.0 ; there was 2 + proteinuria and microscopic examination revealed 710 wbcs / hpf without casts . trans tubular potassium gradient ( ttkg ) was 12.5 , suggestive of renal potassium loss . patient was given intravenous ringer 's lactate along with potassium supplements through ryle 's tube with serum potassium monitored 12 hourly . ecg was repeated which showed reversion to normal sinus rhythm . following improvement in gag reflex and ability to take orally , oral sodamint tablets ( sodium bicarbonate ) and patient 's weakness showed significant improvement in 24 h and complete recovery within 48 h of admission . serum anti ss - a / ro : positive ( above 200 ) and serum anti ss - b / la : positive ( 157 ) suggesting the possibility of sjogren syndrome . schirmer i test was negative in both eyes ( > 5 mm at 5 min ) . finally the definitive diagnosis was hypokalemic quadriparesis with bulbar weakness with distal ( type 1 ) rta with hypergammaglobulinemia with probable sjogren syndrome . patient was discharged on sodamint , two tablets four times a day and potassium citrate syrup two teaspoons thrice a day . in distal ( type 1 ) rta the nephrons lack the ability to secrete h ions and hence acidify the urine normally during spontaneous or induced metabolic acidosis . inherited forms include autosomal - dominant , autosomal - recessive , or x - linked recessive , of which autosomal - dominant form causing mutations in the basolateral chloride - bicarbonate exchanger ( ae1 ) has been identified as the most common form of inheritance . acquired causes include hypergammaglobulinemic states , such as hyperglobulinemic purpura , cryoglobulinemia , fibrosing alveolitis , sjogren syndrome , lupus , chronic active hepatitis , thyroiditis , graves disease , primary biliary cirrhosis ; disorders of calcium metabolism , e.g. , primary hyperparathyroidism , vitamin d intoxication , idiopathic hypercalciuria , familial absorptive hypercalciuria , medullary sponge kidney . tubulointerstitial diseases include leprosy , hyperoxaluria , chronic pyelonephritis , obstructive uropathy ; and genetic diseases like ehler danlos syndrome , hereditary eliptocytosis , south asian ovalocytosis , sickle cell disease , carbonic anhydrase ii deficiency . in distal rta , the urinary ammonium excretion is inappropriately low for the level of acidosis as the defect in acidification decreases ion trapping required for ammonia secretion . this differentiates from chronic diarrhea in which the uag is negative due to enhanced renal ammonium excretion . also ttkg in diarrhea is < 4 due to renal conservation of potassium , hence excluded in our case . in distal rta , there is a tendency for renal calculi , nephrocalcinosis due to hypercalciuria , and hypocitraturia . severely depressed plasma bicarbonate levels with a corresponding inappropriate urinary ph > 5.5 differentiates from type 2 rta . finally the requirement of the patient to maintain plasma bicarbonate levels near normal was less than 1meq / kg body weight pointed toward distal rta . the interesting feature of this case was the presentation with severe hypokalemia , causing acute flaccid quadriparesis with bulbar weakness with cardiac arrhythmia which is extremely rare . this might have been precipitated by underlying urinary tract infection , which may increase the bicarbonate requirement and cause volume depletion and potassium loss . sudden life - threatening hypokalemia with muscle paralysis is the most serious clinical consequence of distal rta . warning symptoms of muscle weakness , psychic apathy , and lethargy are often present for months or years before muscle paralysis occurs . to our knowledge , only 14 cases of distal rta with hypokalemic muscle paralysis have been previously reported in sjogren syndrome . except for one patient delays of anywhere from 3 months to 5 years from the onset of initial clinical manifestation have been reported before a diagnosis of sjogren syndrome is established . urolithiasis and distal rta can precede subjective sicca symptoms , and patients with drta may have sjogren syndrome in the absence of subjective sicca symptoms . anti - ss - a antibodies are common in patients with urolithiasis and distal rta . at least six patients with distal rta and objective signs of sjogren syndrome , but without subjective sicca symptoms , have been reported . thus , sjogren syndrome can be diagnosed in drta patients even in the absence of subjective sicca symptoms . although the diagnostic criteria for sjogren syndrome were not satisfied and that our patient was not having signs and symptoms of xerophthalmia or xerostomia , whether distal rta could be a manifestation of evolving sjogen syndrome is a matter of debate .

civilization progress is associated with unfavorable changes in the lifestyle of the majority of human population . improper diet , rich in fat and carbohydrates , and simultaneous limitation of physical activity can lead to the development of a severe metabolic disorder , diabetes . according to who estimates the worldwide population of patients with diabetes will reach 20 million by 2030 ( 1 ) . diabetes mellitus is a chronic metabolic disorder that can result in multiple long - term micro- and macro - vascular complications . diabetes is well known as the leading cause of blindness , end - stage renal disease ( esrd ) and limb amputation . in molecular level , this disease is the sixth leading cause of death in the world and developing countries . based on various studies , diabetes can lower the life expectancy in humans by about 5 to 10 years ( 2 ) . wide prevalence of diabetes , along with acute and chronic complications that are associated with this disorder has created many potential threats to the human race . statistics show that 14 to 23 percent of the iranian population over the age of 30 years has diabetes ( 3 , 4 ) . one of every twenty iranian people is affected with diabetes and half of these numbers are not aware of their disease . every 30 seconds one person loses their feet in the world because of the diabetes ( 3 , 5 ) . vascular complications of diabetes are classically divided into micro - vascular ( caused by damage to small blood vessels ) and macro - vascular ( caused by damage to larger blood vessels ) . diabetic retinopathy involves changes to retinal blood vessels that can cause them to bleed or leak fluid , distorting vision . diabetic retinopathy is the most common cause of vision loss among people with diabetes and a leading cause of blindness among working - age adults . it should also be noted that people with diabetes are at higher risk of developing glaucoma , cataracts and age - related macular degradation ( amd ) ( 6 , 7 ) . diabetic retinopathy is a common cause of vision loss and blindness ( 25% of cases of blindness in western countries ) . the presence and severity of diabetic retinopathy mostly depends on the age of patient at the time of diagnosis and duration of diabetes other than severity of the disease and control of blood sugar . 100% of patients with type i diabetes and 60% to 80% of patients with type ii diabetes develop retinopathy in the first 20 years of disorder . with improvements in methods of treatment and diagnosis of both diabetes and retinopathy , only a small percentage of diabetic patients will develop serious vision problems , provided that they undergo examinations in the proper time ( 8 , 9 ) . there are two main types of diabetic retinopathy , including background diabetic retinopathy ( small artery aneurysms , infarcts or interrupt blood flow to part of the retina ) and proliferative retinopathy . however macular edema or proliferative retinopathy ( especially new blood vessels near the optic disc ) requires immediate treatment with laser photocoagulation in order to prevent loss of vision ( 10 ) . refraction , the determination of the refractive error of an eye , is an essential part of eye care . refraction is used clinically to determine the spectacle prescription so that the best possible acuity can be achieved . this method is used to measure the visual acuity ( va ) in diabetic patients . patient perception of quality and resolution of vision determines the status of the eye , while in the second type , autorefractometer , as the name suggests , an automatic instrument determines the patient s eye number . for many years , clinical research studies have utilized the manual refrectometry testing method for standardizing refraction and subsequent measurement of va . however , this method requires substantial investment in training and certification of refractionists , and the procedure itself can be time consuming . thus , an acceptable , less time intensive alternative to the rigorous manual refrectometry procedure might result in substantial savings of cost and time for clinical trials in diabetic retinopathy . it might also improve clinical trial subject recruitment and retention because of shorter and less technically burdensome clinic visits . this technique utilizes a computer - controlled device to provide an objective measure of an individual s refractive error without the need for a skilled refractionist . since first being described and validated against manual refraction in the early 1970 ( 11 ) , autorefractors have come into widespread clinical use due to the ease and speed of the semi - automated autorefraction procedure , the lack of need for a trained refractionist , and commercial availability . in clinical trials , the role of autorefraction has been limited to providing starting information for subsequent manual refraction . however , results from a recent single site study sponsored by the diabetic retinopathy clinical research network ( drcr.net ) suggest that autorefraction using certain devices may be an acceptable substitute for the manual refraction in obtaining best corrected va in eyes of patients with diabetes ( 12 ) . the purpose of the study was to evaluate the comparison of manual refraction versus autorefraction in diabetic retinopathy patients . number of samples was determined according to available sources and sample size calculation formula ( n=2(z1-/2+z1- ) 22d2 ) which is based on comparison of average sample size . according to these preconceptions in period of three months study of manual refraction and automatic refraction and comparison of results from these two methods . in manual refraction , patient perception of quality and resolution of pictures was questioned and based on this perceptions the status of the patient s eye was determined . in auto refraction , patient were sat in front of automatic refractometer and at any moment an image was displayed in front of one eye , subsequently the device determined status of patient s eye and therefor the whole process is completely objective . also in this study age , sex , marital status , location , diet , amount of daily exercise , weight and disease history of all participants was recorded . all patients with diabetic retinopathy who had a blood sugar below 200 were included in the study and patients with history of trauma , ocular surgery , cataract , glaucoma , uveitis and other eye diseases were excluded . the average age of the participants in this study was 47.3 years with standard deviation of 13.58 years , the youngest participants was 30 years old and the oldest one was 68 years old . of the 60 patients , 20 patients ( 33.3% ) were affected with grade 1 , 26 patients ( 43.3% ) with grade 2 and 14 patients ( 23.3% ) with grade 3 retinopathy , respectively ( figure 1 ) . the mean of va score for autorefraction and manual refraction was 0.28 and 0.24 with standard deviations of 0.23 and 0.22 respectively . mean of se score for autorefraction and manual refraction was 1.47 and 1.51 with standard deviations of 2.73 and 2.3 respectively ( figure 2 ) . results of independent t - test on these two variables ( mean of va and se ) showed that , although differences in va between the two groups of patients was significant ( p=0.001 ) , no statistically meaningful difference exists in spherical equivalent ( se ) score of the patients ( p=0.539 ) . in addition , the numbers for these two variables were very close to each other . comparison of mean of va and se values for auto versus manual refraction method in diabetic retinopathy patients . chronic diseases such as diabetes mellitus and its complications has now become one of the most important health problems in society . diabetic retinopathy is one of the microvascular complications of diabetes which is under influence of many factors ( 6 ) . comparison of retinopathy in different countries is quite complicated because of differences in the types of diabetes in each country , the number of patients studied in different investigations and differences in examination methods ( 13 ) . diabetic retinopathy is a common cause of vision loss and blindness ( 25% of cases of blindness in western countries ) . those who have untreated diabetes , are 25 times more at risk of blindness than other people ( 10 ) . the presence and severity of diabetic retinopathy is mostly related to age of the patient at the time of diagnosis and duration of diabetes , rather than severity of the disease and control of blood sugar . after 20 years , retinopathy occurs in 100% of patients with type i diabetes and 60% to 80% of patients with type ii diabetes ( 14 ) . on the other hand , the training and certification of examiners to accurately refract study participants for the determination of best corrected va is both time - consuming and expensive . the rigorous manual refraction protocol currently in use also is time consuming to perform and lengthens visits for study participants . thus , the ability to substitute an automated refraction for manual refraction could streamline study visits and result in substantial savings of time and cost for clinical staff . there are a huge body of literature regarding diabetic retinopathy , its prevalence and diagnosis . in a study by funatsu et al to assess retinopathy patients awareness of their disorder , 1333 patients with type ii diabetes were measured by a questionnaire . results of this study showed that although more than 98 percent of patients were aware of their disease , about 30.5% of them refuse to undergo periodic ocular examination because they were not familiar with visual complications of diabetes ( 15 ) . in 2001 orr et al studied 29 patients affected with sub foveal choroidal neovascularization ( cnv ) . on average , manual refraction was reported to be one spherical ( equivalent to 1.04 diopter ) more than auto refraction . moreover , average score of perspicuity in manual refraction was 1.5 times higher than auto refraction ( 2 ) . rein et al studied costs of treatment of over 10 million diabetic patients in the age of 30 to 48 years . results of this work indicate that one - year treatment and examinations of the patients are more affordable than those of two - year , because in the one year excess period , patients may become affected with microaneurysms ( 16 ) . the aim of this study was to compare manual refraction with automatic refraction in patients suffering from diabetic retinopathy . our results showed that , despite significant differences in va between the two groups ( p=0.001 ) , there was no statistically significant difference in se score of the patients ( p=0.539 ) . in addition , the numbers for these two variables were very close to each other . these findings are in contrast with orr s study which was concluded that amount of se in autorefraction is considerably higher than manual refraction . other studies are mostly in support of peggy et al and in contrast to ours ( 2 , 12 ) . the most probable explanation for this difference could be variation in the type of underlying disease . for example , peggy et al studied patients with subfoveal choroidal neovascularization . studies by canon et al and lake success et al are also done on the same subtype of disorder and are in favor of peggys study . in 2006 chung et al studied the accuracy of autorefraction in comparison with manual refraction in diagnosis of children with refractive error . in this study , total number of 117 children was examined by three types of autorefraction machine and manual refraction instrument with and without cycloplegia . results of this study showed that in non - cycloplasia condition , all three types of instruments have more false positive diagnosis for myopia , but with inclusion of cycloplasia diagnosis was associated with more accuracy and hence less false positive results ( 17 ) . in a prospective study by pesudovs et al in 2004 on 190 patients , differences in using auto versus this study showed that results of diagnosis by both types of autorefraction instruments , nidek ark-700a ( fremont , ca ) and topcon kr-8000 ( paramus , nj ) , are completely comparable with results from manual refraction methods . despite a significant difference in the average amount of aspherical ( 0.14 diopters ) , almost the same limitations of the proposed clinical similarities was present in both methods ( 18 ) . for conclusion , results of this study show that although use of autorefraction is comparable with manual refraction in evaluating se scores in diabetic patients affected with retinopathy but in the case of va results from these two methods are not close to each other . moreover , with exception of grade ii , there was no significant difference between va scores in different retinopathy grades . although in general autorefraction may not be an acceptable substitute for manual refraction , specific elements of study design including increased sample size may allow limited substitution of autorefraction for manual refraction in some studies and even in the clinical practice .

about 40% of patients have extra - nodal involvement of the skin , orbit , central nervous system , visceral organs and respiratory system . we report a challenging case of extranodal rosai dorfman disease in a five - year - old child , initially misdiagnosed as orbital inflammatory disease . the child 's first orbital biopsy showed non - specific chronic inflammation and he was treated with oral steroids for at least one year . a previously healthy two - year - old male presented to a pediatric ophthalmologist with a history of acute proptosis of the left eye associated with fever . he had prominent proptosis of the left eye and no palpable lymph node enlargement was noted . a computerized tomography ( ct ) scan of the orbits done at that time revealed a left retro - orbital soft tissue mass with minimal contrast enhancement . he was diagnosed with orbital cellulitis and treated with a course of intravenous ciprofloxacin with little improvement . histopathology showed tissue infiltrated by lymphocytes , plasma cells , hemosiderin - laden macrophages and histiocytes with mild stromal fibrosis . a presumptive diagnosis of fibrosing orbital inflammatory disease was made and he prescribed oral prednisone ( 30 mg / day ) 1 mg per kilogram with some improvement . three years later the patient presented to our unit with recurrent proptosis of the left eye and lid swelling , which could be felt as two firm palpable masses in the upper lid and one in the lower lid [ figure 1 ] . best corrected visual acuity was 20/40 in both eyes and he had mild limitation of abduction of the left eye . magnetic resonance imaging ( mri ) of the orbits showed extensive soft retro - orbital tissue mass , extending to the left cavernous sinus , iso - intense on t1 and t2 with diffuse post - contrast enhancement [ figure 2 ] . lid swelling of the left upper and lower lid with globe dystopia axial t1-weighted gadolinium - enhanced mri showing proptosis and enhancing a retrobulbar lesion involving left lacrimal gland and subcutaneous tissue another orbital biopsy was performed through an upper and lower lid skin approach , along with an adenoidectomy and biopsy of the left inferior turbinate . histopathological examination revealed an infiltrate of lymphocytes ( both b and t ) , polyclonal plasma cells and histiocytes ; however , no eosinophils were present . some of the histiocytes contained numerous intact mononuclear cells , plasma cells and erythrocytes in their cytoplasm . no reed - sternberg cells , dutcher bodies or micro - organisms ( using periodic - acid schiff , gram , ziehl neelson and wade fite stains ) were seen . barr virus staining , bcl-2 and cd1a . however , it was positive for s100 and weakly positive for cd68 . all of the histopathological and immunohistochemistry characteristics ruled out possible malignancy or infection and the findings were consistent with rdd . due to the large extension of the lesion and the cosmetic appearance , rdd ( sinus histiocytosis with massive lymphadenopathy ) is characterized by massive painless bilateral lymph node enlargement in the neck . it was first described by destombes and later by azoury and reed ; however , the condition was named after rosai and dorfman , who described four cases in 1969.1 this entity usually affects males in their first to second decade of life . however , some studies suggest that human herpes virus type 6 ( hhv-6 ) and epstein - barr virus may play a role.2 most patients present with massive cervical lymphadenopathy , but about 40% of patients will have extra - nodal manifestations involving the skin , nasal cavity and paranasal sinus , eye and ocular adnexa , bone , central nervous system , liver and heart.3 systemic manifestations include fever , leukocytosis , elevated erythrocyte sedimentation rate and hypergammaglobulinemia . ophthalmological manifestations occur in 10% of cases , and these include eyelid thickening , swelling of the lacrimal gland , proptosis and anterior granulomatous uveitis.4 a definite diagnosis of rdd requires histopathological and immunohistochemical confirmation . the disease is characterized clinically by massive lymph node enlargement showing sinus histiocytosis . however in extranodal disease , the infiltrate can be polymorphous , composing of small mature lymphocytes , plasma cells and histiocytes . emperipolesis , in which the histiocyte 's cytoplasm contains small lymphocytes , is a very typical pathological finding of rdd [ figure 3 ] . the histiocytes are usually positive for s100 protein and cd68 , but negative for cd1a , which is a differentiating feature from langerhans cell histiocytosis . in addition , langerhans cells usually show a distinctive nuclear grooving and contain birbeck granules when examined under electron microscopy . pathological section of the patient demonstrating emperipolesis ( arrow ) rdd is considered benign and mild and localized cases can be observed . in more severe cases , surgical excision , radiation therapy , corticosteroid and other immunosuppressive therapies should be considered . in contrast to idiopathic orbital inflammatory syndrome in which medical treatment is the initial management of choice , surgical excision can be considered as a first - line therapy in rdd and may be curative.5 corticosteroids have been found to produce resolution after surgical excision.6 various chemotherapeutic agents have been used , with vinca alkaloids being reported as the most effective in combination with corticosteroids.7 our patient presented with a picture resembling orbital inflammatory disease , which led to diagnostic confusion , even after radiographic imaging . hence , even patients diagnosed with orbital inflammatory disease should be closely followed and evaluated to ensure that it is not a masquerading disease . other possibilities considered in our patient were infectious causes and neoplasms such as rhabdomyosarcoma , leukemia and lymphoma . initial biopsy was suggestive of non - specific inflammation and hence the patient was treated as a case of orbital inflammatory disease . due to absence of follow - up for a considerable amount of time the inflammatory lesion had extended into adjacent tissues and intracranially making complete surgical excision impossible . hence the role of a repeated biopsy if necessary in such difficult clinical situations can not be overemphasized .

eckert & ziegler bebig , gmbh , germany , introduced a new afterloading brachytherapy machine ( multisource ) . it has two options to use either co or ir source for high dose rate ( hdr ) brachytherapy . the co source has an advantage due to its longer half lives ( 5.27 years ) . miniaturized co source with sufficient activity ( 70 gbq ) is available on afterloading equipment , dedicated to hdr brachytherapy . the new co source ( model co0.a86 ) referred as new bebig co hdr source is a modified version of the old co source ( model gk60m21 ) from bebig . this study is aimed at obtaining the dosimetry parameters with the tg-43u1 formalism of the american association of physicists in medicine ( aapm ) , and with prerequisites of hebd report . this study is performed using the egsnrc monte carlo transport code ( version v4-r2 - 3 - 1 ) with appropriate electron cutoff energy 0.521 mev ( rest of electron energy + 10 kev as kinetic energy ) , referred as true dose calculation for all subsequent calculations . richter et al . have reported a comparison of co and ir sources using egs - ray monte carlo based calculations , and only photon emission has been considered for the simulations . recently , selvam et al . have published egsnrc monte carlo based dosimetry data except anisotropy functions , and collision kerma is approximated the dose at the close surface of the source . moreover , ballester et al . and granero et al . have reported geant4 based monte carlo dosimetry data in accordance with tg-43u1 formalism for the same source . recently , aapm and estro published consensus data set for photon - emitting brachytherapy sources and the dosimetric data for the bebig co source with same model are available which was taken from granero et al . and selvam et al . . this report consist of radial dose function data , and anisotropy function data are apart from the 0.1 cm and 0.25 cm of radial distances , respectively from the source . this study , the radial dose function data , and anisotropy function data are calculated apart from the 0.06 cm and 0.2 cm of radial distances , respectively from the source . the radial dose function data are calculated with electron cutoff energy 2 mev and 0.521 mev , and the calculated data are compared . 2d dose rate data are calculated for cartesian and polar coordinate system using the dosrznrc user code . the radial dose function and anisotropy function are calculated from the dose rate table of polar coordinate system with tg-43u1 formalism . the specifications of the source geometry and materials are taken from islam et al . . the bebig co hdr source consists of pure cobalt metal ( density of 8.9 g cm ) , and is kept inside the source cylinder having diameter 0.05 cm and length 0.35 cm . the source core is encapsulated with an aisi 316l stainless steel capsule with 0.1 cm in outer diameter and 0.07 cm in inner diameter . the capsule is 0.5 cm long and connected to a 0.2 cm long steel cable . the capsule thickness is 0.075 cm for both longitudinal side of the co core and the steel thickness of axial side is 0.015 cm . there is an air gap of 0.01 cm around the axial side of the active source core . the rounded source tip of the real source geometry is modeled as flat with uniform thickness of 0.075 cm stainless steel , because a rounded tip can not be simulated in dosrznrc . the diameter of the source cable is modeled as same as the diameter of source capsule for simplicity . figure 1a shows the geometry of the real bebig co hdr source , and figure 1b shows the model of it used in the monte carlo calculations . a ) schematic diagram of the new bebig 60co hdr source and ( b ) the model diagram of the source used in monte carlo simulations the dosrznrc is a user code for absorbed dose calculation of egsnrc based monte carlo transport code system . this code is used for the calculation of absorbed dose rate distribution around the new bebig co source . two photon energies , 1.17 and 1.33 mev are considered for co source , and in each disintegration two photons/(bq s ) are generated on average . the photon energy spectrum file ( bareco60.spectrum ) has been used for these simulations . for the absorbed dose rate calculation , the source is positioned at the centre of a cylindrical unbounded water phantom of dimensions 200 cm ( diameter ) 200 cm ( height ) . the density of water considered is 0.998 g / cm at 22c . in order to provide adequate spatial resolution , the cells are 0.01 cm thickness for r < 2 cm , 0.05 cm for 2 < r < 5 cm , 0.1 cm for 5 < r < 10 cm and 0.2 cm for r > 10 cm from the source [ 2 , 10 ] . the dose rate values are calculated in different positions of the water phantom with polar and cartesian co - ordinates for different position of the water phantom . the true dose rate is calculated for all points of interest , and these values are used to calculate tg-43u1 parameters e.g. , radial dose function and anisotropy function . up to 5 10 primary photon histories are simulated to obtain dose rate data . the cut - off energy for photon and electron transport are 0.001 mev , and 0.521 mev , respectively , as maintained in the dose rate calculations for all radial distances . consequently , photoelectric effect , pair production , rayleigh scattering and bound compton scattering are included in simulation . the contribution of primary electron to the dose is not considered , i.e. no beta spectrum is simulated . the value of air - kerma strength , sk / a (= 3.039 10 0.41% u bq ) and dose rate constant , (= 1.097 0.12% cgy h u ) are taken from islam et al . . the authors calculated the air - kerma per initial particle at 100 cm distance from the center of the source as per aapm tg-43u1 recommendations . the mass energy - absorption coefficient for dry air was taken from the latest nist compilation . the photon fluency spectrum at 5 kev intervals was scored along the transverse axis for the point of 100 cm distance . the user - code flurznrc was used to calculate the differential fluency spectrum in the calculation grid per initial photon in the simulation . to estimate the air - kerma strength , the source is kept in an unbounded cylindrical air phantom , and the kerma was scored for a 0.2 cm thick and 0.1 cm high cylindrical ring cell , located along the transverse source axis . in the present work , the radial dose functions are calculated for the distance of 0.06 cm to 100 cm from the source centre for bebig co hdr source considering electron cutoff energy 0.521 mev and 2 mev . figure 2 shows the comparison for radial dose functions for different cutoff energies of electron . the radial dose function ( fig . 2 ) values ( in case of electron cutoff energy 0.521 mev ) from 0.06 cm to 0.18 cm are lower than the values simulated with electron cutoff energy 2 mev , and the values from 0.2 cm to 0.8 cm are 2.36% ( average ) higher than the values of other radial dose function with ecut = 2 mev . figure 3 shows the graphical comparison of the radial dose function with consensus data set [ 2 , 5 ] . figure 4 shows the polynomial curve along with the data points to present the radial dose function for bebig co hdr source . the anisotropy functions for bebig co hdr source are compared with geant4 monte carlo based published value for different radial distances by granero et al . [ 2 , 7 ] . good agreement with the published data was observed except the values less than 40 angle at 0.5 cm distance . figure 5 shows the graphical comparison of the anisotropy functions for different distances . along - away the statistical uncertainties ( type a ) on the calculation are estimated have a coverage factor k = 1 . this is depending on the location of calculation points , calculating grid size , and number of histories simulated . the uncertainties were obtained 0.1% at r < 0.2 cm , 0.3% at 0.2 < r 1 cm and 0.7% at 1 radial dose function for the bebig co source ( model co0.a86 ) comparison of radial dose functions for bebig co hdr source ; simulated with electron cutoff energy 2 mev and 0.521 mev comparison of radial dose functions for bebig co hdr source with this study and consensus data set [ 2 , 5 ] the polynomial fit curve with radial dose function for bebig co hdr source from 0.06 cm to 100 cm comparison of anisotropy functions for bebig co hdr source at different distances from the center of active source . a ) 0.50 cm , b ) 1.00 cm , c ) 3.00 cm , d ) 5.00 cm , e ) 10.00 cm , f ) 15.00 cm with the literature data from granero et al . calculated 5 order polynomial coefficient values of radial dose function from 0.06 cm to 100 cm of radial distances anisotropy functions f(r , ) for new bebig co hdr source ( model co0.a86 ) . the origin is chosen at the center of the co core and the simulated angle values are increased from the source tip to the cable side of the source along away dose rate data for bebig co hdr source ( cgy h u ) . the origin is chosen at the center of the co core and the negative values of distance are placed on the side from origin to the source tip and the positive values for other side the radial dose function for bebig co hdr source was calculated from 0.06 cm to 100 cm of radial distances . the values of the function from 0.06 cm to 0.16 cm are low and these values gradually increased up to 0.3 cm radial distance . however , it sharply decreased with longer distances . the radial dose function values are compared with the values of consensus data set reported by selvam et al . [ 2 , 5 ] using egsnrc code system , and it is in good agreement with the published data for the range . the data for < 0.1 cm is not available in consensus data set , and extrapolated value is included for 0 distances which is the same as the value of 0.1 cm . in this study , the equation for radial dose function , gl(r ) = a0 + a1r + a2r + a3r + a4r + a5r corrects a typographical error in the original tg-43u1 protocol . here , gl(r ) is denoted the radial dose function for the point r and a0 , a1 , a2 , a3 , a4 , a5 is polynomial coefficients up to 5 order . while table lookup via linear interpolation or any appropriate mathematical model fit to the data may be used to evaluate g x(r ) , some commercial treatment planning systems currently accommodate a fifth - order polynomial fit to the tabulated g(r ) data . since this type of polynomial fit may produce erroneous results with large errors outside the radial range used to determine the fit , alternate fitting equations have been proposed which are less susceptible to this effect . the parameters a0 through a5 are fitted , so that the calculated radial dose function from the polynomial curve within 2% compared with original data in table 1 to the radial distance 0.2 cm to 60 cm . the electron cutoff energy influenced dosimetry at the close region of the source due to considering the contribution of betas . in case of catheter based interstitial brachytherapy , higher values of radial dose function ( 2.36% from 0.2 cm to 0.8 cm of radial distances ) may significant affect in radial dose distribution in clinical relevant situation . some of the points in the table are data blank , which are situated within the source encapsulation or in the source core . the anisotropy functions for bebig co hdr source are compared with geant4 monte carlo based published value for different radial distances by granero et al . . the significant difference is noted with 0.5 cm of radial distance including polar angle less than 40 , which could be mainly derived from the influence of geometry model ( the tips of the source and encapsulation ) . in this case , the authors calculated kerma to approximate the dose for the points where electronic equilibrium exists , and the cutoff energy of 10 kev was used for both photons and electrons . the along - away 2d dose rate data are presented for 20 cm apart from the source center . the comparison of away - along tables from both studies reveals that at y = 0.25 cm and z = 0.25 , z = 0 , and z = 0.25 cm the granero et al . data are underestimated . are 6.5% , 8.5% and 5.2% lower than the values obtained from this study , respectively . in this study , the dosimetric data for new bebig co source ( model co0.a86 ) is obtained for an unbounded liquid water phantom . electron contribution is considered with appropriate cutoff energy for absorbed dose calculations throughout the study using egsnrc - based dosrznrc user code .

the home - made balloon sheath mainly consisted of an introducer sheath and a supporting balloon ( figs . it was divided into four parts : the distal angulated part , the balloon part , the straight part , and the proximal handling part ( fig . the introducer sheath was made of a polytetrafluoroethylene ( ptfe ) tube ( sang - a flontec co. , incheon , korea ) with a 4.2 mm inner diameter , a 4.8 mm outer diameter and a 60 cm length . the distal part of the sheath was 8.0 cm in length and it was bent at a 100 angle . a side supporting balloon ( 8.0 cm in length ) was made of latex tube ( unidus co. , seoul , korea ) , and this was attached to the sheath with nylon strings ( teleflex medical , coventry , ct , usa ) . the balloon was designed to point to the opposite side of the angulated tip when it was inflated . a polyetheretherketon ( peek ) tube ( victrex , orangeburg , sc , usa ) was used as the balloon inflating side tube , and this was connected to the proximal part of the balloon . the polyetheretherketon side tube had a 1.0 mm inner diameter and a 1.3 mm outer diameter . the proximal end of the peek side tube was connected with a side - arm that included a three - way stopcock ( cook , bloomington , in , usa ) . the straight part constituted the main part of the introducer sheath ( 44 cm in length ) and it ran parallel with the peek side tube . the distal angulated part and the straight part of the introducer sheath were covered with a heat - shrinkable tube ( 6.0 mm outer diameter ) ( lg cable , anyang , korea ) . the chemical - resistant heat - shrinkable tube was used to tightly wrap the both ends of the side supporting balloon ( nylon - strings ) as well as to mount the straight part of the introducer sheath and the peek side tube together . three radiopaque gold rings ( 0.1 mm in thickness and 1.0 mm in width ) were attached to the sheath with glue at both ends of the balloon and at the distal tip of the sheath ; these acted as markers to improve the radiopacity of the sheath . last , a plastic handling hub ( s&g biotech , seoul , korea ) was connected to the proximal end of the sheath . two types of silicone phantom models were made for the balloon sheath study : one simulated a normal anatomic condition , and the other simulated a 70% concentric stenosis at the gastroduodenal junction . first , a paper plaster clay model was made according to the anatomy and size of a normal human stomach and duodenum . it included the lower part of the esophagus , the entire stomach , the duodenum and the proximal portion of the jejunum . second , after the model had dried for three days , it was put in a lucent molding box . fluid silicone ( shin - etsu chemical co. , tokyo , japan ) was poured into the molding box , and it was allowed to harden . at last , the gastroduodenal phantom was divided into several portions according to the normal human anatomy , including the lower part of the esophagus , the gastric fundus , the gastric body , the gastric antrum , the duodenum and the proximal portion of the jejunum . the duodenum of the phantom was approximately 35 cm in length and it was divided into four segments : the first duodenal bulb portion segment , the second descending portion segment , the third horizontal portion segment and the forth ascending portion segment ( figs . the gastroduodenal junction was angled at about 90 in a posterior direction relative to the long axis of the gastric antrum ( fig . a 150 cm , 0.035-inch hydrophilic guide wire with an angled tip ( radifocus , m - terumo , tokyo , japan ) and a multifunctional coil catheter ( s&g biotech , seoul , korea ) were used in our phantom study . the multifunctional coil catheter was designed to measure the length of the lesion as well as to inject the contrast medium to opacify the area of interest while the guide wire was in place ( 12 , 13 ) . we used the multifunctional coil catheter in this study because we have been using this catheter for interventional procedures in the gastrointestinal tract since august 2000 . the balloon sheath experiments were classified into four groups and the tests were repeated ten times in each group . during each test , the coil catheter was advanced as far as possible into the duodenum or jejunum of the gastroduodenal phantom . in group i , the coil catheter was advanced only over the guide wire as far as possible into the phantom ( fig . ii , the coil catheter was advanced over the guide wire as far as possible through the deflated balloon sheath into the phantom to simulate the sheath without the balloon ( fig . iii and group iv , the coil catheter was advanced as far as possible over the guide wire through the inflated balloon sheath into the phantom . therefore , the inflated sheath balloon was placed in the gastric fundus in group iii and it was placed in the gastric body in group iv ( figs . because the distal angulated part of the balloon sheath should be located proximal to the stricture area at the gastroduodenal junction , the balloon part of the balloon sheath could not be placed as far as in the gastric antrum . the balloon was inflated with air by using a 50 cc syringe until it impinged on the greater curvature . the balloon diameter that was perpendicular to the long axis of the sheath was 7 cm after injecting 50 cc of air . the surface of the coil catheter was not lubricated with any lubricant . in every test , the total procedure time lasted for two minutes , and the two - minute time started with the insertion of the coil catheter in group i , and the two - minute time started with insertion of the balloon sheath in groups ii - iv . the tests were performed for ten consecutive days , and each group with each type of phantom was tested one time per day to reduce the learning effects . the positions reached by the tip of the catheter were numerically ordered ; the first , second , third and fourth segments , and the proximal portion of the jejunum were encoded as 1 - 4 and 5 , respectively . the kruskal - wallis test was performed to evaluate if there were significant differences among the four groups because the catheter tip position was treated as an ordinal variable . if the kruskal - wallis test showed a statistical difference , the mann - whitney u test was used to compare groups iii and iv to determine the best location of the inflated balloon between the gastric body and fundus for the advancement of the catheter tip . statistical analysis was performed using spss version 10.0 ( spss , chicago , il , usa ) the home - made balloon sheath mainly consisted of an introducer sheath and a supporting balloon ( figs . it was divided into four parts : the distal angulated part , the balloon part , the straight part , and the proximal handling part ( fig . the introducer sheath was made of a polytetrafluoroethylene ( ptfe ) tube ( sang - a flontec co. , incheon , korea ) with a 4.2 mm inner diameter , a 4.8 mm outer diameter and a 60 cm length . the distal part of the sheath was 8.0 cm in length and it was bent at a 100 angle . a side supporting balloon ( 8.0 cm in length ) was made of latex tube ( unidus co. , seoul , korea ) , and this was attached to the sheath with nylon strings ( teleflex medical , coventry , ct , usa ) . the balloon was designed to point to the opposite side of the angulated tip when it was inflated . a polyetheretherketon ( peek ) tube ( victrex , orangeburg , sc , usa ) was used as the balloon inflating side tube , and this was connected to the proximal part of the balloon . the polyetheretherketon side tube had a 1.0 mm inner diameter and a 1.3 mm outer diameter . the proximal end of the peek side tube was connected with a side - arm that included a three - way stopcock ( cook , bloomington , in , usa ) . the straight part constituted the main part of the introducer sheath ( 44 cm in length ) and it ran parallel with the peek side tube . the distal angulated part and the straight part of the introducer sheath were covered with a heat - shrinkable tube ( 6.0 mm outer diameter ) ( lg cable , anyang , korea ) . the chemical - resistant heat - shrinkable tube was used to tightly wrap the both ends of the side supporting balloon ( nylon - strings ) as well as to mount the straight part of the introducer sheath and the peek side tube together . three radiopaque gold rings ( 0.1 mm in thickness and 1.0 mm in width ) were attached to the sheath with glue at both ends of the balloon and at the distal tip of the sheath ; these acted as markers to improve the radiopacity of the sheath . last , a plastic handling hub ( s&g biotech , seoul , korea ) was connected to the proximal end of the sheath . two types of silicone phantom models were made for the balloon sheath study : one simulated a normal anatomic condition , and the other simulated a 70% concentric stenosis at the gastroduodenal junction . first , a paper plaster clay model was made according to the anatomy and size of a normal human stomach and duodenum . it included the lower part of the esophagus , the entire stomach , the duodenum and the proximal portion of the jejunum . second , after the model had dried for three days , it was put in a lucent molding box . fluid silicone ( shin - etsu chemical co. , tokyo , japan ) was poured into the molding box , and it was allowed to harden . at last , the gastroduodenal phantom was divided into several portions according to the normal human anatomy , including the lower part of the esophagus , the gastric fundus , the gastric body , the gastric antrum , the duodenum and the proximal portion of the jejunum . the duodenum of the phantom was approximately 35 cm in length and it was divided into four segments : the first duodenal bulb portion segment , the second descending portion segment , the third horizontal portion segment and the forth ascending portion segment ( figs . the gastroduodenal junction was angled at about 90 in a posterior direction relative to the long axis of the gastric antrum ( fig . a 150 cm , 0.035-inch hydrophilic guide wire with an angled tip ( radifocus , m - terumo , tokyo , japan ) and a multifunctional coil catheter ( s&g biotech , seoul , korea ) were used in our phantom study . the multifunctional coil catheter was designed to measure the length of the lesion as well as to inject the contrast medium to opacify the area of interest while the guide wire was in place ( 12 , 13 ) . we used the multifunctional coil catheter in this study because we have been using this catheter for interventional procedures in the gastrointestinal tract since august 2000 . the balloon sheath experiments were classified into four groups and the tests were repeated ten times in each group . during each test , the coil catheter was advanced as far as possible into the duodenum or jejunum of the gastroduodenal phantom . in group i , the coil catheter was advanced only over the guide wire as far as possible into the phantom ( fig . ii , the coil catheter was advanced over the guide wire as far as possible through the deflated balloon sheath into the phantom to simulate the sheath without the balloon ( fig . iii and group iv , the coil catheter was advanced as far as possible over the guide wire through the inflated balloon sheath into the phantom . therefore , the inflated sheath balloon was placed in the gastric fundus in group iii and it was placed in the gastric body in group iv ( figs . because the distal angulated part of the balloon sheath should be located proximal to the stricture area at the gastroduodenal junction , the balloon part of the balloon sheath could not be placed as far as in the gastric antrum . the balloon was inflated with air by using a 50 cc syringe until it impinged on the greater curvature . the balloon diameter that was perpendicular to the long axis of the sheath was 7 cm after injecting 50 cc of air . the surface of the coil catheter was not lubricated with any lubricant . in every test , the total procedure time lasted for two minutes , and the position that was reached by the tip of the coil catheter was recorded . the two - minute time started with the insertion of the coil catheter in group i , and the two - minute time started with insertion of the balloon sheath in groups ii - iv . the tests were performed for ten consecutive days , and each group with each type of phantom was tested one time per day to reduce the learning effects . the positions reached by the tip of the catheter were numerically ordered ; the first , second , third and fourth segments , and the proximal portion of the jejunum were encoded as 1 - 4 and 5 , respectively . the kruskal - wallis test was performed to evaluate if there were significant differences among the four groups because the catheter tip position was treated as an ordinal variable . if the kruskal - wallis test showed a statistical difference , the mann - whitney u test was used to compare groups iii and iv to determine the best location of the inflated balloon between the gastric body and fundus for the advancement of the catheter tip . statistical analysis was performed using spss version 10.0 ( spss , chicago , il , usa ) both researchers had better performances with both phantoms in order of group iv , iii , ii and i. in group iv , both researchers advanced the tip of coil catheter through the fourth segment of the duodenum in both phantoms . in group iii , each researcher advanced the tip of the coil catheter as far as the fourth segment of the duodenum 10 and eight times , respectively , in the phantoms without stricture , and eight and seven times , respectively , in the phantoms with stricture . in group ii , each researcher succeeded in catheterizing the fourth segment of the duodenum twice and three times , respectively , in the phantoms without stricture , and once and once , respectively , in the phantoms with stricture . in group i , however , both researchers failed to reach the third segment of the dueodenum in both phantoms with the tip of the coil catheter . the overall performance for the phantom with stricture was similar with that for the phantom without stricture , although each researcher failed to catheterize the stricture once and three times , respectively . statistical analysis of the differences in the positions reached by the tip of the catheter among the four groups in each phantom showed a significant difference ( kruskal - wallis test , p < 0.001 ) . when comparing the mean values of the catheter tip advancement between groups iii and iv in each phantom , there were significantly higher mean values for group iv than for group iii ( mann - whitney u test , p < 0.001 ) . the results showed that the tip of the coil catheter could easily reach the proximal portion of the jejunum when we worked through an inflated balloon sheath , especially when the inflated balloon was placed in the gastric body . the newly designed balloon sheath was helpful to negotiate a catheter and guide wire through the stomach and into the duodenum or proximal jejunum . negotiation of the catheter and guide wire was best when the inflated balloon sheath was placed in the gastric body . the distal angulated part of the balloon sheath that was 8 cm in length and it had a 100 angle that effectively allowed us to direct the devices toward the pylorus because the balloon , which was designed to point to the opposite side of the angulated tip , could push the lower part of the sheath toward the pyloric canal and against the greater curvature of the stomach wall when the balloon was inflated with air . during gastrointestinal interventional procedures , one of the most important steps for a successful procedure is to pass a guide wire and a catheter through the gastric outlet or through the duodenal obstructions ( 4 , 10 - 11 , 14 - 17 ) . however , when the strictures are severe , the gastric contents may accumulate in the stomach and the gastric cavity becomes dilated and flaccid . when a guide wire and a catheter can not negotiate through the strictures , they are readily curved in the dilated stomach . in order to overcome this technical problem , various angiographic catheters have been used in combination with a torque - control guide wire to traverse the strictures . however , the reported failure rate of fluoroscopically guided stent placement for gastroduodenal strictures has been reported to be between 1% and 5% , even after additional endoscopic assistance , and the average time needed for fluoroscopically guided stent placement has been reported to be from 36 to 45 minutes ( 1 , 5 , 17 ) . the newly designed balloon sheath seems to have several advantages . first , as the outer diameter of the balloon sheath ( maximum outer diameter ; 7.3 mm ) is smaller than that of the routine gastroduodenal endoscope ( outer diameter ; 9.6 mm ) ( gif 100 ; olympus , southend - on - sea , uk ) , the balloon sheath could be well tolerated during the procedure . meanwhile , the inner diameter of the working channel of the same endoscope is 2.7 mm , which is smaller than that of the balloon sheath ( 4.2 mm ) . second , the inflated supporting balloon can give extra - stability to the tip of the balloon sheath in order to prevent it from looping and moving backward in the stomach while the guide wire , the catheter or the delivery system is passing through . the ideal gastric sheath should have such appropriate physical characteristics as suitable torque and supportability . if the sheath is too rigid , it can injure the mucosa of the upper digestive tract and the patients may not be able to tolerate the device during the procedure . third , the balloon sheath can be easily inserted perorally into the stomach , and it is effective in guiding the interventional devices to the duodenum or jejunum . in group i , while the coil catheter was advanced over a guide wire into the duodenum , it usually looped along the wall of the greater curvature and the gastric fundus , and so it could not be advanced further . in group ii , while the coil catheter was advanced over a guide wire into the duodenum through the deflated balloon sheath , the devices could not be advanced further than the greater curvature of the stomach wall . in group iii and group iv , while the coil catheter was advanced over a guide wire into the distal duodenum or proximal jejunum through the inflated balloon sheath , the inflated balloon gave extra - stability against the wall of the gastric fundus or the gastric body , so that the tip of the coil catheter could reach as far as the proximal jejunum . when the inflated balloon was placed in gastric body rather than being placed in the gastric fundus , it was easier to advance the catheter as far as the proximal jejunum . we think that the balloon sheath enables the devices to advance into the more distal areas adjacent to the pylorus by changing the direction of the tip of the devices more towards the gastroduodenal junction . although a gastroduodenal phantom was useful for testing the newly designed balloon sheath , there were some limitations for this study . first , the gastroduodenal phantom model that we made out of silicone had no elasticity like a human gastroduodenal wall . it would have been better to use an ideal model that mimicked the real human gastroduodenum ; however , it was difficult to make a satisfactory gastroduodenal phantom model because there is a lot of variability in curvature , size and shape of the stomach . third , we could not place the balloon part of the balloon sheath in the gastric antrum because the stricture was made at the gastroduodenal junction and the distal angulated part had to be located proximal to the stricture in this experiment . if we could have made the stricture at the second or third segment of duodenum , we could have compared the results according to the location of inflated balloon between the gastric antrum and gastric body . in conclusion , the new balloon sheath displayed good results in the gastroduodenal phantom study , especially when the inflated balloon was placed in the gastric body . the new balloon sheath seems to be feasible for use and it has good clinical potential for fluoroscopically guided gastrointestinal guidance and access .

kyrle disease ( kd ; or hyperkeratosis follicularis et parafollicularis in cutem penetrans ) is a perforating dermatosis , first described in 1916 . apart from kd , the perforating dermatoses include elastosis perforans serpiginosa ( eps ) , perforating folliculitis ( pf ) , and acquired perforating collagenosis ( apc ; or reactive perforating collagenosis , early / late - onset type ) secondary to chronic renal failure and/or diabetes mellitus . the common pathophysiological principle in these disorders is supposedly transepidermal elimination ( tee ) of dermal substances , predominantly collagen in apc , keratin in kd and pf , or thickened elastic fibers in eps . however , features may vary and the composition of the tee material may contain collagen , elastin , and keratin in one patient . a 59-year - old man was diagnosed with perforating dermatosis with lesions resembling both kd and late - onset type apc . the patient had first noticed skin lesions two years before presentation and he reported no pruritus . additionally , the patient had diabetes mellitus type 2 and chronic renal insufficiency . , there were multiple follicular and some non - follicular hyperkeratotic papules , predominantly on the shins and the calves . some of the larger papules exhibited a central depression with an adherent keratotic plug ( fig . a punch biopsy showed epidermal acanthosis and papillomatosis , a central ulceration with an overlying serum crust , deposits of degraded collagen and some elastic fibers in the upper dermis , and some lymphocytic infiltration ( fig . the microscopy images combined with diabetes mellitus with chronic renal insufficiency pointed to the diagnosis of apc , but the mainly follicular , smaller keratotic papules seemed to fit better with a diagnosis of kd . patterson proposed the following classification system : ( i ) perforation as an incidental histological finding ; ( ii ) secondary perforation ( e.g. in granuloma annulare , pseudoxanthoma elasticum , and chondrodermatitis nodularis chronica helicis ) , and ( iii ) primary perforating dermatoses . the latter group includes kd , late - onset apc , and pf in subgroup iiic , which is also termed acquired perforating diseases other than eps and early - onset apc . due to the fact that perforating dermatoses are quite rare , little however , new findings point to the fact that the interaction of keratinocytes with altered structural proteins plays a decisive role , e.g. 67-kda elastin receptor overexpression in eps . in apc , tgf-3 , mmp-1 and timp-1 are overexpressed in lesional skin , indicating changes in extracellular matrix composition . the case of kd and late - onset apc in our patient supports the idea of a common pathomechanism in primary perforating dermatoses ( group iiic according to patterson ) . kd and late - onset apc may be considered morphological variants of the same underlying disease process . however , the question remains whether perforation really exists : in most cases , pruritus leads to scratching , epidermal damage , and alteration of dermal substances ( e.g. collagen or elastin ) . the exposure of altered subepidermal substances through epidermal gaps ( e.g. by scratching ) may appear as epidermal perforation , i.e. tee . continuous imaging of this process remains impossible at the moment ; therefore , the tee concept can neither be proven nor negated .

bacterial spot of tomato and pepper plants has been observed in areas with high humidity and warm climate . the disease causes significant losses , estimated of about 1020% per year and it is one of the economically most important diseases in all continents where capsicum annuum and solanum lycopersicum l. are cultivated . during the last 20 years , the causative agents of bacterial spot have been a subject to many studies and reclassifications [ 15 ] . until 1990s , it was considered that the group of strains causing bacterial spot belonged to a single species , xanthomonas campestris pv . vesicatoria , although several phenotypically and phylogenetically distinct groups were observed . in the 1990s , vauterin et al . transferred groups a and c into xanthomonas axonopodis pv . vesicatoria on the basis of dna homology among a large number of xanthomonads , and group b was separated at species level as xanthomonas vesicatoria . the group d which was firstly isolated in former yugoslavia was given a species status as xanthomonas gardneri [ 4 , 5 ] . a new species was proposed for the weakly amylolytic group a strains , xanthomonas euvesicatoria . currently , the pathogen belongs to four widely distributed species : x. euvesicatoria , x. vesicatoria , x. gardneri , and x. perforans [ 5 , 8 ] . according to the current data , species - specific primers have been designed for detection of each of the xanthomonads ; however , for the purposes of the routine identification , a more common approach is much preferable . biolog system which is based on ability for utilization of different carbon sources has proved to be very convenient to complete diagnostics . this study aims to provide the full metabolic profiles of the bacterial spot 's xanthomonads based on a large selection of strains from bulgaria with accent on their major differentiating characteristics which could be used for species identification by metabolic profiles . the results are compared to the data in the literature in order to clarify the strong features of each species and distinguish the variable ones . eighty - three of them were isolated from pepper ( 19992012 ) and seventy - four from tomato ( 19852012 ) plants with symptoms of bacterial spot . twenty - seven strains were derived from the resident phase of the pathogen on weeds ( 19891999 ) from and around tomato fields . the bacteria were identified by pcr amplification with species - specific primers as xanthomonas euvesicatoria ( 54 strains from pepper ) , xanthomonas vesicatoria ( 115 strains : 29 from pepper , 59 from tomato , and 27 from weeds ) and xanthomonas gardneri ( 15 from tomato ) ( [ 9 , 10 ] , kizheva et al . , unpublished ) . the bacteria were incubated on bug agar at 28c for 24 h prior to analysis . the analysis was performed through the spss hierarchical cluster analysis procedure by ward 's method . the matrix of similarity between the isolates was calculated using the squared euclidean distance [ 1114 ] . the type cultures x. vesicatoria nbimcc 2427 , x. euvesicatoria nbimcc 8731 , and x. gardneri nbimcc 8730 were used . the key phenotypic characteristics described by jones et al . , amylase activity and utilization of cis - aconitic acid for differentiation of the xanthomonas species that cause bacterial spot of tomato and pepper , were determined ( table 1 ) . although some variation among the species is observed , these properties are not stably positive or negative . according to the metabolic patterns obtained by biolog microplates , -cyclodextrine , n - acetyl - galactosamine , adonitol , inositol , d - glucosaminic acid , sebacic acid , and histidine were not utilized . more than 93% of all strains did not utilize also i - erythritol , l - rhamnose , d - sorbitol , d - galactonic acid lactone , d - gluconic acid , -hydroxybutyric acid , p - hydroxy phenylacetic acid , -keto - valeric acid , quinic acid , saccharic acid , l - leucine , l - ornithine , l - phenylalanine , l - pyroglutamic acid , d - serine , thymidine , phenylethylamine , and putrescine and utilized -d - glucose , l - fucose , and sucrose . the biolog metabolic data distributes the strains into three clusters at 70% similarity ( figure 1 ) . the major cluster consists of only x. vesicatoria strains ; the second big cluster is predominantly formed by strains of x. euvesicatoria ; the smallest cluster comprises several miniclusters each of which is composed by the three xanthomonas species ( figure 1 ) . the strains separate in different clusters after analysis only on the basis of a selection of substrates which tend to be utilized differently by the three species ( table 3 ; figure 2 ) . simple characteristics like amylase activity and utilization of cis - aconitic acid showed some variances and according to our data can not be used solely for species differentiation . clusters based on the metabolic fingerprints of the bulgarian xanthomonas isolates from tomato , pepper , and weeds reveal that x. euvesicatoria , x. vesicatoria , and x. gardneri distribute comparatively separated according to the species . however , the smallest cluster includes all the three species and some x. gardneri isolates are grouped together with most of the x. euvesicatoria strains which refers to the insufficient number of strongly differentiating metabolic features among the species . detailed overview of the data of the utilization patterns ( table 2 ) shows that none of the substrates included in the biolog microplates is strongly utilized by one of the species and is indifferent for the others , and vice versa . however , on the basis of a multiple comparison of the nutrition properties , 27 of the substrates ( table 3 ) can serve for species differentiation of the causal agents of bacterial spot of tomato and pepper . since the reaction of x. gardneri to these carbon sources is always negative , this species can easily be distinguished from the other two . they can be differentiated according to mainly 7 substrates toward which the reaction of x. euvesicatoria is consistent . its reaction according to the data for the bulgarian strains can vary towards almost all of the differentiating substrates , though it separates best in the hierarchical cluster analysis based on the full metabolic patterns ( figure 1 ) . cluster analysis based on the selection of 27 substrates ( table 3 ) divides the strains into three distinct groups corresponding to their species ( figure 2 ) and illustrates the high probability for species differentiation based on these metabolic properties . though identification can not be guaranteed using this pattern , generally the three species could be successfully distinguished on the basis of the selected differentiating substrates . the metabolic data obtained shows similarities as well as differences compared to the data available in the literature [ 24 , 16 ] . light differences in the utilization of six substrates were observed between the bulgarian isolates of x. euvesicatoria and the isolates studied by jones et al . . n - acetyl - d - glucosamine , d - galactose , maltose , and bromosuccinic acid were not strongly utilized by all bulgarian strains with 1419% of them being weak positive and 38% negative . lactic acid and -glycerol - phosphate were not strongly negative but were utilized by 2% of our strains and weakly utilized by 82% and 55% , respectively . however , n - acetyl - d - glucosamine , d - galactose , and l - serine were also not utilized by all strains studied by vauterin et al . with a difference between the positive strains of vauterin and ours of less than 10% . great differences were observed between the bulgarian isolates of x. euvesicatoria and the isolates studied by jones et al . in the data for three substrates . glycogen , malonic acid , and l - serine which were utilized by jones ' strains were negative for 78% , 17% , and 15% of our strains , respectively . while 80% of the bulgarian strains still utilized l - serine , the reaction of 55% of our isolates to malonic acid was only weakly positive . malonic acid was also among the carbon sources with variable reaction as stated by vauterin et al . with a difference between the positive strains of vauterin and ours of ~30% . other substrates like melibiose and lactic acid were utilized by more bulgarian strains than those studied by vauterin et al . , and all of our isolates did not show a strong positive reaction to l - aspartic acid , inosine , uridine , glucose-1-phosphate , and glucose-6-phosphate as opposed to vauterin 's ones . x. vesicatoria isolates from bulgaria manifested light differences in ten substrates as compared to the data available in the literature . instead of being positive , only 47% , 77% , and 85% of the bulgarian strains between 1% and 5% of the strains were strongly positive and 10% and 18% of them were only weakly positive to -methyl d - glucoside , -hydroxybutyric acid , itaconic acid , glucuronamide , asparagine , inosine , and uridine instead of being all negative as stated by . great differences were observed in the utilization of l - arabinose , urocanic acid , -glycerol - phosphate , glucose-1-phosphate , and glucose-6-phosphate which were positive for 14% , 18% , 50% , 47% , and 33% of the strains , respectively , instead of not being preferred as a sole carbon source . a great number of strains ( between 29% and 42% ) gave also a weak positive reaction to urocanic acid , -glycerol - phosphate , glucose-1-phosphate , and glucose-6-phosphate . however , a more recent study of x. vesicatoria isolates from tanzania indicated a positive reaction of these strains to glucose-6-phosphate as opposed to bouzar et al . tanzanian isolates manifested some differences in their carbon sources preferences according to the data published before . while most of our strains ( 6078% ) were still positive and 1828% were weakly positive to melibiose , monomethyl succinate , d - alanine , l - alanine , l - alanyl - glycine , and glycerol , only some were positive to glycyl - l - glutamic acid ( 41% strongly and 30% weakly ) , cis - aconitic acid ( 20% strongly and 36% weakly ) , lactic acid ( 17% strongly and 50% weakly ) , l - proline ( 14% strongly and 21% weakly ) , l - aspartic acid ( 27% strongly and 23% weakly ) , hydroxy l - proline ( 34% strongly and 31% weakly ) , and glucose-6-phosphate ( 33% strongly and 36% weakly ) , and the reaction to -methyl d - glucoside and inosine was predominantly negative and positive with only 1% and 3% of the strains , respectively . greatest differences existed between the bulgarian strains of x. gardneri and the data in the literature . the bulgarian strains manifested different preferences from the ones described in the literature to eleven substrates . according to jones et al . , x. gardneri did not use d - alanine , l - alanine , and hydroxy l - proline . seven percent of our strains utilized acetic acid , alaninamide , and l - aspartic acid ; 2733% showed a weak positive reaction to acetic acid , alaninamide , d - alanine , and l - alanine ; and 713% were weakly positive to l - aspartic acid and hydroxy l - proline . l - fucose was stated to be strongly preferred carbon source ; however , 27% of our strains were only weak positive and 13% were negative . methyl - pyruvate and tween40 were not utilized by 27% and 40% of the bulgarian strains , respectively , and 67% of the strains were negative to succinic acid , bromosuccinic acid , succinamic acid , and l - serine as opposed to jones et al . . instead of negative reaction , a great part of the bulgarian strains utilized -d - lactose ( 40% positive ) , lactulose ( 47% positive and 13% weak positive ) , d - raffinose ( 20% positive and 40% weak positive ) , citric acid ( 20% positive and 13% weak positive ) , and l - glutamic acid ( 27% positive ) . the differences manifested by the bulgarian xanthomonas strains compared to the data in the literature [ 2 , 3 , 5 ] can be due to various reasons related to geographical region , climatic conditions , and used cultivars . however , large studies from different regions give the possibility of more accurate evaluation of the strict and the variable bacterial features . although x. euvesicatoria , x. vesicatoria , and x. gardneri can not be distinguished on the basis of simple characteristics like amylase activity and utilization of cis - aconitate or on the basis of individual biochemical tests , the comparison of multiple nutritional properties included in biolog system can serve for species differentiation of the causal agents of bacterial spot .

one - third of the adult americans are obese and 2/3 are overweight [ 1 , 2 ] . obesity increases histrionically and is becoming pandemic worldwide in the past decades , predisposing these populations to great risk for gastric esophageal reflux disease ( gerd ) and subsequent aspiration pneumonia , asthma , obstructive sleep apnea syndrome ( osas ) , and related comorbidities and mortality in lung injury , such as acute lung injury ( ali ) and ards ( acute / adult respiratory distress syndrome ) , even after being adjusted for other risk factors [ 36 ] . this remains true in a large array of studies in patients with ali and critical conditions [ 79 ] . lung injury is a debilitating disease , with mortality close to that of breast cancer , costing our federal government at least 850 million dollars every year [ 10 , 11 ] . as the prevalence of obesity and its comorbidities increases skyrocketing , obesity related lung injury rises significantly in the past decades . this may be mediated by depletion of the antioxidants , destroyed lung endothelium , reduced lung volume and chest wall compliance , and increased susceptibility of the lung to injury [ 12 , 13 ] . under obese state , there are changes with fat sites and sizes . furthermore , obesity is a chronic systemic inflammatory process , with infiltration of macrophages and other cells . this inflammatory process is driven by the adipocytokines derived from adipocytes , macrophages , and other cells in adipose tissues , which cause an unbalance between the proinflammatory adipocytokines such as lepin , resistin , vasftin , and tnf and the anti - inflammatory adipocytokines such as adiponectin , omentin , sfrp5 , vaspin , zag , and interleukin-10 ( il-10 ) . this process is accompanied by the polarization of macrophages , from healthy m2 to unhealthy m1 macrophages and the transformation of t helper ( th ) cells from beneficial treg and th2 to harmful th17 and th1 . these form an inflammatory soup , heavy with proinflammatory adipocytokines , which further activates toll - like receptor 4 ( tlr4 ) , nf-b , and other signaling pathways , initiating a cascade of inflammatory process . furthermore , these changes modulate host defense responses , namely , the innate and adaptive immunity , regulating the susceptibility of the lung for injury . when a variety of insults occur , such as ozone ( o3 ) , gastric acid and bacterial and nonbacterial particles , the lung may become more susceptible for injury , depending on the overall balance between the offense and defense , the proinflammatory and anti - inflammatory adipocytokines . yet , limited articles have a comprehensive review of the overall balance of these adipocytokines and their relationship to the pathogenesis of lung injury . in our series of review articles , we will address these adipocytokines and their relationship with lung injury as the good , the bad , and the ugly : the anti - inflammatory ( the good ) , the proinflammatory ( the bad ) and their impact on host defense response , and the immunity ( the ugly ) . these contents will be included in three respective review articles , with the major objective to get a better view of the pathogenesis of lung injury in obesity , the molecular basis of other comorbidities in obesity , the research gaps in oili , and the scientific and therapeutic targets in a more comprehensive and efficient fashion . and thus this important information will direct our research and scientific focus and further personalized medicine in this huge population in the near future . in this review article , by reviewing the articles with animal models and preclinical trials as well as the clinical trials in human being related to oili , we will focus on the anti - inflammatory adipocytokines ( the good ) and address from the following aspects : adipocytokines and obesity , inflammation and other mechanism involved , adipocytokines and lung injury in obesity bridged by inflammation , and some therapeutic potentials . some possible mechanisms involved are illustrated in figure 1 and this review article will be summarized in table 1 . a large array of adipokines , cytokines , chemokines , and other factors were derived from adipose tissues . in this review article the majority of the evidence supported that adiponectin , omentin , and sfrp5 are anti - inflammatory , the good , and are decreased in obesity , which is associated with increased systemic inflammation , indicated by increased circulating tnf , c reactive protein ( crp ) , il-6 , and other proinflammatory cytokines / chemokines [ 17 , 18 ] . other anti - inflammatory adipocytokines beneficial for weight loss are zag , vaspin , il-10 , il-1ra , tgf-1 , and gdf15 . yet regretfully , very limited information is available for their roles in the pathogenesis of lung injury . we will do our best to get valuable information from these limited studies and discuss some possibilities . adiponectin accounts for 0.01% of total protein in circulation , with a normal range of 220 g / ml , and is quickly cleared after secretion ( half - life of 45 to 75 minutes ) [ 28 , 29 ] . despite this fact , adiponectin concentration remains rather stable in plasma . a growing number of studies suggested that adiponectin is decreased in obesity and negatively correlated to visceral fat mass , inflammation , heart disease , injury , and many other diseases but positively to insulin sensitivity and promotes weight loss [ 3033 ] . a positive correlation between adiponectin and fat mass at the lower extremities has been revealed but a negative one with that of the body trunk was typically seen in abdominal obesity . furthermore , adiponectin drives fat deposit in small fat cells and subcutaneous adipose tissue but mobilizes visceral fat , supporting its beneficial effect in variety of organ injury , such as nonalcoholic fatty liver disease and fatty heart in obesity and t2 dm . administration of recombinant adiponectin or overexpression of adiponectin promotes weight loss increases insulin sensitivity and exerts anti - inflammatory effects . adiponectin decreases oxidative stress , inflammation , angiogenesis , apoptosis , and increases mitochondrial biogenesis , locally ( paracrine / autocrine ) and systemically ( endocrine ) . in obesity , the unhealthy adipose tissues and infiltrated macrophages ( more m1 than m2 ) it was suggested that adiponectin reduces inflammation and alleviates disease states , possibly through its suppression of tnf , il-6 , and crp and upregulation of il-10 and il-1ra [ 4446 ] . additionally , adiponectin increases mitochondrial density and biogenesis , adipocyte flexibility , and the host adaptation to stress . the major signaling pathways involved are ampk and ppar , ppar , mek - erk , pi3k - akt , appl1 , t - cadherin , ca2 + and sirt1 , and so forth [ 40 , 4852 ] , which promote fatty acid oxidation and glucose uptake into skeletal muscle and inhibit gluconeogenesis in liver . another important mechanism is the possible polarizing effect of adiponectin on macrophages and t helper cells . it was suggested that adiponectin may polarize macrophage from m1 , proinflammatory state , to m2 , anti - inflammatory state , as well as from harmful this has been supported by both loss and gain of function studies [ 44 , 5358 ] . moreover , adiponectin suppresses the proliferation of bone marrow - derived granulocyte and macrophage progenitors , inhibits phagocytic behavior of macrophages and proinflammatory cytokines secretion , and promotes anti - inflammatory cytokines of macrophages . adiponectin impacts host defense response and immunity , through inhibiting recruitment of leukocytes , increasing the remodeling of the lung , promoting phagocytosis of neutrophils and macrophages , modulating the productions of th2 cytokines , and reducing / inhibiting b cell and natural killer ( nk ) cells in animal models . yet , little is known about the impact of adiponectin on host response in human beings , especially those related to lung injury . this is largely due to the difficulty in conducting large clinical and translational studies , as most of the patients are not in the conditions willing or able to be consented for these trials . adiponectin resembles the structures of complement factor c1q and surfactant proteins spa and spd of the lung , which function as pattern recognition molecules , and is possibly one major mechanism for adiponectin to limit the inflammation of the lung . all three receptors of adiponectin , adipor1 , adipor2 , and t - cadherin , were detected in a variety of cells of the lung . furthermore , adiponectin can be transported from circulation to alveolar through t - cadherin on the endothelium . lung injury is a complicated pathogenesis process , including activation of immune system and inflammation , stimulation of endothelium , increased capillary permeability , neutrophil and macrophage infiltration , and leaking of albumin [ 64 , 65 ] . the function of adiponectin in lung homeostasis is becoming a hot topic in the past few years , but it remains to be further determined and studied in more details . recent data supported that obesity is a major risk factor for lung injury , and the adipose tissue derived adipokines and cytokines seem to play a very important role during this process [ 6670 ] . this may be associated with activation and polarization of macrophages , stimulation of ampk and cox2 , and its effect on endothelium [ 71 , 72 ] . although there were controversial reports [ 73 , 74 ] , most of the evidence supported that reduced adiponectin level is associated with increased morbidity and mortality in critical care patients , lung transplantation , emphysema , asthma , chronic obstructive pulmonary disease ( copd ) , and acute lung injury of other causes [ 7577 ] , in animal models as well as in human beings . these were accompanied by macrophages activation , reduced clearance of apoptotic cells , and perivascular and lung inflammation [ 78 , 79 ] . additionally , in those contradicted reports mentioned above , adiponectin concentrations were tested during the critical illness , suggesting the possibility of the upregulation of adiponectin due to adaptation over time . this speculation was supported by the studies showing that increased adiponectin level and amelioration of the disease in mice with lupus when treated with ppar agonist , regardless of the already elevated adiponectin level in these mice . from this aspect , we could hypothesize that the changes of adiponectin may be more important than its actual concentration during critical illness . in another word , administration of adiponectin may still benefit these patients regardless their elevated adiponectin level . this being said , it is not difficult to understand the controversial results in patients with copd . in patients with copd , due to the long - term hypoxia and human body adaptation , adiponectin concentrations may be high or low , depending on how long and how badly the patients were sick and how the human body is adapting . with similar theory , those different results in patients with critical illness ( e.g. , those from apache ii ) or bacterial pneumonia seem reasonable as well . apparently , studies investigating the relationship of the changes of adiponectin and clinical outcomes , how the human body adapts , and what the host responses are would possibly provide more valuable information for clinical applications and further personalized medicine , both as a biomarker for a variety of diseases , severity , and prognosis and as a therapeutic potential . interestingly , it was found that total adiponectin levels and its active form , high molecular weight ( hmw ) isoform , are lower in men than their peer women at children - bearing age , which seems be associated with the high testosterone level in men . overall , adiponectin promotes anti - inflammation through inhibiting proinflammatory response , polarizing macrophages ( from m1 to m2 ) , and t helper cells ( from th1/17 to th2/treg ) , inhibiting tlr4-mediated nf-b activation , and protecting endothelium , suggesting that obesity may prime lung toward proinflammatory condition and more susceptible for injury due to hypoadiponectinemia , at least partially . yet many drugs exert their effect via adiponectin and its receptors , decreased ceramide , and antiapoptotic sphingosine-1-phosphate ( s1p ) , via its impact on insulin sensitivity and anti - inflammatory effects . this suggested that adiponectin could be a potential therapeutic target in obesity , metabolic syndrome , and its comorbidities , all of which are regarded as inflammatory processes . yet , it remains unclear if adiponectin can be a potential therapeutic target for lung injury in human subjects . with the newly synthesized adiponectin receptor agonist , adp355 , and the defined adiponectin receptors in the lung , the role of adiponectin in the aforementioned inflammatory states , and its function as pattern recognition molecules apparently , more preclinical and clinical trials are warranted in the near future , for its function , mechanism , and potential therapeutic and preventive applications . particularly , as adiponectin promotes weight loss and reduces inflammation and has receptors in the lung , studies targeting its role in oili would be greatly beneficial for these populations . omentin was initially found in intestinal cell ( called intelectin ) and then omental adipose tissue and human adipocytes ( especially stromal vascular cells of visceral adipose tissue ) , but it is also expressed in lung , heart , placenta , and ovary [ 18 , 83 ] . there are two forms , omentin 1 and omentin 2 , which share 83% of amino acid sequences . it was suggested that omentin level was lower in obese subjects , which is inversely associated with body mass index ( bmi ) and insulin resistance and positively with hdl and adiponectin . moreover , treatment for obesity with bariatric surgery or metformin increases serum level of omentin , which is associated with weight loss and improved insulin sensitivity , possibly through activating akt signaling pathway . studies showed the similarity of omentin and adiponectin [ 8587 ] , especially the effect on weight loss , insulin sensitivity , and type 2 diabetes ( t2 dm ) [ 17 , 8892 ] . it was also reported that omentin level is low in crohn 's disease , synovial fluid of patients with rheumatoid arthritis , polycystic ovary syndrome ( pcos ) , and other inflammatory diseases [ 90 , 93 , 94 ] . paradoxically , one recent study showed that increased omentin level was associated with nonalcoholic fatty liver disease ( nafld ) , the very common comorbidity in obesity and t2 dm . as obesity , t2 dm and nafld were all regarded as inflammatory process ; these contradicted results may indicate an adaptation response . as shown in some studies with adiponectin , treating patients with nafld may still increase omentin level as well as reducing inflammation . further studies are warranted to elucidate this phenomenon , the possible mechanism , and the changes with intervention . as shown in figure 3 , omentin activates ampk and enos , blocks akt pathways , inhibits crp , tnf , and nf-b signaling pathways , reduces adhesion molecules , and thus has anti - inflammatory effect on smooth muscle cells and endothelium [ 9699 ] . administration with recombinant human omentin inhibits tnf , decreases inflammation , and dilates vascular vessels , suggesting its potential therapeutic role in inflammation related conditions . no study has assessed the possible impact of omentin on host defense response or immunity . three studies were conducted in patients with obstructive sleep apnea syndrome ( osas ) [ 101103 ] . it indicated that decreased serum omentin-1 levels could be regarded as an independent predictive marker for the presence and severity of osas . it was reported that intelectin-1 was secreted from malignant pleural mesothelioma and can be detected in pleural effusion , suggesting that it can be a biomarker for this malignancy . furthermore , intelectin is needed for mcp-1 production in lung epithelium and causes airway inflammation in mice with asthma . if the receptor can be further determined , one may test if these effects are through paracrine / autocrine besides endocrine . as osas and asthma are highly associated with obesity , inflammation , and lung injury , this may suggest the association of omentin and lung injury . additionally , given the fact that omentin blocks proinflammatory cytokines tnf , and signaling pathway nf-b , it may be protective in lung injury . furthermore , considering the similarity of omentin and adiponectin , we hypothesize that omentin exerts anti - inflammatory effect in lung injury . however , the possible proinflammatory effect of omentin may not be ignored as well . with the availability of recombinant human omentin , it would be greatly helpful to know if there are receptors for omentin in the lung , if omentin is anti - inflammatory in lung injury , and if omentin exerts its effect through adiponectin or independently , all of which may direct the therapeutic development in oili and other related diseases . sfrp5 was first discovered in adipocytes couple of years ago and the data was published in science . in this study , it was shown that sfrp5-deficient mice fed on high - fat diet aggravated fat accumulation , inflammation , and systemic oxidative stress . administration of sfrp5 reduced inflammation and attenuated insulin resistance , through decoying wnt mediated jnk activation in macrophages and adipocytes , and thus has systemic effects . overexpression of sfrp5 promotes adiponectin and decreases tnf , il-6 , and mcp-1 , suggesting its anti - inflammatory effect . a recent study in chinese subjects showed that sfrp5 is low in patients with t2 dm . furthermore , calorie restriction in obese subjects promoted weight loss and increased insulin sensitivity , which is correlated with improved sfrp5 level . one recent study showed that sfrp1 but not sfrp 25 was found to be decreased in obesity and this is associated with insulin resistance . however , in this study , it did show that sfrp1 increased adiponectin and reduced il-6 and mcp-1 levels , which is consistent with the previous studies . other isoforms should be further tested . perhaps , it is the ratio of sfrp5 to other isoforms that matters . another contradicted study also showed increased sfrp5 expression in diet - induced obesity . in this study , the authors argued that this might be due to the fact that sfrp5 inhibits wnt signaling pathway and thus suppresses adipocytes mitochondrial metabolism and promotes oxidative stress . combed with the previous data , it is confirmed that sfrp5 exerts its effect via inhibiting wnt signaling . this brought up the possibility that the isoforms of sfrp may vary cross species and ethics groups . furthermore , the wnt at different compartments has different effects , which may partially explain these controversial results . apparently , sfrp exerts its effects mainly through inhibiting wnt and jnk signaling pathways , which further inhibits the production of proinflammatory cytokines tnf , il-6 , and mcp-1 , and so forth . one recent study suggested that sfrps might promote or suppress wnt/-catenin signaling , possibly depending on its receptors . additionally , sfrp5 regulates p53 and is a hedgehog target to confine canonical wnt signaling . limited information suggested that sfrp5 was low in pleura mesothelioma , and methylation of sfrp5 was associated with overall survival of lung cancer . patients with unmethylated sfrp5 are more likely to benefit from egfr - tki therapy in non - small - cell lung cancer [ 109111 ] . based on its role in obesity and inflammation , we expect that sfrp5 exerts anti - inflammatory effect in obesity related lung injury . but it may depend on the compartments , the species , the ethnic groups , and other factors . with the availability of the recombinant sfrp5 , more preclinical and clinical trials were needed to explore the effect of sfrp5 on oili , as well as other comorbidities of obesity . vaspin is visceral adipose tissue - derived serpin ( serpina12 ) , and it is also rich in hypothalamus , skin , stomach , and subcutaneous adipose tissues . vaspin level is low in obesity , insulin resistance , and type 2 diabetes and increases with the attenuation of these conditions . furthermore , administration of vaspin suppresses leptin , tnf , and resistin , reduces food intake , and improves glucose control and insulin sensitivity in obesity . yet , two recent studies with bariatric surgery in obese subjects revealed that vaspin decreased after surgery [ 116 , 117 ] , and the reduction was associated with leptin , hba1c , and insulin sensitivity . this may suggest that there is a period of adaptation . apparently , more detailed studies are needed to illustrate the time and impact of vaspin changes . furthermore , vaspin was elevated in ulcerative colitis and other inflammatory conditions , suggesting that it may exert proinflammatory effect as well . it was shown that vaspin is associated differently with metabolic syndrome in males and females , indicating its potential interaction or regulation by sex hormones . yet , these are observed phenomenon and the mechanism remains to be determined in detail . although the mechanism is largely unknown , it has been shown that vaspin inhibits vascular smooth muscle cells proliferation through inhibiting reactive oxidative species ( ros ) , mapk , pi3k / akt , and nf-b signaling pathways . one recent study suggested that the inhibition of vaspin on ros may be through nadph oxidase , which is part of mechanism for cardiovascular disease ( cvd ) . a cell membrane glucose - regulated protein ( grp78 ) was identified and regarded as a liver - specific receptor for vaspin , suggesting its potential role in liver diseases . one study showed that high body fat mass with low cardiorespiratory fitness may be associated with increased vaspin in korean population , suggesting its possible role in lung . no receptor for vaspin was defined in lung yet . as vaspin inhibits ros and nf-b signaling pathways , activating ampk and akt pathways , along with its inverse relationship with respiratory fitness , we believe that vaspin may have a protective role in lung injury , through its anti - inflammatory effect . the important information would be to identify if there is a receptor for vaspin in the lung , if there is paracrine / autocrine effect of vaspin in lung , if the changes of vaspin is associated with less or worse lung injury in obesity , and if administration of vaspin attenuate lung injury . additionally , it is worth the effort to determine if weight loss increases vaspin and if this is correlated with ameliorated lung injury . zag is expressed in adipose tissue , liver , breast , prostate , and so forth . it was identified as a lipid mobilizer in patients with cancer cachexia and obese mice , mediated by 3 adrenoreceptor through activating cyclic amp ( camp ) pathway , increasing energy expenditure and lipolysis [ 124127 ] . zag was expressed in visceral and subcutaneous adipose tissue and presented in stromal vascular cells and mature adipocytes . so far , the majority of the evidence supported that zag level is lower in obesity and insulin resistance in mice with genetic defect or fed on high - fat diet as well as in human beings , and that there is an inverse relationship of zag with bmi and insulin resistance [ 129 , 130 ] . treatment for obesity and insulin resistance with liraglutide for 12 weeks increased zag level , indicating that zag may have a similar pattern as adiponectin . additionally , overexpression of zag promoted weight loss and increased insulin sensitivity , through stimulating fatty acid oxidation . however , some studies [ 132 , 133 ] revealed higher zag level in serum and white adipose tissue of obese / overweight individuals , as well as patients with chronic kidney disease , suggesting a possibility of zag resistance , like leptin resistance . furthermore , it appeared that zag exerts its function as a lipid mobilizer in cancer cachexia more significantly . zag was downregulated by tnf and other proinflammatory cytokines in obesity , suggesting that its pattern is similar to that of adiponectin [ 128 , 134 ] . furthermore , studies in patients with ckd showed that zag is negatively correlated with tnf and vcam-1 , suggesting its inverse relationship with systemic inflammation . a negative association of reduced zag and increased crp or mcp-1 was also reported in obesity , insulin resistance , and metabolic syndrome [ 136 , 137 ] . recent studies also demonstrated a positive correlation between zag and adiponectin and a negative one with leptin in human subjects . it is possible that zag may act in paracrine / autocrine manner and facilitate adiponectin secretion from adipocytes . yet based on the aforementioned , we think that zag may have anti - inflammatory effect on a variety of diseases , including lung injury . considering its lipid mobilization in cancer , it may be valuable to find out what zag does in lung cancer , and if this is associated with the prognosis and clinical outcomes . moreover , the fat mobilizing effect of zag was mediated by 3 adrenergic receptor , indicating its potential role in thermogenesis . . it would be greatly helpful if its receptor can be further identified . as the recombinant zag becomes available , both preclinical and clinical studies were needed to explore its function , mechanism , and potential therapeutic indications of zag . interleukin-10 ( il-10 ) was initially identified as a product of th2 cell and known as an anti - inflammatory cytokines inhibiting th1 cell activity . it is derived from a variety of cells including monocytes , dendritic cells , lymphocytes , macrophages , and t cells . although there were controversial reports , the majority of the evidence supported that il-10 is negatively correlated to bmi , obesity , insulin resistance , and t2 dm ; furthermore , overexpression of il-10 or administration of il-10 reduces body weight , improves insulin sensitivity , and augments glucose control [ 139 , 140 ] . il-10 polarizes macrophages from classically activated m1 to alternatively activated m2 phenotype and th1/17 to th2/treg , upregulates il-1 receptor and tgf- , inhibits phagocytosis and proinflammatory cytokines and chemokines , which further blocks tlr4 , nf-b , and other signaling pathways [ 15 , 141 , 142 ] , and activates jak / stat signaling pathway . this results in decreased production of tnf , il-12 , and other proinflammatory cytokines . additionally , il-10 is a switch factor for igg1 and igg3 and for iga1 and iga2 , which has better protective effect for mucosa . furthermore , therapy with mesenchymal stem cells ( msc ) reprograms toward the polarization of macrophage m2 and increases il-10 levels and thus has a protective role in sepsis , other infections , and acute lung injury . studies performed in lung transplantation showed that il-10 decreases inos , il-2 , and tnf , prevents ischemic - reperfusion injury , and inhibits acute rejection in animal models . early phase clinical trials suggested that il-10 attenuates acute colitis , increases the tumor sensitivity of nk cells in rabbits with melanoma , promotes monocytes differentiating toward to tolerogenic dcs , and thus may have potential therapeutic value in autoimmune and transplantation related immune - compromised conditions . interestingly , these studies suggested that only a small segment at c - terminal of il-10 is responsible for its bioactivity . a synthetic il-10 agonist , it 9302 , was administered to the rabbits with acute lung injury in acute necrotizing pancreatitis [ 149 , 150 ] . it revealed that it9302 reduced the mortality and the incidence of acute lung injury in rabbits with acute necrotizing pancreatitis , possibly by suppressing the productions of tnf , il-8 , mcp-1 , and adhesion molecule complex cd11b / cd18 , as well as increasing serum il-1 ra level . this is very encouraging , as most of the lung injury is related to inflammation and reduced immunity , such as oili . in line with the aforementioned mechanism , along with the available agonists / analogues such as am0010 , sch52000 , rn1003 , and it9302 , and its downstream signaling blockers such as cp-690 and cp-550 , we hypothesized that il-10 may have a protective role in lung injury , and more specifically , in acid aspiration induced lung injury in obesity . related clinical trials are highly recommended to further define this , its bioactivity , safety , efficacy , and therapeutic indications . interleukin-1 receptor antagonist ( il-1ra ) was secreted naturally to encounter the effect of il-1 and neutralize the proinflammatory effect of il-1 , by competitively binding to il-1 receptor i ( il-1ri ) . as it secrets at the time of il-1 secretion , which is generally increased at the states of inflammation such as obesity , t2 dm , and lung injury , it is understandable that il-1ra is elevated in obese and diabetic subjects in whitehall ii cohorts and a few other clinical trials . however , administration of recombinant il-1ra ( anakinra ) lowers body weight and glucose level and decreases inflammation in patients with metabolic syndrome and t2 dm [ 152 , 153 ] . il-1ra competitively binds to il-1ri with il-1 and thus decoys the inflammatory effects of il-1 . deletion of il-1ra leaves il-1 unopposed and thus causes fetal inflammation systemically . under conditions with lung injury , il-1 releases and triggers inflammation and il-1ra releases to encounter this process . there are some ongoing / complete trials in subjects with rheumatoid arthritis , heart failure , pulmonary hypertension , diabetes , and other inflammatory conditions with recombinant il-1ra anakinra . no ongoing / complete clinical trial in oili was reported per the best of our knowledge . tgf- shows anti - inflammatory effect and has interaction with il-10 [ 156 , 157 ] . gene knockout of tgf- confirmed its anti - inflammatory effect presented at the early stage and before the major attack of bacteria . tgf-1 has a very short half - life in circulation and this may contribute to these diverse results . some clinical trials with tgf-1 antibodies such as gc1008 , cat-192 , and ly2382770 are ongoing or complete in subjects with diabetes , diabetic kidney disease , and other inflammatory diseases . no ongoing / complete clinical trial in oili was reported per the best of our knowledge . gdf15 , a member of tgf- family , also known as macrophage inhibitory cytokine-1 ( mic-1 ) , shares similarity with tgf- [ 159 , 160 ] . gdf15 increases in obesity but also suppresses food intake and reduces body weight in obese rodents . gdf15 can be a biomarker for severity of lung diseases as well as inhibitor for cancer development . no study was reported in oili so far . although there are studies showing the anti - inflammatory effect of leptin , there are leptin receptors in lung , alveolar epithelium , and macrophages , and leptin plays very important roles in immunity and host defense response , especially for activation of cell mediated immunity , as leptin is regarded as a proinflammatory adipokine in obesity and lung injury , supported by the majority of the clinical trials and animal studies . as addressed previously , due to the delayed discovery of the receptor for adiponectin , there is no clinical utilization of adiponectin . yet , based on what we reviewed here , adiponectin showed a strong anti - inflammatory effect in obesity , through its activation of ampk and stimulation of mitochondrial biogenesis , as well as its inhibition of nf-b signaling pathways and oxidative stress ; we believe that adiponectin and adiponectin receptor agonist as well as ampk activator would greatly benefit patients from a variety of aspects , including lung injury in obesity . with the availability of adiponectin receptor agonist , adp355 , we expect that more preclinical and clinical interventional trials in oili will be conducted . someday , patients with oili and other inflammatory diseases will be greatly benefited , especially those with obesity . one major obstacle is the route and form of the agents . for lung injury , inhalation and intravenous injection or infusion would be appropriate . details for getting the active molecule into the system and the modification after administration need to work out . alternates would be other agents promoting adiponectin production , such as ppar agonist , the market - available thiazolidinediones ( tzds ) , omega-3 , and dietary modifications . as the definitive receptor of omentin has not yet been identified in the lung , it is difficult to define the exact role of omentin in obesity related lung injury . however , based on its inhibition to tnf , il-6 , and other proinflammatory cytokines , its blocking on nf-b and tlr4 signaling pathways , its potential role in osas , as well as its association with inflammatory states such as crohn 's disease , rheumatoid arthritis , and pcos , we believe that it favors anti - inflammation and may have therapeutic potential in obesity and its comorbidities including lung injury . yet , most exploration of its therapeutic role is still in the preclinical stage , and there is no complete or ongoing clinical trial . with the availability of recombinant omentin , we believe that further studies from these aspects would provide valuable information in the near future . based on the effect of sfrp5 on weight loss , its signaling pathway , and the availability of the recombinant sfrp5 , we expect more preclinical study and clinical trials in related area . as sfrp5 does reduce production of proinflammatory tnf , il-6 , and mcp-1 , we expect it to exert anti - inflammatory effect in obesity related lung injury . based on the switch effects of il-10 on macrophages , th cells , igg , iga , and inhibition on proinflammation and th1 , il-10 may have a great therapeutic potential in treating infections , inflammation , and related diseases including lung injury in obesity . as mentioned , synthetic interleukin-10 agonist such as it9302 varnishes acute lung injury in rabbits with acute necrotizing pancreatitis and promotes monocytes differentiation to tolerogenic dcs . this suggested its therapeutic potential for autoimmune and transplantation - related disease , as well as its potential therapeutic benefit in oili and other inflammatory diseases . clinical trials with human synthetic interleukin-10 are still in the early phase , such as phase 1 trial with sch 52000 in patients with wegener 's granulomatosis , phase 2 trial with rn1003 for scar reduction , phase 2 trial with recombinant human interleukin-10 for psoriasis , and phase 2 trial with tenovil tm in prevention of post - ercp acute pancreatitis . as shown in table 1 , we sum up this review article as follows.the majority of evidence supported that adiponectin , omentin , and sfrp5 were reduced significantly in obesity , which is associated with increased inflammation and possible lung injury , indicated by increase of tnf and il-6 , through activation of tlr4 and nf-b signaling pathways.administration of these adipocytokines promotes weight loss and reduces inflammation.il-10 , zag , vaspin , il-1ra , tgf-1 , and gdf15 seem to be anti-inflammatory.there were controversial reports , though.yet , there is a huge lack of studies for obesity related lung injury . some groups investigated the effect of adiponectin on lung transplantation and subsequent changes for graft function , asthma , copd , and pneumonia , supporting its anti - inflammatory effects and protective role . synthetic il-10 agonist reduces mortality of acute lung injury in rabbits with acute necrotizing pancreatitis , possibly through its inhibition of proinflammatory and promotion of anti - inflammatory adipocytokines , as well as its augmentation of host immunity . more preclinical and clinical trials in wider area with larger population are warranted.for other adipocytokines , there are very limited studies in obesity related lung injury.in oili , there is not much information available for clinical trials and translational research because most of the agonists were recently synthesized . translational studies focusing on the mechanism should reveal valuable information for further investigation and therapeutic potentials . the early phase trials would need to focus on safety , efficacy , and bioavailability at this time point . in the near future , the majority of evidence supported that adiponectin , omentin , and sfrp5 were reduced significantly in obesity , which is associated with increased inflammation and possible lung injury , indicated by increase of tnf and il-6 , through activation of tlr4 and nf-b signaling pathways . il-10 , zag , vaspin , il-1ra , tgf-1 , and gdf15 seem to be anti - inflammatory . some groups investigated the effect of adiponectin on lung transplantation and subsequent changes for graft function , asthma , copd , and pneumonia , supporting its anti - inflammatory effects and protective role . synthetic il-10 agonist reduces mortality of acute lung injury in rabbits with acute necrotizing pancreatitis , possibly through its inhibition of proinflammatory and promotion of anti - inflammatory adipocytokines , as well as its augmentation of host immunity . more preclinical and clinical trials in wider area with larger population are warranted . for other adipocytokines , there are very limited studies in obesity related lung injury . in oili , there is not much information available for clinical trials and translational research because most of the agonists were recently synthesized . translational studies focusing on the mechanism should reveal valuable information for further investigation and therapeutic potentials . the early phase trials would need to focus on safety , efficacy , and bioavailability at this time point . in the near future ,

venous leg ulcers ( vlus ) pose a serious clinical dilemma and an economic burden on health services . they are frequently associated with morbidity , pain , and decreased quality of life in affected patients . about 60% of patients with vlus will experience pain related to their wounds ( hofman et al 1997 ) , which may be constant or intermittent ( ryan et al 2003 ) . the pain can cause depression and a feeling of constant tiredness ( price et al 2007 ) and may also interfere with the healing process ( soon and acton , 2006 ) . these patients may have highly exuding ulcers that could lead to wound malodour and its associated social stigma , resulting in further detrimental psychological effects . vlus also provide an ideal environment for the growth of pathogenic bacteria , with wound colonisation / infection prevalent in immunocompromised patients . vlus are associated with significant treatment costs ( bandages , dressings , and adjunctive therapies ) and nursing resource . about 1%2% of the whole population ( anderson 2006 ) and 3%5% of the population over 65 years of age ( mekkes et al 2003 ) will suffer from a leg ulcer during their lifetime . however , in a typical western population where the average age is steadily increasing , the burden placed upon the health economy by vlus looks set to increase proportionally . back in the 1960s , george winter published his landmark paper in which he demonstrated that , contrary to the belief at the time that wounds should be allowed to dry out and form scabs to promote healing , wounds heal faster if kept moist ( winter et al 1962 ) . since then , the use of cheap gauze dressings and bandages has given way to more technically advanced dressings , such as foams and hydrofibres , which interact with and manage the wound environment . the use of these now established dressings in combination with the gold standard high compression therapy is generally effective in the management of vlus , although up to 20% of vlus fail to heal despite being treated with this regime ( white 2006 ) . the reasons for the lack of response to treatment of hard - to - heal vlus have not yet been fully elucidated , although scientific and clinical research indicates that , instead of progressing through the four distinct but overlapping phases of healing ( hemostasis , inflammation , proliferation , and remodeling ) , these wounds become stuck in a prolonged inflammatory phase ( timmons 2006 ) . control of the processes within the phases of wound healing is complex and involves the interaction of several different cell types ( eg , neutrophils , lymphocytes , macrophages , fibroblasts ) , regulatory mediators ( eg , cytokines , growth factors ) , extracellular matrix ( ecm ) components ( eg , fibronectin , fibrin , collagen , elastin , laminin , proteoglycans , glycosaminoglycans ) , proteases and their inhibitors ( eg , matrix metalloproteinases [ mmps ] and tissue inhibitors of metalloproteinases [ timps ] ) ( schultz et al 2005 ) . early in the wound healing process there is a requirement for the synthesis and deposition of ecm proteins such as fibrin and fibronectin ( hodde and johnson 2007 ) and a sub - set of matrix proteins that are expressed transiently during the wound healing process , eg , galectins , osteopontin , sparc , syndecans , tenascins , thrombospondins , and vitronectin ( agren and werthen 2007 ) . all of these matrix proteins form a provisional wound matrix which provides a scaffold that directs cells into the wound as well as stimulating them to proliferate , differentiate and synthesise new ecm to facilitate the proliferation and remodeling phases of the healing process ( schultz et al 2005 ) . in many chronic wounds , however , the normal healing process is disrupted by extensive tissue damage accompanied by biochemical and cellular imbalances , or by an underlying pathological state ( eg , venous insufficiency ) that can impair or even prevent healing . for example , in the case of hard - to - heal vlus , venous hypertension causes disturbed microcirculation and pathological changes to capillaries , which ultimately locks the condition in a self - amplifying cascade with persistent elevated levels of pro - inflammatory cytokines and proteases that appear to degrade the ecm components , growth factors and receptors that are essential for healing ( agren et al 2000 ; schultz et al 2005 ) the ecm is a vital component of the healing process ; intact or fragmented ecm molecules play a central role in modulating cells through transduction of a variety of signaling mechanisms ( agren et al 2000 ) . additionally , ecm plays a role in angiogenesis , recruitment of circulating progenitor cells , rapid scaffold degradation and constructive remodeling of damaged or missing tissues ( badylak 2002 ) . in studies investigating the mechanisms that lead to venous leg ulceration , it has been demonstrated that intrinsic ecm degradation processes , prevalent in this disease state , are caused by the mmp family of enzymes ( herouy et al 2000 ; bogaczewicz et al 2005 ) . specifically , it has been shown that fibronectin is absent in the base of nonhealing ulcers ( herrick et al 1992 ) although fibroblasts in such wounds synthesize fibronectin normally ( herrick et al 1996 ) . it is hypothesized , therefore , that the reduced levels of fibronectin in nonhealing ulcers is due to excessive degradation of fibronectin by proteases in the ulcers , rather than decreased synthesis ( agren and werthen 2007 ) . on this basis , there is a clear need for new and advanced interventions that can interact with hard - to - heal vlus that are locked in the inflammatory phase of the healing process and progress them to the subsequent proliferative stage of the process . a specific focus of attention for the development of advanced wound therapies should be ( and has been in recent years ) the ecm , either by inducing its synthesis through growth factors , preventing its damage with sacrificial proteins or replacing it with autologous / homologous ecm proteins or their like . one mechanism by which this can be achieved is to provide hard - to - heal ulcers with surrogate ecms which , although not homologous to the patient s own ecm , will provide transient structures that allow cellular adhesion and facilitate tissue regeneration by advancing the wounds to the proliferative phase of the healing process . amelogenin has been identified as an ecm biocompatible protein that can be used as a surrogate scaffold protein . at physiological conditions , amelogenin self - assembles into globular aggregates up to micron - sizes . when applied to the wound bed it provides a temporary matrix for cell attachment and promotes wound healing amelogenin has been used successfully to treat patients with hard - to - heal vlus and other chronic wound types . amelogenin has been used in periodontal applications for some time and there a number of experimental and clinical studies supporting its use in this application . amelogenin has been used as a therapy for the regeneration of alveolar bone by inducing the formation of acellular extrinsic fiber cementum ( kim et al 2005 ) . this protein has also been used to aid in soft tissue root coverage where it has been shown to enhance cellular activity during healing and improve periodontal regeneration ( giannobile and somerman 2003 ; young 2003 ) . more recently , it has been shown that the combination of amelogenin and platelet - derived growth factor - bb ( pdgf - bb ) stimulated proliferation of primary human periodontal ligament fibroblasts and caused enhanced wound fill in an in vitro model ( chong et al 2006 ) . experimental studies designed to elucidate the mechanism by which amelogenin works have shown that it interacts with a variety of cells and chemical messengers involved in the wound healing process . the studies indicate that amelogenin can reduce levels of pro - inflammatory cytokines ( myhre et al 2006 ) . . this cell synthesizes matrix components ( eg , collagen ) that are designed to fill the wound cavity and a variety of cell signals that modulate many different cellular responses such as angiogenesis and epithelialization . in experimental studies , amelogenin has been shown to : augment fibroblast - driven collagen matrix remodeling , increase dermal contraction and fibroblast proliferation ( grayson et al 2006 ) increase synthesis of growth factors , eg , transforming growth factor-1 ( grayson et al 2006 ) and vascular endothelial growth factor ( mirastschijski et al 2004 ) convert chronic fibroblasts into acute fibroblast phenotypes , and the restitution of proliferation and chemokine expression favoring an acute inflammatory response . amelogenin has also been shown to be effective in inducing an angiogenic response in a variety of in vitro models ( yuan et al 2003 ; schlueter et al 2007 ; gren and kleinman 2007 ) . angiogenesis , a key component of the healing process , is significantly reduced in the chronic wound ( drinkwater et al 2002 ; ulrich et al 2005 ) , thereby affecting the healing response . the fact that amelogenin can be used as a temporary ecm protein scaffold provides a sound basis for its use in the treatment of wounds , particularly chronic wounds that are deficient in normal ecm components . it was , therefore , proposed that amelogenin could be used to treat hard - to - heal vlus . initially , a preliminary clinical study was undertaken to ascertain the optimum number of applications of amelogenin ( xelma , molnlycke health care , gothenburg , sweden ) that are required to be applied to a vlu to produce a healing response . the study compared once weekly applications of amelogenin in conjunction with high compression therapy to the hard - to - heal vlus ( duration > 6 months ) of patients over 3 , 6 and 12 week periods ( romanelli et al 2006 ) the wounds were assessed every second week up to week 8 , at final visit ( week 12 ) , and at follow - up visit ( 12 weeks after the final visit ) . the results showed that patients who received the 12-weeks treatment had a larger ulcer reduction ( 72% ) compared with patients who received the 3 or 6-weeks ( 22 and 48% respectively ) treatment with amelogenin ( figure 1 and table 1 ) . percentage change in vlu size following 3 , 6 and 12 weeks of treatment with amelogenin . reproduced with permission from romanelli m , ellervee t , jarve h , et al 2006 . amelogenins ( xelma ) in hard - to - heal venous leg ulcers , an open regime investigation [ poster ] . percentage change in vlu size following 3 , 6 and 12 weeks of treatment with amelogenin . copyright 2006 . reproduced with permission from romanelli m , ellervee t , jarve h , et al 2006 . amelogenins ( xelma ) in hard - to - heal venous leg ulcers , an open regime investigation [ poster ] . european wound management association conference , prague , czech republic the results of this preliminary study showed that hard - to - heal vlus elicited a positive healing response to a defined period ( ie , 12 weeks ) treatment with amelogenin . using this information , a single - blinded , randomized , multi - centre study involving 117 patients was undertaken to assess the effect of amelogenin on hard - to - heal vlus ( vowden et al 2006 ) . to satisfy the inclusion criteria for the study , patients had to have a vlu with a size between 5 and 25 cm and duration of at least six months that had been treated with controlled compression therapy for at least one month prior to enrolment . patients were randomised to receive either amelogenin ( xelma , molnlycke health care , gothenburg , sweden ) plus high compression ( n = 62 ) or a control treatment consisting of a placebo gel ( the amelogenin carrier vehicle ) plus high compression ( n = 61 ) . the amelogenin was applied weekly under secondary dressings for up to a maximum of 12 weeks . although no statistical differences could be identified between the groups as a whole , the percentage wound size reduction was greater in the group treated with amelogenin . a more detailed sub - group analysis was undertaken on patients with ulcers of greater than 10 cm at baseline and for ulcers of duration greater than 6 months . the results of this analysis demonstrated that there was a difference in percentage wound reduction in the group treated with amelogenin ( 33.8% vs 25.6% , respectively ) . it was highlighted that this difference was greatest for the group of patients ( n = 61 ) with the larger ulcers ( > 10 cm ) ( amelogenin 25% vs control 7.9% ) ( figure 2 ) . median wound size reduction in the itt population and sub - group itts following treatment with amelogenin and control . reproduced with permission from vowden p , romanelli m , peter r , et al 2006 . the effect of amelogenins ( xelma ) on hard - to - heal venous leg ulcers . overall the results of this study showed that amelogenin was well tolerated by the patients , with a trend towards less pain and a reduction in the volume of exudate produced by the ulcers in favor of the group treated with amelogenin . this study highlighted the problems associated with undertaking clinical trials involving vlus , the main ones being the wide variations in wound demographics ( eg , sizes and ages ) and their response to treatments . in addition , there was a significant amount of variation between the pre - study treatment regimens employed at different centers , a phenomenon which if not accounted for by allowing a run - in period , may alter the outcome of a clinical trial . as such , the authors concluded that amelogenin could be clinically useful in the treatment of patients with ulcers that could be classified as hard - to - heal ( eg , of duration longer than 6 months and a size greater than 10 cm ) . another clinical trial was undertaken using the lessons learnt from the previous study : this was an open , randomized , comparative , parallel group , multi - centre study involving patients with hard - to - heal vlus ( vowden et al 2007 ) . the inclusion criteria incorporated ulcer size of greater than 10 cm and duration of longer than 6 months , both of which have previously been reported in the literature as prognostic indicators of venous ulcers that are unlikely to respond to treatment and may be classed as hard - to - heal ( european wound management association 2002 ) . the primary objective of this study was to compare the results of 12 consecutive weeks of treatment with amelogenin ( xelma , molnlycke health care , gothenburg , sweden ) plus high compression versus compression therapy alone in hard - to - heal vlus . percentage reduction of wound size from baseline , the percentage of improved ulcers , and parameters such as pain related to the disease , pain at dressing changes and the amount and nature of exudate were evaluated . eligibility for inclusion included adult , mobile patients with hard - to - heal vlus that had been treated with compression therapy for at least 1 month prior to screening . the ulcers had to be at least 6 months old , with a surface area at inclusion of at least 10 cm , but not exceeding 30 cm , and not demonstrating excessive exudate or signs of infection . at the end of the run in period additional criteria for eligibility , eg , change in wound area ( increase / decrease ) of greater than or equal to 50% and a wound area between 8 and 36 cm were applied . in total , 83 patients were randomized to receive either amelogenin plus high compression bandaging ( n = 42 ) or high compression bandaging ( control ) alone ( n = 41 ) . all participants received high compression bandaging therapy one month prior to , during the investigational period of 3-weeks run - in and throughout the 12 weeks of active treatment the results of this study demonstrated that the amelogenin group showed a greater percentage reduction in ulcer size ( mean 33.11% ) compared to the control group ( mean 11.07% ) from baseline to the last visit ( p = 0.03 ) . the number of ulcers showing an improvement was significantly greater ( p = 0.01 ) in the amelogenin treated group than the control group . statistically significant differences in favor of the amelogenin group were also found the proportion of patients with none or low levels of exudate ( p = 0.01 ) . pain was evaluated in this study as related to the disease or to the ulcer . it was evaluated as part of the protocol by interviewing the patients who were asked to rate their pain on a scale with 11 steps ; from 0 = no pain to 10 = unbearable pain ( hartrick et al 2003 ) . the results showed that a reduction in pain related to the disease and a reduction in pain at dressing change were more apparent in the amelogenin treated group . statistical analysis showed that the amelogenin group had significantly ( p = 0.01 ) greater ulcer pain reduction . the mean and 95% ci for the difference between the amelogenin and the control group at final visit was 1.59 ( 2.84 0.34 ) . pain is considered to be a significant problem in relation to patients with vlus , and has been highlighted as cause for major concern when treating patients ( price et al 2007 ) . the european wound management association ( 2002 ) has developed a position document that provides clinical recommendations to assess and manage wound pain , especially at dressing change . pain management has therefore become a major part of wound care with many organizations and care providers incorporating pain management into standards , guidelines and clinical practice ( price et al 1997 ; young 2007 ) . thus the fact that amelogenin therapy , in a way as yet undetermined , was shown to significantly reduce the pain that patients with hard - to - heal vlus experienced is noteworthy and perhaps requires further investigation . as well as the study participants having hard - to - heal wounds , the population evaluated represents possibly the worst case scenario patients , eg , some patients within the amelogenin group had very old ulcers ( 10 years or more ) . this highlights the potential advantages of using advanced therapies for the treatment of ulcers within this patient population . the safety of amelogenin has been established by completing the extensive testing that is required for medical devices . the safety profile of amelogenin is underlined by the results of this study with few adverse events reported , and no significant difference between the treatment and control groups . a series of case studies has been presented in which a number of patients with hard - to - heal vlus were treated with amelogenin ( xelma , molnlycke health care , gothenburg , sweden ) therapy ( huldt - nystrom et al 2007 ) . the healing of these wounds was evaluated by comparing the wound area at baseline ( before treatment ) and upon completion of treatment . in this study , the amelogenin was applied to a clean wound bed , on a weekly basis , up to a maximum of 12 weeks in conjunction with standard compression therapy . some of these patients were treated with compression for four weeks prior to being included in the evaluations , in order to ensure that ulcers were truly nonhealing . the results showed that the success rate with amelogenin therapy was high , with approximately 80% of the ulcers either healing ( 60% ) or showing a reduction in ulcer size ( 20% ) after treatment . only two ulcers ( 10% ) remained unchanged and two ulcers deteriorated ( 10% ) during the treatment period . pseudomonas colonization / infection was present in some wounds after treatment had been started , but this was treated with appropriate antibiotic therapy , and did not affect the successful healing outcome of the wounds . a small study in the united kingdom ( acton 2007 ) has demonstrated similar results in that amelogenin ( xelma , molnlycke health care , gothenburg , sweden ) stimulated a healing response . even if the wounds did not progress to complete healing , they were significantly healthier than before the amelogenin treatment had started and were able to go on to be grafted . in addition , an improvement in quality of life was observed in the patients who received amelogenin therapy . another study undertaken in the united kingdom evaluated amelogenin for the treatment of patients ( n = 17 ) with a variety of complex hard - to - heal ulcers , including rheumatoid ulcers ( n = 2 ) , wounds complicated by rheumatoid arthritis ( n = 3 ) , neuropathic foot ulcers ( n = 4 ) , venous ulcers ( n = 4 ) , and a single ulcer of mixed etiology . the results from this study followed a similar trend to that seen in the treatment of other hard - to - heal ulcers , in that an early reduction in wound pain and exudate were apparent . overall , six wounds healed after a mean of 8 applications of ( xelma , molnlycke health care , gothenburg , sweden ) ( range 316 applications ) , six patients showed an improvement of a greater than 50% reduction in ulcer size , and were continuing to receive amelogenin therapy . only two patients were discontinued due to infection and wound deterioration ( vowden et al 2007a ) . in a case study series involving eight patients with a total of 10 vlus ( mean duration of 9.3 years ) , amelogenin ( xelma , molnlycke health care , gothenburg , sweden ) was applied weekly and covered with a secondary dressing . treatment duration with amelogenin was 12 weeks and patients were followed up for a period of 24 weeks . an overall healing rate of 50% was reported , including one patient with a wound duration of 65 years . the other ulcers were in a healing state at 24 weeks , with a mean reduction in size of 60% compared with baseline values . exudate levels and wound odor also reduced over the treatment period ( hampton et al 2007 ) . amelogenin has recently been evaluated in a study consisting of a mixed population of patients with both diabetic foot ulcers ( n = 5 ) and vlus ( n = 5 ) . both sets of patients demonstrated initiation of healing with a high percentage ( 80% ) of ulcers healing completely ( meuleneire 2007 ) . diabetic foot ulcers have a different aetiology to that of vlus , they are a significant complication of diabetes mellitus and often precede lower - extremity amputation . the most frequent underlying aetiologies are neuropathy , trauma , deformity , high plantar pressures , and peripheral arterial disease . the primary treatment is usually offloading to reduce pressure to the sensitive areas and then management of the wound with appropriate dressings . infection and high levels of wound exudate may be complicating factors in the treatment of these wounds , but the early indications are that amelogenin therapy , in conjunction with appropriate wound management techniques , can be beneficial . pyoderma gangrenosum ( pg ) is an inflammatory ulcerative condition of unknown etiology , although an autoimmune mechanism including immune complex - mediated neutrophilic vascular reactions has been suggested . pg is frequently associated with various diseases , but up to 50% of cases are idiopathic . it is a disease that causes tissue to become necrotic , causing deep ulcers that usually occur on the legs . there are two main types of ulcers that occur : the normal ulcerative form which occurs on the legs , and an atypical form that is more superficial and occurs on the hands and other parts of the body . though the etiology is not well understood , the disease is thought to be due to immune system dysfunction and , in particular , improper functioning of neutrophils . at least half of all pg patients also suffer from illnesses that affect their systemic function . classical ulcerative pg is characterized by the appearance of nodules with pustules that enlarge and lead to chronic ulcers violaceous and undermined borders ( dini et al 2007 ) amelogenin therapy has been used to treat two female patients with recalcitrant pg of the lower leg lasting an average of 11 months ( dini et al 2007a ) . the treatment was applied weekly under an occlusive dressing for a maximum of 8 weeks in conjunction with systemic immunosuppressive therapy before and during the topical treatment . the lesions improved , showing advances in granulation tissue formation and wound size reduction ( figures 3a - c ) . additionally , pain control was reported and the therapy was well - tolerated with no adverse effects . the clinical evidence suggests that in order for advanced therapies like amelogenin to be cost effective in for example the treatment of vlus , then suitable patients must be carefully identified and according to prognostic indicators of delayed healing such as wound size ( larger than 10 cm ) and wound age ( duration of greater than 6 months ) . additionally , if wounds do not show a decrease in size of 40% or more during 4 weeks of compression therapy , then these wounds can be identified as hard - to - heal ( phillips et al 2000 ) and consideration should be given to their treatment with advanced therapies such as amelogenin . this is because it treats the underlying cause of venous hypertension and enables blood to be returned from the lower limbs , thereby reducing oedema and the consequences of stasis . infection in the wound must be treated prior to application of amelogenin , although it has been seen that concurrent treatment of infection with antibacterial therapy is not detrimental to the amelogenin protein as a therapeutic agent . additionally , wounds that exudate highly do not appear to benefit as well from amelogenin therapy as wounds that have low to moderate levels of exudate , implicit in this therefore is the requirement that exudate should be managed with compression and appropriate dressings that can absorb the fluid . this tissue impedes the growth of healthy granulation tissue and re - epithelisation and therefore must be removed . it is also thought that the presence of such dead tissue may prevent the amelogenin protein integrating into the fabric of the wound bed and thus providing the temporary scaffolding to which cells can attach and subsequently stimulate the healing process . qualitatively , vlus treated with amelogenin appear to heal much better than with high compression alone , with no evidence of hypertrophic scarring or excessive wound contracture . the fact that amelogenin can be used as a temporary ecm protein scaffold provides a sound basis for its use in the treatment of wounds , particularly chronic wounds that are deficient in normal ecm components . it was , therefore , proposed that amelogenin could be used to treat hard - to - heal vlus . initially , a preliminary clinical study was undertaken to ascertain the optimum number of applications of amelogenin ( xelma , molnlycke health care , gothenburg , sweden ) that are required to be applied to a vlu to produce a healing response . the study compared once weekly applications of amelogenin in conjunction with high compression therapy to the hard - to - heal vlus ( duration > 6 months ) of patients over 3 , 6 and 12 week periods ( romanelli et al 2006 ) the wounds were assessed every second week up to week 8 , at final visit ( week 12 ) , and at follow - up visit ( 12 weeks after the final visit ) . the results showed that patients who received the 12-weeks treatment had a larger ulcer reduction ( 72% ) compared with patients who received the 3 or 6-weeks ( 22 and 48% respectively ) treatment with amelogenin ( figure 1 and table 1 ) . percentage change in vlu size following 3 , 6 and 12 weeks of treatment with amelogenin . reproduced with permission from romanelli m , ellervee t , jarve h , et al 2006 . amelogenins ( xelma ) in hard - to - heal venous leg ulcers , an open regime investigation [ poster ] . percentage change in vlu size following 3 , 6 and 12 weeks of treatment with amelogenin . copyright 2006 . reproduced with permission from romanelli m , ellervee t , jarve h , et al 2006 . amelogenins ( xelma ) in hard - to - heal venous leg ulcers , an open regime investigation [ poster ] . european wound management association conference , prague , czech republic the results of this preliminary study showed that hard - to - heal vlus elicited a positive healing response to a defined period ( ie , 12 weeks ) treatment with amelogenin . using this information , a single - blinded , randomized , multi - centre study involving 117 patients was undertaken to assess the effect of amelogenin on hard - to - heal vlus ( vowden et al 2006 ) . to satisfy the inclusion criteria for the study , patients had to have a vlu with a size between 5 and 25 cm and duration of at least six months that had been treated with controlled compression therapy for at least one month prior to enrolment . patients were randomised to receive either amelogenin ( xelma , molnlycke health care , gothenburg , sweden ) plus high compression ( n = 62 ) or a control treatment consisting of a placebo gel ( the amelogenin carrier vehicle ) plus high compression ( n = 61 ) . the amelogenin was applied weekly under secondary dressings for up to a maximum of 12 weeks . although no statistical differences could be identified between the groups as a whole , the percentage wound size reduction was greater in the group treated with amelogenin . a more detailed sub - group analysis was undertaken on patients with ulcers of greater than 10 cm at baseline and for ulcers of duration greater than 6 months . the results of this analysis demonstrated that there was a difference in percentage wound reduction in the group treated with amelogenin ( 33.8% vs 25.6% , respectively ) . it was highlighted that this difference was greatest for the group of patients ( n = 61 ) with the larger ulcers ( > 10 cm ) ( amelogenin 25% vs control 7.9% ) ( figure 2 ) . median wound size reduction in the itt population and sub - group itts following treatment with amelogenin and control . reproduced with permission from vowden p , romanelli m , peter r , et al 2006 . the effect of amelogenins ( xelma ) on hard - to - heal venous leg ulcers . overall the results of this study showed that amelogenin was well tolerated by the patients , with a trend towards less pain and a reduction in the volume of exudate produced by the ulcers in favor of the group treated with amelogenin . this study highlighted the problems associated with undertaking clinical trials involving vlus , the main ones being the wide variations in wound demographics ( eg , sizes and ages ) and their response to treatments . in addition , there was a significant amount of variation between the pre - study treatment regimens employed at different centers , a phenomenon which if not accounted for by allowing a run - in period , may alter the outcome of a clinical trial . as such , the authors concluded that amelogenin could be clinically useful in the treatment of patients with ulcers that could be classified as hard - to - heal ( eg , of duration longer than 6 months and a size greater than 10 cm ) . another clinical trial was undertaken using the lessons learnt from the previous study : this was an open , randomized , comparative , parallel group , multi - centre study involving patients with hard - to - heal vlus ( vowden et al 2007 ) . the inclusion criteria incorporated ulcer size of greater than 10 cm and duration of longer than 6 months , both of which have previously been reported in the literature as prognostic indicators of venous ulcers that are unlikely to respond to treatment and may be classed as hard - to - heal ( european wound management association 2002 ) . the primary objective of this study was to compare the results of 12 consecutive weeks of treatment with amelogenin ( xelma , molnlycke health care , gothenburg , sweden ) plus high compression versus compression therapy alone in hard - to - heal vlus . percentage reduction of wound size from baseline , the percentage of improved ulcers , and parameters such as pain related to the disease , pain at dressing changes and the amount and nature of exudate were evaluated . eligibility for inclusion included adult , mobile patients with hard - to - heal vlus that had been treated with compression therapy for at least 1 month prior to screening . the ulcers had to be at least 6 months old , with a surface area at inclusion of at least 10 cm , but not exceeding 30 cm , and not demonstrating excessive exudate or signs of infection . at the end of the run in period additional criteria for eligibility , eg , change in wound area ( increase / decrease ) of greater than or equal to 50% and a wound area between 8 and 36 cm were applied . in total , 83 patients were randomized to receive either amelogenin plus high compression bandaging ( n = 42 ) or high compression bandaging ( control ) alone ( n = 41 ) . all participants received high compression bandaging therapy one month prior to , during the investigational period of 3-weeks run - in and throughout the 12 weeks of active treatment the results of this study demonstrated that the amelogenin group showed a greater percentage reduction in ulcer size ( mean 33.11% ) compared to the control group ( mean 11.07% ) from baseline to the last visit ( p = 0.03 ) . the number of ulcers showing an improvement was significantly greater ( p = 0.01 ) in the amelogenin treated group than the control group . statistically significant differences in favor of the amelogenin group were also found the proportion of patients with none or low levels of exudate ( p = 0.01 ) . pain was evaluated in this study as related to the disease or to the ulcer . it was evaluated as part of the protocol by interviewing the patients who were asked to rate their pain on a scale with 11 steps ; from 0 = no pain to 10 = unbearable pain ( hartrick et al 2003 ) . the results showed that a reduction in pain related to the disease and a reduction in pain at dressing change were more apparent in the amelogenin treated group . statistical analysis showed that the amelogenin group had significantly ( p = 0.01 ) greater ulcer pain reduction . the mean and 95% ci for the difference between the amelogenin and the control group at final visit was 1.59 ( 2.84 0.34 ) . pain is considered to be a significant problem in relation to patients with vlus , and has been highlighted as cause for major concern when treating patients ( price et al 2007 ) . the european wound management association ( 2002 ) has developed a position document that provides clinical recommendations to assess and manage wound pain , especially at dressing change . pain management has therefore become a major part of wound care with many organizations and care providers incorporating pain management into standards , guidelines and clinical practice ( price et al 1997 ; young 2007 ) . thus the fact that amelogenin therapy , in a way as yet undetermined , was shown to significantly reduce the pain that patients with hard - to - heal vlus experienced is noteworthy and perhaps requires further investigation . as well as the study participants having hard - to - heal wounds , the population evaluated represents possibly the worst case scenario patients , eg , some patients within the amelogenin group had very old ulcers ( 10 years or more ) . this highlights the potential advantages of using advanced therapies for the treatment of ulcers within this patient population . the safety of amelogenin has been established by completing the extensive testing that is required for medical devices . the safety profile of amelogenin is underlined by the results of this study with few adverse events reported , and no significant difference between the treatment and control groups . a series of case studies has been presented in which a number of patients with hard - to - heal vlus were treated with amelogenin ( xelma , molnlycke health care , gothenburg , sweden ) therapy ( huldt - nystrom et al 2007 ) . the healing of these wounds was evaluated by comparing the wound area at baseline ( before treatment ) and upon completion of treatment . in this study , the amelogenin was applied to a clean wound bed , on a weekly basis , up to a maximum of 12 weeks in conjunction with standard compression therapy . some of these patients were treated with compression for four weeks prior to being included in the evaluations , in order to ensure that ulcers were truly nonhealing . the results showed that the success rate with amelogenin therapy was high , with approximately 80% of the ulcers either healing ( 60% ) or showing a reduction in ulcer size ( 20% ) after treatment . only two ulcers ( 10% ) remained unchanged and two ulcers deteriorated ( 10% ) during the treatment period . pseudomonas colonization / infection was present in some wounds after treatment had been started , but this was treated with appropriate antibiotic therapy , and did not affect the successful healing outcome of the wounds . a small study in the united kingdom ( acton 2007 ) has demonstrated similar results in that amelogenin ( xelma , molnlycke health care , gothenburg , sweden ) stimulated a healing response . even if the wounds did not progress to complete healing , they were significantly healthier than before the amelogenin treatment had started and were able to go on to be grafted . in addition , an improvement in quality of life was observed in the patients who received amelogenin therapy . another study undertaken in the united kingdom evaluated amelogenin for the treatment of patients ( n = 17 ) with a variety of complex hard - to - heal ulcers , including rheumatoid ulcers ( n = 2 ) , wounds complicated by rheumatoid arthritis ( n = 3 ) , neuropathic foot ulcers ( n = 4 ) , venous ulcers ( n = 4 ) , and a single ulcer of mixed etiology . the results from this study followed a similar trend to that seen in the treatment of other hard - to - heal ulcers , in that an early reduction in wound pain and exudate were apparent . overall , six wounds healed after a mean of 8 applications of ( xelma , molnlycke health care , gothenburg , sweden ) ( range 316 applications ) , six patients showed an improvement of a greater than 50% reduction in ulcer size , and were continuing to receive amelogenin therapy . only two patients were discontinued due to infection and wound deterioration ( vowden et al 2007a ) . in a case study series involving eight patients with a total of 10 vlus ( mean duration of 9.3 years ) , amelogenin ( xelma , molnlycke health care , gothenburg , sweden ) was applied weekly and covered with a secondary dressing . treatment duration with amelogenin was 12 weeks and patients were followed up for a period of 24 weeks . an overall healing rate of 50% was reported , including one patient with a wound duration of 65 years . the other ulcers were in a healing state at 24 weeks , with a mean reduction in size of 60% compared with baseline values . exudate levels and wound odor also reduced over the treatment period ( hampton et al 2007 ) . amelogenin has recently been evaluated in a study consisting of a mixed population of patients with both diabetic foot ulcers ( n = 5 ) and vlus ( n = 5 ) . both sets of patients demonstrated initiation of healing with a high percentage ( 80% ) of ulcers healing completely ( meuleneire 2007 ) . diabetic foot ulcers have a different aetiology to that of vlus , they are a significant complication of diabetes mellitus and often precede lower - extremity amputation . the most frequent underlying aetiologies are neuropathy , trauma , deformity , high plantar pressures , and peripheral arterial disease . the primary treatment is usually offloading to reduce pressure to the sensitive areas and then management of the wound with appropriate dressings . infection and high levels of wound exudate may be complicating factors in the treatment of these wounds , but the early indications are that amelogenin therapy , in conjunction with appropriate wound management techniques , can be beneficial . pyoderma gangrenosum ( pg ) is an inflammatory ulcerative condition of unknown etiology , although an autoimmune mechanism including immune complex - mediated neutrophilic vascular reactions has been suggested . pg is frequently associated with various diseases , but up to 50% of cases are idiopathic . it is a disease that causes tissue to become necrotic , causing deep ulcers that usually occur on the legs . there are two main types of ulcers that occur : the normal ulcerative form which occurs on the legs , and an atypical form that is more superficial and occurs on the hands and other parts of the body . though the etiology is not well understood , the disease is thought to be due to immune system dysfunction and , in particular , improper functioning of neutrophils . at least half of all pg patients also suffer from illnesses that affect their systemic function . classical ulcerative pg is characterized by the appearance of nodules with pustules that enlarge and lead to chronic ulcers violaceous and undermined borders ( dini et al 2007 ) amelogenin therapy has been used to treat two female patients with recalcitrant pg of the lower leg lasting an average of 11 months ( dini et al 2007a ) . the treatment was applied weekly under an occlusive dressing for a maximum of 8 weeks in conjunction with systemic immunosuppressive therapy before and during the topical treatment . the lesions improved , showing advances in granulation tissue formation and wound size reduction ( figures 3a - c ) . additionally , pain control was reported and the therapy was well - tolerated with no adverse effects . the clinical evidence suggests that in order for advanced therapies like amelogenin to be cost effective in for example the treatment of vlus , then suitable patients must be carefully identified and according to prognostic indicators of delayed healing such as wound size ( larger than 10 cm ) and wound age ( duration of greater than 6 months ) . additionally , if wounds do not show a decrease in size of 40% or more during 4 weeks of compression therapy , then these wounds can be identified as hard - to - heal ( phillips et al 2000 ) and consideration should be given to their treatment with advanced therapies such as amelogenin . this is because it treats the underlying cause of venous hypertension and enables blood to be returned from the lower limbs , thereby reducing oedema and the consequences of stasis . infection in the wound must be treated prior to application of amelogenin , although it has been seen that concurrent treatment of infection with antibacterial therapy is not detrimental to the amelogenin protein as a therapeutic agent . additionally , wounds that exudate highly do not appear to benefit as well from amelogenin therapy as wounds that have low to moderate levels of exudate , implicit in this therefore is the requirement that exudate should be managed with compression and appropriate dressings that can absorb the fluid . this tissue impedes the growth of healthy granulation tissue and re - epithelisation and therefore must be removed . it is also thought that the presence of such dead tissue may prevent the amelogenin protein integrating into the fabric of the wound bed and thus providing the temporary scaffolding to which cells can attach and subsequently stimulate the healing process . qualitatively , vlus treated with amelogenin appear to heal much better than with high compression alone , with no evidence of hypertrophic scarring or excessive wound contracture . there is a raft of experimental evidence that supports a mechanism for amelogenin in facilitating the wound healing process . for example , it appears that amelogenin provides a temporary scaffold to allow cellular adherence of fibroblasts and epithelial cells . this in turn will stimulate various functions of cells with regards to the wound healing process , eg , migration ( into the wound ) , proliferation ( increasing the number of cells and filling the wound ) , and synthesis of mediators ( growth factors and cytokines ) that can regulate processes that are relevant to wound healing such as angiogenesis . this review highlights that a number of well controlled trials have established that amelogenin treatment has a statistically beneficial effect in the clinical environment . it has been shown that amelogenin can significantly reduce ulcer size and also significantly increase the number of patients showing a greater than 50% reduction in wound size , compared with comparator controls . pain is a problem in patients with vlus , but it has been shown that amelogenin therapy can reduce the level of pain experienced by patients with hard - to - heal vlus , compared with comparator controls . it is interesting to conjecture whether this is simply due to healing of the wound or whether amelogenin has a direct remedial effect upon pain , future studies may elucidate this mechanism .

adiponectin is a 30-kda ( acrp30 ) protein predominantly produced in the adipose tissue and circulates in the plasma as a trimer ( low molecular weight ) , a hexamer generated from two trimers , or as multimers consisting of 1218 hexamers ( high molecular weight ; hmw ) . levels of adiponectin are decreased in obesity , coronary artery disease , and type 2 diabetes mellitus . in our prior studies , adiponectin levels were found to be inversely correlated with low - grade albuminuria in obese african - american subjects . a similar relationship was found in patients with hypertension from europe and in patients from japan . interestingly , this relationship was primarily found in subjects with elevated body mass index and with low - grade albuminuria . a large study with 440 subjects recently found the same inverse relationship between adiponectin levels and albuminuria in subjects with obesity , even after adjusting for other risk factors . however , in patients with established diabetes or chronic kidney disease ( ckd ) , adiponectin is positively correlated with albuminuria or proteinuria . it remains controversial whether serum adiponectin levels predict future cardiovascular risk factors in ckd subjects . a potential role for adiponectin to act on podocytes has been identified based on expression studies of the receptors for adiponectin . adiponectin receptor 1 and adiponectin receptor 2 are the two major receptors for adiponectin and are described as a new class of heptahelix receptors structurally and functionally distinct from g - protein - coupled receptors . adiponectin receptor 1 gene expression is expressed in the mouse kidney and podocytes to a similar degree as in liver , whereas adiponectin receptor 2 gene expression of kidney and podocyte is much less than liver . protein studies are difficult to interpret as the commercially available antibodies may not be specific and sensitive . presently , based on the published epidemiologic studies there is convincing evidence that in subjects who are obese ( without diabetes or ckd ) there is a relationship between circulating adiponectin levels and low - grade albuminuria , however , the role for adiponectin to mechanistically contribute to albuminuria will be difficult to establish in human studies . a cause and effect role for adiponectin in the development of kidney disease has been supported by several independent studies using different mouse models of manipulating adiponectin . studies in one strain of adiponectin knockout ( ko ) mice by our group identified that the ko mice have elevated levels of albuminuria but only two- to threefold greater than controls on a c57bl6 background . with addition of hyperglycemia , there was a progressive increase in albuminuria in the ko mice , but not in the wild - type diabetic mice . treatment with exogenous adiponectin was found to attenuate albuminuria and restore podocyte foot process effacement . in a similar vein , 5/6 nephrectomy was found to lead to an accelerated disease in a different adiponectin ko mouse . in a recent study , mice with a genetically engineered inducible podocyte injury thus , the available studies are in agreement that , in models of glomerular injury adiponectin has a protective role . however , , adiponectin - deficient mice were protected from ischemia - reperfusion injury , whereas an independent study found the reverse , i.e. , that adiponectin deficiency exacerbated acute kidney injury after ischemia reperfusion . the role of the specific adiponectin receptors in chronic or acute kidney injury remains to be established . the major signaling pathways by which adiponectin appears to confer its effects is via stimulation of ampk , akt , rab5 , and phospholipase c. ampk is a stress - activated kinase that is activated in response to depleting atp or a relative increase in the intracellular amp / atp ratio to preserve cell survival under a low - caloric environment . ampk was established to have a central role in the effects of adiponectin based on studies in the adiponectin receptor ko mice . as obesity is associated with a reduction in adiponectin and an excess of calories will lead to a reduced amp / atp ratio , it would follow that ampk would be reduced with obesity - related kidney disease . we first reported that renal ampk was reduced in a mouse model of high - fat - induced obesity ( diet - induced obesity ) within 1 week of the onset of the high - fat diet . surprisingly , there was evidence of renal inflammation ( elevated urine hydrogen peroxide , urine , and glomerular monocyte chemoattractant protein-1 induction ) by 1 week of the high - fat diet , and the inflammation preceded any increase in albuminuria . stimulation of ampk by 5-aminoimidazole-4-carbox - amide-1-beta - d - ribofuranoside ( aicar ) was able to completely suppress the inflammatory markers and reduced mesangial cell production of monocyte chemoattractant protein-1 in response to palmitate . more recently , chronic stimulation of ampk by aicar ( for 12 weeks ) was also successful to reduce renal inflammation , albuminuria , and matrix accumulation with the high - fat diet ( decleves , kidney int 2014 ; 85 : 611623 ) . furthermore , ampk activation was able to completely reduce lipid vacuolization in proximal tubular cells as well . part of the basis for the latter finding may be due to reduction of hmgcoa reductase activity with ampk activation and reduced cholesterol production . the overall numbers of macrophages infiltrating the kidney with a high - fat diet was completely normalized with ampk activation . furthermore , ampk activation lowered the cd11c / cd11b ratio indicating a reduction in m1 macrophages ( decleves , kidney int 2014 ; 85 : 611623 ) . a role for ampk in regulating macrophage activation has been highlighted recently and will be an active area of research in future studies . ampk also appears to have a key role in regulating the nadph oxidase ( nox ) system . of the major nox isoforms that have been identified , it appears that nox1 , 2 , and 4 may have a role in mediating the oxidative stress involved in ckd . we have previously identified that nox4 was prominent in podocytes and that high glucose - induced upregulation of nox4 can be blocked with adiponectin or activation of ampk . in separate studies , ampk was found to inhibit nox2 subunits via upregulating ib and blocking nf-b - induced stimulation of nox subunits ( p67 , p47 ) in endothelial cells . a role for ampk regulation of nox4 was demonstrated in diabetic kidney disease by several groups and there is a growing consensus that nox4 may be the most critical nox linked to progression of diabetic kidney disease . our group found that mice with nox2 deficiency have the same degree of hyperglycemia and weight loss with streptozotocin - induced diabetes , however , the degree of diabetic kidney disease was not affected in the nox2 ko diabetic group . the degree of albuminuria , glomerular matrix expansion , and urine hydrogen peroxide was essentially the same in the wild - type and nox2 ko diabetic groups . as there was a marked increase in nox4 in the nox2 ko diabetic kidney , it is possible that nox4 may compensate for nox2 and be sufficient to promote diabetic kidney disease . in other studies , inhibition of nox1/nox4 was found to be protective with liver disease and cardiac disease . these studies have taken on added importance , as new phase ii studies are underway to evaluate the role of nox inhibition for diabetic kidney disease . in addition to inflammation , ampk has also been closely linked to fibrosis promoting pathways . in the high - fat diet model , chronic ampk activation with aicar was able to reduce mesangial matrix expansion and reduce urinary levels of tgf-1 ( decleves , kidney int 2014 ; 85 : 611623 ) . recently , we found that ampk activation also markedly reduced glomerular tgf- , collagen , and fibronectin accumulation in several mouse models of diabetic kidney disease ( dugan , j clin invest 2013 ; 123 : 48884899 ) . a prior study found that adiponectin and ampk reduced tgf--induced matrix and myofibroblast transformation , however , smad2/3 phosphorylation was not affected . recently , we found that a key transcription factor usf1 was translocated to the nucleus with high glucose exposure and completely blocked by ampk activation . as usf1 has been found to mediate glucose - induced stimulation of the tgf-1 gene transcription , there could be an important effect of ampk to regulate usf1-induced tgf-1 synthesis . in addition to downstream effects by ampk to regulate nox and tgf- , there is a well - established pathway by which ampk inhibits mtor activity . however , deletion of mtor in podocytes also contributes to disease , and treatment with rapamycin has been found to enhance proteinuria in some patients , thus limiting its utility as a therapeutic for diabetic kidney disease . a recent study found that mtor inhibition also led to reduced nox4 levels in podocytes , suggesting that mtor may have a direct effect to regulate nox4 independent of ampk . a key pathway by which ampk stimulation protects cells in a calorie - deprived state is to stimulate the master regulator of mitochondrial biogenesis , pgc-1. this transcriptional co - activator is a potent stimulator of many mitochondrial proteins and increases mitochondrial content . in states of reduced ampk activation indeed , pgc-1 was found to be markedly reduced in the muscle of patients with diabetes and may be due partly to epigenetic modification of the pgc-1 promoter . recently , we found that the diabetic kidney also had reduced pgc-1 levels in association with reduced ampk , reduced mitochondrial content , and reduced mitochondrial complex activity ( dugan , j clin invest 2013 ; 123 : 48884899 ) . this led to the question that , if there is reduced mitochondrial complex activity in the electron transport chain would there be a concomitant change in mitochondrial superoxide production ? indeed , we found that there was reduced superoxide production in the diabetic kidney using a real - time imaging protocol and further verified by ex vivo studies with electron paramagnetic resonance measurements . thus , we found that the diabetic kidney is actually in a state of reduced mitochondrial activity and reduced mitochondrial superoxide production . this is in direct contrast to the prevailing notion that diabetic complications are due to an excess of mitochondrial superoxide ! we further sought to ask this question in patients with established diabetic kidney disease . to get an index of mitochondrial activity , we performed quantitative measurements of a variety of metabolites linked to various biochemical pathways linked to human disorders ( sharma , j am soc nephrol 2013 ; 24 : 19011912 ) . the predominant signature that was identified was a reduction of metabolites produced by mitochondrial enzymes . semi - quantitative analysis of mitochondrial complex iv revealed reduction in kidney biopsies from patients with diabetic nephropathy . furthermore , there was a reduction of gene expression for pgc-1 in diabetic kidney tissues , but not in minimal change disease . these set of studies help to establish a new paradigm for understanding diabetic kidney disease . an early and progressive reduction in mitochondrial content , potentially driven by reduced ampk / pgc-1 , is linked to early renal inflammation and pro - fibrotic pathways ( see figure 1 ) . chronic exposure of cells to caloric excess , from excess glucose and/or high fat , is linked to reduction of ampk , possibly due to transient or sustained reduction in the amp / atp ratio . persistent reduction of ampk activity allows for stimulation of inflammatory pathways mediated by nfb and pro - fibrotic pathways mediated by usf1 . downstream of the transcription factors , nox and tgf- are stimulated and directly contributing to inflammation and fibrosis in the kidney and heart . pathways that mitigate ampk reduction are the adiponectin - lkb1 pathway as well as direct and indirect activators of ampk , including aicar , metformin , weight loss , and exercise . on the basis of several animal studies , stimulation of ampk stimulation of ampk via pharmacologic and non - pharmacologic interventions may well be beneficial in human kidney disease as well . of note , non - pharmacologic means of increasing ampk has been identified by exercise and food restriction , and exercise has been shown to reduce diabetic kidney disease independently of weight loss and glucose lowering . in conclusion , recent studies in the past 23 years on the basis of inflammation and fibrosis via the ampk pathway has led to new insights and paradigms in our understanding of diabetic kidney disease . additional investigation to understand the mechanistic underpinnings as to how reduced mitochondrial function is linked to inflammation and fibrosis will likely be an exciting and rewarding path to identify new biomarkers and therapeutics for obesity - related and diabetic ckd .

afflictions of shoulder by tuberculosis is rare and when it occurs its more commonly a dry lesion ( caries sicca ) . wet lesions in shoulder are rare and we report this case for the rarity of its occurrence . a 55yrs old female patient presented with a painful swelling with restriction of movements of the right shoulder since six months . patient had taken various treatments without any relief ; there was no history of trauma , weight loss , recent infection in the past or any history of tuberculosis in family or contact with tubercular patient . antero - posterior and axial x - rays of the right shoulder showed no bony involvement however , ultrasonography showed lipoma . serological investigations showed a markedly raised erythrocyte sedimentation rate ( 73 mm / hr ) and a positive c - reactive protein . dna pcr was positive for tuberculosis and patient was started on anti - tubercular treatment(category i ) for six months . any patient coming with the complaints of long standing painful restriction of the movements of the shoulder associated with or without complaints swelling , shall be evaluated to rule out skeletal tuberculosis along with other differential diagnosis of periarthritis of shoulder and adhesive capsulitis . most of the patients with skeletal tuberculosis may not necessarily present with the constitutional symptoms of fever , weight loss , etc and also because of the widespread prevalence of the organism in india . out of all the skeletal tuberculosis , 50% accounts for the spinal tuberculosis and rest 50% accounts for the tuberculosis of the joints ( sacroiliac joint , hip joint , knee joint , shoulder joint , elbow joint , wrist joint ) tuberculosis of the small bones(metatarsus , metacarpus and the phalanges ) , tuberculosis of the tendon sheaths and bursae and the tuberculous osteomyelitis . it varies from an incidence rate of 0.9 to 1.7% . out of the total extra pulmonary tuberculosis . the variants of extra pulmonary or skeletal tuberculosis are the classical dry type / atrophic type ( caries sicca ) 2 or the fulminating or caseating type of shoulder tuberculosis associated with cold abscesses or sinus formation . the atrophic type is further observed into 4 various types depending on the affections , i.e. type i , the caries sicca the atrophic form , type ii the caries exudate with swelling and cold abscess formation and type iii the caries mobile with good range of passive movements . a 55yrs old female patient who presented with a painful swelling with restriction of movements of the right shoulder since six months . patient had taken various treatments without any relief ; there was no history of trauma , weight loss , recent infection in the past or any history of tuberculosis in family or contact with tubercular patient . right shoulder revealed restricted movements ; no local rise in temperature , tenderness was present over anterior and posterior aspect of the right shoulder diffusely . external rotation and abduction movements antero - posterior and axial x - rays of the right shoulder showed no bony involvement however , ultrasonography showed lipoma . serological investigations showed a markedly raised erythrocyte sedimentation rate ( 73 mm of fall in 1 hr ) and a positive c - reactive protein . after pre - anaesthetic evaluation the patient was posted for excision of the mass for which supine position was given with the bolster under the right shoulder [ fig.1 ] , deltopectoral approach was taken[fig . the fluid released along with the rice bodies was also collected and were sent for histopathological examination which showed bursitis with loose boodies and dna pcr ( positive ) testing respectively , following which the patient was given a shoulder immobilizer and was started empirically on anti - tubercular treatment(category i ) for six months . patient in supine position with bolster und er the right shoulder joint deltopectoral approach taken with the internervous plain between the deltoid and pectoralis major soft tissue dissection with capsular release of the swelling rice bodies seen on further dissection of the swelling mycobacterium tuberculosis is responsible for almost all the cases of osteo - articular tuberculosis in india . atypical mycobacteria , other than m. tuberculosis fiumanis or bovis have also been reported in bony lesions . certain precipating factors responsible for transmission of atypical mycobacteria are trauma , local steroidal injection , iatrogenic , diabetes mellitus , poor nutrition , poor hygienic conditions , use of immuno- suppressive drugs , acquired immuno - deficiency syndrome . the gold standards for the diagnosis of osseous tuberculosis are culture of mycobacterium tuberculosis from bone tissue , positive ziehl - neelsen staining and positive dna pcr ( as in this case ) . treatment includes standard antituberculosis drugs for six months or category - i under revised national tuberculosis control programme as per as world health organisation guideline for management of tuberculosis . though swelling associated with restriction of range of movements and pain is a rare clinical presentation of form of tuberculosis of the shoulder but can not be ruled out completely without proper further evaluation of the condition with the help of serological as well as radiological means available . a clear knowledge of the mechanism of injury coupled with a thorough clinical examination can help us in the diagnosis of such rare injuries at the earliest .

congenital epulis ( also known as congenital gingival granular cell tumour ) is a rare benign congenital growth of the newborn . it was first described in 1871 by neumann , hence the alternative name is newmanns ' tumour . it usually presents at birth with an obvious mass arising from the gingival mucosa of the maxilla or mandible . the size of the mass varies from a few millimetres to 9 cm in diameter . the recommended treatment is surgical excision under local or general anaesthetic , although spontaneous regression has been reported . there are no reports of recurrence , even if incomplete margins are excised , malignant change , or future disruption to teeth or gums . an otherwise healthy 1-day - old girl was referred to a large teaching hospital in cardiff for diagnosis and treatment of two large masses protruding from her mouth . the baby had normal antenatal scans at weeks 12 and 20 , and pregnancy had been unremarkable , other than mother being group b streptococcus positive from a high vaginal swab . mother was fit and well gravida 2 para 1 , with no drug history or family history of note . baby was born at term plus eight days weighing 3.85 kg , pink and breathing spontaneously ( apgar : 9 - 10 ) . on examination there were two fleshy , pedunculated masses arising from the upper and lower alveolar ridges measuring 4 3 3 cm just to the right of the midline . she was booked for excision of these masses under general anaesthesia ( figure 1 ) . both masses were removed with an eliptical insion to the peduncles ( figure 2 ) . the child was breastfeeding the day after surgery , and discharged home the following day . the two masses were fixed and examined histologically ( figures 3 and 4 ) . they showed sheets and clusters of cells containing abundant granular eosinophlic cytoplasm and small uninform nuclei , along with some myxoid areas and areas of haemorrhage and ulceration , confirming the diagnosis of congenital epulis . it commonly presents in the neonate , although prenatal diagnosis with ultrasound has been reported as early as 26 weeks gestation . the lesion usually arises over the incisor - canine region of the maxilla ( maxillary / mandibular ratio 3:1 ) . simultaneous involvement of both maxillary and mandibular alveolar ridges occurs in approximately 10% of reported cases . the diagnosis is usually made on clinical grounds alone , although difficulties may arise when the size of the lesion is small , or the index of suspicion is low . mri is useful for diagnosis , and superior to ultrasound , showing the gingival origin of congenital epulis without local extension . epulis is a greek term literally meaning of the gums and is used to describe a wide variety of gum lesions , regardless of their pathological origin . histologically , congenial epulis shows remarkable similarity with the more common granular cell tumours ( gcts ) [ 2 , 11 ] . there are , however , many distinguishing features , such as occurrence solely in the neonate , typical location , plexiform arrangement of capillaries , and lack of pseudoepitheliomatous hyperplasia . gcts are ubiquitous neoplasms occurring in all age groups , very rarely affecting the gingiva , and can occasionally show malignant change . immunohistochemical studies have revealed further differences , demonstrating the reactivity of gcts to s-100 protein and laminin , and their absence in congenital epulis . vered et al . have also recently expanded the immunophenotypic distinction between the two , showing gcts stain positive for ngfr / p75 and inhibin- , whereas congenital epulis does not . cgts are considered to arise from schwann cells , and hence show strong reactivity to s-100 protein . various theories of the origin of congenital epulis include myoblastic , neurogenic , odontogenic , fibroblastic , and histocytic . it has been suggested that the occurrence of congenital epulis solely in neonates , and more commonly in females , implies a hormonal mechanism of development . however , numerous reports have shown no evidence of either oestrogen or progesterone receptors , and as such suggest an alternative histogenesis [ 11 , 14 ] . in a review of 33 lesions , conclude that the immunohistochemical profile does not imply any specific cell types for the histogenetic origin of congenital epulis . no estimation of incidence of congenital epulis has been made to date , to the best of our knowledge . one centre in the usa saw only two cases over the period of 21 years . in university hospital of wales , a tertiary referral centre for otolaryngology and neonatology , this is the only recorded case of congenital epulis since 1980 , a total of 28 years . using incidence of live births ( 157,454 ) within that time period , we calculate an incidence of 0.0006% ( upper 95% confidence interval : 0.0035% , the inverse of the cumulative beta distribution ) . although most likely an underestimate , this calculation will serve as an approximation of incidence before a more thorough estimation can be undertaken .

ten to twenty percent of pancreatic cysts are neoplastic , while only 1% of pancreatic cysts are malignant . serous and mucinous cystic neoplasms are two types of tumors of the exocrine pancreas , and have different biological behaviors . mucinous cystic tumors are typically slow - growing , but carry a significant potential for malignancy , and thus resection is often indicated . in contrast , serous cystadenomas are considered benign tumors with almost no malignant potential . in 1989 , george et al . published the first well - documented serous cystadenocarcinoma and proposed it as a new entity . we report herein a patient with a pancreatic serous cystadenocarcinoma that was initially considered a tumor originating from the colon . she had dizziness and hematochezia for several months , and sought evaluation at the emergency room . an abdomen - pelvic computed tomography ( ct ) was obtained and routine laboratory testing was performed . the abdomen - pelvic ct showed a 12 9 cm mass adjacent to the stomach that invaded the spleen and pancreas . in addition , there was a tract formation between the mass and transverse colon ( fig . 1 ) . the origin of the mass was not determined , but the transverse colon was suspected . several small nodules in the liver were thought to represent metastases , so a liver magnetic resonance imaging ( mri ) was obtained . on the liver mri , the patient underwent segmental resection of the colon , a distal pancreatectomy , and a splenectomy . the gross examination revealed a large hemorrhagic mass of the pancreatic tail , with adhesions to the parenchyma of the spleen and all layers of the colon . microscopically , the tumor consisted of numerous microcysts of various sizes with clear cytoplasm ( fig . the histopathologic findings were compatible with serous cystadenocarcinoma of the pancreas , showing invasive growth into the spleen and colon . there are two categories of pancreatic cystic neoplams ( serous and mucinous cystic pancreatic tumors ) . mucinous cystic neoplasms of the pancreas have a relatively high potential for malignancy , thus surgical removal is the standard treatment . the prevalence of cancer among serous cystic tumors has been reported to be 3% since 1989 . however , serous cystic neoplasms , despite their low prevalence and benign histologic features , have malignant potential and can fulfill all criteria of cancer , including local infiltrative growth and formation of distant metastasis . the pre - operative differentiation between a benign serous cystadenoma and a malignant serous cystadenocarcinoma is difficult . indeed , the correct diagnosis of serous cystadenocarcinoma was not made pre - operatively in any of the cases , including the current case . the benign and malignant variants appear identical histologically , with the only distinguishing feature being gross or microscopic evidence of invasiveness . thus , the utility of cytology or histology obtained from a core needle biopsy is limited . the pre - operative differentiation of benign from malignant cystic lesions of the pancreas using radiologic examinations is not reliable , except for cases with metastatic disease . it is difficult to diagnose as a malignant tumor at the time of primary surgery in the absence of invasion to adjacent organs or metastases . in some patients , other organ metastases have been reported to occur several years after the primary procedure . in general , the tumors are very slow - growing and patients have a long life expectancy from the time of diagnosis . the opinion that serous cystic neoplasms of the pancreas are always benign may have contributed to the recommendation that resection should be performed only when there is evidence of local invasiveness , distant metastasis , or related symptoms . even when metastatic lesions are present , surgical resection of primary tumors and metastatic lesions is the most effective treatment . serous cystadenocarcinomas of the pancreas invade adjacent organs , such as the spleen , regional lymph nodes , and duodenum . in several cases , the tumor spreads to distant organs like the liver , lung , and bone marrow . in the current case , post - operatively , a serous cystadenocarcinoma was diagnosed without evidence of distant metastasis , but with extensive tumor invasion into surrounding structures ; it was unusual that there was fistula formation to the transverse colon .

the consideration of institution of end - of - life care is an emotionally charged issue for the patient , their families and caregivers . decisions are influenced by the beliefs of the caregivers , patients and their families . given the heterogeneity of many societies including our own , it is worthwhile to examine the differences in approaches to palliative care and end - of - life issues in different cultures and religions . even within individual countries , examples of the differences in attitudes on the part of both the caregivers and patients will be presented . a literature search was performed on palliative care , culture , and several religions ( christianity , judaism , islam , hindu , buddhism ) . articles were selected by the author that seemed representative of mainstream thought within those cultures and religions for presentation in this manuscript . cohen and colleagues report on end - of - life attitudes in latino and cambodian patients living in the united states in the boston area . they evaluated the effect of religion , sense of destiny , quality of life , and process preferences regarding end - of - life decision - making . the majority of both groups believed that the inevitability of dying made discussion of advanced directives a moot point . as evidence of this , only 15% of the patients had ever discussed advanced directives with their physician . both the cambodian and latino patients expressed that if quality of life was poor then they saw no point in continuing medical care that would likely only prolong the inevitable . however , when religion was considered there was a significant dichotomy between the two groups . the strong belief in the latino group was that removing a patient from life support was tantamount to killing them . the cambodian cohort separated religion from end - of - life decision - making and did not perceive a connection between the two . there were also subtle differences in the two groups as to the involvement of family in end - of - life decisions . the cambodian group seemed to delegate primarily to their children and spouse , while the latino patients indicated that a more extended family structure was frequently involved and needed to reach consensus on these sorts of decisions . cohen and her co - authors concluded that two main themes had emerged from their focus group study of latino and cambodian patients . integration of belief systems and process preferences were both important and that , specifically , emphasis on quality - of - life , the role of destiny and understanding the role of family in end - of - life discussions were extremely important . ball and colleagues described the results of a large multi - country and multi - cultural survey on end - of - life care in trauma patients . a survey was sent to trauma surgeons , intensivists , bioethicists , and rehabilitation experts in the united states , canada , south africa , europe , asia , and australia / new zealand . the respondents were a diverse group in relation to country of practice , religion , and cultural background . there were significant differences in practice pattern , with most american respondents classifying themselves as both trauma surgeons and intensivists . physicians from other regions were more apt to classify themselves as either a trauma surgeon or intensivist , which probably represents a difference in practice in other areas of the world where the intensivist is more likely not to be a surgeon . most of the physicians indicated that they believed that their views of end - of - life matters were similar to their local colleagues as well as the patients they cared for . the need to transfer a patient to another physician 's care because of a conflict over end - of - life issues was quite rare . except in the united states where the admitting physician was most likely to direct end - of - life discussions with the family , the intensivist was most likely to be the main caregiver involved in those discussions . the respondents indicated that very few were subject to formal medical futility laws to direct their practice . when used , the majority of physicians found that they were either typically or occasionally helpful . physicians in south africa and asia believed that resource limitations affected end - of - life decision - making whereas respondents from other regions rarely thought that was the case . it also seems that one 's perception of god may correlate with patients coping mechanisms in end - of - life situations . van laarhoven and his colleagues surveyed dutch patients with cancer who had entered the palliative care period as to their image of god and their coping strategies . a non - personal image of god may include such beliefs as god is unknowable or something higher , both of which imply the god surpasses all of our powers of imagination . comparatively , a personal image of god is one in which the individual views god as interacting with individuals . the authors found that patients who had a more non - personal image of god were more likely to be subject to denial and were more likely to use the coping mechanisms of seeking advice and information from others , and seeking moral support while they were less likely to rely on a sense of humor . those patients who had a personal image of god were most likely to use religion as a coping mechanism . those who thought that god was unknowable were not likely to use any of the coping mechanisms . the issues become even more complex when specific religions are considered , both from the perspective of the patient and the caregivers . clearly , in judaism , suicide , assisted - suicide , and euthanasia are not permitted . however , the jewish religion recognizes that life is of limited duration and that in end - of - life situations , treatments to provide comfort are permitted even if they have some risk of shortening life . there is no requirement for a jewish patient to accept any treatment not viewed as curative . pain and suffering should be minimized even if there is some risk of shortening life . similarly , babgi describes in great detail the organization of islamic society and beliefs in saudi arabia as they relate to health care and end - of - life issues . in saudi arabia , its constitution and legal system is based on sharia , the system of islamic law . interestingly , do not resuscitate ( dnr ) orders are allowable , but only under certain very proscribed conditions . the family is not consulted as they are viewed as unqualified to make such decisions . living wills and advanced directives are not recognized in islamic law , as it is believed that only allah can make decisions on life and death . bradley presents an essay on roman catholic doctrine as it pertains to end - of - life matters . the catholic church makes a distinction between ordinary and extraordinary treatment and care . as an example , the provision of nutrition and hydration to patients in persistent vegetative states is considered ordinary care and therefore must be administered . euthanasia is not permitted and catholic doctrine addresses euthanasia by omission and clearly states that this is prohibited . treatments are viewed as disproportionate or extraordinary when they either do not offer a reasonable hope of benefit or are excessively burdensome to patient or community . pain and suffering are viewed as part of karma , which is the unfolding of events based on a person 's current and previous lives . put succinctly , pain and suffering are viewed as the state the individual is supposed to be in . the two hindu concepts that are most operational for patients with either acute or chronic pain are acceptance and detachment . acceptance is the accepting of suffering as a natural consequence of karma along with the realization that suffering is temporary and not solely negative . detachment from the world , with its pain and suffering , in order to concentrate on god , is the ultimate goal . in summary , culture and religion at least partially affect one 's perception of palliative care and the decision - making that occurs at end - of - life . it is important to realize that there is an entire system at play that includes the patient , their family , their physicians , and other healthcare providers . in order to provide the best possible care to patients and families in end - of - life situations , it is important to understand their cultural constructs as well as their individual preferences . it is also important for each caregiver to realize that we each bring our own set of biases to these discussions based on our cultural and religious background and personal experiences .

various studies conducted on different photo anthropometry , but each one included some deficiencies and during the years , which were resolved . the anthropometry studies can be divided into three categories : ( 1 ) manual anthropometry , ( 2 ) two - dimensional ( 2d ) photography and ( 3 ) three - dimensional ( 3d ) photography . a review in literature shows that these methods have adequate precision and of course each one has some advantages and disadvantages . the examiner should possess adequate skill . during the measurement some errors may occur , due to pressure of measuring tools on soft - tissues . now - a - days , most of the anthropometry studies are carried out by imaging and computer software analysis and we know modern advanced methods , such as 3d scanning are very expensive . the science of image processing has resulted special attractions to anthropometry and has expended its applications in various areas , specially forensics , anthropology , clothing industry , designing work space , designing manual tools , etc . in medical science , the first accurate anthropometry was done on a human body and dipping the limbs was used to determine the volume of the body , which may not give correct values due to changes after death . the anthropometry of the face is also used in medical sciences , dental sciences , face cosmetic surgeries and hence forth and also for determining the face characteristics like checking the patient condition before the cosmetic surgeries . since the measurement from photo is much simpler some spots may be covered and remain hidden by the skin and adipose tissue that need to be touched . these places can be located before photography . despite the high costs , a significant difference between the 3d scanning techniques and manual methods compared the results of traditional and 3d scanning and found that the values round the body have the most difference , which has increased with the increase of body mass index . meunier and yin simultaneously used two cameras and 2d image processing software and measured six different dimensions of the body including around the neck , around the chest , around the buttock , around the loin , height and length of the sleeve . they came to this conclusion that linear measurements like height are more accurate than peripheral measurements and this method can replace with overall traditional methods . from the standpoint of biomechanics , there is a direct relationship between musculoskeletal injuries and occupational risk factors and working with unsuitable hand tools can exacerbate these symptoms . in a study performed on colombian floriculture workers , showed that the hand size in the present study population appears to be significantly different from those of other populations hand size . and there is not much consistency between their hand size and their hand tools ; therefore , this matter can lead to serious injuries to hand . thus , with respect to the importance of ergonomics of tools , existence of an anthropometry database is essential in every society and this data should be up - to - date . as the size of some body parts may alter during years . hence , developing simple and quicker anthropometry methods would encourage researchers to gather this data . according to the survey , most of the iranian anthropometric studies have been manual . and considering that the design tools should be based on the physical dimensions of user , consequently , providing bank of iranian population is essential . however , due to the difficulty of manual methods so far scattered information is provided in the context of iranian population . unfortunately , most of the industrial designers are forced to use anthropometric data of european or american countries ; it ll cause the maladjustment between the device and the user . hence , the purpose of this study is to compare the accuracy of 2d image processing software with the direct method and present its results to ergonomic specialist , designers and producers . people with bodybuilding background , deformations and swollen hands were excluded from the research . a total of 76% of participants the other demographic information is summarized in table 1 . in this study , 14 dimensions of the hand were measured with both manual method digital caliper and 2d photo anthropometry method as described in figure 1 . the measured dimensions of the hand were selected based on national aeronautics and space administration recommendation . demographic characteristic of participants measured dimensions of the hand digital caliper ( model : mitutoyo corp . digital caliper resolution was 0.01 mm and its precision was 0.01 mm . in anthropometric method , four pictures were taken from people 's hand by a sony dsc - w35 , 7.2 mega pixels digital camera as shown in figure 1 . in order to maintain the distance between the hand and camera , photo anthropometry set an on - screen ruler was also used as stage which their hands could be placed on it . this set was designed to measure also the other dimensions of hand such as internal and external diameter of grip for future . digimizer [ figure 3 ] is a very flexible and simple software package , which is very useful for analyzing the images . this software was compatible with windows xp and vista and it was based on counting the pixels , per unit of length and values entered into the right table . then , after opening the image in the software , using the ruler next to hand , the number of pixels / unit of length was defined and then by drawing a line between the desired points , we obtained the distance between them . the table with the sizes was compatible to microsoft office excel . in this way , the error while entering the numbers into analyses software like excel or spss was resolved and a lot of time saved . the t - test shows [ table 2 ] no significant difference between the two manual and photo anthropometry methods ( p > 0.05 ) . for example , the average of length of hand in photography method is 19.68 2.08 cm and in manual method it is 19.56 2.23 cm ; is not a significant difference using t - test statistical exam ( p = 0.085 ) . descriptive indicators of dimensions in two manual and photo anthropometry methods correlation coefficients between hand dimensions in the two methods are the same shown in the range of 0.71 - 0.95 [ table 3 ] . the correlation between hand dimensions ( l1 , p1 ) and palm dimensions ( l2 , p2 ) is shown in a scatter plot [ figure 4 ] . level of correlation between measured dimensions in the two methods the scatter plot between dimensions of length of hand ( l1 , p1 ) and dimensions of palm ( l2 , p2 ) its application for hand anthropometry was used in this research , for the very first time . the findings imply that the average of hand dimensions , has no significant difference in the two methods ( p > 0.05 ) , in other words , the dimensions of the hand are the same in each method . in this study , designing , we tried to fix the angle and distance of the camera in all images . manual anthropometry is a simple , low cost , time consuming method and needs the cooperation of the individual who is being tested and at the end , it provides a list consisting numbers for us . however , this method may not be feasible to perform for some patients in medical centers . designed an anthropometry computer system and the results of their study demonstrated that some peripheral dimensions like around the neck has a significant difference in manual method , but linear dimensions like height of arm had a high degree of accuracy . they found out that the reasons for this difference were because of missing some key spots in the image and also the weak contrast between the individual 's clothes and their background . also used software to determine the angles and proportions of patient 's face before and after face surgeries . as the basis of this software is the proportion between the two parts , thus he could not determine the height of an individual part or the distance between two parts ( e.g. eyes ) . although , this study showed that the length of a particular member is easy to determine . in another survey , the measurement of foot 's dimensions is studied in two methods : digital photography and manual photography . the results showed that the measured size of the foot is the same in both ways . in a similar research , some of the body 's linear dimensions were measured by an aluminum frame as scale and also with computer software . the important point in photo anthropometric is to evaluate the appropriate scale . in the study which was done by das and kozey , two colored ropes were used as scale in body 's length and width and made a meaningful error in our measurement . however in this study , number of pixels in length unit was used as scale . in anthropometry method , it was observed that because it was less time consuming and also because there was nt any body contact with measuring tools , people were more willing to cooperate . in this method , a photo archive was developed that could be reviewed in the future . among the capabilities of this software , ability to edit images , ability to set the contrast and brightness , ability to change the background image , ability to change images to grayscale mode , ability to define the unit of measurement even in nanoscale , measure angles , determine the center of segment , reduce image noises and exporting excel files for quicker statistical analyses , can be named . the dimensions under study in this research were linear and in comparison to manual methods had adequate accuracy . and this study can be a start of reviewing the accuracy of this software in measuring peripheral dimensions of the body , such as around the wrist , chest and so forth . with respect to the widespread use of different kinds of safety gloves , this study can be used for determine the percentiles of the hands for efficient sizing of safety gloves . this study can also be a preface for more investigations for the development of an anthropometry database . furthermore , since the hand anthropometry is a necessary input for tool design , this survey can be used for determining the percentiles of workers hands . ehsanollah habibi received a b.sc . in industrial safety , central missouri state university in 1984 and he received m.sc in occupational health and safety , from central missouri stateuniversity , in 1986 . in 1992 he obtained the degree of ph.d . in occupational health and safety , from bradford university , england and post doctoral in occupational ergonomic , 1992 . school of health , isfahan university of medical sciences , and the m.sc in occupational health , school of health , isfahan university of medical sciences , isfahan , iran . akbar hasan zadeh received a b.sc statistics from tehran university , in 1985 and m.sc tehran university 1990 .

fusarium head blight ( fhb ) is a recurrent disease of wheat , barley , and other small grains across the world , also including northern europe , mainly caused by the fungal plant pathogens fusarium graminearum and fusarium culmorum ( bottalico and perrone 2002 ) . fhb results not only in the premature bleaching of the spikes giving rise to white or pink kernels and lowered grain yields but also in the accumulation of mycotoxins ( van der fels - klerx et al . f. graminearum and f. culmorum are also the causal species for the formation of deoxynivalenol ( don ) and its acetylated derivatives , as well as other trichothecenes also including the estrogenic mycotoxin zearalenone ( zen ) ( rodrigues and naehrer 2012 ; tiemann and dnicke 2007 ) . the fhb disease cycle starts with the germination of overwintering chlamydospores or mycelia in soil or crop residues , giving rise to the primary inoculum in the spring . the ascospores and/or conidia are then released from the perithecia and are spread by wind or splashing water . when fungal spores land on developing spikes at the time of flowering particularly during moist and warm weather conditions , spore germination and infection of the plant may occur . the considered monocyclic nature of fhb is thought to limit the infection to the primary inoculum released during spring ( wegulo 2012 ) . however , despite substantial knowledge about the pathogenicity of this plant disease , the present intervention strategies seem to have limited effects on disease mitigation and subsequent accumulation of mycotoxins ( van der fels - klerx et al . 2012 ) . don accumulation in cereals were reported in spindles , glumes , stems , and leaves of the plant , raising the question of systemic fungal growth following fhb infection being responsible for the mycotoxin accumulation ( brinkmeyer et al . 2006 ; cowger and arellano 2013 ) . several field and experimental studies have shown a positive correlation between don levels in wheat kernels and the amount of f. graminearum dna ( wegulo 2012 ) . other studies have shown no colonization of f. graminearum or f. culmorum in wheat heads despite high don levels following inoculation of seeds or crowns . recently , moretti et al . ( 2014 ) in growth chamber experiments showed that f. graminearum , inoculated in soil or seeds , can grow systemically in the plant with the exception of kernels and heads . high levels of don and f. graminearum dna were found in crowns , stems , and straw in contrast to low levels of don and no fungal dna in the heads and kernels . ( 2005 ) , following ear infection of spring wheat with f. graminearum , showed the don content of kernels to be less than detected in rachis and straw . another factor which may influence the distribution of don in plant tissues several studies have demonstrated translocation of the toxin in the sieve tubes via the xylem or phloem ( kang and buchenauer 1999 ; snijders 2004 ) . in the last years , different fusarium species infected common cereals particularly in the western part of sweden and resulted in serious challenges for the cereal and pig industry because of reduced yield and mycotoxin accumulation ( fredlund et al . don - producing mold species such as f. graminearum and f. culmorum were shown to be present , and don was frequently reported in oats and other cereals . in a recent survey , we sampled cereals and straw at swedish pig farms and analyzed for don , zen , t-2 , and ht-2 toxin ( nordkvist and haggblom 2014 ) . the data revealed that don was almost ubiquitous with 89 % of the samples being contaminated , while zen was detected in 54 % of the samples where oats were the cereal grain most frequently contaminated . higher don and zen concentrations were detected in straw compared to grain harvested in 2011 and 2012 . the results clearly indicated straw to be a significant source of don and zen in addition to cereals , however , with large variations between farms . trichothecenes have a multitude of effects on eukaryotic cells where the most important seems to be inhibition of protein synthesis ( pinton et al . also , don was shown to inhibit the absorption of nutrients by human epithelial cells in vitro clinical symptoms of trichothecene exposure in animals include feed refusal and weight loss , hemorrhage , emesis , and necrosis of different tissues ( mostrom and raisbeck 2007 ) . pigs seem to be the most sensitive animal species to both don and zen exposure from the feed ( dmello et al . the intake of wheat straw was on average 13 % of the diet ( van barneveld 2003 ) , while it was estimated that gestating sows , which are fed restrictively , most likely consume larger amounts . in a recent study , the bioavailability of don from wheat straw and chaff was investigated in pigs ( rohweder et al . , the results clearly indicated that the bioavailability was not affected significantly by feeding straw or a grain matrix . clearly , the intake of don and zen from straw may significantly contribute to the mycotoxin exposure in pigs and there will be an increased risk of an exposure exceeding acceptable levels when both the grain and straw are contaminated . the complexity of fusarium mycotoxin exposure in man and animals is further complicated by the fact that plants are able to modify the mycotoxins into masked mycotoxins , i.e. , not extracted by conventional extraction solvents used in the analyses ( berthiller et al . 2005 ) . the possible hydrolysis of masked mycotoxins , which may be present in high amounts during mammalian digestion , raises concern that the parent toxin may be released and absorbed in the intestines and thus contributes to the exposure . in a recent report , a nearly complete hydrolysis of deoxynivalenol-3--d - glucoside in the intestinal tract of pigs was observed ( nagl et al . for animal welfare reasons , straw is commonly used as bedding material in animal production and access to straw in the pig production is laid down in the swedish animal welfare regulation ( sjvfs 2010:15 ) where attention is being paid to the amount as well as the hygienic quality of the bedding material . because of the documented don and zen contamination of straw , there is a need for practical sampling methods that can be used at pig farms in order to select straw which present no mycotoxin harm to the animals . the fact that sampling is generally conceived as a step of the analytical chain which gives the largest contribution to the measurement uncertainty ( reiter et al . the sampling uncertainty may be even more pronounced for forages and straw than for grains because of irregular shape , density , and the different anatomical fractions of the plant as opposed to grain being more uniform . in sampling of forages , two strategies to obtain a large number of incremental samples were reported , namely manual grab sampling ( grimsbo jewett et al . 2001 ) and core sampling by drilling into the bales ( schaeffer et al . the aims of the present study were to ( a ) study the occurrence of don and zen and the presence of f. graminearum and f. culmorum in straw bales from wheat , barley , and oats at different pig farms and ( b ) develop a practical sampling method that could be used at the farm to prevent the use of bedding material with elevated mycotoxin levels that may affect pig health . the 2013 growing season was characterized by less rainfall and higher temperatures compared to both 2011 and 2012 which coincided with a smaller number of reports indicating elevated don levels from the pre - harvest surveillance in cereals organized by the swedish grain industry . for that reason , the identification of possible fields suitable for the straw sampling study was based on previous surveillance data for don from 2011 to 2012 . suitable fields were localized by collecting 30 to 40 heads by hand from 17 fields in july or august 1 to 2 weeks prior to harvest , respectively . samples of wheat ( spring and winter varieties ) , triticale , barley , and oats from the southern ( county of skne ) and western ( county of vrmland ) regions of sweden were collected . the grains were recovered by manual threshing and dried at room temperature , ground in a shearing mill ( laboratory mill 3610 , perten instruments , sweden ) , and analyzed for don following the procedure by the supplier of the lateral flow device ridaquick don ( r - biopharm ag , darmstadt ) within 2 to 3 days . fields where don was detected in kernels prior to harvest in the county of vrmland were selected for the straw sampling . the fields were harvested by a combine harvester , and straw was subsequently pressed in round or square big bales within 23 days according to the normal practice of the farmer . sampling was carried out in late august to mid - september of bales ( approximately 150350 kg / bale ) from fields of winter wheat ( 45 round bales ) , barley ( 27 square bales ) , and oats ( 18 round bales ) within 2 days after baling , respectively . the round bales were 1.2 m wide with a diameter of 1.21.4 m , and the square bales were 0.8 0.9 2.5 m. in addition , 7 round bales of oat straw , from one field in the same region , were sampled inside a shelter after 2 weeks of storage . the straw samples were collected using a hay probe bale sampler sampling probe , 18-mm diameter 550-mm length ( best harvest , largo , fl , usa ) , powered by an 18-v cordless electric drill . the sampling of round bales was carried out by drilling twice radially 550 mm into the bale , and the straw was mixed to 15- to 30-g dry matter samples . accordingly , square bales were sampled by drilling once from each short side of the bale . the water content of the samples was estimated after drying at 60 c overnight ( 16 h ) in a ventilated drying cupboard . all samples were ground on a hammer mill to pass a 1-mm screen before analysis of mycotoxins . the analyses of don and zen were carried out by elisa ( ridascreen don and zearalenone , respectively , r - biopharm ag , darmstadt ) . limits of detection ( lod ) and limits of quantification ( loq ) were 37 and 110 g / kg for don and 3.5 and 10.5 g / kg for zen , respectively . mycotoxin concentrations exceeding the range of the calibration curve ( don 500 g / kg , zen 280 g / kg ) were diluted accordingly . a subset of seven samples of wheat straw , five samples of barley straw , and eight samples of oat straw were selected to cover the range of don and zen concentrations and were subject to specific dna analysis for quantification of f. graminearum and f. culmorum . before analyses dna was extracted and quantified in 0.2 g straw , in parallel as previously described , using the taqman exogenous internal positive control ( applied biosystems , ca , usa ) ( fredlund et al . the internal amplification control was amplified to the same level as the negative control , showing that the dna extract did not contain inhibitory substances influencing the quantification . the lowest dna concentration with linear amplification was 9 pg / mg straw for f. graminearum and 15 pg / mg straw for f. culmorum . for regression analysis of the dna data versus the contents of don and zen , respectively , all data was log ( 10)-transformed . the three zen concentrations below the quantification limit were treated as upper level , i.e. , 3.5 g / kg . the different data sets were evaluated by descriptive statistics ( mean , median , min , and max values ) ; distribution was studied graphically by the use of histograms . the relationship between contents of don and dna from f. graminearum was modelled by linear regression after log ( 10 ) transformation . a study of sampling frequency was made on data from the wheat field ( about 8 ha ) . average estimates of don content were calculated by random sampling of subsets of 2 to 45 ( all ) bales from the data set . the procedure was repeated 20 times ; mean values and standard errors of the mean ( sem ) were calculated and evaluated graphically . results from the screening study revealed don concentrations of kernels below the detection limit of the test kit < 500 g / kg in the county of skne and 6001800 g / kg in wheat , oats , and barley from the county of vrmland . the water content of the collected wheat straw samples was between 37 and 64 % ; don was detected in all samples ( fig . 1a ) with an average don concentration of 976 g / kg and a median of 525 g / kg , while in four bales , the concentrations were above 3000 g / kg . for zen , eight samples were below the detection limit ; however , the concentrations were more uniform compared to corresponding don data with an average concentration of 11 g / kg and a median of 13 g / kg ( fig . 1 a deoxynivalenol ( don ) content ( g / kg ) in individual straw bales from one field ( 8 ha ) of winter wheat . average content is shown as a dashed line and median value is represented by a dotted line . b zearalenone ( zen ) content ( g / kg ) in individual straw bales from one field ( 8 ha ) of winter wheat . average content is shown as a dashed line and the median value is represented by a dotted line a deoxynivalenol ( don ) content ( g / kg ) in individual straw bales from one field ( 8 ha ) of winter wheat . average content is shown as a dashed line and median value is represented by a dotted line . b zearalenone ( zen ) content ( g / kg ) in individual straw bales from one field ( 8 ha ) of winter wheat . average content is shown as a dashed line and the median value is represented by a dotted line the samples from the barley straw bales contained 1833 % water and were all positive for don with an average concentration of 449 g / kg , and three bales contained levels above 800 g / kg ( fig . zen was detected in three bales with one sample above 100 g / kg ( fig . 2b).fig . 2 a deoxynivalenol ( don ) content ( g / kg ) in individual straw bales from one field ( 8 ha ) of barley . average content is shown as a dashed line and the median value is represented by a dotted line . b zearalenone ( zen ) content ( g / kg ) in individual straw bales from one field ( 8 ha ) of barley . average content is shown as a dashed line and the median value is represented by a dotted line a deoxynivalenol ( don ) content ( g / kg ) in individual straw bales from one field ( 8 ha ) of barley . average content is shown as a dashed line and the median value is represented by a dotted line . b zearalenone ( zen ) content ( g / kg ) in individual straw bales from one field ( 8 ha ) of barley . average content is shown as a dashed line and the median value is represented by a dotted line in the oat straw samples , the water content was between 17 and 23 % and the average don concentration was 6719 g / kg ( median 6841 g / kg ) , with the lowest concentration at 2614 g / kg and eight samples above 8000 g / kg ( fig . zen contamination was detected in all bales with an average concentration of 53 g / kg with the highest concentration at 219 g / kg ( fig . the water content was 1314 % and 5 out of 7 bales were above 15,000 g don / kg with an average concentration of 16,382 g / kg ( data not shown ) . zen concentrations in the oat bales were on average 153 g / kg with a maximum of 284 g / kg and a minimum at 69 g / kg ( data not shown ) . in fig . 4 it can be seen that sampling only 2 bales at random from the field results in a very high sampling uncertainty . sampling 5 bales 3 a deoxynivalenol ( don ) content ( g / kg ) in individual straw bales from one field ( 5 ha ) of oats . average content is shown as a dashed line and the median value is represented by a dotted line . b zearalenone ( zen ) content ( g / kg ) in individual straw bales from one field ( 5 ha ) of oats . average content is shown as a dashed line and the median value is represented by a dotted line fig . population grand average and median are plotted at respective don levels ( see fig . 1a ) a deoxynivalenol ( don ) content ( g / kg ) in individual straw bales from one field ( 5 ha ) of oats . average content is shown as a dashed line and the median value is represented by a dotted line . b zearalenone ( zen ) content ( g / kg ) in individual straw bales from one field ( 5 ha ) of oats . average content is shown as a dashed line and the median value is represented by a dotted line sampling frequency related to number of sampled bales . population grand average and median are plotted at respective don levels ( see fig . 1a ) f. graminearum dna was present at levels above loq in all investigated samples of wheat straw except one , while f. culmorum dna was detected at levels above loq in two samples . levels of f. graminearum dna were higher in oat straw ( max 6444 pg dna / mg straw ) compared to wheat and barley . the analyses of don / zen levels and f. graminearum dna levels showed that the mycotoxin levels were significantly correlated with f. graminearum dna ( fig . 5regression of don ( filled circles ) and zen levels ( filled triangles ) against levels of dna from f. graminearum in straw from winter wheat , barley , and oats ( n = 20 ) regression of don ( filled circles ) and zen levels ( filled triangles ) against levels of dna from f. graminearum in straw from winter wheat , barley , and oats ( n = 20 ) the results of the present field study support previous information that don and zen may be present in straw ( nordkvist and haggblom 2014 ; brinkmeyer et al . 2006 ; dnicke et al . 2006 ) even at high levels despite good agricultural practice during the growing and harvest seasons . interestingly , none of the pig farmers taking part in the study had made observations of fhb symptoms in their crops and the harvested straw were judged suitable for bedding material . the water content of the oat straw samples was the lowest followed by barley and winter wheat straw . because the sampling was carried out only a few days after harvest , except for the stored oat straw , it suggests that the measured straw concentrations of mycotoxins were most likely present at the time of harvest . in experimental studies , 2011 ) observed decreased don levels and increased zen levels when inoculated straw was stored outdoors for longer periods . the uneven distribution of mycotoxins in the straw bales from some fields indicated large variations in fungal infections within the field . in winter wheat , four bales contained elevated don levels ( > 4000 g / kg ) compared to most bales . interestingly , those bales were from a slightly lower part of the field possibly with a higher moisture level in the soil . for zen , no obvious correlation with the don data could be seen , and in about 10 % of the samples , the levels were below loq . in barley , don levels were more uniform in the bales in contrast to zen where three samples were above loq and one sample above 100 g / kg . in contrast to bales from other cereals , the don and zen levels in oats were higher and also the distribution of mycotoxins in oats seemed to be more uniform . again , the levels of mycotoxins could vary a lot between individual bales with no obvious correlation between don and zen levels , making the sampling rather difficult . results from the present field study did not reveal which part of the straw that may harbor mycotoxins at the time of harvest . in experimental studies where winter wheat was inoculated with spores of f. culmorum , the mean don and zen concentrations were , however , shown to be significantly higher in glumes and spindles compared to the straw ( brinkmeyer et al . further studies should reveal if glumes and spike tissues are the main sources of don and zen in the straw also under field conditions . several studies have shown that the amount of don produced by f. graminearum in grain is positively correlated with fungal biomass ( demeke et al . 2010 ) . other field studies have also shown a positive linear relationship between don concentrations and the fhb intensity as reviewed in wegulo ( 2012 ) . results of specific f. graminearum dna showed extensive fungal growth in straw from all tested cereals with the highest concentrations in oats , suggesting a correlation between the amounts of fungal hyphae in the plant and don levels . when comparing the results in the present study with the data from fredlund et al . ( 2013 ) , considerably higher levels of f. graminearum - specific dna were detected in straw compared to the kernels . in the present study , the range of f. graminearum dna concentrations in oat straw was between 661 and 6444 pg / mg compared to the range in oat kernels of 474 and 917 pg / mg in 2010 and 2011 , respectively . the results clearly suggest a massive fungal colonization in other segments of the plant but the kernel . the results also demonstrate that at the time of harvest , fusarium - infected straw and chaff represent a great risk for spreading f. graminearum inoculum to the soil until the next growing season . whether the demonstrated straw colonization by f. graminearum was the result of the mold being present in the soil or the seeds or infections during flowering is difficult to find out because of diverging reports ( ludewig et al . the farmers participating in the study were all using seeds produced on the farm which may have influenced the infection . in the present study , four fields were studied to give a rationale for a recommended procedure to be used in practical straw sampling and commercially available equipment designed for sampling of forages was used . sampling of individual bales on the respective fields made it possible to get information of the in - field variation of the don and zen contamination as the degree of heterogeneity has a great impact on the sampling plan . the use of drilled core sampling has two main advantages , a core sample collected from a cross section of a big bale represents a large proportion of the bale and , secondly , the sample is cut into short pieces that allows for immediate grinding without further preparation of the sample . in the review by miraglia et al . ( 2005 ) , it was pointed out that following the sampling variance , the sample preparation variance ( resulting from subsampling and grinding of the collected ( aggregate ) sample ) was the most significant contributor to the total analytical variation . thus , grinding the total sampled material will reduce the analytical variation . in the wheat field , 45 bales were sampled and the distribution was very heterogeneous , as illustrated in fig . half of the bales had a don content less than 525 g / kg , while the average content ( 976 g / kg ) was strongly influenced by four highly contaminated bales . ( 2005 ) concluded that sampling variance increases with lowering of the toxin concentration which is in agreement with the findings in the present work where mean don level was lower in wheat and barley straw compared to oat straw . for wheat and barley straw , the mean and median values of don differed greatly with the mean greater than median , reflecting a strong deviation from the normal distribution . it also implies that the average may be of limited use for describing the degree of contamination of individual bales and thus for risk assessment of animal exposure to don . the more highly contaminated oat straw was less heterogeneous , but still the don content varied fourfold between the lowest and highest concentrations within one field . a calculation of sampling uncertainty was made for the wheat field . in fig . 4 , the estimates of don content based on different numbers of sampled bales are shown , where it can be seen that , for this field , sampling 5 bales will result in acceptable sampling uncertainty . however , an estimated mean value of about 1000 don g / kg does not imply the absence of highly contaminated individual bales . also , the zen content of straw varied a lot even if mean and median values were close within the 45 bales of wheat ; the distribution was far from normal with < loq ( 3.5 g / kg ) as mode . in the barley field , the contamination was found to be the lowest where the toxin was distributed in three out of 27 bales , giving an average contamination of 5 g / kg while the median remained at < loq . additional exposure to don and zen from straw , when used as bedding material , can not be excluded in pig production particularly as the bioavailability of don for pigs was shown to be similar to the availability from kernels ( rohweder et al . the significance of mycotoxin exposure from straw at pig farms with cereal production should for those reasons not be neglected particularly in years when high levels of don and zen are also detected in the feed grain . the present study has demonstrated that estimating the concentrations of don and zen in straw requires a careful sampling plan . however , with a limited number of samples , preferably using a sampling probe , it is possible to distinguish lots of straw that should not be used as bedding material for pigs .

it is widely accepted that reactivation of hepatitis b virus ( hbv ) may occur along with an acute hepatitis after chemotherapy or immunosuppressive therapy.1 - 3 current recommendation is to give antiviral agents before starting these therapies.4 - 7 however , most of the reactivation cases are associated with high doses of immunosuppressive agents including steroids or chemotherapeutic agents . although there are some case reports of acute flare - up of hbv after high dose steroid therapy , there have been very few reports of acute severe flare - up after long - term administration of low dose steroid in an inactive hbv carrier . since a vast number of patients are at some point in need of low dose steroid administration , including most rheumatologic diseases such as rheumatoid arthritis ( ra ) , this will be of important concerns in those sectors which low dose steroid is an essential part of prescription . we report a case of previously inactive 86-year - old hbv carrier with an acute severe flare - up during a long term very low dose steroid treatment due to ra , which despite decreased viral activity after prompt initiation of antiviral therapy against hbv , ultimately led to progressive liver failure and death . in september 2009 , a 86-year - old korean woman visited the out - patient department complaining of recent development of both lower extremity and facial edema . poor oral intake was evident for nearly a month , and she appeared chronically ill . she was underweight with a body mass index of 18.2 ( height 151 cm and weight 41 kg ) . she was previously diagnosed as having ra in november 2006 . from the time of diagnosis to september 2009 she made regular visits to the out - patient department of rheumatology and received several treatments consisting of different disease modifying antirheumatic drugs ( dmards ) such as sulfasalazine and hydroxychloroquine , in addition to very low dose oral steroids ( 2.5 mg / day of prednisone ) and cox-2 inhibitor . at that time her serum was positive for hbsag and hbeab with undetectable hbv dna ( < 86 iu / ml ) but negative for hbsab , hbeag , and hcv ab . serum aspartate aminotransferase ( ast ) and alanine aminotransferase ( alt ) remained persistently normal . all other laboratory tests were unremarkable except a mild degree of anemia ( hemoglobin 11.2 g / dl ) . the follow - up test for liver function obtained at december 2008 was completely within normal limit , with a platelet count of 25510/mm . at september 2009 , markedly increased hbv dna titer ( > 170,000,000 iu / ml ) and abnormal results of liver function tests such as serum alt 44 , ast 76 u / l , total bilirubin 2.0 mg / dl , total protein 5.3 , albumin 2.5 g / dl , and prothrombin time ( pt ) ( 16.7 sec , 58% , inr 1.52 ) were first detected . serum creatinine level was 0.9 mg / dl , while routine urinalysis revealed no signs of proteinuria or albuminuria . about two weeks later , she was admitted to the department of gastroenterology for further work - up and management . pitting edema of both lower extremities were evident on physical examination , with mildly distended abdomen . there were no evidences of hepatitis a and c virus , epstein - barr virus , cytomegalovirus , or herpes simplex virus infection . abdominal ultrasonography revealed a mildly coarse parenchymal echogenicity and moderate amount of ascites , with no evidence of liver cirrhosis . since her serum and ascites albumin levels at the time of ascites tapping were 2.2 and 0.3 g / dl respectively , serum ascites albumin gradient ( saag ) was 1.9 , higher than 1.1 , indicating that the ascites was transudate . thus , acute flare - up of hepatitis b caused by hbv reactivation was diagnosed . immediate antiviral therapy with clevudine at a daily dose of 30 mg was initiated along with hepatotonics and diuretics . despite prompt administration of antiviral agent and some virological response toward the reactivated hbv evidenced by a great decrease in hbv dna titer ( 64,500 iu / ml ) at one month iu / l ) , increased total ( 5.9 mg / dl ) and direct ( 3.3 mg / dl ) bilirubin , decreased total protein ( 4.6 g / dl ) and albumin ( 2.5 g / dl ) , decreased cholesterol ( 49 mg / dl ) and prolonged pt ( 24.8 sec , 34% , inr 2.66 ) , aggravated anemia ( hgb 9.0 g / dl ) and first signs of thrombocytopenia ( 10810/mm ) , indicating the progression of liver failure . 2 ) and multi - organ failure eventually developed , and she died after 50 days since administration of antiviral agent . hbv infection is by far the most common chronic viral infection affecting the liver in the world , with over 400 million subjects infected , and it is the leading cause of cirrhosis and hepatocellular carcinoma.8 reactivation of hbv replication in patients undergoing immunosuppressive therapy is a well recognized and frequently reported complication of considerable clinical importance.1 - 3,9 - 10 not surprisingly , most of these reports have come from the fields of oncology and transplantation , but there have been a growing number of cases reported in patients with rheumatic disease undergoing immunosuppressive therapy as well.11 - 13 among the factors that have been shown to precipitate acute flares of chronic hbv infection is the administration of immunosuppressive drugs . in vitro and in vivo studies clearly indicated that immunosuppression leads to increased hbv replication , assessed by different methods ( serum hbv dna , hbsag , and hbv dna polymerase titers ) . this enhanced replication is attributed to a number of mechanisms , one of which is related to a direct stimulatory effect of these agents on hbv replication . this is particularly the case for corticosteroids , because a corticosteroid responsive element is present in hbv genome and is responsible for increased hbv dna transcriptional activity and viremia in patients receiving corticosteroids.14 glucocorticoid is implicated as an important predisposing factor for hbv reactivation . in patients with chronic hepatitis b , long - term prednisolone treatment increases levels of hbsag , hbcag , and hbv dna in hepatocytes,15 as well as decreases t - cell function . in addition , glucocorticoids may cause a direct reactivation of the latent hbv infection.16 the findings of a glucocorticoid responsive element in hbv genome and the demonstration that glucocorticoids specifically activate hbv gene expression in cultured human hepatocellular carcinoma cells lend support to the direct reactivation theory.16 despite these findings , limited information is available on the effect of the different immunosuppressive regimens given for longer periods of time in lower doses in patients with chronic hbv infection . data from the long term administration of immunosuppressive drugs in hbsag positive renal transplant recipients have shown a high incidence of hepatitis and liver related mortality ( 10 - 30%).17 there is a paucity of information for other diseases where long term administration of immunosuppression is required - for example , patients with inflammatory bowel diseases,18 severe asthma , and various different rheumatic diseases including rheumatoid arthritis.11 - 13 although there have been some case reports of acute flare - up of hbv after high dose steroid therapy , only a few patients with rheumatic disease and hbv reactivation associated with both non - biological and biological immunosuppressive therapies have been reported . in addition , all of the previously reported hbv reactivation cases with low dose steroid administration have coadministered methotrexate in some degree ( varying from 4 mg / week to 15 mg / week ) , with no reported case of low dose steroid administration alone . it is also worthy to note that the steroid dose administered in the present case was possibly the lowest dose applicable ( 2.5 mg / day ) . to the best of our knowledge , there has been no previous report of hepatitis b virus reactivation with acute severe flare - up in an inactive hepatitis b virus carrier , especially while taking continuous low dose oral steroid as long as 4-years for maintenance treatment of rheumatoid arthritis . the clinical spectrum of the hbv reactivation can vary from an asymptomatic to acute flare - up and fulminant course . hbv reactivation leading to a fulminant course is a serious condition and results in death or transplantation in most cases . in this situation , the goals of aggressive antiviral therapy prevent the need for liver transplantation , decrease post - transplantation hbv recurrence , and sustain viral suppression and viral activity reduction.19 the patient was not a candidate for transplantation , and we immediately decided to initiate antiviral treatment . although the patient underwent prompt antiviral treatment with clevudine , and despite some response toward the viral activity , the liver function gradually failed , ultimately leading to multi - organ failure and death . the limitation of this case could be that spontaneous reactivation of hbv can not be wholly excluded . moreover , since the patient was visiting only the department of rheumatology at a regular base , the viral markers and hbv dna which represents the activity of the hbv were not recorded as often as it should have been . however , since the incidence of such spontaneous reactivation of hbv is very rare in an old - aged patient such as in this case , and the fact that viral load of the patient surged dramatically , it suggests that the etiology of hbv reactivation may be long - term steroid administration . it is also true to say that the possibility of inactive cirrhosis can not be fully excluded , in which steroid administration might have caused a fatal hbv reactivation . our case suggests that even an administration of non - combination , minimal dosage of single oral steroid can cause reactivation of indolent , inactive hbv . since a vast number of patients are at some point in need of low - dose steroid administration , including most rheumatologic diseases such as rheumatoid arthritis , this will be of important concerns in the sectors where low dose steroid is an essential part of prescription . in conclusion , in an inactive hbv carrier , if a long - term administration of steroid is planned , an adequate evaluation of the liver function and disease status should be evaluated by specialists . once steroid has been administered , regular hbv dna monitoring is mandatory , and early antiviral therapy is required if the serum hbv dna level begins to rise , which may lead to serious liver damage .

origin of t. cruzi isolates - the 77 t. cruzi isolates used in this study were deposited and have been maintained cryopreserved at trypanosoma from wild and domestic mammals and vectors . more information concerning the landscape physiognomy of pda , ra and una may be found in the studies by lisboa et al . 1:collection sites of wild free - ranging leontopithecus chrysomelas from una biological reserve ( una ) [ municipality of una , state of bahia ( ba ) ] and leontopithecus rosalia from poo das antas biological reserve ( pda ) [ municipality of silva jardim , state of rio de janeiro ( rj ) ] and reintroduction areas ( ra ) [ environmental protection area ( apa ) of bacia do rio so joo ( rj ) ] in the atlantic coastal rainforest of brazil . t. cruzi infection patterns in small mammals ( rodents and marsupials ) examined in the golden lion tamarin living areas ( ra ) - the santa helena farm is one of 21 privately owned forest fragments that comprise the ra . these areas consist mainly of pasture , plantation and small forest fragments where wild tamarins are born and living ( beck et al . small wild mammals ( rodents and marsupials ) were noninjuriously captured using live traps ( sherman , hb sherman traps , usa ; tomahawk , tomahawk live traps , usa ) baited with a mixture of banana , peanut butter , oat and bacon / sardines . traps were set for five consecutive nights along linear transects placed on the ground at 10-m intervals and alternating between trap type in three field expeditions ( july 2003 , january 2004 and august 2004 ) . the trapped animals were taken to the field laboratory and , for each captured species , morphological identification , reproductive condition and age estimation ( body weight for rodents and degree of tooth eruption for marsupials ) were recorded . total capture effort was 1,000 traps - nights , equally distributed among the three expeditions . we calculated the relative abundance of small mammals as the number of individuals of each species divided by the total number of individuals multiplied by 100 . parasitological tests were based on examination of fresh blood smears ( microscopic analysis ) and hc , the latter performed as follows : 0.3 ml of blood from each animal was cultured in 2 tubes containing novy - mcneal - nicole medium with liver infusion tryptose overlay . tubes were examined every 15 days up to five months . for the detection of anti - t . cruzi igg antibodies in sera , we used the indirect fluorescent antibody test ( ifat ) as previously described by camargo ( 1966 ) . rodent sera were tested with anti - rat igg fluorescein isothiocyanate ( fitc ) ( sigma - aldrich , usa ) . didelphis were tested with specific intermediary anti - didelphis antibodies raised in rabbits to visualise the reaction with an anti - rabbit igg conjugate ( fitc ) . the cut - off values adopted were 1:40 for marsupials and 1:10 for rodents , as described previously ( herrera et al . prevalence of infection ( hc and ifat ) was calculated based on the total of infected individuals divided by the total individuals sampled . ethics - permission for the study and a license were obtained from the brazilian government environmental agency , brazilian institute of environment and renewable natural resources ( biodiversity authorization and information system license 098/2002 ) and the study was endorsed by the ethical committee of experimental animal research committee / oswaldo cruz foundation ( ceua l-015/04 ) in accordance with brazilian regulations . appropriate biosecurity techniques and individual protection equipment were used during all procedures involving sylvatic animals sample collecting and handling . evaluation of the data obtained during 11 years follow - up of t. cruzi infection in golden and golden - headed lion tamarins ( 1995 - 2005 ) - the data on t. cruzi infection were obtained from golden and golden - headed lion tamarins from three fragments : pda , ra and una . the following parameters were tested : ( i ) annual tamarin population infection rates - total number of examined tamarins per year and number of animals testing positive for t. cruzi by ifat and hc , respectively , ( ii ) serological conversion - total number of tamarin testing positive according to ifat each year and number of re - examined animals with negative ifat , and ( iii ) prevalence of t. cruzi infection within each social group - total number of infected animals ( ifat and hc ) in each group in relation to the total number of primates examined per group . molecular characterised of 77 t. cruzi isolates - total genomic dna was prepared from logarithmic phase cultures using standard phenol - chloroform protocols ( sambrook et al . characterisation was carried out in three steps : ( i ) multiplex pcr amplification of the mini - exon gene following conditions described by fernandes et al . ( 2001 ) for the identification of three dtu t. cruzi groups : { tc1 [ tci - 200 base pairs ( bp ) ] , tc2 ( tcii / tcv / tcvi - 250 bp ) and zymodeme 3 ( tciii/ tciv - 150 bp ) } in addition to trypanosoma rangeli ( 100 bp ) or mixed infections were used ; ( ii ) ribosomal rna ( rrna ) gene analysis - pcr amplification of the d7 divergent domain of the 24s rrna gene ( lsu rdna ) was amplified as described by brisse et al . amplicons of 110-bp are characteristic of tci / tciii dtu , 125-bp of tcii / tcvi , 120 or 130-bp of tciv and both 110 and 125 bp for tcv ; ( iii ) pcr - rflp analysis of the three target loci and restriction enzyme combinations : 1f8 gene ( 1f8/alw21i ) , hsp60/ecorv and gpi / hhai genes was accessed using the methodology described by rozas et al . each reaction included a negative control and positive control samples from those t. cruzi strains representing the dtus to be typed : 1f8 - tci / ii , hsp60 - tci / iii / iv and gpi - tci / iii . this long lasting study of t. cruzi infection in free - ranging tamarins in the atlantic rainforest showed that : ( i ) the enzootical picture of the transmission cycle of t. cruzi in a given forest fragment may modify over time , ( ii ) the distinct enzootical scenarios with the same t. cruzi dtutcii were observed in single but proximate forest fragments of the atlantic rainforest and ( iii ) the high parasitaemia by t. cruzi as expressed by positive hcs occurred in waves of approximately two years ( pda ) . t. cruzi infection pattern in rodents and marsupial examined in golden lion tamarin living areas ( ra ) - the diversity of rodent and marsupial species was low since only five species , akodon sp . , didelphis aurita , nectomys squamipes , oryzomys sp . and t. cruzi infection detected by ifat was observed in n. squamipes ( 15/18 - 83% ) , d. aurita ( 17/47 - 36% ) and akodon sp . ( 3/20 - 15% ) , but the parasite was isolated only from two out of 17 big - eared opossums ( d. aurita ) that also exhibited positive serological ifat ( table i ) . no patent parasitaemia was observed in fresh blood smears ( microscopic analysis ) . table iprevalence of infection by trypanosoma cruzi in rodents and marsupials of the santa helena farm ( reintroduction area ) in the atlantic coastal rainforest of state of rio de janeiro , brazilorder of mammalspeciespositive ifat/ examined animals n / n ( % ) didelphimorphia didelphis aurita 17/47 ( 36)2/17 ( 12)rodentia - cricetidae akodon sp.3/20 ( 15)0/3rodentia - cricetidae nectomys squamipes 15/18 ( 83)0/15rodentia - cricetidae oryzomys sp.0/20/0rodentia - echimydae trynomys sp.0/10/0 total - 35/88 ( 40)2/35 ( 6)hc : haemoculture ; ifat : indirect fluorescent antibody test . t. cruzi infection follow - up of golden and golden - headed lion tamarins between 1995 - 2005 - both tamarin species demonstrated their ability to maintain a stable and well - established t. cruzi ( dtu tcii ) transmission cycle in two geographical regions of the atlantic coastal rainforest , based on their the high parasitological and serological t. cruzi infection prevalence . during the 11-year - long study , we also observed a high serum conversion rate in golden and golden - headed lion tamarins as indicated in table ii . as far as we know , this is the most stable and expressive transmission cycle of t. cruzi ii ever observed in a wild environment . moreover , herein we are demonstrating that in addition to tcii , nine tamarins maintained long - lasting infections by dtu tci , as confirmed with recaptured and retested primates . table iifollow - up of trypanosoma cruzi infection in the free - ranging populations of leontopithecus rosalia ( golden lion tamarin ) and leontopithecus chrysomelas ( golden - headed lion tamarin ) from southeast atlantic coastal rainforest of brazil ( 1995 - 2005)speciesstudies areaspositive ifat/ examined tamarins n / n ( % ) serological conversion/ tamarins tested n / n ( % ) leontopithecus rosalia pda140/249 ( 56)95/140 ( 68)35/107 ( 33)ra 8/54 ( 11)8/8 ( 100)1/6 ( 17 ) leontopithecus chrysomelas una58/76 ( 76)39/58 ( 65)19/34 ( 56 ) a : includes 21 privately owned farms ; hc : haemoculture ; ifat : indirect fluorescent antibody test ; pda : poo das antas biological reserve [ municipality of silva jardim , state of rio de janeiro ( rj ) ] ; ra : reintroduction areas [ municipalities of rio bonito , silva jardim and casimiro de abreu ( rj ) ] ; una : una biological reserve ( municipality of una , state of bahia ) . a : includes 21 privately owned farms ; hc : haemoculture ; ifat : indirect fluorescent antibody test ; pda : poo das antas biological reserve [ municipality of silva jardim , state of rio de janeiro ( rj ) ] ; ra : reintroduction areas [ municipalities of rio bonito , silva jardim and casimiro de abreu ( rj ) ] ; una : una biological reserve ( municipality of una , state of bahia ) . the prevalence of positivity for the parasitological and serological tests with the golden lion tamarin populations exhibited fluctuation with a peak prevalence every three years ( fig . 2:graphical representation of the peaks in the annual prevalence of infection according to indirect fluorescent antibody test ( ifat ) and haemoculture ( hc ) in wild free - ranging populations of golden lion tamarin ( leontopithecus rosalia ) from poo das antas biological reserve , municipality of silva jardim , state of rio de janeiro , brazil . thus , in two ra ( santa helena farm and rio vermelho farm ) where the t. cruzi infection of the animals was always subpatent and only detectable by serology , eight animals displayed positive blood cultures , ie . thus , it was possible to observe three infection patterns according to social group : ( i ) groups in which the individuals never seroconverted , i.e. , never became infected during follow - up , ( ii ) groups in which the individuals seroconverted and had positive hc or had only positive serological tests ( ifat ) , and ( iii ) individuals that exhibited high rates of positive ifat and hc tests . the absence of trypomastigotes in the peripheral blood was a common trait in all infected animals . molecular characterised of 77 t. cruzi isolates - ra - three golden lion tamarins isolates from rio vermelho farm were characterised as tci , while five golden lion tamarins isolates from santa helena farm exhibited the tcii profile ( fig . 3:trypanosoma cruzi discrete typing unit of wild free - ranging leontopithecus rosalia ( golden lion tamarin ) and leontopithecus chrysomelas ( golden - headed lion tamarin ) from atlantic coastal rainforest , brazil . pda - molecular analysis permitted the confirmation of dtu tcii ( tciib ) for 33 out of 42 tamarins formerly typed ( mini - exon gene ) as tcii ( lisboa et al . moreover , nine golden lion tamarin isolates that originally tested as tcii ( fernandes et al . una - in the una , the mini - exon gene characterisation of 24 golden - headed lion tamarins isolates resulted in a tcii profile , while one tamarin isolate corresponded to tci and another was demonstrated to be a mixed infection by tci and tcii . analyses by 24s rdna and pcr - rflp with hsp60 and gpi markers confirmed the mini - exon genotyping ( fig . the majority of epidemiological studies are based on one single report of animal infection prevalence and without regard for ecological variables involved in the analysed ecosystem that certainly influence the dynamics of a multi - host - parasite system . indeed , long - lasting studies of t. cruzi transmission among free - ranging sylvatic primates species are scarce , as they necessitate significant financial support , skilled personnel , long studies about the social behaviours of primates and a multidisciplinary team . in brazil , natural infection by t. cruzi has been reported in primates included in the following families : cebidae ( capuchins , tamarins , marmosets and squirrel monkeys ) , nyctipithecidae ( night monkeys ) , pitheciidae ( sakis and uakaris ) and atelidae ( spider monkeys and howler monkeys ) ( ziccardi et al . the prevalence of t. cruzi infection in endemic primates from amazon ranged from 10.3% using parasitological methods ( blood smear microscopy , hc and xenodiagnosis ) ( ziccardi & loureno - de - oliveira 1997 , ziccardi et al . 2000 ) to 46% detected by serology , i.e. , ifat ( lisboa et al . no studies of the t. cruzi infection in primates have been conducted in the other brazilian biomes . regarding the molecular identification of t. cruzi isolates obtained from primates in the amazon region only tci and/or tciv have been observed ( miles et al . the assemblage of our data showed that a stable transmission cycle of dtu tcii was efficiently maintained in the wild environment by a free - ranging golden lion tamarin s population for at least for 11 years ( fig . t. cruzi serum conversion ( > 51% ) and hc positivity of originally noninfected , re - examined tamarins demonstrated active transmission occurring throughout our 11-year long follow - up period . this enzootic scenario confirms a very well established and robust sylvatic transmission cycle of tcii , a dtu previously associated with human infection , in the atlantic forest . figure 4:the golden lion tamarin ( leontopithecus rosalia ) of poo das antas biological reserve , municipality of silva jardim , state of rio de janeiro , brazil . photo by rodrigo mxas ( oswaldo cruz institute / oswaldo cruz foundation ) . the reproductive strategy of mammals ( k / r ) apparently does not influences t. cruzi reservoir competence in its different mammal host species as long - lived mammalian species ( such as lion tamarins and coatis ) and short - lived ( such as opossums and rodents ) may maintain high and long - lasting parasitaemias , being excellent sources of transmission as attested by the high rates of positive t. cruzi hcs observed in infected exemplars ( macarthur & wilson 1967 , lisboa et al . a very interesting aspect was the wave - like profiles of the infective competence of the t. cruzi - infected tamarin populations that can not be explained be serial reinfection , as experimentally infected mammals do not again exhibit an increase in parasitaemia upon re - infection ( machado et al . however , climatic or seasonal events may explain , at least partially , peaks in parasitaemia among vulnerable mammalian species . interestingly , the high parasitaemia peak of the golden lion tamarin population occurred in 1999 soon after the severe enso event in 1997 - 1998 ( lima et al . the same climatic event affected the prevalence of t. cruzi infection in octodon degus from chile , where the highest peaks of infectivity were observed in the years 1999 - 2000 ( previtali et al . overall , it has already been proposed that enhanced vector - borne and specifically t. cruzi transmission correlated to el nio events ( chaves & pascual 2006 , keesing et al . seasonal influence has been described to modulate reservoir competence for t. cruzi in nasua nasua , which exhibits higher parasitaemia during the dry season in the region of pantanal ( alves et al . our results suggested that vertical transmission does not occur , i.e. , infected females do not transmit the parasite to the newborns , which are born free of t. cruzi infection , as demonstrated by 11 offspring of infected females that were not infected by the parasite , as determined by hc and ifat as previously described in lisboa et al . more details related to the age , gender and reproductive status of golden lion tamarins and t. cruzi infection can be found in lisboa et al . two nonexclusive hypotheses may explain why certain golden lion tamarins did not acquire infection ( during our follow - up ) and other golden lion tamarins became infected soon after weaning ( lisboa et al . 2004 ) , even in the same social group : ( i ) microenvironmental differences , which result in cumulative distribution of triatomines and/or ( ii ) individual behavioural characteristics due to different roles played within the social group . risk of exposure to infection may also be associated with aggressiveness and boldness that lead to agonistic encounters ( kortet & hedrick 2007 , kortet et al . 2010 ) . dominance status in social animals may affect both exposure to parasites and immune functions , as dominant individuals have better access to food resources than subordinate individuals ( stahl et al . in fact both dominants and subordinates golden lion tamarins were infected with t. cruzi . moreover and very important , all noninfected golden lion tamarins belonged to the subordinate class , suggesting at least a tendency towards reduced exposure to t. cruzi infection for less aggressive individuals . individual variation in parasitaemia as expressed by positive or negative hcs is well known in other model hosts , including under laboratory conditions and is probably the consequence of individual differences in nutritional or energetic state , which can have strong effect on the host response to parasites ( michael & bundy 1992 , chandra 2002 , demas 2004 , houston & mcnamara 2013 ) . an environmental or behavioural difference as explanation for these differences is reinforced by the homogeneity of the genetic background of both the parasite and the hosts . the golden - headed lion tamarin from una can also be considered an amplifier host of t. cruzi infection . moreover , we could not forecast the evolution of t. cruzi infection patterns in golden - headed lion tamarin . indeed , for two species of didelphiomorphia , d. aurita and philander frenata , that were observed to have high positive hcs of t. cruzi in 1995 in the city of terespolis ( atlantic forest of altitude ) , no positive hcs were detected 10 years later in serologically positive marsupials ( pinho et al . dtu tcii was exclusively found in primates ; no other mammal species infected with tcii in the pda were observed . this may be partially explained by the absence of exposure of these mammals to this t. cruzi dtu or by their death due to infection . the nine isolates of golden lion tamarins , which were demonstrated to be dtu tci based on the second molecular characterisation ( pcr - rflp ) , suggested that these animals were infected with both subpopulations ( tci and tcii ) . thus , selective forces during the first amplification procedures of these isolates selected only the tcii subpopulation instead of both tcii and tci . it is likely that infection by tci result in low parasitaemia in golden lion tamarin ; therefore , the parasite can not be isolated by blood culture due to the low sensibility of the method . golden lion tamarin sera samples that do not test positive for blood culture eventually may be positive for tci . the occurrence of the golden lion tamarins infected with both tci and tcii in pda shows that the transmission cycles of both t. cruzi dtus occur sympatrically in almost independent ways . the change of enzootic scenery observed in two ra confirms the dynamic character of transmission cycles ; thus , both t. cruzi dtus ( tci and tcii ) may coexist in the same ra forest fragment , in this context , tci infected only d. aurita and tcii infected golden lion tamarins . wild rodents demonstrated to be infected by t. cruzi with low reservoir competence , as positive blood cultures were not observed . investigation of the triatomine fauna in the studied areas was performed by actively searching in hollow trees , bird and opossum nests and armadillo hollows , in addition to use the spray pirisa 1080-l1 and light traps . however , we captured only two triatomines vectors : triatoma vitticeps and panstrongylus megistus , respectively . both were infected with the same genotype of lion tamarin , tcii , demonstrating that these species were involved in the sylvatic transmission cycle of t. cruzi in wild population of golden lion tamarins . the infection of tamarins may have occurred due to presence of the triatomine in the tamarin sleeping sites ( hollow tree cavities , bamboo thickets , dense vine , palm crowns ) as suggested by lisboa et al . another possibility is the infection by the oral route , which is the main route of infection in a wild environment , as bromeliads and palms , which are triatomine habitats , are also frequently used by golden lion tamarin in search for animal prey . it is worth remembering that the vegetation typical of the atlantic rainforest is rich in palms , mostly astrocaryum spp and attalea spp ( pires & fernandez 1999 ) , which are palm species associated with the presence of triatomines . it is very likely that these endemic primates of the atlantic rainforest had already been exposed to tcii ( and tci ) long before the creation of the pda ( 1974 ) and/or una ( 1980 ) in a continuous strip of atlantic forest before the fragmentation process . the six dtu of t. cruzi apparently display different strategies for their maintenance and dispersion in nature ; thus , one can assume that tcii is a successful t. cruzi subpopulation . indeed , this genotype may be found dispersed on almost all brazilian biomes , infecting several mammalian taxa , as already mentioned , and establishing a peculiar interaction with each host species . thus , four - eyed opossums ( p. frenata ) , coatis , but principally lion tamarins may maintain high parasitaemias by this genotype as evidenced by high rates of recovery of the parasite in hc . wild and domestic canidae exhibit only a short period of high infectivity ( rocha et al . 2013 ) . considering that all these animal species are included in a trophic network , transmission will be enhanced or diminished according to local mammalian faunal composition . finally , the data presented herein point to the necessity for long - term studies of host - parasite interactions if a solid knowledge of the ecology of a given parasite species is desired . all together , the data also indicates how far we still are from understanding the dispersal strategies of each t. cruzi dtu ecology .

chronic hepatitis c is a chronic disease and whose global prevalence is estimated at 3% of the world s population ( 1 ) . according to the word health organization , the assumed prevalence of chronic hepatitis c in bosnia and herzegovina is around 1.5% . the current standard treatment of chronic hepatitis c in bosnia and herzegovina consists of the use of pegylated interferon alpha , subcutaneously , once a week , combination with ribavirin , daily ( peg - inf--2a+rbv ) . interferon therapy is associated with many psychiatric significant adverse effects ( insomnia , tension , irritability , dispersion , forgetfulness ) , and the most prominent form is depressive symptomatology ( 3 , 4 ) . in 30 - 50% of cases depression is the most common adverse effect of interferon therapy and it can compromise the effect of treatment ( 5 ) . since this kind of study has never been conducted in bosnia and herzegovina before , the aim was to provide a better screening of a group of patients with a high risk of manifestation of depressive symptoms during interferon therapy . the study would help identify the risk factors for the development of depression and enable prevention of deepening of depressive symptoms . the study population consisted of 46 subjects , 15 subjects treated for hepatitis c with combination therapy , 15 subjects diagnosed but not treated for hepatitis c with combination therapy and 16 healthy controls , aged 18 to 65 years , gender equated , different educational background , with different stages of the disease . prior to the study , the subjects had to sign a consent form in accordance with the previous authorization by the ethics committee . the eligibility criteria were as follows : subjects with a certified clinical and diagnosed chronic hepatitis c. the exclusion criteria were as follows : other liver diseases , abuse of psychoactive substances in the previous six months , attempted suicide in the history , subjects with psychiatric disorders such as psychosis from the schizophrenic specter , mood disorders , delusional disorders , dementia and other serious organic brain disorders , subjects with neurological diseases such as epilepsy , parkinson s disease , cardiovascular diseases ( decompensated heart failure ) , coinfection with hepatitis b or hiv , autoimmune disorders , unregulated diabetes , number of neutrophils under 1,500/cm and number of thrombocytes under 75,000/cm . the assessment and level of depression were based on the structural clinical interview ( scid ) , montgomery - asberg depression rating scale and zung self - rating depression scale . the assessments were conducted before interferon therapy ( on the day 0 ) , after 4 and 8 weeks of therapy . the differences between the subjects and other characteristics were assessed using the following tests : a ) parametric tests ( student s t - test , one - way analysis of variance - anova and pearson s chi - square test ) and b ) nonparametric tests ( chi - square test of homogeneity , mann whitney u test , kruskal - wallis test , wilcoxon singed - rank test and friedman two - way analysis of variance by ranks ) . the strength of the connection between the characteristics of observation was assessed using spearman s rank correlation coefficient . socio - demographic characteristics of the subjects are presented in table 1 ( gender , age , marital status , education , employment , presence of psychiatric disorders in the history ) . there were more male subjects ( 66.7% ) in the peg - inf--2a+rbv subgroup , and ( 93.3% ) in the no treatment subgroup , and least in the healthy subgroup ( 6.2% ) . there was statistical significance on gender match between the groups ( treatment , no treatment , healthy controls ) , ( p= 0.000 < 0.05 ) . there was statistical significance between the subgroups on age match ( treatment , no treatment , healthy controls ) . the mean age was 43.47 , 32.27 and 47.06 years ( p= 0.000 < 0.05 ) , respectively . there was statistical significance between the tested groups on employment match ( p= 0.000 < 0.05 ) . subjects with high school education prevailed in the treatment and no treatment subgroups , while the healthy controls were mostly college / university graduates . there was statistical significance between the subgroups on education match ( p=0.000 < 0.05 ) . majority of the subjects in the treatment subgroup were married ( 60% ) , the healthy subgroup ( 75% ) respectively , while in the no treatment subgroup singles prevailed . there was no statistical significance between the tested groups on marital status match ( p=0.140 > 0.05 ) . in the treatment subgroup 46.7% of the subjects had some sort of psychiatric disorder in the history , and 53.3% in the no treatment subgroup , respectively . madrs scoring showed that the number of depressed subjects in the treatment subgroup increased after 8 weeks ( 46.7% ) , and a rise after 4 and a decrease after 8 weeks in the no treatment subgroup , respectively . there was statistical significance between the subgroups after 4 and 8 weeks ( table 2 ) . the findings further showed the presence of mild depressive symptomatology in the subgroups , while severe depression was not recorded . there was no statistical significance between the subgroups on depression degree match at any week . frequency of depression across the subgroups ( madrs scale ) the findings showed a rise in the number of depressed subjects in the treatment subgroup after 8 weeks ( 73.3% ) , and a rise after 4 and a decrease after 8 weeks in the no treatment subgroup , respectively . there was statistical significance between the subgroups on the day 0 , after 4 and 8 weeks ( table 3 ) . the findings showed that depression in remission was present most across the subgroups , while severe depression ( hospitalization ) was less present . frequency of depression across the subgroups ( zung scale ) assessment of the significance of individual factors on the occurrence of depression was based on the binary logistic regression model . this model was used for gender , age , marital status , education , employment , psychiatric disorder in the history match . the model showed 54% of variance in the dependent variable ( occurrence of depression ) based on the nagelkerke r square indicators . if independent variable were used in predicting the occurrence of depression , 85% of those cases would be correct . according to the model , depression affects men more . respectively , men are 7.7 times likely to develop depression than women . persons who had some sort of psychiatric disorder in the history were 5.9 times likely to develop depression . however , these interpretations are not reliable due to the width of confidence interval , as a direct result of a small sample size . the findings showed that there was a correlation between the assessment instruments in all tests . this study showed that prior to treatment a seventh of the subjects in the treatment subgroup suffered from depressive symptomatology ( 13.3% ) , with the same number in the no treatment subgroup ( 13.3% ) . furthermore , nearly half of the subjects ( 46.7% ) displayed depressive symptomatology during interferon therapy ( ifn ) . the degree of depression in subjects infected with chronic hepatitis c taking ifn varied from 0% ( 6 ) to middle and high values ( 7 ) , and in some cases even more than 80% ( 8) . however , there are other findings too . the elshahawi study from 2011 , which comprised 400 subjects , showed that 30% of the infected subjects who did not take interferon suffered from depression thus pointing to the importance of chc on the manifestation of depression even in the absence of interferon ( 9 ) . this broad spectrum and huge variation results may occur because of different approaches to methodology , including the use of instruments for the assessment of depressive disorder ( 10 ) . in this study , depending on the time of manifestation , the highest level of depression in the peg - ifn--2a subgroup was found after 8 weeks , while in the no treatment group a rise of depression was found after 4 and a decrease after 8 weeks . the only rationale for the increase in depression in the subgroup with no treatment is a reactive state as a consequence of adjusting to and coping with the new situation ( news of serious disease and awaiting treatment ) . symptomatology in the context of adjustment disorder is highest in the first 4 weeks , followed by withdrawal of symptoms . therefore , as far as the frequency of depressions is concerned , madrs scoring has shown statistical significance between the subgroups after 4 and 8 weeks . on the other hand , findings on the frequency of depression on the zung scale indicated there was statistical significance between the subgroups in all weeks . the explanation of difference in the frequency results between these two scales can be explained by the fact that the threshold for the diagnosis of depression is lower on the zung scale . respectively , the use of self - assessment scales could generate higher degrees of depression , mainly due to the overvaluation of symptomatology . next , this study showed that the majority of the subjects infected with chronic hepatitis c were a younger population . other findings ( 11 , 12 ) also show that the majority of persons infected with chc are in the third or fourth decade of their life . when it comes to the degree of depression , according to the mards scale , mild depression was most present in both subgroups , with moderate depression less present and severe depression not present at all . according to the zung scale , both subgroups showed the signs of depression in remission , while a small number of subjects showed signs of severe depression requiring hospital treatment . elshahawi also in his study found that mild depression was the most prominent and severe depression least prominent form ( 9 ) . the question is why are some persons with chc more susceptible to depressive symptomatology during interferon treatment . recognizing risk factors that lead to psychiatric adverse effects can help identify patients at a high risk and thus provide them with additional psychological assessment and support ( 13 ) . gender difference is one of the most consistent findings in the prevalence and incidence in psychiatric epidemiology . studied the occurrence of depressive symptomatology in 71 chc patients during and after interferon alpha treatment and found that the independent factor , which can be regarded as a predictor of interferon alpha caused depression , is female gender ( 14 ) . however , there are conflicting findings proposed by bonaccorso and martin santoz who found no difference in the occurrence of depression on gender match ( 15 , 7 ) . subsequently , this study showed that 46.7% of the subjects in the treatment subgroup had some sort of psychiatric disorder in the history , while this percentage was 53.3% in the no treatment subgroup . psychiatric disorders which were recorded in history were as follows : anxiety disorders , personality disorders and abuse of pas . persons who had some sort of psychiatric disorder in the history were 5.9 times likely to develop depression . this can be explained by the fact that these persons are more vulnerable to different stressors ( psychosocial stress , biological stress ) or react to biological changes through psychiatric symptomatology . in an effort to determine depression risks , lang carried out a study on assessing the safety of interferon treatment with chc patients and he found out that the rate of psychiatric disorders was higher in those patients who had some sort of psychiatric disorder in their history ( 78% ) ( 16 ) . for the assessment of depression , both types of instruments were used in the study ( the madrs and the zung scale ) . a correlation between the two scales this was expected since there is usually a statistically significant correlation in the diagnosis of depression between these two scales , particularly in the assessment of the affect of depression and sleep as a relevant equivalent of depressive symptom . we found in our study that depression was more present in persons infected with chronic hepatitis c treated with pegylated interferon -2a in combination with ribavirin compared to persons infected with the same virus but without treatment ; mild depression was the most common form . our study showed that male gender and psychiatric disorders in the history can be regarded as predictors of depression in patients with chronic hepatitis c receiving interferon treatment . however , the deficiencies of the study are as follows : findings interpretations are not reliable due to a small sample size and short monitoring period of the study subgroups , which greatly affects the hypothesis that depression is a direct result of interferon treatment .

neck pain may originate from muscle , ligament , bursa , disc , neuromuscular junction , or from other problems that result in pain . neck pain can develop in any individual , generally first presenting as a headache , but also sometimes as shoulder pain . it is a cumulative condition occurring because of musculoskeletal damage to the cervical muscles , joints , nerves and is often caused by stressful work related conditions and long working hours . changes in working conditions in recent years have led to an increase in the number of patients being referred to the hospital for neck pain1 . when applying the scapula calibration strategy for patients with neck pain , improving the position of the scapula and trapezius creates an activity ratio of the upper parts that is close to the ratio of normal subjects2 . modifying the position of the scapula in patients with neck pain passively reduces pain levels . additionally , the improvement range of motion and rotation of the neck and the proprioceptive have been reported to improve the position in relation to the position of the shoulder3 . although clear evidence exists for the efficacy of stabilization exercises in reducing neck pain , clear evidence for the other effects shown by several previous studies is lacking4 . in addition , results from previous studies remain controversial because most of the studies on the effects of shoulder stabilization exercises for reducing neck pain are cross - sectional studies with no control group for comparison5 . for this reason , exercise treatments for neck pain patients tend to be based on the subjective judgment of the therapist , rather than on scientific evidence6 . this study examined , it was necessary to consider the impact of the neck muscles around the scapula that can contribute to neck pain , the control of pain , the function of the neck , and whether shoulder stabilization exercises to strengthen the large muscles of the shoulder have any effect on the symptoms of neck pain . the effects of the addition of shoulder exercises to standard neck exercises were also examined in order to present a more effective rehabilitation method for the treatment of neck pain5 . this study involved 30 patients between 20 and 60 years old who were diagnosed with neck pain by rehabilitation specialists at hospital , dae - jeon . patient inclusion criteria was a new diagnosis with complaints of neck pain of 37 on a visual analog scale ( vas ) , a neck disability index ( ndi ) of 15 points or more , and the ability to clearly hear and understand the exercises . subjects were excluded if they had neurological symptoms due to problems with their cervical vertebrae , pain caused by vascular problems , severe orthopedic problems in the shoulder , other medical conditions , or if they were involved in another experiment for the duration of the study . the pressure pain threshold ( ppt ) was measured using the vas and the pressure that reproduced that same level of pain in the neck was quantified . function was measured using the cervical ndi and cervical range of motion ( crom ) was determined using a cervical tilt sensor to directly record the range of motion7 . upper limb stability was measured using a closed kinetic chain test ( ckct ) to test the stability of the shoulder8 . the subjects were randomized into either the shoulder stabilization exercise group ( sseg ) or the control group , with 15 people in each . at the end of the experiment , the sseg included 14 people , as one person dropped out due to poor physical condition , and the control group included 12 people , with three people being excluded because of low participation rates . prior to the subject s participation , all procedures were explained , and each subject provided his or her written informed consent to participate . this study was approved by the ethics committees of the sahm - yook university institutions . the shoulder stabilization exercise and the control groups both received conservative physical therapy , involving a 10-minute hot pack application , 10-minute electrical treatment , 3-minute ultrasound treatment , and 15-minute neck stability exercise rehimen , for a total of up to 40 minutes of treatment9 . the sseg received 20-minutes of shoulder stabilization exercises in addition to the 40-minute conservative physical therapy , for a total of up to 60 minutes of treatment . the sseg and the control group all received the same number of sessions in a similar environment . the kolmogorov - smirnov test was used to determine the normality of the general characteristics and the parameters of the test subjects homogeneity were tested by using an independent samples t - test and test . changes in the dependent variable between the pre - test and post - test values were analyzed using a paired t - test . in order to compare the group effects , the level of significance for all results was set at p - value < 0.05 . the subjects general characteristics are presented in table 1table 1.characteristics of study participantsparameterssseg ( n=14)control group ( n=12)age ( years)67.160.4height ( cm)166.3162.7weight ( kg)48.243.5values are mean standard deviation ( sd).sseg : shoulder stabilization exercise group . no significant differences in general characteristics were observed between the sseg and the control group ( age : 67.14 and 60.41 years ; height : 166.35 and 162.75 cm ; weight : 48.21 and 43.50 kg ; respectively ) . differences in pre- and post - test values within groups and between groups are summarized in table 2 - 1table 2-1.comparison of pain , function and shoulder stability within groups and between groupsparametersvaluesdifferent in valuessseg ( n=14 ) control ( n=12)sseg ( n=14)control ( n=12)prepostprepostpost - prepost - prevas ( score)5.21 ( 1.05)2.42 ( 1.39)4.75 ( 1.05)3.75 ( 3.07)2.78 ( 1.25)1.00 ( 3.27)ppt ( lb / cm)rightupper trapezius8.89 ( 2.20)12.92 ( 1.97)10.58 ( 2.43)10.66 ( 3.05)4.35 ( 2.30)0.08 ( 1.57)levator scapular8.85 ( 2.61)12.21 ( 2.42)9.72 ( 2.38)10.33 ( 2.53)3.35 ( 1.65)0.54 ( 1.52)splenius capitis3.57 ( 3.22)7.64 ( 1.33)4.95 ( 3.91)5.50 ( 3.50)4.07 ( 2.52)1.45 ( 5.44)left upper trapezius8.35 ( 2.21)11.92 ( 2.26)9.12 ( 2.23)10.33 ( 2.53)3.57 ( 2.28)1.20 ( 0.27)levator scapular 9.21 ( 1.63)12.07 ( 2.30)10.50 ( 2.27)11.58 ( 2.35)2.86 ( 1.73)1.08 ( 0.79)splenius capitis 3.00 ( 3.11)6.92 ( 0.99)4.75 ( 2.92)6.33 ( 2.22)3.92 ( 0.63)1.58 ( 2.23)values are mean standard deviation ( sd ) , * p<0.05vas : visual analog scale ; ppt : pressure pain threshold ; sseg : shoulder stabilization exercise group , table 2 - 2table 2-2.comparison of pain , function and shoulder stability within groups and between groupsparametersvaluesdifferent in valuessseg ( n=14 ) control ( n=12)sseg ( n=14)control ( n=12)prepostprepostpost - prepost - prendi ( score)21.42 ( 4.61)12.58 ( 4.53)22.58 ( 6.00)17.66 ( 6.62)8.57 ( 3.73)4.91 ( 4.58)crom ( degree)flexion32.21 ( 7.36)38.92 ( 4.26)32.91 ( 2.23)37.08 ( 5.83)6.71 ( 4.49)4.16 ( 5.18)extension36.42 ( 8.71)40.07 ( 5.90)38.75 ( 12.75)41.08 ( 8.55)3.64 ( 4.51)2.33 ( 5.74)right side banding26.14 ( 6.57)34.85 ( 3.57)30.25 ( 8.86)33.33 ( 7.83)8.71 ( 5.31)3.08 ( 3.50)left side banding29.14 ( 5.60)34.35 ( 6.05)33.00 ( 9.95)35.25 ( 8.12)5.21 ( 0.89)2.25 ( 2.79)right rotation32.78 ( 9.97)38.00 ( 6.93)31.33 ( 5.83)35.16 ( 5.58)5.21 ( 4.66)3.83 ( 1.99)left rotation34.21 ( 8.44)38.42 ( 5.66)37.83 ( 8.37)39.00 ( 6.68)4.21 ( 4.00)1.16 ( 3.32)ckct ( count)4.14 ( 0.41)*6.35 ( 1.27)3.91 ( 0.66)5.58 ( 0.66)2.21 ( 1.10)1.66 ( 0.77)values are mean standard deviation ( sd ) . ndi : neck disability index ; crom : cervical range of motion ; ckct : closed kinetic chain test ; sseg : shoulder stabilization exercise group . first , the ppt in both groups were significantly reduced in the upper trapezius , levator scapulae and splenius capitis , after exercise . second , the ndi values were significantly lower in the sseg compared to the control group and crom , measured by right lateral flexion , left lateral flexion , and left rotation , was significantly higher in the sseg than in the control group . flexion , extension , and right rotation also increased to a greater degree in the sseg than in the control group . third , there were no significant differences in upper limb stability between the control group and sseg ( p<0.05 ) , but there was a significant difference between the control group and the sseg ( p<0.05 ) . sseg : shoulder stabilization exercise group values are mean standard deviation ( sd ) , * p<0.05 vas : visual analog scale ; ppt : pressure pain threshold ; sseg : shoulder stabilization exercise group values are mean standard deviation ( sd ) . ndi : neck disability index ; crom : cervical range of motion ; ckct : closed kinetic chain test ; sseg : shoulder stabilization exercise group neck pain patients with more severe levels of upper limb dysfunction show a strong positive correlation with increased levels of neck pain . in a study in the united kingdom of 151 patients with non - specific neck pain , neck pain and upper limb dysfunction showed a high positive correlation , with a correlation coefficient of 0.799 between the two variables12 . there are a number of explanations for the correlation between neck pain , upper limb dysfunction , and reduced upper extremity strength . the bones and muscles of the cervical spine and the shoulders are connected to each other mechanically , thus a continuously applied mechanical load on the shoulders directly increases the load on the cervical region , which may cause joint and ligament pain . this can be seen in certain occupational groups who use their shoulders more often than most workers . the brachial nerves are linked structures that pass from the cervical spine to the shoulders . if shoulder movements result in cervical tissue elongation and sliding , this may cause symptoms typically seen in neck pain suffers . sustained and repetitive movements of the shoulder and elongation of the nerves11 may cause damage to the fine neural tissue leading to it becoming brittle and causing neck pain . in addition , movement of the shoulder may be limited because it induces neck pain in patients . neck pain leads to a rediction in the levels of activities that require the use of the neck and shoulder . this can lead to physical deterioration through a decrease in muscle strength , cardiovascular endurance , and ability13 . in this study13 , the neck disability index rates patients ability to eat , to do housework , and to carry heavy objects . another study found that , when evaluating the performance of activities of daily living in patients with more severe neck pain it was found that they had more difficulty in performing the activities mentioned in the survey14 . in other studies , on the treatment and management of patients with severe neck pain , the effects of shoulder calibration and other current treatment approaches , have already been presented . this comprehensive foundation can be seen as an opportunity to examine the effects of these different approaches15 . research has shown that the active range of cervical rotation increases significantly when the shoulder of a healthy young adult is supported by placing the scapula in the neutral position15.this maneuver can also reduce symptoms of neck pain16 . another study , examining cervicogenic headaches , compared the activity of the middle trapezius , lower trapezius , and serratus anterior when 30 , 60 , 90 , and 120 of flexion were applied with the shoulder in a flexion , extension position17 . interventional approaches aimed at reducing uneven alignment , including repositioning of the scapula , have shown of a significantly decreased ndi score after 3 months , and a significantly increased crom18 . this , in relation to the present study , can be best explained by the increased stabilization of that occurs during cervical rotation . in a study on healthy young adults , it was shown that , by supporting the upper limbs the active cervical rotation range was significantly increased when release the scapula was released to the neutral position . therefore , when a neck pain patient raise their scapula , thereby increasing stability and supporting the weight of the upper limb , this rotation relieves the symptoms of cervical neck pain , indicating that the cause of the neck pain is through an injury affecting the scapular muscles . comparing these results to those of the study , the previous results showed that there were no significant differences between groups in the stability of the upper limb , whereas in the current study , there was a significant difference between the sseg and the control group . the current study shows that the occurs due to the manipulation of the scapula performed in the shoulder stabilization exercise and massage groups , compared to upper trapezius elongation per during shoulder stabilization exercise , provides greater relief to neck pain patients . this conclusion is supported by a study that shows a link between the cervical and scapular muscles20 . previous studies have reported increased levels of stability in groups undergoing stretching exercises and massage , but these differences were not significantly different20 .

95% of tuberculosis ( tb ) of the female genital tract ( fgt ) is located in tissues other than the cervix . a rare case of primary tb of the cervix which was diagnosed coincidently in a patient of endometrioma was reported in this study . a 34 year old nullipara , a diagnosed case of endometrioma had a small cervical growth . the patient was started on antituberculosis under directly observed therapy along with oral contraceptive pills . although cervical tuberculosis is very rare but for an abnormal looking cervix , cervical tuberculosis should be considered in the differential diagnosis in woman of all the age groups especially in areas where tuberculosis is rampant since these cases are potentially curable with medical therapy . 95% of tb of the female genital tract ( fgt ) is located in tissues other than the cervix . a rare case of primary tb of the cervix which was diagnosed coincidently in a patient with endometrioma was reported in this study . a 34 year old unmarried , nulligravida presented ( somewhere july august 2014 ) to gynecology opd in north eastern indira , gandhi regional institute of health and medical sciences , shillong , meghalaya with chief complaint of pain in lower abdomen for 56 years . her general physical examination was normal . in her first visit , in speculum examination , cervix and vagina were healthy . in bimanual examination , her uterus was normal size , retroverted , right fornix was tender and an ill defined mass about 5 cm was present with restricted mobility . left fornix was clear . in ultrasonographic examination , 6 by 5 cm endometrioma was present in right ovary . as the patient was not willing for further surgery and fertility was not an issue , she was put on continuous oral contraceptive pill with three monthly regimens . her endometrioma decreased in size by 4 by 3 cm in repeat ultrasonographic examination after nine months of therapy . as she was on oral contraceptive pill , this time about after nine months ( on 6th april 2015 ) in speculum examination , a small growth about 0.5 cm , pink , smooth , firm , non - forcible and non bleeding on touch , a cervical polyp was present on anterior lip of cervix . in retrospective interrogation , she denied any history of chronic cough , loss of weight or fever . she did not have menstrual irregularity , post coital bleeding , discharge per vaginum , any bowel or bladder complaint . antibody test for hiv was negative . on the basis of histopathological examination and afb staining report , the patient was started on anti - tubercular drugs under directly observed therapy along with oral contraceptive pills . she was asymptomatic before diagnosis and treatment of cervical tuberculosis and her growth was already removed during biopsy . on follow - up , h&e , 4x - section showing endocervical epithelium with epitheloid granuloma in the stroma h&e , 20x epitheloid granuloma with langerhans giant cells endometriosis and cervical tuberculosis have no common risk factors except that both are seen in reproductive age group and both can lead to infertility , although in the present case , infertility was not an issue . here , cervical tuberculosis was just a coincidental finding . although pulmonary tuberculosis is the most common form , genitourinary tb is also common in developing countries . it usually involves fallopian tube ( 95100% ) , endometrium ( 5060% ) and ovary ( 2030% ) . tb of cervix is extremely rare and accounts for 0.10.65% of all cases of tb and 524% of genital tract tb ( 15 ) . genital involvement is usually secondary to extra genital tb which in most cases arises from pulmonary focus ( 2 , 4 , 5 ) . spread of infection to the cervix is either by hematogenous or lymphatic route or by direct local extension from tubercular salpingitis or endometritis ( 2 , 4 , 5 ) . rarely cervical involvement may occur via sexual contact with a partner who is affected by genitourinary tb ( 1 , 2 , 46 ) . it has been suggested that sputum used as sexual lubricant may also be a route of transmission ( 4 , 6 ) . in extremely rare cases , primary cervical tuberculosis may occur if a person sits on infected sputum . in the present case , there is no personal history of tb and her partner also did nt have any obvious urogenital manifestation . genital tract tb usually presents with abnormal vaginal bleeding or discharge , menstrual irregularities , abdominal pain , infertility , postmenopausal bleeding and other constitutional symptoms ( 19 ) . in clinical presentation , it may be papillary , endophytic , exophytic , ulcerative growth which may simulate cervical cancer ( 25 ) . in the present case , the diagnosis of cervical tb is often established by histopathological examination of cervical biopsy specimen ( 2 , 4 , 7 ) and sometimes by staining for afb as happened in the present case . although culture of mycobacterium is considered the gold standard for diagnosis , but in 30% of cases , the culture may be negative ( 25 , 7 , 10 ) . therefore , presence of typical granuloma is enough for diagnosis ( 25 , 7 , 10 ) . although cervical tuberculosis is very rare but for an abnormal looking cervix , cervical tuberculosis should be considered in the differential diagnosis in woman of all age groups especially in areas where tuberculosis is rampant since these cases are potentially curable with medical therapy .

we investigate the growth of graphene on polycrystalline pt foils ( 25 m , 99.99% , alfa aesar ) by cvd using in situ sem ( see the methods section ) . the samples are first annealed in h2 ( 10 mbar ) at 9001000 c for 15 min to promote pt grain growth and remove adventitious carbon from the surface of the foil and then exposed to c2h4 ( 1010 mbar ) at temperatures of 9001000 c , with sem images ( 0.1 hz frame rate ) acquired throughout . figure 1 shows se micrographs of the pt surface during c2h4 exposure at different times during the growth . prior to the introduction of the precursor ( figure 1a ) , the polycrystalline nature of the catalyst surface is apparent from the variations in contrast between different grains related to electron - channeling contrast and the topographical contrast arising from the network of grain boundaries separating them . the few small ( < 1 m ) , bright features visible within some of the pt grains are attributed to residual oxygen contamination of the pt surface ( as also observed in corresponding x - ray photoelectron spectroscopy measurements ; not shown ) and disappear early in the precursor exposure as they presumably react with carbonaceous species arriving at the surface . although the precursor pressure within the chamber is reached within 15s ( as confirmed by a residual gas analyzer ) , there is a distinct incubation period during which no graphene forms on any of the pt grains . as the precursor exposure continues , faceted graphene domains appear on several pt grains ( figure 1b ) . their darker contrast compared to the bare pt surface is related to the lower secondary electron generation of graphene . the graphene domains grow in size with time and merge with the other graphene domains upon which they impinge . meanwhile , the nucleation of new graphene domains occurs on other pt grains that show longer incubation times ( figure 1c ) . the incubation times of different pt grains vary widely , with graphene domains nucleating on several grains within 90 s of the precursor being introduced , while others show no nucleation events even after > 2500 s and only become covered with graphene due to the expansion of domains from adjacent pt grains across grain boundaries ( figure 1d ) . this is attributable to grain orientation dependent variations in the precursor dissociation rate , graphene nucleation barrier , or both , which are likely to be affected by the density of low - coordination sites such as step edges . during graphene growth , we note that ripening of the pt grains is also observed ( compare panels a and d of figure 1 ) , again highlighting the need for in situ measurements when considering the relationship between the microstructure of the catalyst and 2d material . ( a d ) sequence of in situ sem images of pt ( 25 m ) during c2h4 ( 10 mbar ) exposure at 900 c , acquired 0 s ( a ) , 150 s ( b ) , 1500 s ( c ) , or 6000 s ( d ) after precursor introduction . the approximate orientations of the pt grains determined by ebsd analysis are indicated within the respective grains in ( a ) . ( e ) plot of the areal coverage of graphene , a , with c2h4 exposure time , t , for the regions marked with red and green boxes in ( a ) . after the first graphene domain appears , the nucleation of other domains occurs within a relatively short time frame ( < 60 s ) . the first nuclei typically form near the center of a pt grain away from grain boundaries , and the nucleation density is also observed to be lower close to pt grain boundaries ( see , for example , figure 1b ) . figure 1e considers a single pt grain ( indicated by the red polygon in figure 1a ) and plots the areal graphene coverage , a , with time , t , for the entire grain ( red ) and for a region close to the center of the pt grain ( green square in figure 1a ) . in both cases , a rises rapidly following the incubation period , but the rate of increase in a reduces over time , and complete single - layer graphene coverage is only slowly approached . the jmak equation is widely applied to describe nucleation and growth during phase transformations and has previously been used to interpret ex situ graphene growth results on polycrystalline cu . the inset of figure 1e therefore plots the evolution of graphene areal coverage in terms of avrami coordinates , ln ( ln ( 1 a ) ) versus ln ( t ) ; however , the clearly nonlinear shape confirms that the jmak equation does not adequately describe the increase in the area fraction with time . in interpreting the observed growth behavior , it is instructive to consider the key processes involved in the growth of graphene on polycrystalline catalyst surfaces during cvd , as outlined in figure 2 . the dissociation of the precursor delivers carbon to the catalyst surface ( process 1 ) , which can readily diffuse on the surface ( process 2 ) . this carbon can attach to a graphene domain it encounters contributing to growth ( process 3 ) or can be removed from the surface by diffusion into the bulk ( process 4 ) or grain boundaries ( process 5 ) , which serve as more rapid pathways for diffusion . while processes 4 and 5 can also contribute to delivering carbon to the growing graphene domain , the lower activation barrier for surface diffusion means process 2 is expected to dominate.figure 2 indicates the net directions of mass transport associated with these processes for typical graphene cvd conditions , as used herein , where the catalyst bulk is not initially filled with carbon and a net flux of carbon is delivered to the catalyst surface by precursor exposure . we note , however , that the net directions of mass transport associated with each of the processes can also be reversed depending on the processing conditions , corresponding to removal of surface carbon into the gas phase by etching , shrinking of graphene domains due to carbon removal , and the diffusion of carbon out from the bulk or grain boundaries . process 4 : carbon diffusion into the bulk of the catalyst removes carbon from the surface . process 5 : grain boundary diffusion serves as a more - rapid pathway for carbon removal from the surface . while the jmak equation is found to be inadequate in describing the behavior observed in figure 1e , the evolution of a within an individual catalyst grain qualitatively resembles that obtained by consideration of an idealized single - crystalline surface with a semi - infinite bulk on which graphene growth is fed by the net flux arising from the supply and removal of carbon by processes 1 and 4 , respectively . the supply of carbon by process 1 can be modeled based on the kinetic theory of gases , as being constant for a given grain orientation , precursor partial pressure , and temperature but reducing in proportion to the bare catalyst area ( 1 a ) as graphene coverage isolates the catalyst from the precursor supply . the removal of carbon by process 4 can be modeled based on 1d fickian diffusion perpendicular to the catalyst surface . this yields the following general behavior for a catalyst whose bulk is not initially filled with carbon : upon the introduction of the precursor , the supply of carbon to the surface is matched by bulk diffusion , leading to an incubation period during which the carbon concentration close to the catalyst surface increases until the solubility limit is reached and a local supersaturation develops . this supersaturation feeds the nucleation and subsequent growth of graphene domains and is maintained by precursor dissociation on bare areas of the catalyst . as the graphene coverage increases , the supply of carbon to the surface by precursor dissociation is reduced while diffusion into the catalyst bulk still continues , meaning that growth slows and complete coverage is only gradually approached . the dependence of the area fraction on time for the entire grain ( red ; figure 1e ) deviates slightly from that obtained from just the central region ( green ; figure 1e ) , showing a slower initial growth rate and with full coverage approached more slowly . this is attributed to the contribution of process 5 in which grain boundaries serve as pathways for rapid diffusion of carbon away from the catalyst surface . this additional pathway for carbon removal also reduces the supersaturation that develops in these areas , which accounts for the lower nucleation densities observed close to grain boundaries . notably lower nucleation densities are observed close to the grain boundaries toward the top left of the grain compared to those toward the bottom right ( see figure 1b ) , consistent with the dependence of grain boundary diffusion coefficient on the structure of the grain boundary . while consideration of the balance of fluxes between processes 1 , 4 , and 5 is useful in describing how the overall graphene coverage evolves within a platinum grain , i.e. , the collective contribution of many graphene domains , this assumes process 1 to be the rate - limiting step in growth and thus does not take processes 2 and 3 into account , nor does it provide insights into the localized behavior of individual graphene domains such as how their shape and growth rate evolve . in modeling these processes , we consider the edge of a single graphene domain growing on the pt surface where there exists a flux of carbon from the pt surface to the graphene domain , jsg , and vice versa , jgs . this yields a net flux j that feeds the growth of the graphene edge , which by conservation of mass is related to the growth velocity normal to this edge , v:1cg is the concentration of carbon in graphene , and ci is the carbon concentration at the interface between the growing graphene domain and the bare pt surface . considering that attachment of carbon to the graphene edge is impeded by an energy barrier , ga , and that for growth to proceed there must be a driving force , g , for carbon attachment , we obtain the following expression ( see the supporting information for detailed derivation):2 m(, ) is the interfacial mobility and incorporates the energy barrier associated with carbon attachment , which can depend on the angle of the edge relative to the graphene lattice , , and to the orientation of the underlying metal substrate , , as we will discuss further below . (ci ) and are , respectively , the carbon chemical potentials of the pt surface at the edge of the graphene domain and the graphene itself . for the case in which (ci ) , eq 2 reveals that j m(, ) and , therefore , growth is interface - attachment - limited . this situation arises when a large supersaturation exists , such as immediately following nucleation . when m(, ) is small , j is also small , and initially the supersaturation close to the growing edge can be readily replenished , such that ci is maintained near its far - field value , and growth remains interface - attachment - limited . thus , it follows from eq 1 that v remains constant . when m(, ) is large , however , the supersaturation near the growing edge is quickly depleted and can not be replenished fast enough to maintain ci near its far - field value , and thus , (ci ) and local equilibrium is approached . the driving force for growth is reduced , such that , with ci determined by diffusion of carbon across the catalyst surface and growth becoming diffusion - limited . v is therefore now time - dependent , varying as a function of ci . to precisely determine this variation in v , the carbon diffusion field around the graphene domain would need to be calculated ( e.g. , via the phase field method ) ; however , we can nevertheless consider its general trend at different stages of growth . when the graphene domain is small , the concentration gradients around the domain are steep , and as the domain becomes larger , these gradients become smaller , meaning v will decrease with time in the diffusion - controlled limit . in the context of this model , and by measuring the interfacial velocities from the real - time data , we now consider the growth evolution of individual graphene domains in a region of the same pt grain . figure 3a shows how the graphene domains evolve across several time steps ( 160 , 170 , 180 , and 190 s ) for a region within the green square indicated in figure 1a . the growth velocities of facets growing toward the top left ( colored cyan ) are notably slower than those growing toward the bottom right ( colored red ) , as is apparent from the different spacing of facets between isochrones . figure 3b shows a histogram weighted according to facet length , revealing the angular distribution of the facets at a growth time of 170 s. the four major facets are aligned with 0 , 110 , 220 , and 290 and are found to remain dominant even as the graphene domains merge , although the histogram intensities vary somewhat , and thus , the domain morphology is self - similar between time stamps ( see video s1 ) . most significantly , the differences between the facet angles are not multiples of 30 , as might be expected if the graphene domain shapes were primarily determined by zigzag , armchair , or some other intermediate termination of the edges being most energetically favorable . therefore , as the faceted shapes of the graphene domains can not be attributed to the edge termination alone , the role of the underlying substrate must be considered . electron backscatter diffraction ( ebsd ) patterns collected postgrowth reveal the bulk orientation of the underlying pt grain whose surface lies close to the ( 522 ) crystallographic plane . previous leed studies reveal that similarly oriented surfaces remain stable , maintaining their nominal structure when clean and under vacuum conditions and when covered with graphitic carbon following precursor exposure.figure 3c therefore shows a ball model of the corresponding unreconstructed pt surface consisting of ( 111 ) terraces with ( 100)-like steps , with the step edges indicated by dashed red lines and the uphill direction indicated with an arrow . while these ( 111 ) terraces are not expected to reconstruct under the growth conditions used herein , we note that reconstruction of pt(111 ) surfaces has been reported under certain conditions , albeit while maintaining hexagonal symmetry . for other pt grain orientations , faceting , reconstruction of the surface , including changes in symmetry , or both may occur as for pt(100 ) , which is known to undergo a hexagonal reconstruction . ( a ) sequence of overlaid isochrones extracted from sem images taken 160 , 170 , 180 , and 190 s after c2h4 introduction , showing domain edges for a region within the green box marked in figure 1a . the orientations of the facet normals ( defined as pointing out from the graphene domain to the substrate ) are indicated by the line color . ( b ) plot of the angular distribution of the graphene facet normals corresponding to the image taken after 170s in ( a ) . ( c ) ball model of the unreconstructed ( 522 ) surface orientation of the underlying pt grain , determined by ebsd measurements . arrows indicate the six < 111 > directions that lie closest to the plane of the surface ( see figure s2a for other directions ) , with the four colored arrows corresponding to the similarly colored dominant graphene facet directions in ( a ) . red dashed lines highlight the step edges , with the uphill direction indicated by the labeled arrow . consideration of the low - index crystallographic directions of the pt surface reveals a clear relationship with the dominant graphene facet orientations , which all lie normal to < 111 > directions . under kinetic control , the domain shape will be dominated by the slowest growth directions . given the dependence of v on m(, ) and ci exemplified in eqs 1 and 2 , these < 111 > directions are therefore expected to correspond to directions of low interfacial mobility , slow surface diffusion , or both . it is also apparent that the growth of the slower velocity facets toward the top left of the images ( colored cyan ) corresponds with growth in the uphill direction . this slow uphill growth is consistent with various surface science studies of graphene growth on low - index surfaces of single - crystalline substrates , which reveal that the uphill graphene facets can be embedded in the step edge and thus grow by a metal - etching mechanism . the slow velocity of these uphill facets accounts for the trapezoidal shape of the graphene domains , as they will dominate over the facets perpendicular to the two adjacent < 111 > directions ( black arrows in the inset of figure 2c ) . this difference in uphill and downhill growth rate can not be attributed to the delivery of carbon across the catalyst surface , as it persists even as the uphill and downhill facets of different domains approach one another , and an asymmetry in the uphill and downhill diffusivity of carbon species would violate the onsager principle of microscopic reversibility , which holds for transport properties such as diffusion . instead , it is attributable to a larger barrier for attachment to embedded facets , which presumably relates to the need to eject metal atoms from the step for carbon to be incorporated . this confirms that the interfacial mobility , m(, ) , varies between different facets and must be considered a function of both the facet direction relative to the graphene lattice , , and to the substrate orientation , . this can account for the more - diverse ( including nonsymmetric ) domain shapes that are experimentally observed , in contrast to previous models that typically assume an epitaxial alignment between the graphene lattice and substrate orientation and thus predict domain shapes with at least 2-fold rotational symmetry . given the anisotropy in interfacial mobility that we have observed , it is insightful to consider the kinetics of each facet individually . figure 4a shows the growth evolution of a single graphene domain that was grown under conditions of higher growth temperature and lower precursor partial pressure and maintains an irregular hexagonal shape throughout ( see video s2 ) . the slow growth rate and large size of this domain , which results from the growth conditions , gives the necessary spatial and temporal resolution to more precisely consider the growth kinetics of each facet individually . a total of six major facets colored blue , navy , red , orange , green , and cyan ( clockwise from the top facet ) are apparent from early on and persist throughout the ensuing growth period . figure 4b shows the variation in velocity of each of these facets with time based on the analysis of 100 frames that were acquired 9.5 s apart ( see the methods section ) . the velocities of the red and orange facets are seen to be constant throughout the growth period , indicating that their growth is interface - attachment - limited and independent of domain size . conversely , the velocities of the blue and navy facets reduce over time as the particle grows , indicating that the growth of these facets is instead diffusion - limited . the cyan and green facets initially show constant growth velocities , but after some time , their velocities start to reduce , indicating that they transition from interface - attachment - limited to diffusion - limited growth . this clearly highlights that different facets within a single grain can show different rate - limiting steps that may evolve during growth . ( a ) sequence of isochrones ( spaced by 47.5 s ) of the domain edges obtained from sem images of pt ( 25 m ) during c2h4 ( 10 mbar ) exposure at 1000 c , colored according to the orientations of the facet normals ( defined as pointing out from the graphene domain to the substrate ) . ( b ) plot of the facet velocities with time for the six major facet orientations apparent in a. ( c ) ball model showing the unreconstructed ( 635 ) surface orientation of the underlying pt grain , determined by ebsd measurements . arrows indicate the six < 111 > directions that lie closest to the plane of the surface ( see figure s2b for other directions ) with the four colored arrows corresponding to the similarly colored dominant graphene facet directions in ( a ) . red dashed lines highlight the step edges , with the uphill direction indicated by the labeled arrow . the bulk orientation of the underlying pt grain is determined from postgrowth ebsd patterns , revealing that the surface lies close to the ( 635 ) crystallographic plane , which again corresponds to a surface orientation that is not expected to facet or undergo a surface reconstruction either when clean and under vacuum or when covered with graphitic carbon . a sketch of the corresponding unreconstructed surface is therefore shown in figure 4c , with the step edges indicated by red dashed lines . this reveals a close alignment of four of the domain s facets ( green , navy , cyan , and red ) perpendicular to certain < 111 > directions ( similarly colored arrows in the inset of figure 4c ) , similar to that noted earlier for the multiple domain growth of figure 3 . we again observe a lower growth velocity for those facets growing uphill ( green and cyan ) , i.e. , the facets expected to be embedded in pt steps , compared to those growing downhill ( red and navy ) . the navy facet is the fastest growing at the start of growth and thus corresponds to the highest mobility facet . its diffusion - limited behavior throughout growth is consistent with the local carbon supersaturation ahead of it being rapidly depleted . the uphill growing facets ( green and cyan ) show much lower growth velocities at the start of growth and have correspondingly lower mobilities consistent with there being a larger barrier for attachment to embedded graphene facets . they thus remain interface - attachment - limited for a longer time , with the carbon supersaturation developed prior to nucleation feeding their growth , and transition to diffusion - limited growth as this supersaturation becomes depleted . interestingly , the red and orange facets toward the bottom of the domain have higher mobilities but show interface - attachment - limited behavior throughout growth , suggesting that a relatively high local carbon supersaturation is maintained . conversely , the blue facet toward the top of the domain has the lowest growth velocity and yet shows a diffusion - limited growth behavior consistent with the local carbon supersaturation being rather low . while sem does not directly reveal the carbon supersaturation around the graphene domain , lower - magnification sem images ( not shown ) show that no other domains nucleate near the top - left of the domain of interest , while other domains do nucleate toward the bottom - right . this would be consistent with a gradient in carbon supersaturation existing across the pt grain increasing from the top - left to the bottom - right , which may relate to variations in the rates of carbon removal by different grain boundaries , differences in the precursor dissociation rates between adjacent pt grains , or both . alternatively , such differences in local carbon supersaturation could arise due to the shape of the graphene domain itself , as the local carbon supersaturation experienced by a facet is not only influenced by its own growth but also by the growth of neighboring facets . having considered the growth behavior of graphene domains within pt grains , we now focus on the growth across grain boundaries , which is key to forming a continuous graphene layer over a polycrystalline catalyst surface . figure 5 shows two sequences of sem images ( a e and f j ) in which graphene domains that have nucleated on different pt grains grow across pt grain boundaries onto different areas of the same pt grain , whose surface orientation is determined as ( 431 ) by ex situ ebsd measurements . for both sequences , as the initially sharp apexes of each graphene domain ( figure 5a , f ) approach the pt grain boundaries , they are seen to drastically widen in the directions parallel to the grain boundaries ( see , for example , figure 4c , h ) , leading to a change in the facet directions even before the grain boundaries have been crossed . after crossing the grain boundary , the growing domains both adopt new facet orientations , as indicated by the dotted lines in figure 5e , j . notably , the facet orientations adopted by both domains after crossing onto the same pt grain are closely aligned ( compare the red , green , and blue dotted lines ) despite the two domains nucleating on differently oriented pt grains and thus presumably having different crystallographic orientations , . this similarity in graphene domain shapes can not be attributed to the orientation of graphene lattice , i.e. , any epitaxial relationship , but instead indicates that their shapes are predominantly determined by the underlying structure of the catalyst grain on which they both end up . many of the facet normals again appear to align with certain < 111 > directions of the catalyst grains both before and after crossing the grain boundary , as indicated by the arrows colored orange in figure 5a , purple in figure 5f , and green and blue in figure 5e , j . this highlights the importance of the substrate in influencing both interfacial mobility and diffusion of carbon species , with these < 111 > directions again expected to correspond to directions of low mobility , slow diffusion , or both . it is also apparent from figure 5 that apexes of the graphene domains coincide with the positions of the pt grain boundaries , indicating fast diffusion directions parallel to these boundaries . the grain boundaries are clearly visible in all images during growth due to the topographic contrast resulting from their high local curvature . this curvature corresponds to a high density of step edges close to the grain boundary , which run approximately parallel to it , as indicated in figure 5k . the lower barrier to diffusion along the terraces compared to that of crossing step edges leads to anisotropic surface diffusion , which increases as the density of step edges increases ( see figure 5k ) . this increased anisotropy in surface diffusion accounts for the drastic widening of the graphene domains parallel to the grain boundary as it is approached . we note that for pt , which has an fcc crystal structure , no anisotropy in bulk diffusivity is expected , and thus , these observations again highlight the dominant role of surface diffusion in determining domain shape . this higher density of step edges could also lead to a local increase in hydrocarbon dissociation rate ; however , while we do not exclude this , we note that this alone can not account for the faster growth rate parallel to the grain boundary , and in any case , the rapid removal of carbon by grain boundary diffusion is likely to dominate . graphene growth across grain boundaries . ( a e ) sequence of sem images of a graphene domain ( facets indicated with orange dotted lines in ( a ) that nucleated on a pt grain with a ( 512 ) oriented surface , showing its growth across a grain boundary onto a pt grain with a ( 431 ) orientated surface . ( f j ) sequence of sem images of a graphene domain ( facets indicated with purple dotted lines in f ) that nucleated on a pt grain with a ( 632 ) oriented surface , showing its growth across a grain boundary onto a different region of the same ( 431 ) orientated pt grain as identified in ( a ) ( surrounded by cyan dashed lines in ( a ) and ( f ) ) . the red , green , and blue dotted lines in ( e ) and ( j ) indicate similar facet orientations established on this pt grain after the graphene domains have crossed from the differently oriented pt grains where they nucleated . arrows indicate the < 111 > directions of each catalyst grain ( see figure s2c e ) , with the colored arrows corresponding to those < 111 > directions that lie normal to highlighted graphene facets . ( k ) schematic of graphene growth across grain boundaries : ( 1 ) the higher barrier to surface diffusion of carbon across step edges than along terraces leads to an anisotropy in surface diffusion . ( 2 ) as a grain boundary is approached , the much - higher density of step edges increases this anisotropy in surface diffusion . ( 3 ) grain boundary diffusion serves as a rapid pathway for carbon removal from the surface , reducing the local carbon concentration and slowing the growth rate of the graphene domain as the grain boundary is approached . our data reveal that processes 15 identified in figure 2 all play an influential role in determining the final growth outcome during graphene cvd . we therefore outline the following consistent picture of how these processes contribute to the growth behavior on realistic polycrystalline catalyst surfaces observed herein , as summarized in figure 6 : carbon is delivered to the catalyst surface by precursor dissociation , whose rate may vary across differently oriented , or reconstructed catalyst grains . this carbon supply is initially matched by diffusion into the catalyst bulk , giving a notable incubation time during which the catalyst becomes locally filled with carbon close to the surface , and a supersaturation then starts to develop ( figure 6b ) . grain boundaries serve as rapid pathways for the diffusion of carbon away from the catalyst surface , leading to a lower supersaturation developing close to grain boundaries . the carbon supersaturation feeds the nucleation of graphene domains , with higher nucleation densities occurring in regions of higher supersaturation , such as the middle of catalyst grains ( figure 6c ) . initially , the ready supply of carbon from the supersaturation results in growth of the nucleated domains being interface - attachment - limited , with facets growing at constant velocities determined by the attachment barrier ( figure 6d ) . the shape of the graphene domains is thus determined by the interfacial mobility , which is a function of both the orientation of the graphene lattice and the underlying catalyst , with the lowest mobility facets persisting . over time , the local carbon supersaturation in front of a growing facet can become depleted if it is not sufficiently replenished , and growth will then transition toward being diffusion - limited ( figure 6e ) . in this regime , the interfacial mobility will still influence the growth velocity in a given direction ; however , the role of any anisotropy in surface diffusion will be increasingly dominant , and the domain shape can evolve toward having facet normals aligned with slow diffusion directions . this is exemplified close to catalyst grain boundaries where the local curvature of the surface results in a high density of step edges aligned with the grain boundary , which are barriers to surface diffusion , leading to rapid diffusion parallel to the boundary and the dramatic widening of the graphene domain in this direction ( figure 5k ) . as growth proceeds and neighboring graphene domains grow close to one another , their diffusion fields overlap , and the bare catalyst surface available for precursor dissociation reduces , leading to growth of facets becoming dissociation - limited ( figure 6f ) . ( a c ) on the initially clean catalyst surface ( a ) , carbon precursor dissociation delivers a flux of carbon , which is initially matched by diffusion into the catalyst bulk ( as indicated by red arrows ) , giving a notable incubation time ( b ) . as exposure continues , the catalyst becomes locally filled with carbon close to the surface and a supersaturation develops , which feeds the nucleation of graphene domains ( c ) . red arrows illustrate the fluxes of carbon arriving at the catalyst surface and diffusing into the bulk and grain boundaries . rapid grain boundary diffusion ( large red arrows ) lowers the supersaturation and , thus , nucleation density close to grain boundaries . ( d f ) the supersaturation developed prior to nucleation provides a ready supply of carbon that results in domain growth initially being interface - attachment - limited ( d ) , but as this supersaturation is locally depleted in front of a growing facet , surface diffusion becomes rate - limiting ( e ) . as the graphene domains grow further and begin to merge , the catalyst surface available for precursor dissociation diminishes and growth becomes dissociation - limited ( f ) . the colored arrows represent precursor dissociation , surface diffusion , and interface attachment ( processes 13 identified in figure 2 ) , with the red arrows indicating which of these processes is rate - limiting at each stage . importantly for polycrystalline catalysts , the variations in precursor dissociation rate with grain orientation and the rate of carbon removal at different grain boundaries lead to an inhomogeneous carbon distribution across the catalyst surface , meaning that the rate - limiting growth process not only varies with time but can vary across different regions of the catalyst . this is observed even on very local scales , for different graphene domains within a catalyst grain , and even for individual graphene domains where the growth of different facets can show different rate - limiting steps . this has wider implications for the interpretation of growth dynamics , where it can not necessarily be assumed that a certain process will be rate - limiting throughout growth or even across the catalyst surface . therefore , care must be taken when using global metrics ( e.g. , areal growth rate ) to quantify growth dynamics as the contributions of facets with different rate - limiting steps may be convolved , and the underlying origin of the growth behavior may only be apparent from more local measures . the inhomogeneous carbon distribution on the catalyst surface and the dependence of interfacial mobility on graphene lattice and catalyst orientation violates major assumptions of the established jmak equation for crystal growth , including that the nucleation rate must remain constant and homogeneous across the surface and that the growth rate must be constant and isotropic . indeed , our results show that even for selected regions of the catalyst surface , the jmak equation does not adequately describe growth . we therefore suggest that in modeling cvd graphene growth on polycrystalline catalysts , orientation - dependent precursor dissociation , grain boundary diffusion , and the different rate - limiting steps that can exist during growth must all be taken into account . while our results herein relate to polycrystalline pt , which has a notable carbon solubility ( 1.1 atom % at 1000 c ) at the growth temperatures used , polycrystalline cu remains the most widely used catalyst for graphene cvd and has a much lower bulk solubility ( 0.00070.0280 atom % at 1000 c ) . for both catalysts , isothermal graphene growth is observed , with the supply of carbon by precursor dissociation filling the catalyst with carbon close to its surface until a supersaturation develops that feeds graphene nucleation . the associated incubation time will depend on the catalyst s carbon solubility , the rate of carbon supply by precursor dissociation , and the rate of carbon removal into the catalyst , determined by its permeability ( the product of solubility and diffusivity ) . despite the low solubility of carbon in cu , recent literature suggests a relatively large carbon diffusivity , and thus , the permeability and therefore the rate of carbon removal from the surface of cu may not be so drastically different from that of pt . nevertheless , for all catalyst surfaces , a carbon supersaturation will develop prior to graphene nucleation , which feeds subsequent growth . the surface carbon concentration close to a graphene facet will therefore decrease with time as the facet grows , leading to time - dependent interfacial velocities and , thus , violation of a key assumption of the jmak equation . for cu in particular , the numerous observations of nonisotropic graphene domain shapes further highlights that the jmak equation is inappropriate for describing growth . instead , we emphasize that in modeling growth on cu , grain boundary and surface diffusion are expected to play important roles regardless of the precise extent of carbon removal through bulk diffusion , and thus , their influence on surface carbon distribution should be carefully considered . in conclusion , we have shown that for polycrystalline catalyst foils under realistic graphene cvd conditions , the rate - limiting steps in growth can vary throughout the process and across different regions of the catalyst surface . the dependence of precursor dissociation on catalyst grain orientation and the different diffusion pathways ( surface , grain boundary , and bulk ) that exist on polycrystalline samples are found to play key roles in the observed growth behavior , affecting the distribution of carbon species across the surface and , thus , the rate - limiting growth process . the supersaturation developed prior to graphene domain nucleation feeds an initial interface - attachment - limited growth period , which transitions to diffusion - limited growth as this supersaturation is depleted . as the domains begin to approach one another and the available bare catalyst diminishes , precursor dissociation becomes limiting . when growth is interface - attachment - limited , the graphene domain shape is defined by the interfacial mobility , which can vary as a function of both graphene lattice orientation and the catalyst grain orientation . in diffusion - limited growth , surface diffusion plays a more - dominant role in determining domain shape and is influenced by the catalyst surface morphology , including the presence of step edges and grain boundaries . this highlights that controlling the catalyst texture and surface morphology is key to controlling the domain structure of the graphene film produced . our observations and analysis provide new insights into how the structure of cvd graphene films is intimately linked to that of the underlying catalyst , and the concepts developed can serve as a general framework for understanding the growth of 2d materials on polycrystalline transition metal catalysts . in situ sem experiments are performed using a commercial environmental sem ( fei quantum 200 , base pressure of 10 mbar ) with a custom ir laser heating stage and with gas supplied through a leak valve . temperatures were measured with a type - k thermocouple spot - welded to the sample and have an estimated uncertainty of 50 c . samples were imaged using an everhart thornley detector and an acceleration voltage of 5.0 kv during pretreatment and growth , while the cvd atmosphere was monitored by a mass spectrometer ( pfeiffer omnistar ) . each full - image frame is acquired by raster scanning from top left to bottom right and takes 9.5 s to acquire . the image sequences analyzed in figures 24 correspond to smaller regions taken from full - image frames and thus have scan times of 1 , 2 , and 0.5 s , respectively . low - magnification images of the samples taken at several points during each growth experiment show that regions around the imaged area have similar extents of growth , indicating that the contribution of electron - beam induced effects does not overwhelm the dominant cvd growth behavior . the assignment of single - layer graphene based on in situ sem contrast is confirmed by extensive growth calibrations in which ex situ sem ( zeiss sigmavp , 12 kv , in - lens detector ) images of the as - grown graphene on pt are correlated with optical microscopy and raman spectroscopy ( renishaw raman invia microscope , 532 nm excitation ; see figure s3 ) measurements following transfer to sio2 ( 300 nm)/si substrates using an electrolysis - based bubbling technique , as previously described elsewhere . ebsd measurements were performed ex situ in a fei helios dual - beam microscope ( 515 kv , current of 5.5 na , working distance of 5.06.5 mm , and sample tilt of 60 with respect to the electron beam ) with an oxford instruments hkl ebsd nordlys ii detector in spot mode using channel 5 software . for quantitative analysis , the sem images were first binarized with an appropriate threshold . domain areas were determined by summing over those pixels belonging to the graphene domains . a pair of copies were made of each binary image , and these were then stacked upon one another to create a pseudo-3d structure . the height of this structure measured one unit , while the length and width were given by the image dimensions . then , the 3d structure was meshed ( represented by a sequence of triangles and vertices ) . to remove any staircasing artifacts , the mesh was smoothed by a minimal number of iterations of mean curvature flow . the normal of a given mesh triangle was given by the curl of its edge vectors , and its velocity was calculated using the nearest - neighbor approach . all codes were written in matlab r2015b and executed on a mac pro 3.5 ghz , 12 core intel xeon system with 64 gb of ram .

down syndrome ( ds ) , trisomy 21 , is the most common chromosomal abnormality in childhood and occurs in 1 in 1,000 births . among several physical and cognitive abnormalities , children with ds also have a 10- to 20-fold increased risk of leukemia relative to other children . additionally , children with ds who are younger than 4 years of age have a 500-fold increased risk for acute myeloid leukemia ( ml - ds ) . nearly half of all diagnosed ml - ds cases are of the acute megakaryoblastic ( amkl ) phenotype . the driver events in ml - ds have long appeared to be unique to patients with ds . reported that nearly all patients with ml - ds share mutations in the essential hematopoietic transcription factor , gata1 , located on the x chromosome . although the specific mutations may vary , all occur in exon 2 , resulting in utilization of an alternate exon 3 initiation site . the functional but truncated protein , gata1s , has a deficient or absent amino - terminal transactivation domain.5 , 6 nearly all children with ds - aml diagnosed before age 4 years have an exon 2 gata1 mutation . although gata1s is sufficient to cause myelodysplasia , progression to ds - aml requires additional mutations , commonly in the cohesin components , ctcf , epigenetic regulators , jak family kinases , mpl , sh2b3 , and ras pathway components . trisomy 21 , gata1s cells are dysplastic and mimic leukemic phenotypes , but are not necessarily leukemic.7 , 8 acquisition of a subsequent leukemogenic mutation is still needed for the malignant transformation . crispr / cas9 causes a blunt - end double - strand break ( dsb ) 3 bp upstream of a pam sequence ( ngg in streptococcus pyogenes cas9 , used in this paper).9 , 10 this dsb is repaired via the natural pathway of non - homologous end joining ( nhej ) , involving resection of individual free ends in a 3 to 5 direction and patching of the break site via alignment and polymerization of microhomology sequences . the effects of crispr / cas9-mediated resection is stochastic , with resection patterns ranging from deletions and insertions ( indels ) 1 bp to hundreds in length.12 , 13 , 14 this variety of gene - editing products is useful in the ablation of whole - gene function , as is the case in creating genetic knockouts . the clinically relevant molecular restructuring of ml - ds presents a unique challenge for gene - editing technologies . instead of the widely used , broad - based strategies of gene knockout or forced insertion , this genetic engineering problem requires a more surgical approach . here , we use the precise gene - targeting capabilities of crispr / cas9 to surgically modify the gata1 gene and alter gene function.16 , 17 , 18 , 19 , 20 herein , we describe the successful creation of gata1s - expressing cells by utilizing a crispr / cas9 system that causes a dsb 2 bp upstream of the met1 initiation site . we also provide detailed analyses for tracking allelic variance after crispr / cas9 editing of exon 2 of gata1 in human cells . the goal of this work is to alter the normal translation start site of the gata1 gene , thereby creating a truncated gata1 variant known as gata1s . the strategy is to use a crispr / cas9 system to disable an atg sequence at the normal met1 translation start site of the gene . this action will enable a second initiation codon to be utilized , one located 84 bases downstream in exon 3 ( met 84 ) . initiation of translation from a transcript starting at met 84 will produce the truncated protein variant known as gata1s . crispr / cas9 activity induces dna cleavage near the met1 site and dna resection through the nhej pathway , eliminating the met1 sequence . this was accomplished through the analysis of two individual transfections performed in k562 cells , producing 74 total clonally expanded populations . figure 1 outlines our experimental strategy and rationale for the mutagenesis of the first translation initiation codon . as seen in the diagram , dna breakage was targeted at a position two bases upstream ( 2 ) from the targeted met 1 site . because nhej occurs on free ends of dsb sites , the adjacency of the crispr / cas9 cleavage to the met1 site is vital to appropriate indel formation and low off - target effects . because the underlying dna sequence is critical for designing active crispr / cas9 , we sequenced 462 bases across the gata1 gene within the region of interest . the dna sequence is presented in figure 2a , and the target atg sequence is shaded . by integrating this information into the analytical crispr design tool website ( http://crispr.mit.edu ) , we generated several possibilities for the crispr / cas9 system ( figures 2b and 2c ) . in addition , information was also gathered that defined a ranking of potential off - target sites for crispr / cas9 . this ranking is based on the equation published by hsu et al . , which utilizes as factors : ( 1 ) weighted combination of mismatches , ( 2 ) location of these sequences within genome ( sequences located within exonic regions of genes are weighted more heavily than those located within intronic sequences ) , and ( 3 ) the steric hindrance created by individual and consecutive mismatches at their specific locations within the overall 20 bp sequence . the off - site target score , out of 100 , and the specific locus are also highlighted in figure 2c . a crispr / cas9 complex that will induce dna cleavage at the 2 bp position relative to the met1 site was chosen because it had the lowest potential for off - site activity , yet would induce a dna break closest to the target site . the crispr / cas9 system , expressed from plasmid px458 , which also carries a single copy of a wild - type egfp gene , was introduced into k562 cells by lipofection . the experimental design is illustrated in figure 3a , with the ultimate analysis of sequence alteration confirmed at both the genotypic and the phenotypic levels . after 72 hr , the cells were sorted for egfp expression using a facsaria , and single cells scoring egfp were placed into individual wells ( figures 3b and 3c ) . individual cells were allowed to expand for a period of 36 weeks , after which time the clonal expansions were isolated for dna and functional protein analyses . figure 3d exhibits a representative sanger sequence trace file , demonstrating the breakdown of the peaks directly downstream of the crispr / cas9 complex cut site . successful dna cleavage of the gata1 gene would be seen if the dna sequence upstream of the cut site appears contiguous without sequence breakdown . in contrast , the dna sequence downstream would be reflected as multiple peaks at the same position , although the spacing of the peaks should remain constant . these predictions are fulfilled and displayed in figure 3d , indicating that specific cleavage by crispr / cas9 at the designated met1 site had occurred . trace files of sanger sequencing obtained from individual clones can be analyzed by the software program tracking of indels by decomposition ( tide ) , to determine the individual sequences within the multi - peaked breakdown product after crispr / cas9 activity . tide utilizes an algorithm that isolates individual sub - sequences determined by similar sub - peak intensity within the breakdown sequence . by aligning each of the sub - sequences with a control parental band , the indel pattern of individual alleles within the clonal population can be determined and exhibited in a simple format . this software allows for the qualitative analysis of individual clonal populations and is a useful intermediate step in overall analysis of the cleavage profile in each clonal population . figure 4 displays the results of sanger sequencing of several individual clonally expanded populations . figures 4a4 g exhibit the variety of altered genomes obtained from clonal populations expanded from individual cells as a result of the crispr / cas9 cleavage activity . g in figures 4 , 5 , 6 , and 7 correspond to various analyses of the same clonal population of crispr / cas9-edited cells ( i.e. , figures 4a , 5a , 6a , and 7a all correspond to analysis from the same expanded population , clone a ) . each clonal population is presented along with its corresponding tide analysis , showing a different indel profile with varying lengths on either or both alleles . the percentage of the mixed - peak sequence falling into each individual sub - sequence is listed above each bar . in our hands and others , crispr / cas9 systems are often efficient enough to execute site - specific dna cleavage in a biallelic fashion , rarely showing activity on only one allele.13 , 24 , 25 figure 4a illustrates the activity of the crispr / cas9 system on a clonal population where one gata1 allele was resected 6 bp , with the other allele resected 4 bp . figure 4b displays another distinct crispr cleavage event , exhibiting one allele apparently uncut , whereas the other allele displays a 22 bp deletion . positioned adjacent to each tide readout is the analysis of sequence decomposition , showing the decomposition of the single - peak sequence in relation to the crispr / cas9 cut site ( indicated by a blue dotted line ) . the increase in sequence breakdown prior to the cut site indicates that resection after crispr / cas9-mediated cleavage occurred both upstream and downstream of the cut site . figure 4c displays another example of biallelic crispr activity , with one allele being resected 4 bp and the other allele 38 bp . this example also reveals signal decomposition prior to the selected cut site , indicating that resection of the 38 bp deletion occurred partially upstream of the cleavage site ( the decomposition extends too far upstream of the cut site to be caused solely by the 4 bp deletion ) . a similar indel pattern can be observed in figure 4d , with both alleles incurring a 5 bp deletion and a 12 bp deletion . figure 4e displays a clone containing only one deletion that could accurately be detected with the tide software . the maximum indel size that can be distinguished by the software is 50 bp , indicating that the second allele contains a deletion larger than 50 bp , with the higher background levels and lower r value being attributed to the tide analysis s attempts to align the second allele to the parental sequence . finally , figures 4f and 4 g both display heterozygosity , with the resection of the affected allele occurring partially upstream of the cut site ; figure 4f contains one 12 bp deletion , and figure 4 g contains a 22 bp deletion . the data given by tide analysis are useful to intermediate data analysis , especially when evaluating clonally expanded populations . however , as seen in figure 5 , utilizing tide as a terminal analytical tool in data analysis can lead to an incomplete analysis of the obtained data . figure 5 demonstrates the further allelic analysis performed using tide data . because resection can occur both upstream and downstream of the cut site , an overall deletion of three or more bases does not implicitly demonstrate that the met1 site was ablated in that allele ; only resection of three or more bases downstream of the cut site is necessary for met1 elimination . this situation is in fact observed in figure 5a ( the individual populations examined in figures 4a4 g correspond with the samples presented in figures 5a5 g ) . tide analysis for figure 5a shows that a 4 bp and a 6 bp deletion occurred in both alleles of the population . this is verified when using the tide data to manually analyze the sanger trace file . by determining which two bases exist at each position in the sequence , the actual , exact sequence of each allele this is an example where , even though the tide analysis alone shows that both alleles showed a deletion greater than 3 bp , one allele exhibited resection entirely upstream of the cut site , leaving the met1 site untouched . this disparity between tide readout and primary data is shown more vividly in figure 5b . the tide analysis determined that one allele has a 22 bp deletion , whereas the other allele is untouched . examining the trace file , however , reveals that the allele determined as unbroken in fact contains a single thymine insertion directly at the cut site , leaving the met1 site intact . the second allele of clone b also displays a different breakage pattern ; the allele contains both a 26 bp deletion combined with a 5 bp insertion ( labeled in orange ) that eliminates the met1 site . clone c , upon initial allelic analysis , exhibited a potential example of a biallelic met1 knockout . allele 1 showed a 3 bp deletion , appearing to eliminate a portion of the met1 site . allele 2 , in comparison , contains a 36 bp deletion , removing a large section of exon 2 including the met1 site . this genetic restructuring , however , was later determined to be incorrect , with allele 1 containing a 2 bp deletion that retained the met1 site . figure 5d shows an example where both alleles contain an insertion and a deletion event . allele 1 contains a 13 bp deletion and a single thymine insertion . however , resection occurred entirely upstream of the cut site , leaving the met1 site intact . the second allele displays a deleted met1 site , with another 13 bp deletion , this time paired with a 6 bp insertion . examining the trace file in figure 5e allows for an accurate determination of the length of the deletion that could not be analyzed with tide . this allele contains a 220 bp deletion , eliminating both met1 and a majority of exon 2 . the other allele contains an intact met1 , with a 5 bp deletion upstream of the cut site , combined with a single thymine insertion . figures 5f and 5 g both show similar sequence decomposition , with one allele with a single thymine insertion and the other allele containing a deletion . figure 5f contains an 11 bp deletion on the second allele , but leaves the met1 site intact . figure 5 g , on the other hand , shows ablation of the met1 site on its second allele , with a 21 bp deletion both upstream and downstream of the cut site . in both of these examples , thymine insertion is seen in every + 1 insertion event analyzed across all 73 isolated clones . the individual allelic analysis of each of the 26 clonally expanded populations is shown in figure s1 . the wild - type gata1 gene transcript is modified into two splice variants in wild - type k562 cells : one splice variant , including exon 2 , encodes full - length gata1 ; and the other , with exon 2 excluded , encodes gata1s.27 , 28 because of this , wild - type k562 cells show expression of both long - form and short - form gata1 mrna ( figure 6 ) , with levels of expression weighted toward the short - form mrna . clones a and e exhibit severe knockdown , although not complete elimination , of full - length gata1 mrna expression , exhibiting highly weighted expression of the gata1s - encoding mrna splice variant . the crispr / cas9-mediated resection shows less effect on the expression profile of clones b , c , and g , which exhibit transcription profiles similar to wild - type k562 cells . clones d and f display similar expression profiles , with relatively equal levels of long - form and short - form transcripts . these variants were sequenced in order to determine which allele is being expressed in each clonally expanded population ( figure s2 ) . sequences obtained from each sample determined that each short - form gata1 mrna sequence encoded an identical gata1s splice variant , lacking exon 2 . the protein expression levels of gata1 and gata1s , analyzed via western blot , are displayed in figure 7 . in wild - type k562 cells , the mrna expression profile is weighted toward expressing short - form mrna ( figure 6 ) . the protein expression , in comparison , is skewed heavily toward full - length gata1 . this demonstrates that in k562 cells , the full - length gata1 mrna is translated at a higher efficiency than the short - form splice variant . clone f shows a similar characteristic ; although both variants exhibit at comparable amounts at the mrna level , the protein expression profile is heavily weighted toward full - length gata1 expression , with very little gata1s formed . another example is observed in data from clones a and e ; although there is virtually no long - form mrna expressed in these populations , the protein levels of gata1 and gata1s are equal . clone b , in comparison , maintains its mrna expression profile at the protein level ; both profiles are weighted toward the short - form transcript and gata1s , respectively . this is also seen in clone d , where equal levels of gata1 and gata1s are observed . finally , clones c and g exhibit high knockdown of full - length gata1 , although their transcription profiles display the presence of low levels of gata1 mrna . figure 8 displays the allelic analysis and protein expression of two important clones , generated from a second transfection under the same protocol , with the tide and allelic analysis of clones 67 and 72 ( figure 8a ) . the dna sequence of each clone reveals an elimination of the gata1 met1 initiation site . clone 67 displays a 36 bp deletion in allele 1 and a 34 bp deletion in allele 2 . again , the discrepancy in deletion length with the tide results is expected , as the spacing of residue outputs of the sanger sequencing was not constant . clone 72 shows a 20 bp deletion in allele 1 and an 18 bp deletion in allele 2 . because spacing of the sanger sequence was constant , the tide results aligned with the actual results of allelic analysis . the top panel in figure 8b displays the complete ablation of long - form gata1 in a patient sample from a child diagnosed with ml - ds , whereas the bottom panel shows complete ablation of long - form gata1 and shift of expression to gata1s in clones 67 and 72 ( normalized values of full - length gata1 : 0.0055 and 0.051 , respectively ) . this result is verified in figure 8c . expression of full - length gata1 and gata1s was measured by densitometry and normalized to the actin levels of their loading control . the full - length gata1 expression of both clones fell within background levels , indicating complete abrogation of the full - length gene product . clone 67 displayed a nearly 10-fold increase in gata1s expression compared with wild - type , and clone 72 displayed a 4-fold increase . a mutation in the gata1 gene is observed in almost all cases of ml - ds diagnosed before 4 years of age.29 , 30 a particular class of mutations that account for a majority of these cases results in the exclusive or near - exclusive expression of the gata1s splice variant of the protein , lacking the first 83 aa of the full - length protein . the main objective of this work was to develop a model cell line for the analysis of downstream genetic instability triggered by the ablation of full - length gata1 expression . the intrinsic pathway that leads to the knockout ability of crispr / cas9 is nhej , a stochastic process . as such , using a specifically designed crispr / cas9 system can lead to a multitude of unique products of dna resection at the dsb site.12 , 13 , 25 , 31 because of this , analysis of genetically modified clones on a cell - by - cell basis is necessary in order to verify products expressing the appropriate met1 deletion . through this process the efficiency of crispr / cas9 comes from the sequence of the sgrna component of the complex . as such , sgrna sequences must have a perfect match to the sequence at the target site , with the dsb as close to the site of change as possible . to minimize the potential of the crispr / cas9 to cleave off - target sites the protospacer used in this experiment ( 5-gttaatccccagaggctccatgg-3 ) was used with an off - target score of 63 . perfect on - target homology results in a score of 100 , with the introduction of mismatches and their location in the seed sequence relative to each other , and the sequence as a whole returning a score lower than 100 . the highest single off - target score obtained was 2.5 , with the highest exonic off - target site scoring a 2.3 . because of its lack of significant off - target sites and proximity to the met1 site , this protospacer was chosen to be used in the experiment . the collection of 25 clones exhibited a variety of different insertion / deletion ( indel ) profiles , as seen in figures 4 , 5 , 8 , and s1 , respectively . although most clones display vastly different resection patterns , even between alleles , there was one recurring indel motif in many clonal populations . of the 25 isolated clones , 19 populations contained at least one allele where a single thymine insertion occurred at the dsb site , with 9 of them being homozygous , containing the single thymine insert on both alleles . four more clones contained a single thymine insertion along with a deletion of various lengths . in every case with a single base insertion , in fact a distinct theme is seen in clones b , f , and g regarding the disparity between tide analysis in figure 4 and allelic analysis in figure 5 . during tide analysis , the mixed - peak sample is broken down into each of the constituent decomposition products , with each of these decomposition products being compared to determine what the indel profile of each clone contains . however , the products are determined based on sequences of homology on either side of the cut site within each of the decomposition products ; any mismatches that occur outside these homology sequences are not utilized in the determination of indel length . because of this , misaligned sanger sequencing has the potential to return tide analysis results that show indels 1 bp shorter or longer than the actual indels . the allelic analysis reveals that clones b , f , and g in fact contain a single thymine insertion in each of the alleles that the tide analysis displayed as wild - type . a related issue can be seen analyzing crispr activity that leads to a combination of insertions and deletions , as seen in clones b and d. because sequence length between each of the alignment windows is the determining factor in indel length , only the net size of the indel is returned in the tide analysis . in clone b , a 22 bp deletion is observed in the tide analysis . allelic analysis reveals that this deletion is actually 27 bp long , compounded with a 5 bp insertion ; the net change in the sequence length is 22 bp . both issues are compounded in clone d , where a combination indel combined with atypically spaced sanger sequencing . this situation leads the tide software to return a deletion of 5 bp when the allele in question obtained a deletion of 13 bp compounded with an insertion of 6 bp . in this clone , the combination of misspaced sanger sequencing , deletion , and insertion leads to a predicted deletion 2 bp shorter than the net change in sequence length and 8 bp shorter than the actual deletion . when utilizing the software to analyze crispr activity in populations of cells , or when looking for overall gene knockouts , the tide software may be able to be used as a terminal stage of data analysis . both of these levels of analysis allow for a degree of latitude in the exact size of indels formed . tide is still incredibly useful when employing crispr / cas9 to create a more exact change , such as in this experiment . however , the tide readout must then be further investigated to obtain a more exact allelic profile and can not be used as the terminal step in data analysis . figure 6 demonstrates the effects of the crispr / cas9-mediated resection on the expression levels gata1 and gata1s in each of clones a gata1 in wild - type k562 cells is expressed in two splice variants , both with and without exon 2 . because exon 2 contains gata1 s met1 site , the smaller splice variant codes for gata1s naturally , whereas the longer variant encodes full - length gata1 . expression of both splice variants of gata1 is seen in cell lines and patient samples.27 , 28 , 32 , 33 , 34 we observe this same expression profile in our samples , with the various crispr / cas9-mediated resections affecting the balance between long - form and short - form gata1 expression consistent with mrna and protein expression profiles seen in various ml - ds patients with a variety of different mutations at the met1 site.27 , 28 , 33 in each clone , the short - form mrna sequence was identical to the wild - type ( the exon 1 to exon 3 splice site is shown in figure s2 ) . the expression of genes on the x chromosomes is tempered through the process of lyonization . this chromosome is replicated normally during s - phase ; furthermore , the same x chromosome is inhibited in each generation of daughter cells . however , this is not the case for all genes ; depending on the cell , up to 15% of x - linked genes can escape inactivation . this effect can be seen in several of the sequences of the long - form mrna amplicons ( data not shown ) . figure 7 shows the protein levels of gata1 and gata1s in each of the clones . when compared with the mrna expression profiles , it becomes apparent that the mrna for full - length gata1 is preferentially expressed as compared with gata1s mrna . clone c highlights an important issue that is often overlooked in the generation of cell lines using this more surgical form of gene editing . when the overall goal is to modify precisely , rather than totally abrogate , gene expression , a detailed allelic profile must be generated at both the genotypic and the phenotypic levels ; a population - based genotyping without clonal expansion could lead to a level of insufficient data with which to make valid conclusions . for example , our own initial allelic analysis of clone c indicated that a 3 bp deletion had occurred , and that the initial adenine residue had been resected . however , mrna and protein expression analysis determined that the clone in fact was affected with only a 2 bp deletion in allele 1 , allowing the met1 site to remain intact . because of this lack of a biallelic knockout in all 25 of the initial clones , a second transfection was performed , with a further 48 clones being successfully isolated . it was from this transfection that clones 67 and 72 , both biallelic knockouts , were generated . expression of full - length gata1 was completely ablated , with the corrected gata1 protein levels falling well within background levels . furthermore , expression of gata1s was augmented greatly in the clone , with a 9.9-fold increase in gata1s compared with wild - type . this profile matches the gata1 expression profile of the ml - ds patient sample , indicating the success of the genetic surgery strategy . clone 72 also displayed complete abrogation of full - length gata1 and a 4.0-fold increase in gata1s expression . the differences in the degree of resection of the gata1 gene between both clones open the possibility of disruption of the 3d orientation of the gata1 promoter sequence . this , combined with the magnification of an individual cell s minor genetic or epigenetic variations as a product of clonal expansion , confound the determination of an exact mechanism for the differing expression profiles . both clones show complete elimination of full - length gata1 , and so both clone 67 and clone 72 are evidence of the success of this genetic surgery . however , the higher levels of gata1s protein expression in clone 67 elevate the clone to the level of replicating the gata1 expression profile of the patient sample . the protein expression profiles of the clones demonstrate that the long - form gata1 mrna is preferentially translated . the exact mechanism behind this is unclear , but may be related to the stronger kozak sequence located proximal to the met1 site , in comparison with the met84 site . at the met1 site , the 4 , 2 , 1 , and + 4 sites of the start sequence match that of the consensus kozak sequence , whereas the met84 site match only at the 4 and + 4 sites.36 , 37 this increased homology to the consensus kozak sequence may be a contributing factor to the preferential translation of full - length gata1 . furthermore , altering of these kozak sequences through crispr / cas9-mediated resection may have altered the translation efficiency of the resulting mrna transcripts , even if the met1 site itself remains intact . in clones a and e , the proportional levels of long - form mrna are much lower than the amount of full - length gata1 protein being expressed . clones c and g , in contrast , showed lower levels of full - length gata1 protein than would be expected , given the amount of long - form mrna seen in figure 6 . interestingly , the mrna expression profiles and the protein expression profiles are identical in clones b and d. the crispr / cas9-mediated resections that occurred led to a variety of combinations of exonic splice - site variations and kozak sequence permutations , leading to such vastly different proportions of full - length gata1 and gata1s . although the variable effects of crispr / cas9-mediated knockouts have been explored extensively , the levels and identity of indel formation on a larger scale require further investigation . these findings may hold value in examining the identity and efficacy of oligonucleotide - mediated gene manipulation or insertion , or in the utilization of crispr / cas9 alone as a surgical tool for gene editing . these cells can now be used to determine downstream mutations as a result of an altered start site in the process of leukemogenesis . furthermore , our experimental approach is of significant value in studies of ds - induced pluripotent stem cells ( ds - ipscs ) to elucidate a pathway that reveals where genetic hits occur to enable ml - ds . our long - term goal is to examine how these hits can occur in non - transformed cells and to develop a more clinically relevant mechanism for identifying druggable pathway targets . k562 cells were treated with a crispr / cas9 system designed to cleave 2 bp upstream of the gata1 met1 initiation codon site . the population was enriched for cells that successfully took up the crispr plasmid , and the cells were clonally expanded after single - cell isolation . upon expansion , genomic dna was extracted from each clonal population and analyzed via sanger sequencing and tide analysis . clones exhibiting varied degrees of crispr / cas9-mediated resection were analyzed for mrna and protein expression via rt - pcr and western blot , respectively . k562 cells were cultured in iscove s modified dulbecco s medium ( atcc ) , supplemented with 10% fetal calf serum and 1% penicillin / streptomycin . for transfections , k562 cells were seeded in six - well plates at a density of 5 10 cells / well in 1.5 ml of complete media . transfection complexes were formed at room temperature in 500 l of opti - mem according to the optimized ltx protocol ( thermo fisher scientific ) and added drop - wise to k562 cells , followed by 72 hr incubation . cells were sorted into a 96-well plate with a facsariaii flow cytometer ( bd biosciences ) , with an individual positively transfected cell sorted to each well . clones were expanded into larger plates as the individual clones reached confluence , with dna isolation occurring when cells reached confluence in a six - well plate ( 1 10 cells / ml ) . the gata1 gene sequence was entered into the zhang lab s online generator ( http://crispr.mit.edu/ ) , and the appropriate crispr guide sequence that binds in close proximity to target ( 2 bp upstream of met1 ) was chosen . the crispr was constructed using standard cloning methods following the latest oligo annealing and backbone cloning protocol with single - step digestion - ligation . the crispr guide sequence was cloned into the px458 backbone vector ( plasmid 48138 ; addgene ) , a human codon optimized pspcas9 and chimeric guide rna expression plasmid with a 2aegfp , purchased through addgene ( https://www.addgene.org ) . following construction , cellular gdna was isolated from pellets of 2 10 k562 cells using the qiagen dneasy blood and tissue kit ( cat . pcr was performed using amplitaq gold fast pcr master mix ( thermo fisher scientific ) on isolated gdna , with amplification parameters optimized for an amplicon size of 462 bp ( forward [ fwd ] primer : 5-gggaggtgggaaggagaaatatggag-3 , reverse [ rev ] primer : 5- cctcacagtggtattctgacctagcc-3 ; obtained from integrated dna technologies ) . amplicon size was verified on 1% agarose gel , and pcr products were verified by dna sequencing by eton bio incorporated . for rt - pcr , total cellular rna was isolated using trizol reagent from thermo fisher scientific ( cat . cdna was synthesized from total rna using iscript cdna synthesis kit from bio - rad laboratories ( cat . purified cdna was then amplified using amplitaq gold fast pcr master mix ( thermo fisher scientific ) , optimized for differentially sized amplification of long - form and short - form gata1 cdna conversions ( fwd rt primer : 5- gatcacactgagcttgccacatcc-3 , rev rt primer : 5- gcactattggggacagggagtg-3 ; obtained from integrated dna technologies ) . mixed amplicons were separated on a 1% agarose gel , and individual bands were excised and purified using a qiaquick gel extraction kit ( cat . protein isolation from individual clones was performed using sds lysis buffer , with protein concentrations determined and normalized via the bio - rad dc protein assay ( cat . western blot was performed using standard procedures and transferred to nitrocellulose membrane using the bio - rad mini transblot cell ( cat . 4591 ; cell signaling technology ) , with tubulin being stained with rabbit anti - tubulin unconjugated antibody ( cat . secondary antibody used to visualize protein was anti - rabbit igg , hrp - linked antibody ( cat . i d rpn2232 ; ge life sciences ) on x - ray film , with an exposure time of 10 s. tide analysis was performed on all sanger sequences obtained from all clones as part of allelic analysis . the p value used to determine significance of potential sequence decompositions was p = 0.001 . designed the experiments . k.m.b . performed the transfection and culturing of the clonal cell populations before and after single - cell sorting .

glaucoma is treated initially with medical therapy or laser procedures . for patients who are unresponsive , surgical treatments may be necessary , and traditionally these include trabeculectomy and tube shunts . if these procedures fail , options to decrease intraocular pressure ( iop ) are limited , and either repeat filtering surgery or cyclophotocoagulation can be considered . common filtering procedures such as trabeculectomy and tube shunts lower iop by shunting aqueous humor from the anterior chamber of the eye to the sub - conjunctival space . unlike these methods , the gold shunt exploits an alternate pathway , shunting fluid from the anterior chamber directly into the suprachoroidal space . the gold shunt is a glaucoma drainage implant made of 99.95% gold , measuring 6 mm by 3.5 mm by 0.12 mm , and contains a series of channels to allow for fluid shunting from the anterior chamber . the head of the device is placed in the anterior chamber with the tail placed into the suprachoroidal space . drainage through this route is possible due to a hydrostatic pressure gradient between these compartments.1,2 the gold shunt has only been approved for use in certain european countries since 2005 . health canada approved gold shunt use in 2009 , and in the united states the gold shunt is currently under phase iii clinical trials . here , we report a unique case of gold shunt surgery with long - term follow - up in a challenging patient with refractory , advanced , low - tension glaucoma , at risk of losing remaining central vision after multiple failed filtration surgeries previously . a 50-year - old male electrical engineer was referred to the glaucoma unit at st . michael s hospital on october 20 , 2010 , for left uncontrolled low - tension glaucoma on maximum medical therapy . his past ocular history included a right eye trabeculectomy , followed by a right ahmed valve placement and right cataract extraction with lens implantation in 2006 . in the left eye , a diagnosis of low - tension glaucoma had been previously established by the referring glaucoma specialist . the patient had progressive visual field loss despite iop in the teens , with multiple failed filtration surgeries . his family history was negative for glaucoma , and he had a history of remote inhaled steroid use of several months duration . past medical history included a resolved tuberculosis infection 16 years earlier . at the time of his visit , he was using brinzolamide 1% tid ou , brimonidine bid ou , timolol 0.5% once am ou , and bimatoprost 0.01% at hs ou . on examination , best corrected visual acuity was 20/30 od and 20/40 os and iops were 12 mmhg od and 16 mmhg os at 12:35 pm . ocular motility was full , and pupils were equally reactive to light with no afferent pupillary defect . corneal thicknesses were 574 and 576 m in the right and left eyes , respectively . the right anterior chamber showed a tube entering at the 11 oclock position and the left anterior chamber showed a tube entering at the 4 oclock position in the presence of iridotomies superiorly . angles were wide open , and right posterior chamber intraocular lens was in good position . right optic nerve head revealed severe cupping , and the left optic nerve revealed a 0.8 cup with superonasal neuroretinal rim remaining . visual field tests showed right superior hemifield loss splitting fixation and left superior arcuate changes approaching fixation . left visual field progression over 2 years was documented ( figure 1a and b ) . in view of uncontrolled , end - stage , low - tension glaucoma with advanced progressive field loss and iop in high teens on maximal medical therapy , the options of trabeculectomy with or without cataract extraction and intraocular lens implantation were discussed in detail with the patient . after discussing the benefits and risks of the procedure , the patient elected to proceed with combined left trabeculectomy , cataract extraction , and intraocular lens implantation with mitomycin c. the surgery was performed on november 30 , 2010 . several weeks after the surgery , he underwent bleb needling due to rapid scarring and remained uncontrolled despite reinstituting maximal medical therapy . the option of gold shunt surgery was discussed to bypass conjunctival scarring issues , including the benefits and risks , and the patient elected to proceed with gold shunt surgery . on february 22 , 2011 , left uncomplicated gold shunt surgery was performed . on the first day after the surgery , best corrected visual acuity was 20/200 os , iop measured 6 mmhg os at 4:20 pm , with trace anterior chamber reaction . left eye visual acuity improved , and iop remained low over the first 3 months . on july 13 , 2011 , best corrected visual acuity was 20/20 od and 20/30 + 2 os . intraocular pressures remained mostly in the low teens , using two glaucoma medications in the right eye only . the gold shunt remained in good position and glaucoma appeared stable ( figure 2a and b ) . over a 5-year follow - up period , visual acuity ( va ) , iops , and glaucoma remained stable . on may 27 , 2015 , best corrected visual acuity was 20/30 + 2 od , 20/252 os , and iop measured 14 mmhg ou at 11:35 am , with no changes in medication . the gold shunt continued to be in good position , and glaucomatous optic neuropathy remained stable ( figure 3a and b ) . visual fields remained stable throughout the period of follow - up ( figure 4a d ) . at his last visit on october 26 , 2015 , iop measured 10 mmhg ou and 12 mmhg os , with no changes in medication . st . michael s hospital research ethics board approval was obtained for this case report , and the patient provided written informed consent . in this challenging case of a young gentleman with low - tension glaucoma , advanced visual field loss , and history of multiple failed glaucoma filtering procedures , the gold shunt effectively reduced iop without need for glaucoma medications , over 5 years . the efficacy and complication rate between standard surgical interventions , such as trabeculectomy and tube shunts , are similar at 68% and 70% , respectively , at 4 years.35 glaucoma care is significantly challenged when either of these procedures fail and particularly in patients with good visual potential . in these cases , information on the success rate of a second filtration surgery is limited . in pediatric populations , however , repeat procedures of either type after failed trabeculectomy report success rates of 69% and 64% for trabeculectomy and ahmed valve placement , respectively.6 cyclophotocoagulation is indicated for patients with refractory glaucoma and limited visual potential due to higher risks of suprachoroid hemorrhage and hypotony.79 sequential tube shunt success is reported to range from 62% to 75% , similar to cyclophotocoagulation success rates of 66% . placement of a second tube shunt after primary tube failure has a low reported success rate of 40%.10 gold shunt surgery is generally reserved for patients with low visual potential and has success rates ranging from 67% to 79%11,12 , comparable to that of ahmed valves.13 shunt failure and complications may be due to a variety of factors , including shunt fibrosis possibly from cytokines , varying surgical techniques , and use of older gold shunt models.1417 this patient continues to be well controlled 5 years after gold shunt surgery . after previous failed traditional filtration surgeries , the option of gold shunt in the suprachoroidal space may also be considered for patients needing lower iop in the presence of good central vision .

public reporting of hospital readmissions for heart failure ( hf ) underscores its importance as a measure of quality of care and as a metric for medicare reimbursements . among different racial / ethnic groups , older black and hispanic patients have been reported to have higher 30day readmission rates compared with whites , in part related to the hospitals where care was provided . although these data indicate racial / ethnic disparities in hf readmissions , lack of longer followup data has limited the evaluation of how shortterm rehospitalizations may impact longterm outcomes . a statewide study documented that minorities were more likely to be rehospitalized and were paradoxically less likely to die within 1 year , yet it remains unknown if these results are similar across the united states . a postulated explanation for these observations is that hf hospitalization in minority populations may not necessarily imply more severe disease but rather may result from poorer access to followup care and/or poorer understanding of outpatient management of their symptoms and medications . understanding racial / ethnic differences in hf rehospitalizations and mortality may be crucial in helping to guide initiatives aimed at promoting health equity , reducing the financial burden of readmissions , and improving outcomes . we sought to evaluate the hypothesis that in contrast to white patients , black , hispanic , and asian patients have more comorbid diseases and higher rates of short and longterm readmissions but better short and longterm survival . our 2 main objectives were to ( 1 ) compare 30day readmission and 1year mortality rates for hf among these 4 race / ethnic groups and ( 2 ) examine the impact of clinical profile and socioeconomic status ( ses ) variables on hf outcomes stratified by race / ethnicity . data for this analysis were derived from the get with the guidelines ( gwtg)hf registry linked with medicare inpatient data . the gwtghf registry is an ongoing national , voluntary program formed to improve quality of care for patients hospitalized with hf . data elements , including patient characteristics , medical history , medications , laboratory data , contraindications to treatment , inpatient care , outcomes , and hospital characteristics , were entered into an online interactive case report form and patient management tool ( outcome sciences , inc , a quintiles company ) . using standardized definitions , race / ethnicity data were recorded by patient selfreporting by admissions or medical staff during registration . race was recorded as part of a multiplechoice data entry tool ( ie , white , black , asian , american indian / alaska native , and native hawaiian / pacific islander ) . a separate data element for hispanic ethnicity ( ie , yes versus all participating institutions were required to comply with local regulatory and privacy guidelines and , if required , to secure institutional review board approval . because data were used primarily at the local site for quality improvement , sites were granted a waiver of informed consent under the common rule . through an internetbased system , the duke clinical research institute served as the data analysis center and examined the aggregate deidentified data for research purposes . the medicare data included part a ( inpatient ) claims and the associated denominator file . medicare inpatient claims data from january 1 , 2005 , through december 31 , 2011 , were linked with data from the gwtghf registry , matching by admission and discharge dates , hospital , date of birth , and sex . the study population for this analysis was based on those patients who ( 1 ) came from hospitals in gwtghf registry that were fully participating , ( 2 ) had a principal hf diagnosis , ( 3 ) were 65 years old with a gwtghf registry hospitalization linked to medicare , ( 4 ) were discharged between january 1 , 2005 , and december 31 , 2011 , ( 5 ) were enrolled in feeforservice medicare at discharge , and ( 6 ) whose race / ethnicity were recorded as white ( nonhispanic white ) , black ( nonhispanic black ) , hispanic , or asian ( nonhispanic asian / pacific islander ) . if multiple hospitalizations exist for a patient , the first hospitalization was selected as the index hospitalization for this study . for outcomes ( ie , length of hospital stay and mortality ) assessed post index admission , all patients after the aforementioned exclusions were analyzed . for outcomes ( ie , readmission , hospital stay , mortality , and composite of mortality / readmission ) post index discharge , the analysis was further limited to those who were discharged alive but did not leave against medical advice and were not discharged / transferred to either another shortterm hospital or hospice or had discharge destination missing . the primary outcome measures were 30day and 1year rehospitalization , length of hospital stay , and mortality . more specifically , we examined allcause readmission post index discharge ( 30day and 1year ) , cardiovascular readmission post index discharge ( 30day and 1year ) , allcause mortality post index admission ( 30day and 1year ) , allcause mortality post index discharge ( 30day and 1year ) , and composite of mortality and allcause readmission post index discharge ( 30day and 1year ) . in addition , we assessed days in hospital for index admission and hospital stay within 30 days or 1 year post index discharge . readmissions excluded the index hospitalization claim , transfers to or from another hospital , and admissions for rehabilitation . there are 2 study subpopulations : 1 for mortality and hospital stay post index admission and the other for mortality , readmission , composite of mortality / readmission , and total hospital days post index discharge . baseline characteristics of study population were described by race / ethnic groups , by using proportions for categorical variables and means with sd values for normally distributed continuous variables and medians with 25th and 75th percentiles for continuous variables with skewed distribution . differences between the 4 race groups were tested by using tests for categorical variables and wilcoxon tests for continuous variables . characteristics of the study population were based on gwtghf registry data and included demographics ( age , sex ) , clinical characteristics , including medical history , vital signs at admission ( systolic blood pressure , heart rate , respiratory rate ) , lab work at admission ( blood urea nitrogen , creatinine , hemoglobin , and sodium ) , ejection fraction , medications at discharge ( angiotensinconverting enzyme inhibitor , angiotensin ii receptor blocker , blocker , digoxin , diuretic , aldosterone antagonist , aspirin , and anticoagulants ) , length of stay > 7 days for the index hospitalization , and year of index hospitalization , and hospital characteristics , namely geographic region , number of beds , teaching status , and rural location . most variables had < 5% missing except brain natriuretic peptide and missing on continuous variables were imputed to median and missing on categorical variables were imputed to dominant level . additionally , observed event rates were described for each race / ethnic group . for mortality , incidence at 30 days and 1 year were calculated by using kaplan the mortality and composite of mortality / readmission were compared between race groups by using logrank tests . for readmissions , incidence at 30 days and 1 year were calculated by using estimates from the cumulative incidence function to account for the competing risk of mortality . the hospital stay days were reported as a continuous variable and compared between the 4 race groups by using wilcoxon tests . finally , the association of race / ethnicity with each outcome was assessed by using adjusted regression modeling for patient and hospital characteristics . for timetoevent outcomes , the adjusted association of race / ethnicity with outcome was assessed by using multivariable cox proportional hazards models , where robust standard errors were used to account for the clustering of patients by hospital . the variables described here , except ejection fraction and discharge medications , were included in addition to the main variable of interest race groups with white as the referent . the adjustment was performed in 3 steps : adjustment for patient characteristics only , adjustment for patient and hospital characteristics together , followed by additional adjustment for ses data . ses variables included mean household income estimate , percentage of persons aged 25 years with 4 years of college ( 20062010 census ) , and percentage of persons aged 25 years with high school diploma or greater ( 20062010 census ) . negative binomial regression model with individual specific offset was performed to examine the association of race / ethnicity and total days rehospitalized . the individual specific offset was determined by logarithm of length of followup until censoring and the hospital stay days outcome was capped by the number of followup days . therefore , the individual specific offset allows model to account for the fact that patients had different length of followup days . other covariates in the model are same as those we used in the analyses of mortality and readmission outcomes . data for this analysis were derived from the get with the guidelines ( gwtg)hf registry linked with medicare inpatient data . the gwtghf registry is an ongoing national , voluntary program formed to improve quality of care for patients hospitalized with hf . data elements , including patient characteristics , medical history , medications , laboratory data , contraindications to treatment , inpatient care , outcomes , and hospital characteristics , were entered into an online interactive case report form and patient management tool ( outcome sciences , inc , a quintiles company ) . using standardized definitions , race / ethnicity data were recorded by patient selfreporting by admissions or medical staff during registration . race was recorded as part of a multiplechoice data entry tool ( ie , white , black , asian , american indian / alaska native , and native hawaiian / pacific islander ) . a separate data element for hispanic ethnicity ( ie , yes versus all participating institutions were required to comply with local regulatory and privacy guidelines and , if required , to secure institutional review board approval . because data were used primarily at the local site for quality improvement , sites were granted a waiver of informed consent under the common rule . through an internetbased system , the duke clinical research institute served as the data analysis center and examined the aggregate deidentified data for research purposes . the medicare data included part a ( inpatient ) claims and the associated denominator file . medicare inpatient claims data from january 1 , 2005 , through december 31 , 2011 , were linked with data from the gwtghf registry , matching by admission and discharge dates , hospital , date of birth , and sex . the study population for this analysis was based on those patients who ( 1 ) came from hospitals in gwtghf registry that were fully participating , ( 2 ) had a principal hf diagnosis , ( 3 ) were 65 years old with a gwtghf registry hospitalization linked to medicare , ( 4 ) were discharged between january 1 , 2005 , and december 31 , 2011 , ( 5 ) were enrolled in feeforservice medicare at discharge , and ( 6 ) whose race / ethnicity were recorded as white ( nonhispanic white ) , black ( nonhispanic black ) , hispanic , or asian ( nonhispanic asian / pacific islander ) . if multiple hospitalizations exist for a patient , the first hospitalization was selected as the index hospitalization for this study . for outcomes ( ie , length of hospital stay and mortality ) assessed post index admission , all patients after the aforementioned exclusions were analyzed . for outcomes ( ie , readmission , hospital stay , mortality , and composite of mortality / readmission ) post index discharge , the analysis was further limited to those who were discharged alive but did not leave against medical advice and were not discharged / transferred to either another shortterm hospital or hospice or had discharge destination missing . the primary outcome measures were 30day and 1year rehospitalization , length of hospital stay , and mortality . more specifically , we examined allcause readmission post index discharge ( 30day and 1year ) , cardiovascular readmission post index discharge ( 30day and 1year ) , allcause mortality post index admission ( 30day and 1year ) , allcause mortality post index discharge ( 30day and 1year ) , and composite of mortality and allcause readmission post index discharge ( 30day and 1year ) . in addition , we assessed days in hospital for index admission and hospital stay within 30 days or 1 year post index discharge . readmissions excluded the index hospitalization claim , transfers to or from another hospital , and admissions for rehabilitation . there are 2 study subpopulations : 1 for mortality and hospital stay post index admission and the other for mortality , readmission , composite of mortality / readmission , and total hospital days post index discharge . baseline characteristics of study population were described by race / ethnic groups , by using proportions for categorical variables and means with sd values for normally distributed continuous variables and medians with 25th and 75th percentiles for continuous variables with skewed distribution . differences between the 4 race groups were tested by using tests for categorical variables and wilcoxon tests for continuous variables . characteristics of the study population were based on gwtghf registry data and included demographics ( age , sex ) , clinical characteristics , including medical history , vital signs at admission ( systolic blood pressure , heart rate , respiratory rate ) , lab work at admission ( blood urea nitrogen , creatinine , hemoglobin , and sodium ) , ejection fraction , medications at discharge ( angiotensinconverting enzyme inhibitor , angiotensin ii receptor blocker , blocker , digoxin , diuretic , aldosterone antagonist , aspirin , and anticoagulants ) , length of stay > 7 days for the index hospitalization , and year of index hospitalization , and hospital characteristics , namely geographic region , number of beds , teaching status , and rural location . most variables had < 5% missing except brain natriuretic peptide and missing on continuous variables were imputed to median and missing on categorical variables were imputed to dominant level . additionally , observed event rates were described for each race / ethnic group . for mortality , incidence at 30 days and 1 year were calculated by using kaplan the mortality and composite of mortality / readmission were compared between race groups by using logrank tests . for readmissions , incidence at 30 days and 1 year were calculated by using estimates from the cumulative incidence function to account for the competing risk of mortality . the hospital stay days were reported as a continuous variable and compared between the 4 race groups by using wilcoxon tests . finally , the association of race / ethnicity with each outcome was assessed by using adjusted regression modeling for patient and hospital characteristics . for timetoevent outcomes , the adjusted association of race / ethnicity with outcome was assessed by using multivariable cox proportional hazards models , where robust standard errors were used to account for the clustering of patients by hospital . the variables described here , except ejection fraction and discharge medications , were included in addition to the main variable of interest race groups with white as the referent . the adjustment was performed in 3 steps : adjustment for patient characteristics only , adjustment for patient and hospital characteristics together , followed by additional adjustment for ses data . ses variables included mean household income estimate , percentage of persons aged 25 years with 4 years of college ( 20062010 census ) , and percentage of persons aged 25 years with high school diploma or greater ( 20062010 census ) . negative binomial regression model with individual specific offset was performed to examine the association of race / ethnicity and total days rehospitalized . the individual specific offset was determined by logarithm of length of followup until censoring and the hospital stay days outcome was capped by the number of followup days . therefore , the individual specific offset allows model to account for the fact that patients had different length of followup days . other covariates in the model are same as those we used in the analyses of mortality and readmission outcomes . after further excluding 358 hospitals with limited participation and data capture ( n=110 656 patients ) ; 68 hospitals with < 25 patients in the cohort ( n=844 patients ) ; patients with no feeforservice eligibility at index discharge ( n=2473 ) ; patients with race missing ( n=1768 ) , unable to determine ( n=1332 ) , or american indian / alaska native ( n=210 ) ; patients without 1 documented admission vital signs ( eg , systolic blood pressure , heart rate , or respiratory rate ) ( n=3979 ) ; and patients without at least 1 documented admission laboratory data result ( eg , serum creatinine , blood urea nitrogen , or sodium ) ( n=7282 ) , the final study population was composed of 47 149 patients from 213 hospitals across the united states . the composition by race / ethnicity was as follows : white ( 83.2% ; n=39 213 ) , black ( 10.5% ; n=4946 ) , hispanic ( 5.0% ; n=2347 ) , and asian / pacific islander patients ( 1.4% ; n=643 ) . from the study population of 47 149 patients , we excluded those who left against medical advice ( n=135 ) , were transferred out ( n=685 ) , were discharged to hospice ( n=1220 ) , died during the index admission ( n=1413 ) , and had ndocumented or undetermined discharge destination ( n=551 ) , and the analysis sample for the outcomes of interest post index discharge included 43 145 patients . asian patients had the highest household income and were most likely to have a college degree ; hispanic patients were least likely to have a high school diploma . compared with other racial / ethnic groups , white patients were more likely to have a history of atrial fibrillation , chronic obstructive pulmonary disease , ischemic etiology of hf , and peripheral vascular disease , whereas nonhispanic black patients were more likely to have anemia , hypertension , renal insufficiency , smoking , and a history of stroke . hispanic patients were more likely to have diabetes , while asian patients had a stronger history of valvular heart disease . on admission , black and asian patients were more likely to have higher brain natriuretic peptide levels ; black and hispanic patients were more likely to have lower left ventricular systolic ejection fraction . at discharge , blockers and angiotensinconverting enzyme inhibitors were more likely to be prescribed to black patients , while angiotensin ii receptor blockers and aldosterone antagonists were more likely to be prescribed to asian and white patients , respectively . geographically , more black and hispanic patients were discharged from hospitals in the south , whereas more asian patients were treated at centers in the western region . baseline characteristics acei indicates angiotensinconverting enzyme inhibitor ; arb , angiotensin receptor blocker ; bnp , brain natriuretic peptide ; bun , blood urea nitrogen ; cabg , coronary artery bypass graft surgery ; cad , coronary artery disease ; copd , chronic obstructive pulmonary disease ; crtd , cardiac resynchronization therapydefibrillator ; cva , cerebrovascular accident ; icd , implantable cardiac defibrillator ; lvef , left ventricular ejection fraction ; med hx , past medical history ; mi , myocardial infarction ; pci , percutaneous coronary intervention ; tia , transient ischemic attack . p value tests the difference across the 4 race groups . the unadjusted 30day mortality post index admission and allcause readmission post index discharge ranged from 5.6% to 9.7% and 22.9% to 26.3% , respectively ( table 2 ) . in contrast to white patients , black and hispanic patients had lower shortterm mortality ( mortality postdischarge : white 6.3% , black 4.3% , hispanic 4.4% ; p<0.001 ) , higher shortterm allcause ( nonhispanic white 23% , black 25.1% , hispanic 26.3% ; p=0.003 ) and cardiovascular readmissions . compared with white patients , asian patients had similar shortterm mortality and allcause readmission but slightly higher shortterm cardiovascular readmission . cumulative incidence of outcomes overall and by race / ethnicity p value tests the difference across the 4 race groups . the unadjusted 1year mortality post index admission ranged from 29.5% to 37.8% , while allcause readmission post index discharge ranged from 66.5% to 72.9% ( table 2 ) . in comparison to white patients , all other groups had lower longterm mortality ( mortality postdischarge : white 34.1% , black 27.7% , hispanic 29.2% , asian 31.2% ; p<0.001 ) . however , black and hispanic patients had higher longterm allcause ( white 66.9% , black 72.9% , hispanic 71.3% ; p<0.001 ) and cardiovascular readmission and slightly higher combined mortality and readmission than nonhispanic white patients . asian patients had similar longterm readmission rates as white patients ( figures 1 through 5 ) . after multivariable adjustment for patient and hospital characteristics , black patients had lower odds of death but showed slightly higher risk of readmission and combined mortality / readmission than did white patients in both short and longterm followup ( table 3 ) . compared with white patients , hispanic patients had similar 30day mortality postadmission , modestly lower 30day mortality postdischarge , similar 1year mortality , and higher short and longterm readmission and combined mortality / readmission . in reference to white patients , asian patients had similar outcomes , except for marginally higher risk of shortterm cardiovascular readmission . associations between race / ethnicity and outcomes adjustment variables in the multivariable models are patient characteristics including age ; sex ; past medical history of atrial fibrillation / flutter , anemia , chronic obstructive pulmonary disease , depression , diabetes mellitus , heart failure , hypertension , implantable cardiac defibrillator , ischemic cause , dyslipidemia , pacemaker , peripheral vascular disease , renal insufficiency , cerebrovascular accident / transient ischemic attack , valvular heart disease , smoker ; systolic blood pressure , heart rate , and respiratory rate ; sodium , serum creatinine , blood urea nitrogen , and hemoglobin ; year of index hospitalization ; and hospital characteristics including region ( midwest , west , south vs northeast ) , number of beds , teaching status , and rural ( vs urban ) . socioeconomic status characteristics adjustment included mean household income estimate , percentage persons aged 25 years with 4 years of college , and percentage of persons aged 25 years with a high school diploma or greater . when ses data were added to the multivariable model , we observed that the majority of mortality end points did not change after adjusting for ses variables . black patients remained at higher risk for rehospitalization compared with white patients , even after accounting for ses . however , the difference in rehospitalization risk , including 30day allcause and cardiovascular readmission and 1year allcause readmission , between white and hispanic patients diminished and became statistically nonsignificant . analysis of total readmission days in the first 30 days postdischarge revealed that there was no difference in this end point across racial / ethnic groups after adjusting for patient , hospital , and ses characteristics ( table 4 ) . for the first 1 year postdischarge , black patients had more rehospitalization stay days within 1 year after patient , hospital , and ses adjustments . hispanics and asians also had more rehospitalization stay days after adjusting for patient and hospital characteristics , but the difference became marginally insignificant after further ses adjustment . analysis of total number of hospital stay days in first 30 days and 1 year after index hospital admission or index hospital discharge by race / ethnicity patient characteristics adjustment included age , gender , pmhx ( past medical history ) , atrial fibrillation / flutter , anemia , chronic obstructive pulmonary disease , depression , diabetes mellitus , heart failure , hypertension , implantable cardiac defibrillator , ischemic cause , dyslipidemia , pacemaker , peripheral vascular disease , renal insufficiency , cerebrovascular accident / transient ischemic attack , valvular heart disease , and smoker , vital signs : systolic blood pressure , heart rate , and respiratory rate , labs : sodium , serum creatinine , blood urea nitrogen , hemoglobin , year of index hospitalization . hospital characteristics adjustment included region ( midwest , west , south vs northeast ) , no . of beds , teaching status , rural ( vs urban ) . socioeconomic status characteristics adjustment included mean household income estimate , percentage persons aged 25 years with 4 years of college , and percentage of persons aged 25 years with a high school diploma or greater . moreover , we examined the interaction by race / ethnicity region and found that compared with white patients , hispanics in the west had lower 1year mortality risk relative to the other regions ( table 5 ) . analysis of interaction between race / ethnicity and geographic region model adjusted for patient , hospital characteristics , and socioeconomic status variables as displayed in table 3 footnote . after further excluding 358 hospitals with limited participation and data capture ( n=110 656 patients ) ; 68 hospitals with < 25 patients in the cohort ( n=844 patients ) ; patients with no feeforservice eligibility at index discharge ( n=2473 ) ; patients with race missing ( n=1768 ) , unable to determine ( n=1332 ) , or american indian / alaska native ( n=210 ) ; patients without 1 documented admission vital signs ( eg , systolic blood pressure , heart rate , or respiratory rate ) ( n=3979 ) ; and patients without at least 1 documented admission laboratory data result ( eg , serum creatinine , blood urea nitrogen , or sodium ) ( n=7282 ) , the final study population was composed of 47 149 patients from 213 hospitals across the united states . the composition by race / ethnicity was as follows : white ( 83.2% ; n=39 213 ) , black ( 10.5% ; n=4946 ) , hispanic ( 5.0% ; n=2347 ) , and asian / pacific islander patients ( 1.4% ; n=643 ) . from the study population of 47 149 patients , we excluded those who left against medical advice ( n=135 ) , were transferred out ( n=685 ) , were discharged to hospice ( n=1220 ) , died during the index admission ( n=1413 ) , and had ndocumented or undetermined discharge destination ( n=551 ) , and the analysis sample for the outcomes of interest post index discharge included 43 145 patients . asian patients had the highest household income and were most likely to have a college degree ; hispanic patients were least likely to have a high school diploma . compared with other racial / ethnic groups , white patients were more likely to have a history of atrial fibrillation , chronic obstructive pulmonary disease , ischemic etiology of hf , and peripheral vascular disease , whereas nonhispanic black patients were more likely to have anemia , hypertension , renal insufficiency , smoking , and a history of stroke . hispanic patients were more likely to have diabetes , while asian patients had a stronger history of valvular heart disease . on admission , black and asian patients were more likely to have higher brain natriuretic peptide levels ; black and hispanic patients were more likely to have lower left ventricular systolic ejection fraction . at discharge , blockers and angiotensinconverting enzyme inhibitors were more likely to be prescribed to black patients , while angiotensin ii receptor blockers and aldosterone antagonists were more likely to be prescribed to asian and white patients , respectively . geographically , more black and hispanic patients were discharged from hospitals in the south , whereas more asian patients were treated at centers in the western region . baseline characteristics acei indicates angiotensinconverting enzyme inhibitor ; arb , angiotensin receptor blocker ; bnp , brain natriuretic peptide ; bun , blood urea nitrogen ; cabg , coronary artery bypass graft surgery ; cad , coronary artery disease ; copd , chronic obstructive pulmonary disease ; crtd , cardiac resynchronization therapydefibrillator ; cva , cerebrovascular accident ; icd , implantable cardiac defibrillator ; lvef , left ventricular ejection fraction ; med hx , past medical history ; mi , myocardial infarction ; pci , percutaneous coronary intervention ; tia , transient ischemic attack . the unadjusted 30day mortality post index admission and allcause readmission post index discharge ranged from 5.6% to 9.7% and 22.9% to 26.3% , respectively ( table 2 ) . in contrast to white patients , black and hispanic patients had lower shortterm mortality ( mortality postdischarge : white 6.3% , black 4.3% , hispanic 4.4% ; p<0.001 ) , higher shortterm allcause ( nonhispanic white 23% , black 25.1% , hispanic 26.3% ; p=0.003 ) and cardiovascular readmissions . compared with white patients , asian patients had similar shortterm mortality and allcause readmission but slightly higher shortterm cardiovascular readmission . cumulative incidence of outcomes overall and by race / ethnicity p value tests the difference across the 4 race groups . the unadjusted 1year mortality post index admission ranged from 29.5% to 37.8% , while allcause readmission post index discharge ranged from 66.5% to 72.9% ( table 2 ) . in comparison to white patients , all other groups had lower longterm mortality ( mortality postdischarge : white 34.1% , black 27.7% , hispanic 29.2% , asian 31.2% ; p<0.001 ) . however , black and hispanic patients had higher longterm allcause ( white 66.9% , black 72.9% , hispanic 71.3% ; p<0.001 ) and cardiovascular readmission and slightly higher combined mortality and readmission than nonhispanic white patients . asian patients had similar longterm readmission rates as white patients ( figures 1 through 5 ) . the unadjusted 30day mortality post index admission and allcause readmission post index discharge ranged from 5.6% to 9.7% and 22.9% to 26.3% , respectively ( table 2 ) . in contrast to white patients , black and hispanic patients had lower shortterm mortality ( mortality postdischarge : white 6.3% , black 4.3% , hispanic 4.4% ; p<0.001 ) , higher shortterm allcause ( nonhispanic white 23% , black 25.1% , hispanic 26.3% ; p=0.003 ) and cardiovascular readmissions . compared with white patients , asian patients had similar shortterm mortality and allcause readmission but slightly higher shortterm cardiovascular readmission . cumulative incidence of outcomes overall and by race / ethnicity p value tests the difference across the 4 race groups . the unadjusted 1year mortality post index admission ranged from 29.5% to 37.8% , while allcause readmission post index discharge ranged from 66.5% to 72.9% ( table 2 ) . in comparison to white patients , all other groups had lower longterm mortality ( mortality postdischarge : white 34.1% , black 27.7% , hispanic 29.2% , asian 31.2% ; p<0.001 ) . however , black and hispanic patients had higher longterm allcause ( white 66.9% , black 72.9% , hispanic 71.3% ; p<0.001 ) and cardiovascular readmission and slightly higher combined mortality and readmission than nonhispanic white patients . asian patients had similar longterm readmission rates as white patients ( figures 1 through 5 ) . after multivariable adjustment for patient and hospital characteristics , black patients had lower odds of death but showed slightly higher risk of readmission and combined mortality / readmission than did white patients in both short and longterm followup ( table 3 ) . compared with white patients , hispanic patients had similar 30day mortality postadmission , modestly lower 30day mortality postdischarge , similar 1year mortality , and higher short and longterm readmission and combined mortality / readmission . in reference to white patients , asian patients had similar outcomes , except for marginally higher risk of shortterm cardiovascular readmission . associations between race / ethnicity and outcomes adjustment variables in the multivariable models are patient characteristics including age ; sex ; past medical history of atrial fibrillation / flutter , anemia , chronic obstructive pulmonary disease , depression , diabetes mellitus , heart failure , hypertension , implantable cardiac defibrillator , ischemic cause , dyslipidemia , pacemaker , peripheral vascular disease , renal insufficiency , cerebrovascular accident / transient ischemic attack , valvular heart disease , smoker ; systolic blood pressure , heart rate , and respiratory rate ; sodium , serum creatinine , blood urea nitrogen , and hemoglobin ; year of index hospitalization ; and hospital characteristics including region ( midwest , west , south vs northeast ) , number of beds , teaching status , and rural ( vs urban ) . socioeconomic status characteristics adjustment included mean household income estimate , percentage persons aged 25 years with 4 years of college , and percentage of persons aged 25 years with a high school diploma or greater . when ses data were added to the multivariable model , we observed that the majority of mortality end points did not change after adjusting for ses variables . black patients remained at higher risk for rehospitalization compared with white patients , even after accounting for ses . however , the difference in rehospitalization risk , including 30day allcause and cardiovascular readmission and 1year allcause readmission , between white and hispanic patients diminished and became statistically nonsignificant . analysis of total readmission days in the first 30 days postdischarge revealed that there was no difference in this end point across racial / ethnic groups after adjusting for patient , hospital , and ses characteristics ( table 4 ) . for the first 1 year postdischarge , black patients had more rehospitalization stay days within 1 year after patient , hospital , and ses adjustments . hispanics and asians also had more rehospitalization stay days after adjusting for patient and hospital characteristics , but the difference became marginally insignificant after further ses adjustment . analysis of total number of hospital stay days in first 30 days and 1 year after index hospital admission or index hospital discharge by race / ethnicity patient characteristics adjustment included age , gender , pmhx ( past medical history ) , atrial fibrillation / flutter , anemia , chronic obstructive pulmonary disease , depression , diabetes mellitus , heart failure , hypertension , implantable cardiac defibrillator , ischemic cause , dyslipidemia , pacemaker , peripheral vascular disease , renal insufficiency , cerebrovascular accident / transient ischemic attack , valvular heart disease , and smoker , vital signs : systolic blood pressure , heart rate , and respiratory rate , labs : sodium , serum creatinine , blood urea nitrogen , hemoglobin , year of index hospitalization . hospital characteristics adjustment included region ( midwest , west , south vs northeast ) , no . of beds , teaching status , rural ( vs urban ) . socioeconomic status characteristics adjustment included mean household income estimate , percentage persons aged 25 years with 4 years of college , and percentage of persons aged 25 years with a high school diploma or greater . moreover , we examined the interaction by race / ethnicity region and found that compared with white patients , hispanics in the west had lower 1year mortality risk relative to the other regions ( table 5 ) . analysis of interaction between race / ethnicity and geographic region model adjusted for patient , hospital characteristics , and socioeconomic status variables as displayed in table 3 footnote . our results highlight several important racial / ethnic differences in hf outcomes . first , black and hispanic hf patients had higher 30day and 1year readmission rates but lower 30day and 1year mortality , whereas asian patients had similar 30day readmission rates but lower 1year mortality relative to white patients . second , adjustment for patient and hospital characteristics resulted in similar outcomes for black patients but significantly diminished the mortality difference between hispanic and white patients . third , after additional adjustment for ses variables , the majority of associations between race / ethnicity group and outcomes persisted , with the exception that there was further reduction in the difference in 30day and 1year readmission rates between white and hispanic patients . a previous analysis of the gwtghf database revealed that black and hispanic patients hospitalized with hf , despite having more cardiovascular risk factors , had lower inhospital mortality rates than white patients . by linking with medicare data for postdischarge outcomes , our results extend these observations by demonstrating that better survival among black patients persists during the 30day and 1year followup periods , although they were more likely to be readmitted during the same followup intervals . these findings are consistent with data from the optimizehf ( organized program to initiate lifesaving treatment in hospitalized patients with heart failure ) registry comparing 60 to 90day outcomes between african americans and nonafrican americans . other reports using medicare data described higher 30day readmission for hf in black and hispanic patients , even after controlling for patient and hospital characteristics . a multiethnic population study using a california database found that ageadjusted hospitalization rates were highest in black patients ; similar between white and hispanic patients ; and lowest among asian men . conversely , black and hispanic patients were found to be more likely than white and asian patients to survive for 12 months after the index hf hospitalization . data from other recent studies support the observation of lower adjusted inhospital and shortterm mortality in black and hispanic patients than whites . racial / ethnic differences in hf outcomes have been previously explained on the basis of comorbid conditions and quality of care . consistent with prior registry data , our findings show lower left ventricular ejection fraction , higher brain natriuretic peptide levels , and higher comorbidity burden in black ( ie , hypertension and renal insufficiency ) and hispanic patients ( ie , diabetes ) than in white patients . in reference to quality of hf care , results from both the optimizehf and gwtg databases demonstrate comparable to better delivery of guidelinerecommended measures of care in blacks and hispanics relative to white patients . apart from these differences , another plausible explanation for the seemingly paradoxical observation of higher hf readmission frequency and lower mortality among minorities is poorer socioeconomic resources . a prior analysis of a national sample of medicare beneficiaries hospitalized with hf between 1998 and 1999 reported that lowerses patients had a higher risk of 1year readmission and 1year mortality compared with higherses patients . patient ses factors , determined based on the sociodemographic characteristics of the patient 's zip code of residence , were not significantly associated with 30day death but were strongly associated with longerterm outcomes in that population . in our study , multivariable adjustment for several ses variables such as household income and markers of higher education reduced intergroup differences in rehospitalization risk ( ie , between white and hispanic patients ) but did not significantly impact most of the mortality end points . how race / ethnicity , as a social determinant , could influence hf outcomes has not been adequately evaluated in prior studies . in this study , our results suggest that when the analyses are limited to feeforservice medicare patients ( aged > 65 years , have insurance and social security ) , the impact of ses parameters on hf outcomes , mortality in particular , may be blunted . relative to its effect on hf outcomes , income may be more important among younger patients and less indicative of ses in older , retired individuals . whereas our multivariable model adjusts for patients ' educational level , conventional definitions of education may not completely correlate with health literacy . race / ethnicity may also be a surrogate for other factors that may account for health disparities . in our and other analyses , collection of ses variables do not incorporate access to care , acculturation , language barrier , healthrelated behaviors and cultural beliefs , patient provider trust , and provider issues of bias and cultural insensitivity . our study has a number of other limitations . as a retrospective observational study , residual measured and unmeasured confounding variables may account for some or all of the findings despite adjustment for multiple variables . because the study population was composed of patients who were feeforservice medicare beneficiaries and who were from hospitals participating in the gwtghf program , our results may not be applicable to other hf populations . in this context , our study population is not reflective of the racial / ethnic distribution of the national us population . the method of recording race and ethnicity by patient selfdesignation as recorded by administrative staff or admitting providers may be less reliable than direct patient reporting . moreover , the existing data entry tool for race / ethnicity recording limited our ability to examine differences at the level of white , black , hispanic , and asian subpopulations . ses was derived from environment level ( ie , residence zip code ) household income rather than individual attributes . among medicare patients hospitalized with hf , there were important differences in patient characteristics as well as short and longterm outcomes among 4 racial / ethnic populations . compared with white patients , black and hispanic patients had lower 30day and 1year mortality but higher 30day and 1year readmission rates . after controlling for patient clinical characteristics , ses , and hospitalrelated variables , black patients had higher 1year readmission rates but lower 30day and 1year mortality ; while hispanic and asian patients had largely similar riskadjusted outcomes , compared with white patients . this study provides evidence of contemporary racial and ethnicbased differences in hf outcomes and highlights the need for further studies to identify underlying causes for these racial and ethnic differences in care and outcomes of hf patients .

mediastinal abscesses ( mas ) in the pediatric population are relatively rare and usually occur as a complication of esophageal perforation , thoracic trauma , or thoracic surgery . ma due to a non - traumatic etiology is extremely rare in childhood and usually results either from direct extension along contiguous anatomic pathways and fascial planes like pulmonary / pleural and buccopharyngeal areas , osteomyelitis of the ribs or vertebrae , or by hematogenous or lymphatic spread from distant sites of infection . this report describes a boy who presented with abscesses in the left axilla and over the right thigh , along with right pleural empyema and pericardial effusion . during the course of the illness , the patient developed a large abscess in the anterior mediastinum which necessitated open surgical drainage . a 5-year - old male child presented with high - grade fever of 1 week duration , associated with swellings over the right thigh and in the left axillary region . for 4 days , the child was having increasing difficulty in breathing . at admission a tender , non - fluctuant swelling was present over the antero - lateral aspect of the right thigh and was associated with limitation of movement of the right hip . another tender and fluctuant swelling measuring 3 cm was present in the left axillary region . the chest examination revealed findings suggestive of right - sided pleural effusion and a pericardial rub with normal heart sounds . provisionally , the possibility of severe pneumonia with right pleural empyema , pericardial effusion , right thigh abscess and left axillary abscess was considered . initial laboratory investigations showed marked leukocytosis with neutrophilia and raised erythrocyte sedimentation rate ( esr ) . a chest radiograph showed right - sided pleural effusion with cardiomegaly [ figure 1 ] . an ultrasound ( us ) chest showed a localized hetero - echoic collection in the right pleural space with maximum thickness of 2.7 cm and pericardial effusion with a fluid thickness of 8 mm . an intercostal drainage tube was inserted to drain the localized empyema on the right side . on day 1 , incision and drainage of the left axillary and thigh abscesses was also performed and the pus sent for culture . in view of the possibility of disseminated staphylococcal sepsis , chest radiograph shows mediastinal widening despite these measures , the child continued to have high - grade fever along with significant respiratory distress and toxicity . repeat us chest after 72 hours showed resolution of the right - sided empyema as well as the pericardial effusion . pus culture from the left axillary abscess showed methicillin - sensitive staphylococcus aureus which was sensitive to amoxicillin a small localized collection of pus became apparent in the epigastric area and this was drained by needle aspiration . a repeat chest radiograph showed superior mediastinal widening ; therefore , a contrast - enhanced computed tomogram ( ct ) chest was performed . this revealed multiple conglomerate anterior and superior mas extending from the thoracic inlet to the diaphragm [ figure 2 ] . pediatric surgery consultation was sought and the child underwent surgical drainage of the ma via right anterolateral thoracotomy through the 6 intercostal space . subsequently , the condition of the child improved dramatically as fever subsided and toxicity disappeared . ma secondary to a non - traumatic etiology is extremely uncommon in childhood , especially since the advent of widespread antibiotic treatment ; in the last two decades , around 13 such cases have been reported . non - traumatic ma is most common in the first 2 years of life with the age at presentation varying from 15 days to 11 years . aureus , although cases caused by streptococcus pneumoniae ] and mixed aerobic and anaerobic bacteria have also been reported . in most reported cases of non - traumatic ma , the abscess was secondary to spread from a local infective focus . in two cases tubercular chronic retropharyngeal abscess secondary to tubercular spondylitis extended to the antero - superior mediastinum . komatsu et al . reported ma in the postero - superior mediastinum , complicating purulent tonsillitis by gram - positive cocci . pneumoniae ma secondary to right - sided pneumonitis and focal thymic infection have also been reported . cases of non - traumatic ma secondary to hematogenous and/or lymphatic spread from distant infection sites have also been described . in a report of a case , tercier et al . described a 12-month - old boy in whom cellulitis of the left hemithorax with multiple abscesses in the left axilla caused by sta . aureus was complicated by pericarditis , a left pleural effusion , and finally , a huge collection in the left antero - posterior mediastinum , necessitating drainage by a suprasternal horizontal cerviotomy . the authors believed that their case could be due to hematogenous / lymphatic spread from the axillary site , with lymphatic spread being the more likely reason since the internal mammary lymphatics communicate extensively with lymphatics in the upper mediastinum . ma secondary to septic arthritis has been reported , while cases of ma developing secondary to an abscess over the hip or over the hand have also been described . gamiao reported a 5-year - old boy who developed an abscess in the antero - superior mediastinum , secondary to a massive pericardial effusion due to sta . the case was also complicated by pleural effusion and the author surmised that the primary bacterial endocarditis in their case might have caused contiguous extension of infection in the mediastinal area via a direct or hematogenous route . in our case , the most likely etiology of ma was lymphatic or haematogenous spread from the abscess in the left axilla and over the right thigh . the epigastric abscess which developed during the hospital stay could have been a separate subcutaneous collection or an extension from the anterior ma through persistent anterior diaphragmatic slits . early diagnosis and aggressive treatment of mediastinal infections is a must to decrease mortality and morbidity . although a chest radiograph can give information regarding a widened mediastinal shadow , the findings may not be confirmatory . the imaging modality of choice is contrast - enhanced ct scan which gives information about the extent of the ma , presence or absence of loculations , and its relationship with vital organs . in infants with deep cervical infections , a close vigilance should be instituted to detect potential mediastinal extension by performing follow - up plain radiograph or ct scan . although it has been stated that for non - traumatic ma , conservative treatment with appropriate antibiotics alone for prolonged periods may be successful , most reported cases have in addition needed a minimal invasive percutaneous drainage procedure which was performed by an extrathoracic approach . this varied from drainage by a suprasternal approach , supraclavicular approach , right - sided thoracocentesis , catheter drainage by a subxiphoid approach , multiple ultrasound - guided needle aspirations , to ct - guided parasternal drainage . no case reported earlier in the literature needed thoracotomy for drainage and it has been suggested that thoracotomy is unnecessary for draining ma in the pediatric population and should be avoided to prevent thoracic wall and hemithoracic spread . in our patient , thoracotomy with open drainage was necessary because of the worsening condition of the child and as the abscess was large with multiple loculi and septations . in conclusion , non - traumatic ma is a rare and dreaded complication of various etiologies , especially deep neck infections or septic foci elsewhere in body .

in dupuytren s disease , the metacarpophalangeal ( mp ) , proximal interphalangeal ( pip ) , and distal interphalangeal ( dip ) joints can develop fixed - flexion deformities due to the development and subsequent contraction of diseased cords in the affected tissue . although pip joints are not affected as often as are mp joints , they can be more disabling for patients , as many routine , daily activities are impaired when these joints are contracted [ 1 , 2 ] . frequently , pip joint contracture is accompanied by mp joint contracture . research has shown that improvements in pip joint contractures correlate positively with improved hand function [ 2 , 4 ] . the pip joint contractures are also more challenging to treat using corrective surgery [ 5 , 6 ] or minimally invasive procedures such as percutaneous needle fasciotomy ( pnf ) and collagenase clostridium histolyticum ( cch ) injections . the cch injection is the first non - surgical , pharmacologic treatment for dupuytren s contracture ( dc ) with a palpable cord approved for use in the usa and europe . clinical trials [ 79 ] and post - marketing studies [ 10 , 11 ] have demonstrated the efficacy and safety of cch for correcting dc . in this secondary analysis of data from four large clinical trials , we explored the efficacy of cch on ( 1 ) pip joint contractures when only the adjacent mp joint was treated ; and ( 2 ) isolated pip joint contractures treated with cch . cord ( collagenase option for reduction of dupuytren s ) i and ii were 90-day , phase iii trials conducted at 16 sites in the usa and five sites in australia , respectively . joint i and ii were 9-month , open - label studies conducted at 14 sites in the usa and 20 sites in europe and australia , respectively . for all four studies , eligible patients ( aged 18 years ) were required to have a fixed - flexion deformity in 1 finger ( other than the thumb ) that was 20 and 100 in an mp joint or 20 and 80 in a pip joint caused by a palpable cord that had not been previously treated with cch . before treatment in all four studies , investigators selected the hand to be treated and prioritized all palpable cords as primary , secondary , and tertiary . the primary cord could affect an mp or pip joint if the contracture occurred solely in these respective joints . if there were contractures in both the mp and pip joints of the same finger , the cord causing the mp contracture was deemed the primary cord . after the primary joint was successfully treated , either an mp or pip joint contracture could be selected . subsequent joints were selected on the basis of providing the patient with full functionality of the treated hand . the primary endpoint was clinical success , defined as a reduction in contracture of the primary joint to 5 of full extension 30 days after injection . secondary endpoints included clinical improvement , defined as a 50 % reduction in contracture of a treated joint , and a spontaneous effect of treatment , defined as a 20 reduction in contracture of any other joint not directly treated with cch . changes in fixed - flexion contractures ( ffc ) and range of motion ( rom ) were also assessed . for this secondary analysis , patients were included if they had 1 pip joint contracture at study entry and received 1 cch injection during the study . to evaluate the indirect and direct effects of cch on pip joint contractures , data for two patient subgroups were analyzed . in group a , patients had mp and pip joint contractures in the same finger , but only the cord affecting the mp joint was treated with cch . in group b , patients had an isolated pip joint contracture ( or pip joint contracture combined with an mp joint contracture < 20 ) , and only the cord affecting the pip joint was treated with cch . for brevity throughout the report , when we refer to a cch - treated joint , the cord contracting the joint received the cch injection(s ) . in keeping with the definitions used in the phase iii studies , in group a , the indirect effects of cch on pip joints after the injection of mp joints were evaluated for correction of contracture , defined as a reduction in ffc to 5 30 days after injection , and as a spontaneous treatment effect ( i.e. , improvement ) , defined as a 20 reduction in ffc 30 days after injection . in group b , the direct effects of cch on pip joints were evaluated for correction of contracture , as defined previously . improvement in contracture was defined as a 50 % reduction in ffc from baseline 30 days after injection . the results for joints that corrected were included in the results for joints that showed improvement . in both groups , the percentage change in ffc and mean change in rom were also assessed . in all four trials , the adverse events ( aes ) were monitored and recorded for the duration of the studies . although the cord and joint studies differed by design ( i.e. , double - blind vs. open - label ) , all four protocols used the same inclusion / exclusion criteria to enroll patients , and the treatment paradigms were virtually identical . moreover , the respective patient populations were relatively homogeneous for baseline demographic ( e.g. , age , sex ) and clinical characteristics ( e.g. , contracture severity , digits / joints affected ) . thus , it was considered appropriate to pool all of the relevant data for this analysis . inferential statistics were not performed on the data ; only descriptive attributes are reported , including means and standard deviation ( sd ) or medians and ranges ( minimum , maximum ) when appropriate . the data are reported at the joint level unless otherwise specified ( i.e. , patient level , finger level ) . cord ( collagenase option for reduction of dupuytren s ) i and ii were 90-day , phase iii trials conducted at 16 sites in the usa and five sites in australia , respectively . joint i and ii were 9-month , open - label studies conducted at 14 sites in the usa and 20 sites in europe and australia , respectively . for all four studies , eligible patients ( aged 18 years ) were required to have a fixed - flexion deformity in 1 finger ( other than the thumb ) that was 20 and 100 in an mp joint or 20 and 80 in a pip joint caused by a palpable cord that had not been previously treated with cch . before treatment in all four studies , investigators selected the hand to be treated and prioritized all palpable cords as primary , secondary , and tertiary . the primary cord could affect an mp or pip joint if the contracture occurred solely in these respective joints . if there were contractures in both the mp and pip joints of the same finger , the cord causing the mp contracture was deemed the primary cord . after the primary joint was successfully treated , either an mp or pip joint contracture could be selected . subsequent joints were selected on the basis of providing the patient with full functionality of the treated hand . the primary endpoint was clinical success , defined as a reduction in contracture of the primary joint to 5 of full extension 30 days after injection . secondary endpoints included clinical improvement , defined as a 50 % reduction in contracture of a treated joint , and a spontaneous effect of treatment , defined as a 20 reduction in contracture of any other joint not directly treated with cch . changes in fixed - flexion contractures ( ffc ) and range of motion ( rom ) were also assessed . for this secondary analysis , patients were included if they had 1 pip joint contracture at study entry and received 1 cch injection during the study . to evaluate the indirect and direct effects of cch on pip joint contractures , data for two patient subgroups were analyzed . in group a , patients had mp and pip joint contractures in the same finger , but only the cord affecting the mp joint was treated with cch . in group b , patients had an isolated pip joint contracture ( or pip joint contracture combined with an mp joint contracture < 20 ) , and only the cord affecting the pip joint was treated with cch . for brevity throughout the report , when we refer to a cch - treated joint , the cord contracting the joint received the cch injection(s ) . in keeping with the definitions used in the phase iii studies , in group a , the indirect effects of cch on pip joints after the injection of mp joints were evaluated for correction of contracture , defined as a reduction in ffc to 5 30 days after injection , and as a spontaneous treatment effect ( i.e. , improvement ) , defined as a 20 reduction in ffc 30 days after injection . in group b , the direct effects of cch on pip joints were evaluated for correction of contracture , as defined previously . improvement in contracture was defined as a 50 % reduction in ffc from baseline 30 days after injection . the results for joints that corrected were included in the results for joints that showed improvement . in both groups , the percentage change in ffc and mean change in rom were also assessed . in all four trials , the adverse events ( aes ) were monitored and recorded for the duration of the studies . although the cord and joint studies differed by design ( i.e. , double - blind vs. open - label ) , all four protocols used the same inclusion / exclusion criteria to enroll patients , and the treatment paradigms were virtually identical . moreover , the respective patient populations were relatively homogeneous for baseline demographic ( e.g. , age , sex ) and clinical characteristics ( e.g. , contracture severity , digits / joints affected ) . thus , it was considered appropriate to pool all of the relevant data for this analysis . inferential statistics were not performed on the data ; only descriptive attributes are reported , including means and standard deviation ( sd ) or medians and ranges ( minimum , maximum ) when appropriate . the data are reported at the joint level unless otherwise specified ( i.e. , patient level , finger level ) . mean sd age was 63 10 years ; nearly 70 % of patients were aged 4474 years . eighty - three percent of patients were male , and 100 % were white . more than 75 % of patients had 2 pip joints affected ; 61 % of patients had 3 joints affected . isolated pip joints in the fifth finger accounted for the largest percentage ( 61 % ) of affected joints treated with cch , followed by combined mp / pip joint contractures on the fifth finger ( 25 % ) . the distribution of affected mp and pip joints by finger is shown in fig . 1 . nearly half ( 46 % ) of patients with mp and pip joint contractures on the same finger received cch injections for the mp joint only ; 18 % received cch injections for the pip joint contracture , and 36 % received both mp and pip joint injections . for pip joint contractures that received direct cch injections ( group b ) , the median ( minimum , maximum ) number of injections per joint overall was 1.0 ( 1.0 , 4.0 ) [ mean sd , 1.6 0.8 ] . the median number of injections per joint for total correction was 1.0 ( 1.0 , 3.0 ) [ mean sd , 1.3 0.6].table 1baseline demographic and clinical characteristicsvariablevaluepatients ( n = 616 ) age ( years ) [ mean sd]63 10 male sex [ n ( % ) ] 508 ( 83 ) joints affected [ mean sd]3.3 2.2 pip joints affected [ mean sd]1.9 1.2same finger mp / pip contractures / patient [ n ( % ) ] 1268 ( 44 ) 260 ( 10 ) 38 ( 1 ) 41 ( 0.2)fingers with mp / pip contractures ( n = 416 ) [ n ( % ) ] only mp treated191 ( 46 ) both mp and pip treated150 ( 36 ) only pip treated75 ( 18)joints treated with cch ( n = 577 ) [ n ( % ) ] mp201 ( 35 ) pip376 ( 65 ) cch collagenase clostridium histolyticum , mp metacarpophalangeal , pip proximal interphalangeal , sd standard deviationfig . mp metacarpophalangeal , pip proximal interphalangeal baseline demographic and clinical characteristics cch collagenase clostridium histolyticum , mp metacarpophalangeal , pip proximal interphalangeal , sd standard deviation distribution of affected metacarpophalangeal and proximal interphalangeal joints by finger . mp metacarpophalangeal , pip proximal interphalangeal in group a , 28 % of pip joint contractures spontaneously corrected after the first cch injection for the mp joint deformity ; 43 % of pip joint contractures were corrected after the last mp joint injection . in group b , 31 % of pip joint contractures were corrected after the first cch injection ; 39 % were corrected after the last injection ( fig . in group a , 40 % of pip joints showed improvement in contracture after the first mp joint injection ; 63 % showed improvement after the last mp joint injection , which for 46 % ( 93/201 ) of cases was also the first injection . in group b , 56 % of pip joints showed improvements in contracture after the first direct cch injection ; 66 % showed improvement after the last injection , which for 44 % ( 209/376 ) of cases was also the first injection ( fig . 2results for fixed - flexion contracture correction ( a ) and improvement ( b ) after collagenase clostridium histolyticum by group . correction = reduction in contracture to 5 30 days after injection for both groups ; improvement = 20 reduction in contracture ( group a ) or 50 % reduction in contracture ( group b ) 30 days after injection . ffc fixed - flexion contracture , mp metacarpophalangeal , pip proximal interphalangeal results for fixed - flexion contracture correction ( a ) and improvement ( b ) after collagenase clostridium histolyticum by group . correction = reduction in contracture to 5 30 days after injection for both groups ; improvement = 20 reduction in contracture ( group a ) or 50 % reduction in contracture ( group b ) 30 days after injection . ffc fixed - flexion contracture , mp metacarpophalangeal , pip proximal interphalangeal for group a , the mean change in ffc was 66 % after the first cch injection and 77 % after the last injection . for group b , the mean change in ffc was 55 % after the first cch injection and 62 % after the last injection ( fig . baseline ffc values were higher ( i.e. , more severe contractures ) among joints that received a second or third injection ( table 2 ) . as indicated by the relative changes , ffc values were lowest after the last injection in both groups . 4a to show the linear relationship between baseline pip joint contracture severity and ffc measures 30 days after each injection in both groups . more severe contractures at baseline received two or three cch injections , and although the change in ffc from baseline was dramatic , day 30 ffc measures remained higher than did those for pip joints that received only one cch injection.fig . 3changes in fixed - flexion contracture ( a ) and range of motion ( b ) of proximal interphalangeal joint contractures by group and collagenase clostridium histolyticum injection . ffc fixed - flexion contracture , mp metacarpophalangeal , pip proximal interphalangeal , rom range of motiontable 2goniometry results for fixed - flexion contracture and range of motion at baseline and day 30 by collagenase clostridium histolyticum injection number and groupinjectiongroup a group b joints ( n)baselineday 30joints ( n)baselineday 30ffc first20148.2 20.219.2 22.437649.7 18.524.4 21.4 second7555.6 19.421.6 20.914757.5 16.928.5 20.0 third3359.7 18.927.4 24.46260.5 14.530.2 19.6 last20148.2 20.213.3 19.437649.7 18.520.1 20.4rom first197 43.1 18.971.0 22.2372 49.9 19.674.0 23.0 second74 22.3 third3333.5 16.864.1 22.961 37.5 16.169.3 20.3 last197 43.1 18.977.5 19.4372 49.9 19.678.8 22.0results are mean sd ( ) cch collagenase clostridium histolyticum , ffc fixed - flexion contracture , rom range of motion group a : patients with mp and pip joint contractures in the same finger , but only the cord affecting the mp joint was treated with cch group b : patients had isolated pip joint contracture ( or pip contracture combined with an mp joint contracture < 20 ) , and only the cord affecting the pip joint was treated with cch day 30 , n = 199 ( first ) , 75 ( second ) , 199 ( last ) day 30 , n = 374 ( first ) , 62 ( third ) , 373 ( last)fig . 4fixed - flexion contracture ( a ) and range of motion ( b ) at baseline and day 30 by group . ffc fixed - flexion contracture , mp metacarpophalangeal , pip proximal interphalangeal , rom range of motion changes in fixed - flexion contracture ( a ) and range of motion ( b ) of proximal interphalangeal joint contractures by group and collagenase clostridium histolyticum injection . ffc fixed - flexion contracture , mp metacarpophalangeal , pip proximal interphalangeal , rom range of motion goniometry results for fixed - flexion contracture and range of motion at baseline and day 30 by collagenase clostridium histolyticum injection number and group results are mean sd ( ) cch collagenase clostridium histolyticum , ffc fixed - flexion contracture , rom range of motion group a : patients with mp and pip joint contractures in the same finger , but only the cord affecting the mp joint was treated with cch group b : patients had isolated pip joint contracture ( or pip contracture combined with an mp joint contracture < 20 ) , and only the cord affecting the pip joint was treated with cch day 30 , n = 199 ( first ) , 75 ( second ) , 199 ( last ) day 30 , n = 374 ( first ) , 62 ( third ) , 373 ( last ) fixed - flexion contracture ( a ) and range of motion ( b ) at baseline and day 30 by group . ffc fixed - flexion contracture , mp metacarpophalangeal , pip proximal interphalangeal , rom range of motion for rom , the mean sd change in group a was 27.8 19.7 after the first cch injection and 34.3 19.8 after the last injection ; changes in group b were 24.4 18.1 after the first cch injection and 29.1 20.0 after the last injection ( fig . baseline rom values were lower among joints that received a second or third injection ( table 2 ) . the rom values were highest after the last injection in group a and after the third injection in group b. figure 4b shows the linear relationship between rom measures at baseline and 30 days after each injection in both groups . again , despite notable increases in rom for all pip contractures , joints with more restricted rom received two or three cch injections and the day 30 values were lower than in pip joint contractures that received only one cch injection . nearly all patients ( 98 % ) in the pip joint contracture subgroup experienced 1 ae during the study in which they were enrolled . the majority of aes were mild , transient , and localized to the injection site , including peripheral edema , pain , hemorrhage , tenderness , and swelling . the aes occurring in 10 % of patients in the pip joint contracture subgroup and compared with all patients from the four studies are summarized in table 3 . with one exception ( injection - site pain ) , slightly larger percentages of patients in the pip subgroups experienced aes compared with all patients from the four clinical studies.table 3adverse events occurring in 10 % of patientsadverse eventgroups a / b ( n = 616)all patients ( n = 961)patients with 1 adverse event604 ( 98)934 ( 97)general and injection - site conditions edema peripheral500 ( 81)736 ( 77 ) injection - site pain239 ( 39)381 ( 40 ) injection - site hemorrhage231 ( 38)359 ( 37 ) tenderness181 ( 29)250 ( 26 ) injection - site swelling170 ( 28)255 ( 27)contusion402 ( 65)574 ( 60)pain in extremity263 ( 43)383 ( 40)pruritus94 ( 15)122 ( 13)ecchymosis87 ( 14)125 ( 13)skin laceration79 ( 13)106 ( 11)blood blister70 ( 11)79 ( 8)lymphadenopathy67 ( 11)86 ( 9)data are given as n ( % ) cch collagenase clostridium histolyticum , mp metacarpophalangeal , pip proximal interphalangeal group a : patients with mp and pip joint contractures in the same finger , but only the cord affecting the mp joint was treated with cch group b : patients had isolated pip joint contracture ( or pip contracture combined with an mp joint contracture < 20 ) , and only the cord affecting the pip joint was treated with cch adverse events occurring in 10 % of patients data are given as n ( % ) cch collagenase clostridium histolyticum , mp metacarpophalangeal , pip proximal interphalangeal group a : patients with mp and pip joint contractures in the same finger , but only the cord affecting the mp joint was treated with cch group b : patients had isolated pip joint contracture ( or pip contracture combined with an mp joint contracture < 20 ) , and only the cord affecting the pip joint was treated with cch nearly all patients ( 98 % ) in the pip joint contracture subgroup experienced 1 ae during the study in which they were enrolled . the majority of aes were mild , transient , and localized to the injection site , including peripheral edema , pain , hemorrhage , tenderness , and swelling . the aes occurring in 10 % of patients in the pip joint contracture subgroup and compared with all patients from the four studies are summarized in table 3 . with one exception ( injection - site pain ) , slightly larger percentages of patients in the pip subgroups experienced aes compared with all patients from the four clinical studies.table 3adverse events occurring in 10 % of patientsadverse eventgroups a / b ( n = 616)all patients ( n = 961)patients with 1 adverse event604 ( 98)934 ( 97)general and injection - site conditions edema peripheral500 ( 81)736 ( 77 ) injection - site pain239 ( 39)381 ( 40 ) injection - site hemorrhage231 ( 38)359 ( 37 ) tenderness181 ( 29)250 ( 26 ) injection - site swelling170 ( 28)255 ( 27)contusion402 ( 65)574 ( 60)pain in extremity263 ( 43)383 ( 40)pruritus94 ( 15)122 ( 13)ecchymosis87 ( 14)125 ( 13)skin laceration79 ( 13)106 ( 11)blood blister70 ( 11)79 ( 8)lymphadenopathy67 ( 11)86 ( 9)data are given as n ( % ) cch collagenase clostridium histolyticum , mp metacarpophalangeal , pip proximal interphalangeal group a : patients with mp and pip joint contractures in the same finger , but only the cord affecting the mp joint was treated with cch group b : patients had isolated pip joint contracture ( or pip contracture combined with an mp joint contracture < 20 ) , and only the cord affecting the pip joint was treated with cch adverse events occurring in 10 % of patients data are given as n ( % ) cch collagenase clostridium histolyticum , mp metacarpophalangeal , pip proximal interphalangeal group a : patients with mp and pip joint contractures in the same finger , but only the cord affecting the mp joint was treated with cch group b : patients had isolated pip joint contracture ( or pip contracture combined with an mp joint contracture < 20 ) , and only the cord affecting the pip joint was treated with cch in this secondary analysis of data from four large clinical trials of cch for dc , we explored treatment effects on pip joint contractures from two perspectives : ( 1 ) spontaneous correction and/or improvement after cch injection for an mp joint contracture affecting the same finger ; and ( 2 ) correction and/or improvement after direct injection into a cord contracting an isolated pip joint . more than 600 patients from the cord i and ii trials and the joint i and ii studies were included ; 201 mp / pip combination contractures and 376 pip contractures were evaluated . isolated pip joints in the fifth finger accounted for the largest percentage ( 61 % ) of affected joints treated with cch . overall , similar percentages of pip joint contractures showed spontaneous correction after cch injection for a contracted mp joint in the same finger or after direct cch injection of an affected pip joint ( 43 and 39 % , respectively ) . although improvement in contracture was defined differently for group a ( i.e. , 20 reduction in contracture 30 days after injection ) and group b ( i.e. , 50 % reduction in contracture 30 days after injection ) , comparable percentages of pip joint contractures met this endpoint after cch injection for a contracted mp joint in the same finger or after direct cch injection of an affected pip joint ( 63 and 66 % , respectively ) . interestingly , in both groups , there was a small decrease in the percentage of pip joint contractures that were corrected or showed improvement after the second cch injection . this could be due at least in part to some residual aes at the injection site , including edema and stiffness . overall , 46 % of mp / pip joint contractures ( group a ) and 44 % of isolated pip contractures ( group b ) received only one cch injection . in both groups a and b , the relative ( percentage ) changes in ffc decreased slightly between the first and third cch injections . pre - injection ffc measures were quite similar between groups a and b , and baseline values increased slightly before the second and then the third injections in both groups . overall , reductions in ffc were slightly larger among pip joints in group a versus group b at each timepoint , as shown in fig . 3 and by the slopes of the lines in fig . similarly , changes in rom were slightly larger in group a versus group b. a potential explanation may be related to the mechanics of the entire digit . by releasing two joints , the overall benefit for movement could be larger than the sole improvement in the measure of ffc . although the treated joint may remain stiff or swollen , the released joint could be moving freely . it is feasible that releasing a proximal cord may reduce some of the tension affecting more distal joints , allowing for improvement along the entire digit . in group overall , regardless of the type or number of cch injections received , all of the changes in rom can be considered clinically meaningful . in all but one instance , the change in rom was twofold larger than the previously demonstrated clinically important difference ( cid ) of 13.5 ( 95 % ci 11.915.1 ) . the cid is calculated statistically , but it can help interpret the clinical relevance of changes in objective measures from the patient s perspective [ 13 , 14 ] . the vast majority of patients experienced 1 ae during the studies ; most events were injection - site reactions , including edema , pain , hemorrhage , and swelling . most aes were mild and transient in nature . although slightly larger percentages of patients in the pip joint contracture subgroups experienced aes compared with all patients , these differences are not likely to be clinically relevant . as reported for the cord [ 7 , 8 ] and joint studies , the types , frequencies , and severity of aes were comparable with those in other published research on the safety and tolerability of cch . to our knowledge , this is the first report of spontaneous correction and improvement in pip joint contractures after treatment of an mp joint contracture in the same finger . thus , it is not possible to discuss the findings as they relate to the extant literature . however , the body of evidence describing the overall results of surgical or non - surgical interventions for dc is large . in a systematic review of fasciotomy and fasciectomy among european patients , more mp than pip joint contractures met the pre - specified outcome , with mean improvements of 80 and 49 % after fasciotomy and 94 and 66 % after fasciectomy , respectively . reported similar results in a systematic review of pnf . in a more recently published study comparing outcomes of pnf versus limited fasciectomy , van rijssen et al . showed that 55 % of mp and 26 % of pip joints corrected to 5 at 6 weeks after pnf ; 94 % of mp and 47 % of pip joints corrected after limited fasciectomy . in another recent study , shin and jones showed that > 90 % of mp and 82 % of pip joints were fully corrected at least 2 weeks after segmental fasciectomies . thus , the relative efficacy of different treatment options for correcting mp versus pip joint contractures is well - established : pip joints are less responsive to intervention , become even more so over time , and are more susceptible to recurrence [ 1 , 3 ] . fundamental anatomical differences [ 3 , 19 ] between the mp and pip joints play a large part ; other factors not related to dupuytren s disease are also involved , including secondary contraction of the volar plate and/or collateral ligaments , arthritic changes and stiffness , and attenuation of the extensor mechanism . any one or more of these processes may hold the pip joint in a flexed position even after correction of the mp joint contracture or partial correction of the pip joint contracture . the irony is that , despite the problematic nature of the contracted pip joint and its relative resilience to corrective intervention , in some cases as demonstrated here the pip joint contracture resolves spontaneously after treating an mp joint contracture in the same finger . a plausible explanation is that some of the cch spreads across multiple cords . alternatively , spontaneous correction of pip joint contractures may be facilitated by the finger - extension procedure . a careful clinical examination is essential for identifying the source and arrangement of the cord or cords causing the pip joint deformity . these and other patient clinical characteristics , including the extent to which hand function is compromised , should be considered when deciding whether or not to treat the deformity . if affirmative , these factors are again considered in deciding on the approach for corrective intervention . in cases in which the affected mp and pip joints are in the same finger , the likelihood of achieving full correction for both joint contractures is high after cch if there is one central , pretendinous cord . if there is a separate cord affecting just the pip joint , spontaneous correction after treating the mp joint contracture is less likely . isolated pip joint contractures are most prevalent in the fifth finger [ 21 , 22 ] . although this is the smallest of the fingers , it contains one of the largest digital branches of the ulnar nerve in the hand , which may make the approach to treatment even more challenging . the abductor digiti minimi cord is commonly observed in the fifth finger . as this type of cord is confined to the finger , pnf is not recommended , although cch would be a viable alternative . moreover , although there is no robust clinical evidence to suggest that pnf has an increased risk of iatrogenic nerve , artery or tendon injury , it is reasonable to imagine that a blind procedure with multiple passes of a sharp needle would place such structures at risk . thus , many surgeons only perform pnf in the palm on well - defined cords . on occasion during open surgery , after the pathological tissue has been removed , the contracted pip joint can be manipulated straight by the surgeon . in such cases , this exploratory analysis is not without limitations , and the findings may be most useful for hypothesis generation and as a resource for the design of future clinical studies . for example , the clinical trial data were not analyzed by finger , and details regarding the nature of the cords contracting the mp and/or pip joints were not available . that said , most surgical studies also do not report on the detailed structure of pathological cords only that they were divided or excised . moreover , multiple cords affecting multiple joints or digits may be excised during a single surgical session . by contrast , the product label for cch stipulates a 30-day interval between injections . of note , a phase iii study evaluating the safety and efficacy of two concurrent injections into the same hand of patients with multiple contractures is in progress ( clinicaltrials.gov identifier no . additional studies that investigate different treatment paradigms may afford clinicians a better understanding of these issues . such designs might include multiple , simultaneous cch injections to treat mp and pip joint contractures of the same finger or close , sequential injections to treat residual pip joint contractures . finally , it is important that future studies provide for the collection of details regarding the pathological anatomy of affected joints and take these configurations into account when analyzing the results . the results of this post hoc analysis suggest that the efficacy of cch for pip contractures , as reflected by measures of clinical success and clinical improvement , was comparable after treatment of an mp joint contracture in the same finger and injection of an isolated pip joint contracture . likewise , improvements in ffc and rom were quite similar across the two groups . in both groups a and b , the changes in rom exceeded the cid , which represents a 1-point change on a 4-point scale for patient - reported improvement . thus , regardless of the specific pathological palmar anatomy , and consistent with previous research using surgical and non - surgical techniques , some pip joints not directly treated with cch will show meaningful spontaneous improvements in contracture . a. bayat has received an investigator award from pfizer ; he has no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed .

chagas ' disease is a zoonosis caused by the protozoan parasite trypanosoma cruzi , and it is a major public health problem in most of latin america and in particular in mexico . t. cruzi infects many cell types , including myocytes , fibroblast , vascular endothelial , and smooth muscle cells among other cells . since the monocyte is a target cell in t. cruzi infection and monocytes play a major role in regulating immune responses , monocyte dysfunction may contribute to host immunosuppression . it has been observed that during the experimental infection with t. cruzi , there is an increased expression of proinflammatory mediators , including cytokines , chemokines , vascular adhesion molecules , and nitric oxide synthase among other molecules , which promotes the inflammatory process and vascular damage . there is evidence that immune mechanisms are involved in the pathogenesis of many parasitic infections . the initial stages of the disease are generally characterized by the induction of a nonspecific lymphoproliferation , which is believed to disrupt antigen recognition and interfere with protective immune responses . this hyporesponsiveness to antigen - specific and polyclonal stimuli in chronic parasitic infections could be related to immunosuppressive cytokines secreted by antigen presenting cells and regulatory t cells . a growing list of parasite - derived molecules able to exert immunomodulatory activities on the cells of the immune system leading to such polarized cytokine secretion has been reported . the intracellular phase of the parasite has been poorly studied , and it is known that t. cruzi amastigote surface antigens induce an immune response [ 9 , 10 ] . however , few such molecules have been thoroughly studied . recently our group has studied a t. cruzi amastigote - specific surface protein ( ssp4 ) , that is bound to the plasma membrane by a gpi anchor , which is released to the culture medium by phospholipase c activity . the gene for this protein ( cdna ) was cloned and partially characterized , obtaining the recombinant protein rmbp::ssp4 . we have reported that this protein is a modulator of humoral and cellular immune responses in murine model , inducing low levels of iga , igm , and igg3 , but high levels of igg1 , igg2a , and igg2b isotypes ; moreover , it is able to modulate nitric oxide production , as well as , to modulate the expression of cytokines in vivo in murine macrophages after immunization , suggesting that rmbp::ssp4 might exert a regulatory influence on macrophages during the immune response against t. cruzi . also , it has been observed that the protein , rmbp::ssp4 activates a population of il-10/ifn--secreting cd4 + t cells , which has been observed to be activated during chronic infections and is responsible for prolonged persistence of parasite and for host protection against severe inflammatory responses . finally , it was observed that immunization with rmbp::ssp4 protein makes mice more susceptible to trypomastigote infection , with high mortality rates , whereas mice immunized with a eukaryotic expression plasmid containing the rmbp::ssp4 cdna were able to control the acute phase of infection . it should be noted that parasite molecules that regulate the host ( human ) immune response during chagas ' disease have not been fully identified , and to date there are few reports about the role of amastigote proteins in the development of the disease ; therefore , it is important to characterize parasite molecules and their involvement in the pathology of the disease . in this work , we analyzed the effect of rmbp::ssp4 , a recombinant protein derived from t. cruzi in cursive ( a major surface glycoprotein ( ssp4 ) that is bound to the plasma membrane by a gpi anchor ) on the induction of nitric oxide ( no ) , cytokines , chemokines , and adhesion molecules ( cams ) using humans ' pbmc . briefly , the tcssp4 ( genebank , embl , and ddj databases accession number af480943 ) was cloned in the eco ri site of the expression vectors pmal - c2 ( new england biolabs ) resulting in the plasmid pmalssp4 . this plasmid and plasmid pmalc2 were used to transform e. coli dh5- , to obtain the fusion proteins rmbp::ssp4 and maltose binding protein ( mbp ) , which were induced and purified according to the manufacturer . either mbp or mbp - fusion protein were eluted by competition with free maltose ( 10 mm maltose in 20 mm tris - hcl ph 7.4 , 200 mm nacl , and 1 mm edta ) , and then acetone - precipitated . protein purification was analyzed by 10% sds - page in reducing conditions and coomassie blue staining . heparinized fresh human whole blood ( 10 iu heparin / ml ) was diluted 1 : 2 with pbs ( 137 mm nacl , 2.7 mm kcl , 4.3 mm na2hpo4 , 1.4 mm kh2po4 , and ph 7.4 ) solution . the pbmc fraction was obtained by ficoll - hypaque centrifugation . the pbmcs were cultured for 24 h at 37c at a density of 1 10 cells / well in dulbecco 's modified eagle ( dmem ) medium supplemented with 10% ( v / v ) fetal calf serum ( fcs ) . the viability of pbmcs was measured by trypan blue dye exclusion and was consistently greater than 98% . pbmc were incubated with dmem containing 10% fcs at 37c in 5% co2 in 24 well plates ( 1 10 cells / ml ) . cells were cultured separately in the presence de 10 g / ml rmbp::ssp4 protein or medium alone . cells and culture supernatants were collected at different times ( 12 , 24 , 48 , 72 , and 96 h ) , cytokine and chemokine concentrations and the expression of genes for cytokines , chemokines , adhesion molecules , and metalloproteinases were determined . nitrite accumulation , an indicator of no synthesis , was measured in the culture medium by griess reaction . in brief , human pbmc were stimulated with either rmbp::ssp4 ( 10 g / ml ) , mbp ( 10 g / ml ) , lps from escherichia coli ( 0.0111:b4 , 4 ng / ml ) ( sigma chemical co ) , ifn- ( 100 u / ml ) ( genzyme diagnostic ) , or lps plus ifn- , respectively . nonstimulated cells were used as a control . in some cases , n - nitro - l - arginine methyl ester ( l - name ; 3 mm ) ( sigma chemical co ) was added separately ; similarly 15 g / ml of polimyxin b sulphate ( pmb ) ( sigma chemical co. ) was added to inhibit the lps present in the recombinant protein derived from the purification process ( data not shown ) . 100 l of cell culture medium was mixed with 100 l of griess reagent and incubated at room temperature for 15 min . absorbance at 540 nm was determined , and nitrite concentration was calculated from a sodium nitrite standard curve . interleukin 1-beta ( il-1 ) , il-6 , il-12 , tnf- , ifn- , and chemokines ccl3 , ccl4 , ccl5 , cxcl10 ( ip-10 ) , cxcl8 ( il-8 ) , and ccl11 were quantified by elisa in culture supernatants of monocyte under different conditions of stimulation , according to the manufacturer 's protocol . briefly , 96-well flat - bottom plates were coated over night with a capture antibody at a final concentration of 2 g / ml , and then plates were blocked with 10% pbs - fcs , washed three times , and incubated with the cell culture supernatant samples or control antigens overnight at 4c . after washing , plates were incubated with the respective biotinilated anti - cytokine antibodies ( r&d system ) at 1 g / ml for 1 h in the dark . plates were washed and streptavidin - alkaline phosphatase at 1 : 2000 was added for 30 min in the dark then washed , and 100 l of abts ( 2,2,-azino - bis ( 3-ethylbenzthiazoline)-6-sulphonic acid ) ( zymed ) was added as substrate and the reaction was allowed to proceed for 20 min at room temperature ( rt ) ; the reaction was stopped with 2% sulphuric acid , and absorbance was read at 415 nm by an elisa reader ( multiscan ms , labsystem ) . total rna from pbmc , cultured in 24-well plates with different treatments for 48 h , was isolated using the trizol system ( life technologies ) . one microgram of rna was reverse transcribed to cdna with an oligonucleotide ( poly(dt)16 ) using the superscript ii reverse transcriptase ( life technologies ) and the cdna used as a template for pcr . pcr sequences and pcr conditions used for amplification of gapdh , il-1 , il-6 , il-12p40 , ifn- , tnf- , ccl3 , ccl5 , cxcl10 , e - selectin , icam-1 , vcam-1 , tnfr - i , and tnfr - ii were as follows : gapdh ( 5-ggt gaa ggt cgg agt caa cgg-3 and 5-ggt cat gag tcc ttc cac gat-3 ) , il-1 ( 5-atg gca gaa gta cct aag ctc gc-3 and 5-aca caa att gca tgg tga agt cag tt-3 ) , il-6 ( 5-atg aac tcc ttc tcc aca agc gc-3 and 5-gaa gag ccc tca ggc tgg act g-3 ) , il-12p40 ( 5-aac ttg cag ctg aag cca tt-3 and 5-tga tgt act tgc agc ctt gc-3 ) , ifn- ( 5-gac cag agc atc caa aag a-3 and 5-cct ttt tcg ctt ccc tgt ttt a-3 ) , tnf- ( 5-ttc tgt cta ctg aac ttc ggg gt-3 and 5-gta tga gat agc aaa tcg gct gac gg-3 ) , ccl3 ( 5-cgc ctg ctg ctt cag cta cac ctc ccg gca-3 and 5-tgg acc cct cag gca ctc agc tcc agg tcg-3 ) , ccl5 ( 5-cgg gat cca tga agg tct ccg cgg ca-3 and 5-cgg aat tcc tag ctc atc tcc aaa ga-3 ) , cxcl10 ( 5-cca cgt gtt gag atc att gct ac-3 and 5-aca tag cac ctc agt aga gct tac-3 ) , e - selectin ( 5-ctc tga cag aag aag cca ag-3 and 5-act tga gtc cac tga agc ca-3 ) , icam-1 ( 5-tat ggc aac gac tcc ttc t-3 and 5-cat tca gcg tca cct tgg-3 ) , vcam-1 ( 5-atg aca tgc ttg agc cag g-3 and 5-gtg tct cct tct ttg aca ct-3 ) , tnfr - i ( 5-tca gtc ccg tgc cca gtt cca cct t-3 and 5-ctg aag ggg gtt ggg gat ggg gtc-3 ) , and tnfr - ii ( 5- gct cgc cgg gcc aat atg c-3 and 5-ggc ttg cac acc acg tct ga-3 ) . pcr conditions were as follows : initial dna denaturation at 94c for 5 min and 35 rounds of denaturation ( 95c for 1 min ) , annealing ( 55c for il-1 , tnf- , il-6 , ccl5 , cxcl10 , icam-1 , vcam-1 , and e - selectin , 58c for tnfr - i , and tnfr - ii , 59c for gapdh , and 60c for ccl3 , ifn- , and il-12p40 for 1 min in each case ) and extension ( 72c for 1 min ) . pcr products were electrophoresed on 1.8% agarose gels containing 0.5 g / ml ethidium bromide and photographed under ultraviolet light . densitometric analyses were done using the image j software ( version 1.43 u ) . analysis of variance ( anova ) followed by tukey 's post - hoc test was performed to compare the mean values among various groups to first test the ability of the protein rmbp::ssp4 to induce nitric oxide production , pbmcs were stimulated with 10 g / ml of protein in vitro . result showed that rmbp::ssp4 protein is capable of inducing no production in pbmc after 48 hours of stimulation ( figure 1 ) , and that production is inhibited by the action of the inhibitor l - name . nitrite values obtained by the stimulation of rmbp::ssp4 protein are statistically significant when compared with the values of nonstimulated cells ( p < 0.0001 ) or with the values obtained from cells stimulated with mbp . cytokines play a fundamental role during the acute phase of t. cruzi infection and affect the function of all cells types involved in an immune response . to investigate whether rmbp::ssp4 protein altered cytokine expression , rt - pcr analysis was performed in pbmc stimulated in vitro with rmbp::ssp4 protein ( figure 2 ) . when pbmc were stimulated with rmbp::ssp4 protein , an increase in the expression of genes for il-1 , il-6 , il-12 , ifn- , ccl3 , ccl5 , and cxcl10 was observed from 12 to 96 h with low expression at 48 h ( figure 2 ) . when pbmcs were stimulated with rmbp::ssp4 protein , the production of il-1 , tnf- , and il-6 significantly increased ( figure 3 ) . for il-1 , the increase was observed at 2472 h , while for tnf- , the increase was from 1248 h and a sustained production of il-6 from 1296 h of interaction , with a maximum production at 2448 h. likewise , we observed an increase in the production of chemokines , such as il-8 , ccl3 , ccl4 , ccl5 , and cxcl10 in pbmc stimulated with rmbp::ssp4 protein . production of il-8 was observed only from 12 to 24 h of interaction , and an increase in the production of ccl3 , ccl5 , and cxcl10 with a maximum production at 48 h and decreased at 96 h of interaction . ccl4 production was also observed with a peak of synthesis at 72 h ( figure 3 ) . to investigate whether rmbp::ssp4 protein was able to induce gene expression of cams and tnf receptors , pbmcs were stimulated with recombinant protein . we observed in pbmc an increased expression of gene for icam-1 ( 1224 h ) and an increase in the expression of genes for e - selectin and vcam-1 , with a maximum expression at 96 and 48 h , respectively . likewise , we observed an increase in the expression of genes for tnfr - i and tnfr - ii of the 12 to 24 h ( figure 4 ) . most of the affected and/or exposed populations are living in developing countries where control measures are lacking or inadequately applied . although significant progress has been made in our understanding of the immune response to parasites , no definitive step has yet been successfully done in terms of operational vaccines against parasitic diseases . pathophysiology of chagas ' disease is not completely defined , although innate and adaptive immune responses are crucial . in acute infection , some parasitic antigens can activate macrophages , and this may result in proinflammatory cytokine production , nitric oxide synthesis , and consequent control of parasitemia and mortality . during the acute phase of infection , t. cruzi replicates extensively and releases immunomodulatory molecules that delay parasite - specific responses mediated by effector t cells . . the disturbed cytokine - chemokine network could play an important role in the onset of diseases with inflammatory processes . the results showed that rmbp::ssp4 protein induced no production in pbmc from 24 to 48 h ( figure 1 ) . we have previously shown that rmbp::ssp4 protein was able to induce nitric oxide ( no ) production by spleen and peritoneal macrophages ( pms ) and macrophages from immunized mice . inhibition of no production by l - name in murine ms , results in a down - regulation of inos expression . our results showed that no production was affected when stimulated pbmcs were incubated in the presence of l - name , thus indicating that the enzyme inos was participating in no synthesis , it is also known that tnf- regulates nos expression and/or activity , which exerts direct effects on no production . according to these observations , and the fact that rmbp::ssp4 protein induces no production by pbmc , the participation of no in the suppression of t cell activation has been reported in a number of biological systems . in the case of t. cruzi , previous studies have shown that ifn- and nonoxidative molecules ( tnf- and no ) could play a role in the control of t. cruzi infection in mice [ 27 , 28 ] . furthermore , a series of experiments supports the notion that ifn- and tnf- mediated activation of macrophages , which leads to increased production of no , and in turn suppresses t cell activation . therefore , it is likely that no production during the initial phase of acute infections might participate in the clearance of parasites by macrophages , whereas its overproduction during the late phase of acute infection would account for the immunosuppression observed . we investigated the cytokine and chemokine gene expression pattern in these cells as well as the production of these molecules in the culture supernatant . results showed that this antigen induces the secretion of several chemokines ( il-8 , ccl3 , ccl4 , ccl5 , and cxcl10 ) and cytokines , such as il-1 , il-6 , ifn- , and tnf- in considerable amounts , whereas il-12 was produced at a very low level suggesting that ssp4 is an immunomodulatory molecule of t. cruzi . furthermore , ifn- is an important th1 proinflammatory cytokine that could participate in the generation of tregs cells during acute phase , thus as has been observed in the mouse model using rmbp::ssp4 . in addition , il-12 has been described to have stimulatory effects on hematopoietic precursor cells and on b lymphocytes . the il-12 produced during this inflammatory phase , both by direct action and , indirectly , by determining the composition of the cytokine milieu at the site of the immune response , induces differentiation of t helper type 1 ( th1 ) cells while inhibiting the generation of th2 cells . thus , because of its double function of a proinflammatory cytokine and an immunoregulatory factor , il-12 plays a key role in the resistance to infections , particularly those mediated by bacteria or intracellular parasites , against which phagocytic cell activation and th1-mediated responses are particularly effective . however , because of the same activities , il-12 also plays a role in pathological situations , such as septic shock , tissue damage during inflammation , and organ - specific autoimmune diseases . accordingly , there are reports in animal models showing that inflammatory cytokines play a central role in acute t. cruzi infection ; upon activation , such cells secrete proinflammatory cytokines and chemokines are promptly released and further activate other inflammatory cells . this pattern of expression has been observed in the inflammatory responses in cardiomyocytes during t. cruzi infection . it was shown that heart tissues collected from t. cruzi - infected rats expressed il-6 , il-1 , tnf- , and inos , moreover , hearts of mice infected and cardiomyocytes express the same pattern of cytokines and chemokines [ 4 , 3234 ] . ssp4 superficial protein is expressed shortly after trypomastigotes begin to transform into amastigotes , in a phase which is released the amastigote - specific ssp4 protein , this protein can interact with and activate pbmc , secreting cytokines , chemokines , no , and other molecules which might attract leukocytes to the inflammatory site after interaction with specific molecules of the parasite , and that rmbp::ssp4 can significantly increase this effect . since it has been observed that pbmc recruitment is a rapid and remarkable phenomenon during acute infection with the intracellular protozoan parasite t. cruzi , the causative agent of chagas ' disease , the functional capabilities of these cells during the infection , however , are poorly understood . the ability of monocyte - derived macrophages to process and present antigens , produce cytokines , and provide costimulatory signals demonstrate their pivotal role in initiating immune responses . activated macrophages exert critical activities in immunity to parasites , playing a key role in the mechanism for halting the acute t. cruzi infection . activation of macrophages by parasite antigens results in proinflammatory cytokine production and consequent control of parasitemia and mortality . on the other hand , it has been observed that this protein induces high production of il-6 [ 13 , 15 ] , according to our results and because of the pleiotropic character of il-6 has made it difficult to obtain a clear answer for the role of this cytokine in this model ; however , the production of il-6 observed in pbmcs could possibly modulate the differentiation of t cells infiltrated through the process of chemotaxis toward a th2 pattern and may later be involved in the maturation process of b cell , during polyclonal activation observed in the acute phase of infection . this inflammatory t cell and antibody response leads to control but not complete elimination of tissue and blood parasitism . we showed that the expression of adhesion molecules and tnf receptors are upregulated in pbmc by the stimulation in vitro with rmbp::ssp4 protein ( figure 4 ) . accumulation of leukocytes at the site of local injury or infection of endothelial cells is dependent on the interaction of circulating leukocytes with vascular adhesion molecules , such as e - selectin , vcam-1 , and icam-1 . likewise , it is known that tnf receptors play a role in inflammatory process ; tnfr - i may have anti - inflammatory and inflammatory effects , depending on the signaling pathway that is activated . expression of tnfr - ii observed in pbmc suggested in the context of infection that parasite probably ensures the survival of the cell to perpetuate the process of infection and their tissue retention , possibly promoted by the action of il-8 . the soluble parasite factors can elicit a complex series of cellular interactions leading to an immunosuppression state , in addition , may have additional roles in driving early immunological events toward th2-type or anti - inflammatory responses fully polarized . these raise the distinct possibility that the production of parasite factors that interact with cell surface receptors may be one mechanism , whereby the parasite is able to interfere with the regulation of the induction / initiation phase of the host immune response that may protect the host from excessive inflammation and may potentiate the parasite 's own survival . finally , inflammatory response that follows the infection with t. cruzi is essential for host resistance to infection but is also responsible for the diverse pathology observed in chagas disease . parasite persistence depends on a combination of factors , including release of molecules that interfere with the immune response . therefore , suppression induced by parasite molecules is more relevant at the acute phase , when the concentration of such molecules can be fairly high . although the amastigote stage is considered essentially as the stage of intracellular replication , this form of the parasite is present in the circulation during the acute phase of infection in mice and can enter and develop in both mammalian phagocytic cells ( in vitro ) and nonmammalian phagocytic cells . in conclusion , all these results suggest that the amastigote ssp4 molecule could play a key role in the inflammatory process , modulating the expression and production of inflammatory molecules , which may represent a mechanism participating in the immunoregulatory and/or immunosuppressive processes carried out by t. cruzi during the development of the acute phase of chagas ' disease .

chronic obstructive pulmonary disease ( copd ) is a rising epidemic which is responsible for one in six deaths currently and is predicted to rise to one in three deaths by 2020.1 the disease is high cost , with advanced copd patients having repeat admissions with average stays of 811 days.2 these admissions are due to exacerbations , with the longest stays for those with hypercapnic respiratory failure ( hrf).3 hrf is common in moderately severe cases with forced expiratory volume in 1 second ( fev1 ) 40% of predicted . upon infection , these patients develop acidosis with co2 retention which can usually be reversed by nasal intermittent positive pressure ventilation ( nippv ) , antibiotics , steroids , nebulized bronchodilators and other standard treatments for copd.4 at our hospital , nippv is commenced in hrf when ph < 7.35 and paco2 > ; 8 kpa or earlier if there is a change in conscious level . according to our local guidelines , inspiratory positive airway pressure ( ipap ) and expiratory positive airway pressure ( epap ) are increased according to responses of the patients ( initial pressures ipap 810 , epap 56 ) . the carbon dioxide levels will often correct after an acute exacerbation , without the need for long - term nippv . for the individual patient at this stage , however , their disease progression and further damage from the exacerbation ( with or without smoking ) will lead them to more frequent admissions and episodes of hrf in the future.5 each admission carries the risk of death , which before the advent of nippv and the use of central nervous system stimulants such as doxapram , carried a very high mortality in the first 48 hours.6 since nippv , the mortality is much lower acutely and lives are saved , but readmissions of the same patients can become more and more frequent , with high hospital bed use.7,8 the use of long - term nippv is expensive in the uk . acute hospital costs of ventilation per patient episode are 13,163 . while home therapy costs 4,909 per annum , with a ventilator life span of only 5 years.9 many patients , with time , become poorly compliant due to the effects of nippv on sleep quality , nasal blockage , and dryness of the airway , with sinus - like headaches.10 as a result , many minimize their use and remove the nippv after an average of 4 hours . in the past , protriptyline was an oral respiratory stimulant that had use in respiratory failure either alone or in combination with nippv or cuirass , for example.11 this drug was suddenly discontinued in 2000 in the uk . modafinil is a successful drug licensed for narcolepsy and acts through the dopaminergic system and also has effects on the sympathetic nervous system.1214 it has multiple sites of action on the central nervous system and is noted to have an effect on respiratory drive.15 this effect is probably via the brain stem respiratory center and mediated by the tuberomammillary nuclei , which activate the cerebral cortex , including the striatum of motor activity.1618 studies have shown actions in obstructive sleep apnea and apnea - hypopnea syndromes , and even shift workers.19 the license for shift work has not been renewed in the uk by cephalon uk ltd since 2010 . the described actions of modafinil at various sites in the central nervous system led us to trial this drug in patients with hrf . modafinil must be avoided in pregnancy , lactation , and uncontrolled moderate to severe hypertension with cardiac arrhythmia . there is evidence for low - level dependence on modafinil.20 modafinil stimulates the brain to increase alertness and also to stimulate the rate of breathing . this latter effect has not been formally studied , although obstructive sleep apnea conditions have been examined;19,21 most studies focus on narcolepsy.2224 we report the use of modafinil in our six most recent cases with hrf , where prolonged or repeat admissions were occurring or nippv treatment was refused , failed , or was not tolerated . modafinil corrected the problem with very significant life quality improvements noted by the patients and their family , which avoided readmissions , as outlined in the case histories . the authors confirm that there is no conflict of interest in the presentation of these cases . this was his third admission for the same condition in 4 weeks . on each occasion , on awakening , he would panic and pull off the nippv mask and refuse to go on with the treatment . he would be observed for approximately 4 days and would remain hypercapnic ( partial pressure of arterial carbon dioxide [ paco2 ] > ; 6.8 kpa ) . once judged clinically stable with optimized bronchodilatation , he would be discharged , only to return 1 week later unconscious in the same condition . on questioning , he denied any additional use of alcohol , sleeping tablets , tranquilizers , or painkillers prior to these episodes . he did not have home oxygen , and these episodes could not be explained by an infectious process . he had a home nebulizer ( air driven ) for salbutamol and ipratropium , a seretide 250 inhaler , frusemide , aspirin , digoxin , and oral theophylline . he was on mirtazapine 45 mg once daily ( od ) for his anxiety , and citalopram 40 mg od for several years without prior respiratory depression . he admitted to anxiety and panic disorders , even associated with using a nebulizer mask , and especially with the nippv mask . he felt that his respiratory condition had generally deteriorated over 6 months , following pneumonia the previous summer . the onset of each episode of respiratory failure was unpredictable , and he would report going to bed well but awake in hospital on the nippv without any warning . low - flow nasal oxygen ( 1 l / min ) rapidly increased the paco2 to > ; 8.3 kpa . to avoid a further admission and the risk of death , he was started on modafinil 200 mg in the morning . by 10 days of treatment , his arterial gases had improved , along with day- and nighttime oximetry ( table 2 ) , and remained stable long - term . modafinil did not affect his previous anxiety , and he felt his breathing and well - being had improved . lung function remained the same . in april 2013 , he developed community - acquired pneumonia and died following admission , without hrf after 15 months of treatment . an 81-year - old man ( table 1 ) was admitted with a left hip fracture after his leg suddenly gave way at home . he had copd and had used a nasal cpap ( continuous positive airway pressure ) machine ( 6 cm pressure without oxygen ) for obstructive sleep apnea for 7 years . medication included seretide ( 250 ) , two puffs twice daily ( bid ) by a spacer , tiotropium 18 g mane , salbutamol mdi ( metered - dose inhaler ) 200 g as required , carbocisteine 375 mg three times a day , and frusemide 40 mg od . upon admission with the hip fracture , his oxygen saturations ( spo2 ) were low ( table 2 ) , with hrf . as a result , surgery was delayed , and nasal ventilation was commenced ( ipap 12 , epap 6 ) with oxygen ( 2 l / min ) . at 48 hours , gases improved , and he proceeded to have his hip replacement . postoperatively , the patient was observed in the high dependency unit , where nasal ventilation continued before transfer to the orthopedic ward for mobilization . by day 3 postop , he was still in hrf despite nippv ( ipap 18 , epap 8) and oxygen 1 l / min . the patient s chest was clear , without evidence of infection clinically or radiologically , but antibiotics and steroids were started in case of benefit . his arterial carbon dioxide levels remained elevated without painkillers , sedatives , oxygen over - prescription , or any obvious explanation for nippv failure . he transferred to the chest ward and continued mobilizing with the physiotherapist , who noticed low spo2 ( 68% ) on exertion . eventually , nippv use became continuous due to increasing episodes of co2-narcosis by day , occurring when the ventilator mask was removed for breakfast , lunch , or supper . he would suddenly be noted by the nurses to be sleepy and unresponsive ( respiratory rate 68 bpm ) , with paco2 values > ; 10 kpa on air . he had been rescued so many times from unconsciousness that there was great concern as to whether we could break this cycle . his family were alarmed and felt he could not be discharged in view of these episodes . nippv continued for over 6 weeks without clear benefit , despite compliance with treatment and increased inspiratory and expiratory pressure adjustments . a request to pharmacy for modafinil 200 mg mane was granted . from the first tablet this completely stopped the episodes of unconsciousness and facilitated his discharge from hospital . by day 10 , he was carefully observed by frequent home visits by our respiratory nurses and has remained very well , without nippv or cpap at any time . on modafinil , there have been no further readmissions , no episodes of loss of consciousness , and no adverse effects in 15 months . the patient claims that he is better by the week and extremely active compared with previously . he is pursuing his hobbies of cooking and gardening , and he partakes in social outings . case 3 is a 68-year - old man with hrf , diagnosed following admission for leg swelling . his hemoglobin was 20.3 g m / dl ( hematocrit 0.66 ) and he admitted to shortness of breath and a cough , with an exercise tolerance of 50 meters . the polycythemia was deemed secondary to smoking ( table 1 ) , and he admitted to lethargy and headache and had a cyanotic appearance . he was commenced on seretide ( 250 ) two puffs bid via a spacer , tiotropium 18 g mane , and salbutamol as required . once clinically improved by bronchodilators , steroids , and diuretics , his arterial gases still showed hrf with significant hypoxemia . on 1 l / min oxygen over 4 hours his life quality had been poor , and he smoked to increase his state of alertness . he was commenced on modafinil 200 mg in the morning , and by 14 days , he felt better , more alert , and stopped smoking . his forced vital capacity ( fvc ) improved on treatment and smoking cessation to 63% predicted . he no longer appears cyanosed , has had no further admissions in 21 months , and went on to complete pulmonary rehabilitation . a 64-year - old man ( table 1 ) was referred by the hematologists in 2009 for polycythemia considered secondary to his respiratory disease . he was generally cyanosed , and an echocardiogram confirmed a dilated right heart consistent with cor pulmonale . a computed tomography scan had ruled out pulmonary emboli but showed hyperinflation without significant emphysematous changes . the patient was inactive , overweight , and smoking , with daily malaise and headaches . he took regular symbicort ( astra - zeneca , london , uk ) ( 400/12 ) two puffs bid , tiotropium 18 g mane , and bumetanide . oxygen studies showed hrf ( table 2 ) and low nocturnal spo2 . an oxygen trial ( on 1l / min nasal prongs ) showed an improvement in oxygen levels to 77% , with a small rise in paco2 at 2 hours to 6.91 kpa ; but the overnight trial produced severe headache , increased drowsiness , and a further rise in paco2 . in the uk , his active smoking would preclude any use of home oxygen even with a nasal ventilator . day- and nighttime spo2 improved ( table 2 ) , with complete alleviation of malaise , headache , and inactivity . his fev1 improved marginally ( from 33% to 38% ) , and also his fvc ( from 61% to 85% ) , but smoking continued . his wife reported that he developed an interest in things , and was much more alert and chatty for the first time in several years . he continued to smoke but had no admissions in 3 years remaining on modafinil without requiring further venesections . in december 2012 , the patient developed a chest infection with leg edema . this did not improve over several weeks despite antibiotics , steroids , and diuretics , and there was an increase in hypoxia and drowsiness leading to admission . gases showed ph 7.37 , paco2 7.94 , pao2 ( partial pressure of arterial oxygen ) 5.6 , be ( base excess ) 9 , hco3 ( bicarbonate level , mmol / l ) 34.7 , and spo2 63% . he was treated with antibiotics , steroids , bronchodilators , mucolytics , physiotherapy , short - term nippv , and diuretics for right heart failure to which he responded . since discharge he has remained on modafinil now for 4 years , with arterial gases remaining stable , apart from a single admission at 36 months . nocturnal spo2 is now 90% on air , with only a mild obstructive sleep apnea pattern . daytime saturations remain at 92% on air . with smoking cessation and further weight loss , fev1 was 1.23 l ( 42% predicted ) and fvc 2.28 l ( 88% predicted ) . a 78-year - old woman ( table 1 ) was admitted with hrf , with a presumed infective exacerbation . she had been on long - term antipsychotic therapy and was taking the following drugs : frusemide 40 mg / day , losartan 25 mg / day , olanzapine 10 mg / day , mirtazapine 30 mg / day , seretide ( 250 ) two puffs bid , and tiotropium bromide 18 g / day . the patient s lung function was poor ( table 1 ) , and following treatment , she was readmitted later the same month with a second exacerbation of hrf ( c - reactive protein 68 ) . although she responded to nippv each time , she was intolerant due to claustrophobia associated with wearing the mask , removing it as soon as she was alert . once she was alert and more stable , overnight oximetry showed mean nocturnal spo2 of 72% on air . oxygen studies even at low flow 1 l / min ( nasal cannulae for 2 hours ) gave a rise in paco2 to 11.0 kpa . to facilitate discharge and prevent a third admission , modafinil 200 mg mane was commenced with monitoring in view of her blood pressure , anxiety , and psychosis . at 10 days following discharge on modafinil her arterial gases had improved ( table 2 ) . the prescription was continued , and the patient remained entirely well and out of hospital . there were no adverse events of modafinil on her anxiety or her mental health , and ongoing follow - up continued for 10 months . the improvement was maintained despite no discernable change in her lung function . in december 2012 , the patient developed community - acquired pneumonia with congestive cardiac failure . on hospital transfer , the ambulance crew gave 60% oxygen , resulting in a rise in the paco2 to 10.1 kpa . this was corrected with nippv , and she was treated for heart failure and pneumonia but died within 24 hours . a 66-year - old retired national health service ( nhs ) nursing sister ( table 1 ) was admitted july 2012 unconscious with hrf . she was a nonsmoker , but as a nurse had been exposed to years of passive smoking at work and at home . her spirometry was poor , with a restrictive pattern , probably due to her increased body mass index . clinically , she was considered to have copd , congestive cardiac failure , and pulmonary hypertension . in the past treatment included frusemide 40 mg od , symbicort ( 400/12 ) two puffs bid , tiotropium 18 g / day , and candesartan 4 mg / day . the patient described being unwell and housebound during the last 4 years , ordering shopping online , with lethargy and shortness of breath probably due to chronic elevation in her blood carbon dioxide . she suffered chronic leg edema and was aware that her heart was large on chest x - ray , with dilated pulmonary arteries . her echocardiogram showed a normal left ventricle but a dilated right ventricle , with tricuspid regurgitation but preserved right ventricle function . the patient was obese and had been an alcoholic but had given up drinking alcohol 30 years before . she was commenced on continuous nippv , and after several days , she improved clinically . she then developed severe nose bleeds each night while continuing nocturnal nippv , despite cauterization , oral tranexamic acid , and stopping prophylactic heparin . day and night oximetry recordings improved after 1 week of nippv ( table 2 ) from 75% on air initially . dietary advice and exercise was highlighted . the idea of lifelong nippv did not appeal to her despite the explained benefits to symptoms and survival , along with the improvement in her arterial gases and energy . overnight studies on 1 l / min of oxygen via nasal prongs showed her to be very oxygen sensitive , with a sharp rise in her paco2 to > ; 9 kpa . due to the risks of further hrf , she was commenced on oral modafinil 200 mg mane . at 10 days following discharge from hospital , arterial blood gases the patient felt well , her fatigue and headaches cleared , and she described herself as being champion compared with the last 4 years . her progress continued , and she was out of the house doing her own shopping , which aided weight loss . she has had no further admissions in hrf and remains on modafinil ( 17 months ) . over the last severe winter , she became very inactive , gaining more weight ( 13 kg ) , leading to a fall in spirometry . fev1 was 0.6 l ( 36% ) and fvc was 0.64 l ( 31% ) , but arterial gases remained stable . as a result , she is undergoing pulmonary rehabilitation and further dietary advice . searches of the current medical literature show that there are no described studies or case reports of modafinil use in hrf . our findings show modafinil to have had a beneficial effect on all the described patients , improving their oxygenation and lowering their blood carbon dioxide . this effect occurred without a clear change in lung function , and the likely mechanism is unclear . the patients remained stable and without readmissions with hrf for periods of over 1 year , and we encountered no adverse events . the possible effects on respiratory rate have not been formerly studied , with only one patient ( case 2 ) showing an alkalosis on their arterial gases post - modafinil . in case 2 , around - the - clock treatment with nippv at maximal ventilation over 6 weeks did not correct the blood carbon dioxide level . yet modafinil improved his arterial gases measured by 10 days of treatment , which is the time required for a steady - state blood level . from our cases , the effects of modafinil on hrf would appear useful and needs evaluation , especially with the increase in pressure to reduce admissions and length of stay , especially in advanced copd . it may be useful in hrf unrelated to copd , as in case 6 , especially where the etiology of the hrf was probably mixed . modafinil 200 mg / day has a cost of 745.92 per annum within the nhs ( actual cost per annum is 1,262 ) . the current nhs tariff for patients admitted with copd who require nippv is 2,2273,228 per episode , depending on complications . the real cost of nippv per inpatient episode in the uk , from the leeds study 10 years ago , gave a value of 13,163.9 the cost savings are obvious from the cases we described , and has probably saved lives and hospital bed occupancy in the presence of the high admission rates for advanced copd . additionally , there are calls for current drugs on the market to have their full therapeutic potential investigated instead of a relentless drive to manufacture new agents.2528 this has been highlighted by many serendipitous observations of drug benefit in unrelated conditions , leading to major therapeutic breakthroughs in disease management . one such example is the use of thalidomide in myeloma.29 however , the restrictive practices placed on doctors by evidence - based medicine makes off - license use difficult . in the uk , this includes preliminary observations and testing , which is always advisable prior to time and resources being spent on clinical trials . in the usa , the off - label use of modafinil is very common by doctors and is acknowledged in the medical journals , with a 15-fold increase in off - license use by neurologists , pulmonologists , otolaryngologists , and psychiatrists.30,31 currently , the only evidence - based treatment in hrf is the use of oral acetazolamide which produces a decrease in blood carbon dioxide levels , maybe via actions in the carotid body and elsewhere.32 to take this work forward , we feel that an open study of modafinil in hrf is justified , probably using modafinil added to standard care versus standard care alone . many patients , including those with copd , have abnormalities in sleep architecture and oximetry , which is being increasingly recognized , indicating a more complex picture in copd.33 the speed of patient improvement in arterial gases , overnight oximetry , and hospital length of stay / readmission would no doubt be of interest to future clinical commissioning groups in the national health service . other interesting questions would include the drugs effect on the respiratory rate , quality of life , sleep architecture , and bode ( body mass index , airflow obstruction , dyspnea , and exercise capacity ) score . this would require a formal study in order to take a more systematic approach to data collection .

proteins fold because water is a marginal solvent for peptide polymer chains . changing the quality of the solvent affects solution stability or aggregation and is usually accompanied by changes in protein stability . timasheff and co - workers have analyzed changes in protein stability and solubility in the presence of osmolytes in terms of liquid state correlations among the protein , the solvent , and the osmolyte . the correlations may be described by preferential inclusion or exclusion of cosolvents in the protein solvation shells . other work has parsed the solution data into contributions from the backbone vs the side chains in various solvents and has shown that the backbone contributes significantly to changes in protein stability and solubility . oligoglycines are useful models of the protein backbone and of aspects of protein folding since as their length increases , their solubility in water decreases , resembling the marginal solubility and behavior of many globular proteins in water . they are also idealized models of certain intrinsically disordered proteins ( idps ) or disordered protein domains because of their inherent flexibility and lack of persistent secondary structure . this allows us to use them to examine transitions from extended , disordered states to collapsed , disordered states simulations of dilute oligoglycine models show that they collapse in water , a result that was borne out by single molecule fluorescence correlation spectroscopy experiments on oligoglycines at subnanomolar concentrations . the collapse of long oligoglycine is another manifestation of the experimental poor solubility of short oligoglycines in water . remarkably , this collapse occurs in spite of the favorable decrease in solvation free energy with increase in length that has been calculated at infinite dilution . in both peptide collapse and aggregation , the result is not a solid state but a liquid the occurrence of a separate dense liquid phase has been noted in the precrystallization stage in proteins as well however , such a separation can also be formed by inducing a large dipole moment in the protein or peptide by , for instance , changing the ph of the solvent . ph dependent changes in the solubility of ribonuclease sa resulting in the formation of insoluble amyloid fibrils of the protein have also been reported . change in solubility can also be caused by altering the flanking sequence of aggregating units as has been observed in huntingtin protein . here , we simulate the aggregation of pentaglycines in water as a model of a liquid liquid phase separation analogous to proteins and analyze the molecular basis of the transition . in folding and aggregation , we expect a mechanism dominated by solute - to - solute interactions . generally , protein and peptide structures are thought to be dominated by hydrogen bonding . we analyze the nature and type of interactions responsible for the solubility limit of our model peptides . in the next section , we then present and discuss the results in terms of the interactions and correlations found in the systems . we conclude by drawing comparisons to longer oligomers of glycine and consider the role these mechanisms may play in the function of idps . in order to study the phase separation of glycine oligomers , we built systems of 625 pentaglycines with capped ends ( gly5 ) at two concentrations , 0.3 m ( the concentrated system ) and 0.03 m ( the dilute system ) . both concentrations are above the extrapolated experimental solubility limit of pentaglycines of around or under 1 mm . some liquid state studies have been done on ionic solubilities for common force fields that showed interesting trends with respect to van der waals parameter contributions . the large number of peptides used in this study is necessary to allow for the formation of clusters / aggregates that are large enough to overcome surface tension effects . a large system also ensures that artifacts of the periodic boundary conditions can be made small ; for example , a smaller system would have a smaller heat capacity , which would affect the rate at which aggregation proceeds . the peptides , built with charmm , were arranged on a cubic lattice and solvated using vmd at concentrations of 0.3 and 0.03 m. the concentrated system had 90,940 water molecules and a cubic cell of side length 145 . the dilute system had 1,105,898 water molecules and a cubic cell of side length of 325 . it was briefly heated to 400 k , cooled , and equilibrated at constant pressure and temperature ( npt ) ( pressure = 1 atm ; temperature = 300 k ) for 2 ns , the pressure checked at constant volume for 1 ns , and then run for a 100 ns in npt ( pressure = 1 atm ; pressure = 300 k ) . aggregation began as soon as the temperature was reassigned to 300 k. the other two were run , one for 100 ns and one for 50 ns , in the npt ensemble ( pressure = 1 atm ; temperature = 300 k ) after minimization ; aggregation started immediately in both runs . results reported here are primarily for the nonheated system run for a 100 ns unless otherwise noted . the dilute system was first equilibrated in npt ( pressure = 1 atm ; temperature = 300 k ) for 2 ns and in nvt ( temperature = 300 k ) for 1 ns , before being run in the npt ensemble ( pressure = 1 atm ; temperature = 300 k ) for 100 ns . the namd package was used to run all simulations with the charmm22 force field with cmap correction . particle mesh ewald was used to calculate long - range electrostatic interactions , with a maximum space of 1 between grid points . temperature was controlled with langevin dynamics with a damping coefficient of 4/ps , applied only to non - hydrogen atoms . pressure was controlled by the nose - hoover method with the langevin piston , with an oscillation period of 200 fs and a damping time scale of 100 fs . simulations were run on keeneland , a heterogeneous , high performance computing system with 264 nodes comprised of hp sl250 servers . each node has 32 gb of host memory , two intel sandy bridge cpu s , and three nvidia m2090 gpus . all binary mixtures that are not miscible in all proportions display a solubility limit . liquid phase separation or a liquid solid transition . for the systems here , all evidence both theoretical and experimental points to a liquid liquid separation . experimentally , when density differences between the two phases are large , gravity is sufficient to separate the liquids . when the density difference is less , an ultracentrifuge may be employed . here , we have chosen a route computationally convenient without the presence of another field to separate the phases . we consider a phase separated system in terms of aggregated clusters and supernatant , which has only monomers and small associated clusters . this is based on the expectation that given a standard nucleation barrier , we would expect a few large clusters and some number of solute molecules not in aggregates left in equilibrium in solution . the supernatant by definition is at the concentration of the solubility limit in a phase separated system . on the basis of the cluster size distribution , we can remove the substantial clusters from our supernatant by calculating the number of molecules and the solvent excluded volume of the large clusters and subtracting them from the total system . as the clusters are dynamically in exchange with molecules in the solvent , we average over 1000 time points after the phase separation in completed , in the last 50 ns . , the inherent flexibility of the pentaglycine makes the classification of peptides into clusters based on the distance between the centers of mass or geometry ambiguous . furthermore , a cutoff distance based on these centers leads to erroneous assignment of well solvated peptides into clusters . for our analysis , we have used an all - atom distance criteria . while this is more computationally intensive , it gives us an accurate cluster assignment . we have assigned two peptides to the same cluster if the distance between any two atoms in the peptides is 4 , which is just larger than the donor acceptor distance for a h - bond but small enough that water may not intervene . the conventional belief is that as a peptide collapses ( or aggregates ) and sacrifices solvation free energy , it gains stability through internal interactions , particularly hydrogen bonds . for our purpose an h - bond was defined as present if the distance between o and polar h atoms was less than 2.4 . the h - bond criteria is the same as the one used by default in the charmm program and has been shown to include all h - bonds possible when an angle cutoff was not included . other stabilizing interactions for proteins involving the backbone co dipoles have been considered in the literature . co interaction was considered to be present if the distance between c and o atoms was 4.2 . both cutoff distances were taken from the radial distribution curves of the respective atoms about each other . solvent , solute solute , and intrasolute terms as reflected in the force field . those were further subdivided into van der waals , electrostatics , and bonded interactions . we also monitored the classical h - bonds in the total energy categories . a variety of dipolar interactions were also monitored of which h - bonds are a subset . for the analysis presented below , the h - bond energy between a pair of nh and co atoms was calculated as the sum of the lennard - jones potential and electrostatic interactions : where i defines the donor atoms n and h , and j defines the acceptor atoms c and o. the co co interaction energy was calculated using the same method with a second co atom pair instead of the nh atom pair . a distance cutoff of 10 between the o and h atoms for h - bonds and the o and c atoms for co co interactions was used to exclude long - range interactions that have a negligible contribution to the interaction energy . we calculated the dipole moment of the peptide amide group using the method described by ripoll et al . the peptide group dipole is defined aswhere ri is a charge - weighted average of the positions of the atoms in the dipole , i.e.,correlations between two dipoles are given by the dot product between them . the concentrated systems ( 0.3 m ) begin to phase separate almost immediately during production , and by 10 ns , distinct peptide - rich and water - rich regions are visible . by 20 ns , clusters of tens of peptides are formed for the 300 k runs . over the next 30 ns , these clusters combine to form very large clusters of hundreds of peptides with relatively dry interiors that resemble droplets observed during simulations of phase separation in solvophobic solutions . by 50 ns , these droplets combine to form a single large cluster of over 550 peptides that persists for the remainder of the simulation ( figure 1a ) . all systems at the same concentration showed phase separation , albeit by different pathways . the system that was heated briefly rapidly formed clusters of hundreds of peptides and was stable by 20 ns , a classic example of an increase in temperature leading to an increase in solvation forces driving aggregation . temperature increases leading to increased collapse has recently been observed in several proteins including idps . representative snapshots showing clustering in ( a ) 0.3 m at 100 ns and ( b ) 0.03 m at 40 ns . the color scheme is red , monomers ; yellow , dimers ; green , trimers ; and blue , larger clusters . in the 0.3 m system , almost all of the peptides are in a single large cluster . in the 0.03 m system , there are a few small clusters comprising 47 peptides . where peptides form a classic secondary structure , it is shown as a ribbon . the dilute system ( 0.03 m ) forms clusters of up to 12 peptides , which then break up and reform repeatedly ( figure 1b ) . throughout the simulation , we did not observe phase separation , and large clusters like those observed in the concentrated system are not formed on the time scale allotted for the computer experiment . distribution of average cluster size across the last 50 ns at the two concentrations . the inset shows variation in cluster size in the two concentrations across the entire simulation period ( 100 ns ) . the red squares indicates clusters from the 0.3 m solution , and the blue triangles indicate clusters from the 0.03 m solution . the time averaged distribution of the observed clusters by size , shown in figure 2 , demonstrates the variation of cluster sizes in the two systems in the last 50 ns , the period after phase separation has occurred in the concentrated system . the cluster size distribution over the entire simulation is also shown ( inset ) . two peptides are defined as belonging to the same cluster if the distance between any two atoms in the peptides is 4 , a distance just larger than the donor last 50 ns in the concentrated system ( indicated by red squares ) , most of the peptides ( 600 ) are in a single large cluster , while the remaining peptides are present in the solvent as monomers . the dilute system ( indicated by blue triangles ) has a few hundred monomers . as the cluster size increases , their number of occurrences decreases until when the cluster size reaches 10 , it goes to zero and remains there . the distribution for the dilute system is similar to the distribution during the last 50 ns . across the entire simulation period , however , since these clusters are absent after phase separation , it is clear they are only formed during the initial phase transition stages . if monomers , dimers , and trimers are considered small clusters , the ratio of peptides in large clusters to small clusters is 15.9 in the concentrated system , indicating that at 0.3 m the equilibrium constant of the concentrated systems favors the formation of large clusters . in figure 3 , we have shown a surface rendering of the peptides from a single simulation snapshot . the morphology and topography of the peptide cluster surfaces are reminiscent of those of globular proteins depicted in a similar manner . we categorize the instantaneous solvation into three groups : water on a surface peptide , water near clefts or pores in a cluster , and water excluded from dry interiors of clusters . in the figure , we have shown examples of all three types of waters within 6 of any atom of a representative peptide for each category . we chose an interior dry peptide , which was only one layer of peptides deep , for which only a single water on the surface met the criterion . we also show a channel of waters protruding through the droplet and waters on the surface of an exposed peptide . the difference here is that the peptide droplets are not made of kinetically stable secondary or tertiary structure . thus , the topography the water samples changes on a few picosecond time scale and is generally less stable than the surface of natively folded proteins . surface rendering of peptides from a single snapshot from the 0.3 m system . three examples of all solvation water that is within 6 of a given peptide are shown with red colored water oxygens . the case of waters near a dry , interior peptide is on the left , around a surface peptide is shown in the middle , and a channel of waters penetrating into the cluster is shown on the right . using the surfaces as depicted in figure 3 , we can calculate the concentration of the saturated solution by subtracting the volume and number of the large clusters from the whole system and recalculating the concentration from the remainder as discussed in methods . using the plot in figure 2 we find the concentration of the saturated solution to be 0.016 m 0.003 . this is below both our initial concentrations of 0.3 and 0.03 m. while the aggregation into clusters in our more concentrated system stabilized within a few nanoseconds , no sizable aggregates ( greater than 12 ) formed on the 100 ns time scale for the 0.03 m system . however , when the single , large aggregate formed in the 0.3 m system was placed in a larger box of water , such that its concentration was 0.03 m , the aggregate ( comprising 598 peptides ) was stable for over 15 ns , indicating that when large aggregates are formed in the dilute system , they are very stable , both kinetically and mechanistically . figure 4 depicts the convergence of the solubility as measured by the concentration in the supernatant with respect to time for 0.3 m. a precipitous drop initially from around the starting concentration is seen until we reach a plateau in a few tens of nanoseconds . small fluctuations persist as the shape and makeup of the aggregates evolve in time . the experimental solubility is lower by an order of magnitude , but the force field used and force fields in common usage in the field we now consider the mechanism of cluster formation by computing the number of stabilizing interactions per peptide , both intramolecular and with other peptides within a cluster , versus cluster size . h - bonds feature prominently in the literature of peptide / protein structure stabilization , and those are plotted versus cluster size in figure 5a . on average , we observe less than two interpeptide h - bonds per pentapeptide in the concentrated system , irrespective of cluster size , and the dilute system has even fewer interpeptide h - bonds . the peptides do not form any appreciable intrapeptide h - bonds ( figure 5a ) in either system . this is notable , especially given the loose definition of a h - bond used here , given that the conventional wisdom is that h - bonds are the primary drivers of the stabilization of collapsed states in peptide and protein systems . variation in the number of h - bonds and co co interactions , per peptide versus cluster size for the two concentrations . at the top , panel a shows the number of interpeptide ( empty bars ) and intrapeptide ( filled bars ) h - bonds in 0.3 m ( red ) and 0.03 m ( blue ) concentrations . at the bottom , panel b shows the number of interpeptide ( empty bars ) and intrapeptide ( filled bars ) co co interactions at 0.3 m ( red ) and 0.03 m ( blue ) concentrations . we observe a considerable number of interactions among the co dipoles both intramolecular and with other peptides within a cluster versus cluster size ( figure 5b ) . we have defined a co co interaction as present if the distance between the two atoms is 4.2 ( taken from the first minimum of the radial distribution of c atoms about o atoms ) . all peptides , irrespective of cluster size , form about 5 intrapeptide co co interactions in both systems . most of these interactions occur between adjacent dipoles that are oriented in the same direction . we notice that the number of interpeptide co co interactions increases as cluster size increases in the 0.3 m system as opposed to the steady number of intrapeptide interactions . an interaction between co dipoles is not considered a traditionally stabilizing protein / peptide interaction . however , such an interaction is possible due to the partially positively charged c atom and the partially negatively charged o atom . different groups have seen evidence supporting the stabilizing effect of these effective interactions . some have referred to these correlated non - h - bonding interactions of the peptide group or in terms of quantum chemical descriptors as in n * interactions . others have used a classical electrostatic description of these interactions such as dipolar electrostatic interactions . several such interactions have been observed in crystal structures of small molecules , protein we use are parametrized in part from quantum chemical calculations , they represent similar model energetic concepts . pair energy probability distribution of interaction energy from co to nh groups ( red ) and between co groups ( blue ) between peptides at 0.3 m. figure 6 depicts the pair probability distribution of the energy for traditional h - bonds and the co co interaction between peptides in the 0.3 m system . our pair energy distribution for the h - bond uses only the co to nh interaction . using the whole peptide moiety produces a value a couple of kcal / mol lower for both the traditional h - bonds and the co interactions but does not change the number or probability of the interactions . while the co co interaction is weaker than the co - nh interaction ( the lowest point of the co to co peak occurs at 3.0 kcal / mol in comparison to 4.0 kcal / mol for the co to nh ) , there are far more co co interactions as indicated both by the heights of the peaks and by figure 5 . thus , co co interactions , while weaker , contribute more to the overall stability of the clusters . therefore , it appears that stable clusters are formed with hardly any appreciable role played by the expected peptide other , nontraditional dipolar interactions are seen here to play a significant role holding the clusters together . stable secondary structural elements such as -helices and -sheets are absent in the aggregates in the 0.3 m system ( figure 1a ) . however , we do observe order in the arrangement of atoms about each other by charge . figure 7 shows the radial distribution of positively charged atoms ( h and c ) about negatively charged atoms ( n , o , and c ) , negative about negative , and positive about positive , between peptides . the peak near 2 in the + curve is from the ho interactions of hydrogen bonds . similarly , the peaks at 3 in both the like charge curves ( + + and ) are also due to atoms involved in h - bonding , with the distribution of c atoms about h atoms causing the peak in the + + curve , and the distribution of n about o causing the peak in the curve . beyond these distribution of c atoms about o atoms occurs in this peak . a final layer of like charges occurs at 5 before the correlations deteriorate . a shoulder at 4.0 in the curve is caused by the distribution of oxygen atoms about each other . thus , while the peptides do not interact via h - bonds , they do arrange themselves such that the charged atoms form layers about each other facilitating the formation of other , non - h - bond interactions . we have previously observed charge correlations causing peptides to undergo changes in solubility as well as structure . the structure is not unexpected from the arrangements seen in other charged systems such as fused salts . distribution of atoms by charge between peptides in 0.3 m. distribution of oppositely charged atoms about each other is indicated by the red curve , distribution of positively charged atoms about each other is indicated by the black curve , and distribution of negatively charged atoms about each other is indicated by the blue curve . dipole correlation with respect to the distance between the center of peptide charge , both within an oligopeptide and between oligopeptides , up to a distance of 10 for the concentrated system . the intrapeptide curve shows a strong positive correlation between 2 and 3 followed immediately by a strong negative correlation between 3 and 4 . an example is shown in figure 9a , where the two parallel dipoles are 3 apart , and the two antiparallel dipoles are 3.5 apart . adjacent dipoles oriented in the same direction are usually involved in co co interactions , as we previously observed when we estimated the number of intrapeptide co interpeptide ( red ) and intrapeptide ( blue ) dipole correlations with respect to distance in the 0.3 m system . the correlations between dipoles that have been identified as forming n * interactions in the k - fab complex are shown as black asterisks . examples of ( a ) intrapeptide and ( b ) interpeptide dipoles in the 0.3 m system . co groups on adjacent dipoles have been observed forming n * interactions in the k - fab complex . they occur within our regions of positive intrapeptide dipole correlations for adjacent peptides . it is interesting to note because the interactions identified by barlett et al . occur along a stretch of protein comprising varied sequences . thus , while it could be argued that the absence of side chains in our system allowed the occurrence of these dipole correlations , their presence in protein complexes in regions composed largely of side chains indicates that they can and do occur in full length proteins and conceivably play a functional role there . the third peak in figure 8 ( positive ) occurs in both intra- and interpeptide correlations between 4 and 5 . both h - bonds and co however , since there are no intrapeptide h - bonds to speak of , we find that in the intrapeptide curve at least , all the correlations at this distance are due to the co co interaction . thus , while n *-type interactions as defined by raines and co - workers can occur only between adjacent amides , we find evidence that similar interactions can occur between peptide groups that might be quite far apart sequentially . figure 9b shows examples of two such correlations between three dipoles across three different peptides . the dipoles are oriented such that the o atoms of each are pointed toward c atoms of the other dipole . while this brings the oxygen atoms of each dipole into close proximity , it also allows for the formation of interactions between the o atom of one dipole and the c atom of the other , which in turn hold these three different peptides together in a cluster . glycines are often on the border between polar and nonpolar on many scales of solvophobicity . the oligomers of the glycine phase separate , and clustering the separation seen is similar to that observed in simulations of classically hydrophobic molecules such as neopentane and 2-butoxyethanol , as well as the turbidity observed in protein solutions undergoing liquid liquid phase transitions . in our case , phase separation is accompanied by a decrease in the interpeptide potential energy , with both the van der waal s interaction and the electrostatic interaction contributing favorably . at the same time , the peptide water interaction potential increased across the same period as the water molecules are excluded from the interiors of the peptides , again behavior often considered a part of the hydrophobic effect that drives protein folding . we observe further similarity between aggregation and protein folding in the solvation pattern of the aggregates , with relatively dry interiors , highly solvated surfaces , and channels of water traversing the clusters that resemble structures formed during intermediate stages of protein folding when water is being expelled from the interior of the protein . h - bonding is believed to be necessary for the stabilization of the folded protein , and several studies have found that in the crystalline , three - dimensional native state , all the well - folded proteins are almost completely h - bonded ; that is , few dangling or unsatisfied h - bonds are seen . in this study , we observe a small number of h - bonds per oligoglycine but not enough to explain the presence of the clusters . the absence of h - bonds has been noted before during the aggregation of tetramers of a peptides . here , the clusters are held together by other non - traditional dipolar interactions such as co co interactions . while these interactions are weaker than a h - bond , they are more numerous , allowing them to play a significant role in holding the clusters together . several other groups have noticed similar interactions leading to considerable structural and energetic stabilization of groups of small molecules . these co co dipole interactions have been shown to affect the structures of well - folded proteins in several studies . co dipoles in proteins contributes as much to the direction of the twist in -sheets as steric hindrance between side chains . more recently , others showed that dipoles play a role in stabilizing various protein secondary structures by orienting them so that they are aligned with the external field created by the rest of the protein . our results may be compared to previous observations about the absence of h - bonds in studies on the collapse of longer , single oligoglycines indicating that the same mechanism could be in effect in full - length unfolded , unordered proteins . in other work , raines and collaborators have shown that interactions between the c and o atoms of adjacent amides occur in several classes of protein structure . however , we observe correlations between co atoms of amide groups at up to 6.4 indicating that these effective interactions occur not just between adjacent amide groups but could extend via the mechanism of charge layering throughout proteins . charge layering and dipole correlations across the clusters together facilitate the formation of weaker interactions . this ordering , along with the weaker interactions allow the clusters to remain fluid , allowing them to explore several structural states with ease , an important feature in idps that allows them to perform their functions . we observed phase separation of pentaglycines in water at 0.3 m into peptide clusters and water rich regions . from the water rich supernatant , we measured the saturation concentration for our model as 0.016 m. we did not see phase separation at 0.03 m , even though that concentration is above the solubility limit we measured apparently due to the system being kinetically limited . interestingly , when we briefly heated the concentrated system , the kinetics markedly increased , an effect sometimes associated with the hydrophobic effect . these systems with very negative free energies of solvation are far from the classical definition of the hydrophobic solute . peptide clustering and aggregation in our model study were not driven by h - bond formation but by other dipolar interactions such as co co interactions . energetically , the co co interactions are weaker than a h - bond but are more numerous and so play a significant role in stabilizing the clusters . structural ordering of atoms by charge type and by correlations between amide dipoles facilitated the formation of such interactions .