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brown squard syndrome bss occurs due dysfunction spinothalamic tract typically reflects hemisection spinal cord cervical thoracic level the syndrome mainly occurs result penetrating trauma syringomyelia hematomyelia tumor severe discs blunt trauma among multiple etiologies common cause penetrating trauma gunshot7,8 therefore management guidelines focus penetrating cervical injuries and/or vertebral artery va injury12 non missile penetrating spinal cord and va injuries rare bony structures protect spinal cord va14 thus treatment approach wounds caused non missile penetrating spinal injuries knife power drill bit even pen could different common missile penetrating injuries5,6,13,17).to knowledge reports literature complete obstruction va due penetration foreign body neural foramen spinal canal herein authors report va dissection bss caused penetration electric screw driver bit a 25-year old machine operator involved violent episode stabbed right neck electric screw driver bit thrown opponent arrival emergency department the electric screw driver bit placed right lateral aspect neck zone fig the tip electric screw driver bit located center vertebral canal c3 fig he given high dose methylprednisolone bolus dose 30mg kg followed 5.4mg kg hour 23 hours according protocol spinal cord injury an immediate interventional angiography undertaken without general anesthesia due nature emergency the angiography revealed total occlusion dissection right va level c3 immediate coil embolization proximal distal ends injury site performed fig an attempt manual extraction electric screw driver bit failed great resistance after patient moved operating room electric screw driver bit removed manually muscle dissection general anesthesia venous blood spilled controlled easily application several pieces gelatin sponge no postoperative complications wound dehiscence cerebrospinal fluid csf leakage infection observed the neurological motor function right upper lower extremities recovered 3/5 4/5 respectively persistent decreased sensory function one year fortunately patient experienced neck swelling auscultation neck bruit delayed ischemic complications penetrating injury third frequent cause spinal cord injury adults surpassed traffic accidents falls3,18 stab wounds associated lesser surrounding tissue injury gunshot wounds former delivers less energy missile injuries9 although vascular injury common sequel penetrating neck trauma va injury rare well protected transverse foramen4,10 therefore penetrating injury va mostly caused gunshot wounds deliver large kinetic energy depending upon bullet mass speed12 article report rare case va penetration electric screw driver bit spinal cord insult consequently presenting bss moreover surgical exploration va cause additional damage spine surrounding tissues therefore may reasonable embolize occluded artery unilateral ligation va rarely results brainstem ischemia11,16 there reports regarding treatment traumatic va injury arteriovenous fistulas pseudoaneurysms2 emergent surgical exploration necessary patients hard signs vascular injury hemodynamic instability hemorrhage exsanguinations expanding hematoma15 patients hemodynamically stable without respiratory compromise undergo diagnostic imaging evaluation15 presented case endovascular techniques safe effective method treatment associated significant morbidity mortality1 airway management intubation methods surgical positions points debate anesthesiologists surgeons9 lacerated va successfully obliterated penetrating electric screw driver bit may extracted without general anesthesia nevertheless authors recommend surgeons prepared conversion open surgery extraction performed support surgical team we initially tried extract electric screw driver bit manually without general anesthesia intervention theater va embolization however electric screw driver bit positioned firmly neural foramen patient complained severe pain electric screw driver bit pulled in addition important rationale justified surgical exploration extraction electric screw driver bit extraction electric screw driver bit the authors describe rare case penetrating cervical injury caused electric screw driver bit accompanying va penetration bss

there are few reports in the literature of complete obstruction of the vertebral artery ( va ) due to an electric screw driver bit penetration through the neural foramen into the spinal canal with brown - squard syndrome ( bss ) . a 25-year - old man was admitted to the emergency department with a penetrated neck injury by an electric screw driver bit after a struggle . the patient presented the clinical features of bss . computed tomography scan revealed that the electric screw driver bit penetrated through the right neural foramen at the level of c3 - 4 , and it caused an injury to the right half of the spinal cord . emergent angiography revealed va dissection , which was managed by immediate coil embolization at both proximal and distal ends of the injury site . after occlusion of the va , the electric screw driver bit was extracted under general anesthesia . bleeding was minimal and controlled without difficulties . no postoperative complications , such as wound dehiscence , csf leakage , or infection , were noted . endovascular approaches for occlusion of vertebral artery lesions are safe and effective methods of treatment .

vogt koyanagi harada disease vkh granulomatous inflammatory disorder affecting eyes auditory system meninges skin 1 2 it one common causes uveitis asia accounts approximately 16% uveitis cases china clinically vkh divided four stages prodromal acute convalescent chronic recurrent exudative retinal detachment common cause visual function impairment among vkh patients secondary alterations retinal morphology function also occur 5 6 due advancements retinal imaging fundus autofluorescence faf ) 7 8 optical coherence tomography oct 9 10 important clinical insight pathophysiology retina achieved noninvasively recent studies report significant association inner segment is)/os junction cone outer segment tips cost line various retinal diseases visual function 1113 however majority previous studies used best corrected visual acuity bcva measure visual function recently microperimetry found provide comprehensive assessment macular sensitivity functional changes macula 14 15 we investigated characteristics late stage vkh via oct especially photoreceptor microstructure integrity potential correlation visual function patients late stage vkh defined 12 months disease onset visited uveitis clinic fudan university eye ent hospital shanghai china april 2011 march 2013 enrolled cross sectional study the diagnosis vkh made according revised diagnostic criteria proposed international nomenclature committee the inclusion criteria treatment high dose corticosteroid therapy iv methylprednisolone 1 g 3 followed slow tapering drug dose 6-month period undergoing thorough examination including oct microperimetry providing required information 12-month follow including sex age vkh onset bcva initial presentation current bcva interval onset vkh symptoms initiation high dose corticosteroid treatment current ocular inflammatory status exclusion criteria inadequate mydriasis 7 mm media opacities caused cataract glaucoma defined characteristic optic nerve damage typical visual field defects accompanied compatible optic nerve damage this study conducted accordance tenets declaration helsinki approved local ethics committee fudan university eye ent hospital 1.5.12.0 heidelberg engineering heidelberg germany using volume scan mode 1024 25 25 lines 1024 scans per line line comprising 30 averaged scans obtained using eye tracking covered 30 30 degree area centered fovea microperimetry covered central 12 macula therefore nine scans central macula firstly scan across central fovea found subsequent four scans superior inferior directions picked covers 12 central macula selected analysis changes os junction cost line rpe layer graded experienced ophthalmologist chunhui jiang masked clinical findings patients grading performed using scale 1 3 based standardized protocol table 1 also one part outer nuclear layer onl thinner onl obviously much thinner normal considered thinning onl based 42 staircase strategy set 45 goldmann iii stimulating spots covering central 12 fundus tests performed using automated pattern 1 circle target the intensity stimulus varied according 1-step 0.1log scale 020 decibels db equivalent duration 200 ms per step the software automatically calculated mean sensitivity defined average 45 scores all analyses performed using statistical software package spss windows ver differences mp bcva groups late stage vkh eyes different oct grading examined using mixed linear model test differences prognostic factor sex age onset initial bcva interval onset treatment analyzed using generalized estimation equations test the durations patients still using systemic corticosteroids alone combination immunosuppressant 12 months analyzed using mann whitney u test during study period 34 vkh patients follow longer 12 months visited clinic twenty nine patients 15 males 14 females 58 eyes met inclusion criteria enrolled study mean follow time 12.2 months range 1214 months the mean age onset 34.24 10.67 years range 1265 years mean bcva onset 0.96 0.56 logmar range 2.000 mean bcva 12 months 0.07 0.16 logmar range 0.40 0.08 logmar mean retinal sensitivity 17.05 2.35 db range 9.920 db 12 months 16 29 patients still using systemic corticosteroids alone combination immunosuppressant among 10 patients tapering using low dose prednisone another six patients receiving corticosteroids cyclosporine prescribed 3 mg kg tapering guided severity ocular inflammation the oct findings late stage vkh eyes localized outer retina the findings included thickening rpe layer 28/58 eyes breakage disappearance cost line 51/58 eyes and/or inner segment outer segment os junction 28/58 eyes thinning outer nuclear layer onl 17/58 eyes the grading rpe cost line os conjunction listed table 2 notably eyes displaying intact cost lines also displayed intact os junctions normal rpe layers however 23 30 eyes displaying intact os junctions displayed fragmented grade 1 3 eyes grade 2 20 eyes cost lines 18 51 eyes displaying fragmented cost lines grade 1 2 16 28 eyes displaying fragmented os junctions grade 1 2 central fovea remained intact figures 1(a)1(c our analysis revealed strong correlation retinal sensitivity bcva oct findings cost line os junction rpe layer p 0.01 table 2 figure 2 for clarification patients categorized according cost line os junction characteristics graded intact table 3 the three groups similar gender age distributions table 3 different bcva retinal sensitivities the os+/cost+ group displayed best retinal sensitivity bcva furthermore group displayed relatively normal retinal sensitivity table 3 the oct findings cost line os junction displayed clear correlations interval symptom onset initiation high dose corticosteroid treatment usage systemic corticosteroids and/or immunosuppressant 12 months onset table 3 the os+/cost+ group exhibited smallest interval symptom onset treatment usage systemic corticosteroids and/or immunosuppressant in contrast 11/23 21/28 patients os+/cost os/cost groups using systemic corticosteroids alone combination immunosuppressant patients using systemic corticosteroids alone combination immunosuppressant 12 months longer durations without using versus using median 30 versus 9 p 0.0001 mann whitney u test changes outer retina rpe late stage vkh patients including thickening rpe layer disruption loss cost line os junction thinning onl clearly demonstrated sd oct further analysis revealed close correlation oct findings also correlated bcva retinal sensitivity clinical characteristics better oct findings correlated better vision prompt treatment disease onset less incidence systemic corticosteroids and/or immunosuppressant 12 months onset clinical findings late vkh patients vary greatly da silva et al recently reported late stage vkh eyes 6 months past disease onset grading color fundus photos mainly based diffuse fundus depigmentation subretinal fibrosis correlated well full field electroretinogram erg results however vkh patients diffuse fundus depigmentation subretinal fibrosis therefore objective method higher resolution might useful study vkh patients less fundus change oct routinely used diagnosis follow many retinal diseases 913 reported defects os junction multifocal thickening rpe late stage vkh patients however use oct identify retinal morphological changes late stage vkh patients extensively studied 5 7 8 18 this study comprised relatively large series late stage vkh patients oct clearly demonstrated retinal changes rpe in addition defects os junction thickening rpe thinning onl high prevalence 51/58 defects cost line found thereby indicating oct useful observation late stage vkh eyes all eyes displaying intact cost lines displayed normal os junctions rpe layers whereas displaying intact os junctions normal rpe layers displayed disrupted cost lines it reported central serous chorioretinopathy macular hole patients cost line later recover os line characteristics notably patients displaying cost line os junction defects loss perifovea exhibited intact structures central fovea the mechanism underlying finding fully understood high density photoreceptors fovea may play important role alternatively cones fovea display different configuration arrangement cones regions fundus might also explain findings whether structures preserved first regenerate studying the mechanism behind characteristic might improve understanding vkh neuroprotection neuroregeneration retina our study also demonstrated strong correlation cost line and/or os junction integrity visual function late stage vkh patients a former paper reported similar correlation patients macular holes 12 20 however bcva employed measure visual function previous studies time microperimetry provides quantitative measurement retinal sensitivity macula found os+/cost+ eyes exhibited normal results os+/cost+ group 19.56 0.36 db normal group 19.1 0.9 db these results suggest presence reappearance intact cost line might serve sensitive indicator photoreceptor functional status os junction accordance yokota report higher cone density eyes displaying intact cost lines eyes displaying disrupted cost lines finding supported fujita itoh theory recovery cost line os junction suggests enhanced restoration photoreceptors 11 12 hand might also suggest retinal sensitivity measured microperimeter appeared able provide comprehensive accurate evaluation macular function late stage vkh patients might patients retinal diseases the oct findings also related mean interval symptom onset initiation systemic steroid therapy using systemic corticosteroids and/or immunosuppressant 12 months onset table 3 therefore interval symptom onset initiation systemic steroid therapy important prognostic factor patients late stage vkh chee reported early treatment 2 weeks high dose corticosteroids associated better visual results vkh patients our os+/cost group displayed relatively normal bcva average treatment interval 20.22 13.12 days in contrast cost+/is os+ group exhibited normal retinal sensitivity normal bcva treatment interval 6.71 0.76 days this result suggests prompt treatment might required achieve complete restoration macular structure function the importance prompt treatment also suggested correlation less incidence systemic corticosteroids and/or immunosuppressant 12 months onset the strengths study include well defined population underwent standard long term follow a relatively large sample size achieved using sophisticated statistics like mixed linear model others these methods correct dependence eyes patients eyes patients could included primary outcome measures used study ( oct findings microperimetry retinal sensitivity bcva largely objective measures easily quantifiable the study retrospective design limitation fact longitudinal assessment disease activity visual function could assessed due use cross sectional study design the mechanisms underlying particular findings preservation retinal structure fovea fully understood further follow study might able tell us vkh patients recovered acute episode functional anatomic recovery correlated summary our study demonstrated significant correlation oct findings visual function late stage vkh patients determined via bcva microperimetry the integrity cost line sensitive os junction moreover prompt initiation high dose corticosteroid treatment associated improved prognosis visual function retinal structure less incidence systemic corticosteroids and/or immunosuppressant 12 months

purpose . to characterize the optical coherence tomography ( oct ) findings in late - stage vogt - koyanagi - harada ( vkh ) disease and its correlation with visual function . methods . the records of patients with late - stage vkh disease ( defined as 12 months from disease onset ) were retrospectively reviewed . the analysis focused on the oct findings and microperimetry , in addition to the possible correlation between morphology and functional findings . results . twenty - nine patients ( 58 eyes ) were included . mean age at onset was 34.24 10.67 years . the oct revealed that the outer retina and retinal pigment epithelium ( rpe ) were mainly affected . these effects included rpe thickening and breakage or disappearance of the cone outer segment tip ( cost ) line and/or inner segment / outer segment ( is / os ) junction . the cost line and is / os results were related to macular function and the interval between symptom onset and initiation of high - dose corticosteroid treatment ( all p < 0.01 ) . eyes with intact cost lines demonstrated intact is / os and normal rpe layers as well as better visual function and normal retinal sensitivity . conclusions . the oct findings are strongly correlated with macular function , as well as other clinical findings in late - stage vkh . with respect to the cost line and retinal sensitivity especially , the oct and microperimetry findings may be useful for evaluating later - stage vkh .

past decade protein separation techniques peptide analysis mass spectrometry greatly improved qualitative quantitative proteome comparisons become powerful tools defining composition complex proteomes functions protein complexes.(1 current ion trap mass spectrometers particular sensitive fast analysis times enable identification hundreds proteins single lc ms ms analysis however obtaining comprehensive protein profiles complex samples biological fluids mammalian cell tissue lysates remains challenging due large number proteins present sample wide concentration ranges improve proteome coverage samples typically fractionated either protein level peptide level regardless initial fractionation methods used final step strategies involves analysis tryptic peptides lc ms ms however undersampling occur subset peptides identified complexity tryptic digests exceeds analytical capacity mass spectrometer e.g. peptides elute hplc column per unit time analyzed low abundance peptides instrument detection limit a common observation complex proteomes analyzed lists proteins identified often quite variable raised concerns general value proteomics applications actually poor reproducibility typically identifications abundant proteins reproducible variability observed lower abundance proteins one strategy achieving comprehensive proteome coverage actually exploits variability simply performing repetitive lc ms ms analyses sample other strategies increasing number proteins identified include prefractionating proteome based protein physical properties improving separation peptide mixture hplc separation step modifying esi interface enhance ionization adding ion mobility spectrometry faims interface ion trap.(19 proteome coverage also improved data analysis stage using high mass accuracy data(20 improved data process algorithms other strategies include two step method reanalyzes sample inclusion list eliminating redundant precursor selection second analysis replay run analyzes sample twice single injection targeted analysis undersampled features replay run.(26 although methods achieve increased sensitivity difficult assess relative importance strategies directly compared single study due variations samples study design mass spectrometry platforms data analysis strategies used laboratory laboratory variations consequently number direct comparisons alternative analysis strategies conducted identify best analysis approaches specific types samples serum cell tissue lysates factors considered comparing alternative analysis strategies amount total protein required analysis total amount mass spectrometry instrument time required method it well established repetitive lc ms ms analyses longer hplc gradients additional fractionation via either fractions modes separation complex proteomes usually confer benefit terms depth analysis the key factor determine time- resource effective strategies achieving high depth analysis factors directly affect sample throughput proteome analysis cost study compared effects two commonly used strategies gelc ms ms repetitive lc ms ms analysis additional protein prefractionation assessed effects depth analysis reproducibility using cancer cell lysates neither strategy requires expensive special hardware reagents integrated diverse analyses complex proteomes multiple laboratories because simplicity repetitive analysis often used increase protein identifications different fractionation schemes involve two dimensions separation either peptide protein levels mud pit gelc utilizing cell extract human metastatic melanoma cell line 1205lu,(31 gelc ms ms analysis without repetitive injections compared data sets compared 3-d method included prefractionation cell lysate using microscale solution isoelectrofocusing microsol ief prior sds page separation balance total number lc ms ms runs total instrument time repetitive 2-d method 3-d method 20 fractions sds page fractionation analyzed four times cumulative result compared 80-fraction proteome analysis using 3-d approach other factors hplc gradient mass spectrometry method data processing kept uniform demonstrate clearly relative effects repetitive analyses versus additional prefractionation step protein level these comparisons showed excellent reproducibility methods substantially improved proteome coverage using 3-d method compared repetitive analysis fractions 2-d method rpmi-1640 cell culture medium sypro ruby stain nupage precast gels invitrogen corporation carlsbad ca ultra pure urea thiourea dtt chaps ge healthcare ltd giles u.k the bradford protein assay kit thermo fisher scientific waltham sequencing grade porcine trypsin promega corporation madison wi the human melanoma 1205lu cell line maintained rpmi-1640 medium supplemented 10% fetal calf serum cells harvested ice scraping cold phosphate buffered saline pbs inhibitors 0.3 mm pmsf 2 mm sodium orthovanadate 50 mm sodium fluoride 1 g ml leupeptin 1 g ml pepstatin 7090% confluence reached the tissue culture plates washed three times cold pbs inhibitors washes collected combined initial scraped cells collected centrifugation cell pellet quick frozen liquid nitrogen followed storage 80 c cell lysates prepared using 800 l lysis buffer 25 mm tris ph 8.0 8 urea 2 thiourea 4% chaps 1 mm dtt per cell pellet collected 15-cm tissue culture dish the lysate sonicated probe sonicator ice duty cycle setting 50% output level 5% insoluble material removed centrifugation 100 000 g 60 min estimated 1% total protein based staining intensity colloidal coomassie sds gels the supernatant reduced using dtt alkylated n n dimethylacrylamide dma previously described.(11 total protein supernatant measured using bradford method cell lysates 1.5 mg separated four ph ranges microsol ief using zoom ief fractionator invitrogen corporation carlsbad ca separations the fractionator contained five immobiline gel disks sustaining ph 3.0 5.4 6.2 7.0 12 respectively these disks delineated four separation chambers cell lysate sample buffer consisting 8 urea 2 thiourea 4% chaps 1% dtt 1% ph 37 zoom focusing buffer 1% ph 712 zoom focusing buffer loaded middle two separation chambers the outer chambers loaded sample buffer standard electrode solutions used samples focused constant 750 v maximum 1 3 h. solution sample chamber removed measured chamber rinsed small volume sample buffer combined original solution removed chamber final volume 700 l proteins trapped immobiline gel disks recovered using two sequential 175 l extractions 30 min using 10 mm tris ph 8 1% sds 30 min the cell lysate microsol ief fractions initially analytically separated 10% nupage minigels mes running buffer using standard separation conditions preparative sds page run using unfractionated cell lysate four microsol ief fractions analogous manner analytical run except separation distance limited 40 mm minimize required numbers fractions gel volume per fraction typically six lanes two lanes gelc 2-d method containing 60 g cell lysate one lane 3-d method sliced 4 mm 1 mm 1 mm thick gel pieces two adjacent pieces lane combined digestion well digested overnight trypsin corresponding digestions duplicate 2-d method lanes combined generating total 20 digestions 2-d method trypsin digestions injected 75 i.d 15 cm picofrit new objective inc woburn column packed 5 magic c18 resin peptides separated nano hplc eksigent technologies dublin ca interfaced ltq linear ion trap mass spectrometer thermo fisher scientific waltham analysis 8 l trypsin digest loaded onto column using solvent 0.1% formic acid milli q millipore corporation billerica water peptides subsequently eluted using following gradient conditions 0.1% formic acid acetonitrile solvent b 128% b 42 min 2850% b 25.5 min 5080% b 5 min 80% b 5 min minimize carryover twenty 2-d samples analyzed first followed another replicated injection 2-d samples then 80 3-d samples analyzed followed third fourth replicated injections 2-d samples the ltq mass spectrometer operated dynamic exclusion enabled 30 full scans 4002000 z data dependent ms ms analysis six intense ions 28 rev 13 university washington seattle wa bioworks ver 3.1 thermo fisher scientific waltham human uniref 100 ver may 2007 protein database downloaded protein information resource georgetown university generate decoy database protein amino acid sequence database entry was reversed entire reversed database appended front original forward sequences the data searched combined forward reverse database using partial trypsin specificity 2.5 da precursor mass tolerance 1 da fragment ion mass tolerance consensus protein lists generated dtaselect ver 1.9 licensed scripps research institute la jolla ca applying following filters full tryptic boundaries xcorr 1.8 z 1 2.1 z 2 3.25 z 3 cn 0.05 custom software used ensure unique peptide sequence used assembling protein list identify common unique proteins found 2-d and 3-d methods protein peptide data put relational database mysql matched using custom software the strategy used compare 1 conventional gelc ms ms method protein separation sds gel reverse phase peptide separation 2 gelc ms ms repetitive injections 3 3-d method used microsol ief fractionation prior sds page step protein ief sds gel reverse phase peptide separation summarized figure 1 directly compare relative impact proteome coverage repetitive injections versus additional protein separation step equal amounts cell lysate fractionated equal number lc ms ms analyses used a cell lysate 1205lu cell line processed manner common step comparable the 3-d method consisted microsol ief sds page lc ms ms subsequently final minimum consensus protein lists three data sets compared evaluate ability enhancing sensitivity repetitive analysis additional fractionation strategies microsol ief initially developed prefractionate complex proteomes prior narrow ph range 2-d gels(33 subsequently used prior 1-d sds page part 4-d strategy fractionating serum plasma.(11 study evaluated utility prefractionating cell lysates prior gelc ms ms since separation mode orthogonal sds page suffer many limitations 2-d gels because dynamic range protein concentrations cell lysates substantially less plasma serum microsol ief separation designed yield four final fractions rather larger number fractions typically used plasma the immobiline membrane disks chambers extracted shown previously proteins pi values close ph disk remain disk recovered high yield extracts disks extreme ph values 3.0 12 showed trace amount proteins proteins pi near values extractions disks located separation chambers contained mixture proteins unique disk proteins present adjacent chambers previously observed when similar samples separated microsol ief analyzed 2-d gels comparisons solution fractions membrane fractions showed proteins recovered membrane disks either unique membrane pi slightly lower ph membrane data shown since goal experiment fractionate entire proteome small number fractions microsol ief step disk extracts combined fraction right maximize recovery figure 2 the extraction ph 12 disk discarded contained negligible amount protein thus microsol ief fractionation cancer cell proteome divided four fractions roughly similar complexity simpler protein content separation melanoma 1205lu cell extract microsol ief pooling produce four fractions equal portions fraction separation membrane disk extract analyzed sds page evaluate separation relative amounts total protein fraction the solution recovered individual chambers pooled adjacent membrane disk extract low ph side pool shown bottom gel produce four fractions the extract ph 12 membrane disk negligible protein used conventional gelc ms ms method 60 g original cell lysate close maximum load avoiding band distortion loaded two lanes nupage gel figure 3a proteins separated tracking dye migrated 40 mm staining gel colloidal coomassie 40-mm lane divided 20 equal fractions digested trypsin digests corresponding positions two replicate lanes combined yield sufficient volume four 8 l injections sds page separation melanoma 1205lu cell lysate microsol fractions proteome analysis samples electrophoresed tracking dye migrated 4 cm gels stained colloidal coomassie individual lanes cut 20 equal sized slices shown ( 2-d method unfractionated lysate 1205lu cells separated two lanes 60 g lane ( b microsol ief fractions derived 120 g cell lysate separated 3-d method for 3-d method amount fraction loaded onto preparative gel protein recovered 120 g total cell lysate figure 3b hence total amount protein four gel lanes figure 3b close amount protein two lanes figure 3a provided sample losses microsol ief procedure low previously demonstrated as noted gel lanes microsol fractions cut digested manner gel slices figure 3a except case duplicate gel lanes combined hplc mass spectrometer performance carefully monitored ensure consistent performance experiments to minimize effects minor instrument performance variations samples injected order described materials methods consistent performance autoinjector monitored weighing sample vial injection there significant difference injection amount two methods test p 0.05 all data consistently analyzed filtered described materials methods resulted estimated false positive rates fpr calculated dividing reverse hit peptide counts forward hit peptide counts 1.6% 1.4% 2-d 3-d data respectively figure 4 shows nonredundant peptide protein counts 3-d data set well differing numbers replicates 2-d sample set similar numbers peptide protein identifications obtained individual 2-d data sets data shown expected the total number nonredundant peptides increased moderately additional replicate data sets combined incremental increase diminished new replicate added it evident curve shown figure 4a data set resulting combining four replicate runs approaching plateau parallel trends observed peptide data sets defined proteins identified 3 2 1 peptide(s a total 25 641 nonredundant peptides identified combining four replicates 2-d analysis 26% less 32 216 peptides identified 3-d method ( nonredundant peptide counts single 2-d analysis combined data increasing numbers replicate analyses 3-d method ( b corresponding nonredundant protein counts data sets shown panel a. total number lc ms ms runs data set contains shown bottom figure protein counts showed similar trend peptide counts figure 4b adding second replicate increased number nonredundant proteins markedly adding third fourth replicate for example number proteins identified two peptides increased 594 305 162 two three four replicates combined appropriate comparison 3-d 2-d methods 3-d data set four replicates 2-d samples involve total 80 lc ms ms runs using equal amounts initial cell lysate when protein identifications based two peptides counted 3-d method identified 3486 proteins compared 2850 proteins 2-d method these 636 additional proteins 22.3% increase indicate clear advantage 3-d method compared 2-d replicate run method even equal amounts mass spectrometer time utilized the proteomes produced 2-d four repetitive run 3-d methods compared using lists proteins identified two peptides this comparison facilitated fact data sets searched using database dtaselect lists protein names identified search if protein identification carried one protein name could found data set deduced data sets identified protein this comparison protein name level showed extensive overlap two data sets even first level comparison figure 5 specifically two data sets shared 2555 proteins corresponded 90% smaller 2-d repetitive run proteome the 295 proteins appeared unique less depth 2-d replicate run data set investigated determine basis apparent lack reproducibility depth analyses biological sample further analysis showed 184 proteins present 3-d data set single hit proteins a majority 135 184 proteins two peptide proteins 2-d data indicating 3-d method identified proteins one less peptide when unfiltered data 3-d proteome examined using randomly selected proteins group found substantial number proteins identified second peptide 2-d data set one filtering parameters slightly lower cutoff values selected global data filtering result moderate changes scoring parameters reduced two- three hit protein identification 2-d data set 1-hit protein 3-d data set we also evaluated 111 proteins found exclusively 2-d method three quarters 88 two peptide proteins among identifications the 11 proteins identified three unique peptide sequences particular concern number peptide identifications suggested proteins near detection threshold identified depth 3-d proteome we found majority peptide sequences associated 11 proteins 2-d data set also existed 3-d data set assigned highly homologous proteins supplemental table 1 although large number common sequences proteins observed data sets small number unique sequences led dtaselect program assign peptides different proteins data set of 385 peptides assigned 111 proteins unique 2-d replicate run data set 226 peptides present filtered 3-d data the two methods identified 2555 common proteins two peptides per protein 90% total proteins identified smaller 2-d replicate run data set of 295 apparently unique proteins 2-d method 184 proteins identified 3-d data set one peptide the pie charts lower panels show number peptide hits 2-d method proteins directly identified 3-d data set 111 proteins 3.9% proteins 2-d data set identified single peptide 3-d data set 183 proteins 6.5% proteins 2-d data set these proteins identified two three peptides 2-d replicate data set identified single peptide 3-d data set case second peptide detected 3-d data set failed pass data filter cutoff due slightly lower values xcorr cn bolded values table the filtering cutoff values used xcorr 1.8 1 2.1 2 3.25 3 cn 0.05 bold values indicate cutoff value indicates found to first approximation proteins identified 2-d repetitive runs data set two three peptides regarded likely low abundance proteins sample analyzed since sequence coverage usually rough indicator protein abundance level the relative depth coverage putative low abundance proteins compared 2-d repetitive run 3-d methods shown figure 6 for proteins identified data sets majority showed greater sequence coverage 3-d data set that 491 common proteins identified two peptides 2-d data 3-d method found peptides 317 proteins 64.6% remaining proteins group identified equal number peptides furthermore 114 cases 3-d method found least three additional unique peptides indicating substantially greater depth analysis of 394 common proteins identified 2-d method three peptides 325 proteins 82.5% identified equal larger number peptides 3-d method these data clearly indicate cases 3-d method ability detect peptides low abundance proteins 2-d method comparison number peptides identified 2-d repetitive 3-d methods ( among proteins common data sets 491 proteins identified two peptides 2-d repetitive data set the 3-d method found equal number peptides 174 proteins peptides 317 proteins ( b among 394 proteins identified three peptides 2-d method 3-d method found one less peptide 69 proteins i.e. 1 2-d equal number peptides 99 proteins peptides 226 proteins in study systematically evaluated relative merits repetitive lc ms ms runs compared introduction additional protein level separation step increasing proteome coverage cancer cell lysates factors affect apparent reproducibility proteome analyses performed sample also evaluated basic analysis platform used repetitive analyses was commonly utilized gelc ms ms method considered 2-d proteomics method involves two dimensions separation protein separation using sds page reverse phase hplc separation tryptic peptides this method compared 3-d method consisting solution ief protein level followed gelc ms ms method it important comparing alternative analysis platforms consider total number lc ms ms runs per proteome improved proteome coverage typically achieved lengthening hplc gradient repeating lc ms ms analysis complex samples similarly many separation modes prior lc ms ms least incrementally improved simply increasing number fractions collected provided resolution separation method exceeds initial fraction size used but cases increases protein coverage may small considered advantageous total analysis time per proteome considered hence evaluation merits greater depth analysis particularly small improvements must constantly weighted relative overall throughput furthermore total mass spectrometer instrument time frequently limiting resource fractionation prior lc ms ms step used rate limiting step proteome analysis throughput hence meaningful comparisons total mass spectrometer analysis time per proteome held constant study used consistent gradient time 80 lc ms ms runs 3-d method 2-d repetitive run method four repeat injections similarly experimental variables held constant possible including use replicate aliquots single cell lysate preparation gel separation lengths gel volumes per trypsin digestion reaction instrument tuning data analysis methods our goal determine quantitatively method represents efficient utilization mass spectrometer time analyzing complex proteomes robust proteome analysis methods reproducible addition identifying majority proteins present biological sample one major cause variations proteins identified replicate analyses proteome undersampling mass spectrometer discussed therefore high proteome coverage linked good reproducibility proteome analysis results extensive proteome coverage occur undersampling minimized second factor contribute poor reproducibility proteome protein lists use data filtering conditions result high false peptide protein identification rates since false positives usually random hence data filtering stringency another tradeoff must considered selecting proteome analysis strategy while low stringency filters contribute noise low reproducibility excessively stringent filters greatly diminish number protein identifications hence value experiment current study data filters used yielded peptide false positive rates 1 2% estimated using decoy reverse database thereby minimizing apparent poor reproducibility data sets this level stringency results false positives proteins identified two peptides false positives within one hit protein list majority identifications correct the repetitive analyses 2-d data showed increased peptide protein counts figure 4 indicative undersampling basic gelc ms ms method used the overall gain four repetitive analyses proteins identified two peptides 1061 59% compared initial single analysis similar increase 61% ) was observed repetitive mudpit using nine analyses.(10 expected greatest positive impact proteome coverage use second replicate run increased number proteins identified two peptides 33% doubling instrument time in contrast adding third fourth replicate increased protein coverage 13% 6% respectively these data indicate performing second analysis fraction using gelc ms ms would positive tradeoff instrument time protein coverage however doubling instrument time performing four repetitive runs unlikely represent optimal use instrument time types experiments course alternative performing repetitive analysis gel fractions would obtain slices per gel lane in analogous experiments gel lanes divided 40 60 fractions observed increases number proteins identified similar obtained study duplicate triplicate analyses 20 fractions per gel lane data shown although producing fractions per gel lane increases number gel digestions overall increase total analysis time proteome minor hence generally prefer use fractions per lane rather replicate analyses greater depth analysis desired using gelc ms ms experiments longer gels larger numbers fractions per lane used current study wanted keep total mass spectrometer time per proteome approximately 160 h per proteome within practical limits simultaneously matching gel lengths gel volumes parameters extrapolating 2-d 3-d experiments performed expect total number proteins data set 2-d 2-d repetitive runs 3-d ) would increased moderately would used 40 60 slices per gel lane samples but use 40 60 fractions per gel lane would increased total instrument time 320 480 h per proteome represents impractically low throughput studies interestingly increase number fractions per gel lane 40 60 fractions incremental increases new proteins identified diminish analogous diminishing benefits adding additional replicate repetitive run approach figure 4a although similar trends observed two approaches mechanisms increasing protein coverage quite different using larger number gel fractions increases protein separation simplifies mixture proteins present fraction repetitive runs exploit subtle variations peptide separations replicate hplc runs subtle variations data dependent selection low level ions ms ms fragmentation analysis the 3-d method clearly provided superior protein peptide coverage compared 2-d repetitive method indicates adding additional protein separation step represents efficient use mass spectrometer instrument time this method identified 3486 proteins two peptides 22% 2-d repetitive method used equal instrument time peptide level 3-d method identified 30 385 high confidence nonredundant peptides nearly 2.5 times found single survey using 2-d method 12 160 28% cumulative count four repetitive analyses 23 648 furthermore unique peptides found low abundance proteins 3-d method data compared cumulative 2-d method data figure 6 it surprising adding solution ief additional orthogonal protein separation step gelc ms ms method efficient strategy increasing proteome coverage sequence coverage lower abundance proteins microsol ief separates proteins would normally single gel slice four gel slices see figure 3 simpler samples decrease ion suppression effects reduce dynamic range within digest finally cases improved scores ms2 spectra 3-d method probably resulted lower probability interfering ions isolated target ion fragmentation while repetitive analysis strategy also improved proteome coverage neither built mechanism reduce repeated sampling abundant ions replicates could explore ions ms2 triggering threshold an alterative technique sometimes used improve replicate runs scan different mass ranges replicate however pilot experiments suggested approach less productive simple repetitive analysis method used here one frequent criticism proteomics methods proteins identified repeat analyses often reproducible a recent study suggested good reproducibility achievable across 27 laboratories simple 20-protein mixture uniform data processing used.(35 simple sample abundant proteins concentration reflect real biological complexity hence current study compared reproducibility different analysis methods using complex sample biological interest human cancer cell lysate among four replicate analyses 2-d samples this indicated least 76% proteins observed one analysis reproducibly detected despite significant undersampling more importantly least 90% proteins observed 2-d four replicate data set based two peptides directly matched corresponding protein 3-d data set apparent mismatches caused trivial data analysis issues a rigorous comparison two comprehensive data sets showed greater 96% proteins identified 2-d repetitive run proteome actually observed within complete 3-d data set one reason initially apparent lower reproducibility protein names compared slight variations peptides scores together use rigid data filter cutoff values see figure 5 that 10% proteins apparently unique 2-d repetitive run data set included 184 proteins 6.4% 2-d repetitive protein list identified single peptide 3-d data set reasons proteins identified single peptide 3-d data set include run run variations automated selection low abundance signals ms ms run run variations sequest scores coupled use rigid data filters a second contributing factor initially apparent lower reproducibility protein list level database redundancy limitations current software consistently producing consensus protein lists identified peptides 111 proteins apparently unique 2-d / repetitive run data set identified single hit protein 3-d data set highly homologous proteins identified 3-d data set see results supplemental table 1 among 385 peptides belonging 111 unique proteins 2-d data although 111 unique proteins comprised 4% proteins identified 159 unique peptides 0.7% 2-d filtered peptides this illustrates small variations identified peptides proportionally higher apparent since used unique peptide single time assembly consensus protein lists common sequences assigned protein unique sequences consequently group proteins high sequence identity one two unique peptides could determine protein protein family emerged final consensus protein list this illustrates better software tools needed identifying displaying putative unique proteins within protein families similarly improved databases uniform names labels clearly indicate membership within protein family would beneficial conclusion additional prefractionation microsol ief substantially increased proteome coverage sequence coverage compared gelc ms ms repetitive run method utilized equal amount mass spectrometer time furthermore reproducibility protein lists two methods quite high undersampling data acquisition minimized most apparent differences protein identifications due limitations current sequence databases protein naming conventions well software limitations filtering database search results building consensus protein lists

in - depth , reproducible coverage of complex proteomes is challenging because the complexity of tryptic digests subjected to lc - ms / ms analysis frequently exceeds mass spectrometer analytical capacity , which results in undersampling of data . in this study , we used cancer cell lysates to systematically compare the commonly used gelc - ms / ms ( 1-d protein + 1-d peptide separation ) method using four repetitive injections ( 2-d / repetitive ) with a 3-d method that included solution isoelectric focusing and involved an equal number of lc - ms / ms runs . the 3-d method detected substantially more unique peptides and proteins , including higher numbers of unique peptides from low - abundance proteins , demonstrating that additional fractionation at the protein level is more effective than repetitive analyses at overcoming lc - ms / ms undersampling . importantly , more than 90% of the 2-d / repetitive protein identifications were found in the 3-d method data in a direct protein level comparison , and the reproducibility between data sets increased to greater than 96% when factors such as database redundancy and use of rigid scoring thresholds were considered . hence , high reproducibility of complex proteomes , such as human cancer cell lysates , readily can be achieved when using multidimensional separation methods with good depth of analysis .

increasing evidence clear genetic link phenotypic characteristics dogs adverse drug reactions the fields pharmacogenetics pharmacogenomics become increasingly promising regarding clinical application genetic data aid prevention adverse reactions prediction behaviour drugs discovery new drug targets study the introduction new parasiticide 1980s revealed pre existing mutation dogs predisposes animals potentially fatal neurotoxicosis the drug ivermectin exerts antiparasitic action potentiating ligand gated chloride ion channels peripheral nervous system several invertebrate phyla the mdr1 1 mutation multidrug resistance gene mdr-1 causes ivermectin intoxication carriers this mutation results altered expression p glycoprotein altered behaviour drug collies related dog breeds the mdr-1 cyp2d15 genes studied uruguayan cimarron dog breed however mutations identified 36 uruguayan cimarron animals analysed studies several recent pharmacogenetic findings shown clinically relevant patients veterinary clinics dog population one suitable models examination population genetics clinically collies related dog breeds observed susceptible effects ivermectin central nervous system cns the clinical signs effects include tremors salivation coma depression ataxia moreover small doses 1/100 1/200 standard cause acute severe reactions collie breeds approximately 75% collies united states france australia mutant allele expression modified p glycoprotein furthermore affected breeds similar lineage includes sheepdog breeds old english sheepdogs australian shepherds shelties english shepherds border collies german shepherds longhaired whippets silken windhounds breeds suffer deletion mdr-1 gene likely experience adverse reactions response low doses drug the cyp2b6 enzyme system cyp2b6 member cytochrome p450 group enzymes encoded cyp2b6 gene humans cyp2b11 dogs this class enzymes responsible metabolism wide variety drugs while risk drug drug interactions involving human cyp2b enzymes appears low due minimal involvement drug oxidation low hepatic expression canine cyp2b11 exhibited surprisingly high levels activity vitro toward drugs used dogs benzodiazepines the cyp1a2 enzyme member cytochrome p450 encoded cyp1a2 gene cytochrome p450 cyp superfamily enzymes plays important role oxidative metabolism wide variety xenobiotics endogenous compounds additionally gene constitutively expressed human dog livers involved metabolism many drugs including caffeine phenacetin teophylline tracing drugs single nucleotide polymorphisms snps identified deficiencies canine cyp1a2 gene these deficiencies shown significantly alter pharmacokinetic behaviour two drugs associated large inter individual differences kinetic behaviour third drug the resulting deficiency cyp1a2 found cause significant kinetic variations ac3933 ym-64277 drugs beagles however significance effects genetic polymorphisms canine cyps yet fully explored in contrast snp genetic markers considered recent generation molecular markers any four nucleotides may present position genome therefore must assumed snp four alleles this theoretically possible practice majority snp variants two alleles original sequence single mutated version way appear distributed population this study conducted gain insight characteristics breed population identify snp genetic polymorphisms associated three genes mdr-1 cyp1a2 cyp2b11 involved metabolism drugs used medical treatments several animals belonging four different canine breeds several authors noted further studies required understand regulatory effects polymorphisms potential clinical relevance the rationale identify genetic polymorphisms genes encode proteins enzymes involved drug transport metabolism action predict usefulness particular drug increase numbers responders decrease numbers subjects affected adverse drug reactions the future pharmacogenetics veterinary applications lasting effects clinical decisions made future the goal drug therapy maximise therapeutic effects minimising adverse effects associated drugs drug interactions a total 106 different animals belonging four different canine breeds studied 25 uruguayan cimarrons 23 border collies 29 labrador retrievers 29 german shepherds analysis blood extracted aseptic conditions dogs owners houses dna extracted blood samples using dneasy tissue kit qiagen netherlands according manufacturer instructions dna quality purity evaluated using nanodrop nd1000 spectrophotometer twenty six snps belonging three different genes mdr-1 5 snp cyp1a2 20 snp cyp2b11 one snp investigated according results obtained several authors reference snp cluster report dbsnp ncbi genbank fig 1 table 1 samples sent geneseek neogen usa snp testing a total 5,512 sequences analysed 26 loci two sequences locus 106 animals the resulting sequences analysed using blast programme search similar sequences genbank national center biotechnology information usa snp polymorphisms analysed using genetix arlequin multivariate descriptive statistical analyses correspondence analyses snpstat programmes this analysis allows investigation qualitative variable breed terms qualitative variables alleles classical term inertia assimilated diversity this method conceptually similar principal component analysis applies categorical rather continuous data manner similar principal component analysis method provides means displaying summarising set data three dimensional graphical form the analysis conducted contingency table according breed rows composed dependent variable breed columns set explanatory variables alleles the results allow acquisition relative importance inertias breeds alleles a graphical representation created system points euclidean space degree robustness verified the snpstat programme designed conduct genetic association studies using snps analyse moderate numbers snps 26 dnps paper this programme provides allele genotype frequencies test hardy weinberg equilibrium analysis association response variable based linear logistic regression analysis interactions linkage disequilibrium statistics haplotype frequency estimation analysis associations haplotypes response analysis interactions haplotypes covariate the cumulative frequency also known facilitate selection threshold cut point rare haplotypes grouped analysis the association analyses haplotypes similar genotypes logistic regression results shown either ors 95% cis linear regression results differences means 95% cis the frequent haplotype automatically selected reference category rare haplotypes pooled together group the 26 snps examined 106 animals exhibited different frequencies breed table 2 an exact test hardy weinberg equilibrium performed confirmed uruguayan cimarron population indeed population equilibrium loci snp12 snp14 snp15 snp16 snp18 0.05 p 0.10 therefore used control population shown table 2 snp1 snp2 snp3 snp4 snp 5 exhibited allelic fixation uruguayan cimarron labrador retriever breeds all obtained fis values negative indicating lack inbreeding examined animals 2 cimarron population population 1 exhibited greater genetic distance respect border collie population 2 labrador retriever population 3 german shepherd population 4 breeds correspondence analysis indicated border collie breed exhibited 60% inertia greatest inertia observed four breeds uruguayan cimarron breed exhibited least 8% first axis explained 65.68% total inertia clearly differentiated cimarron breed breeds axis 2 explained 10.42% total inertia third axis explained 23.90% clearly discriminate breeds the uruguayan cimarron breed exhibited greatest genetic difference respect studied breeds the haplotypes individuals analysed using snpstat programme enabled estimation haplotype frequencies although number potential combinations haplotypes high 2 frequent haplotypes frequencies greater 1% included 12 cimarron population 19 border collie population 22 labrador retriever population 18 german shepherd population these haplotypes compared control population exhibited frequencies 1% included rare haplotypes section the programme selected frequent haplotype reference rare haplotypes pooled together group breed 34% individuals exhibited haplotype number 1 21% exhibited haplotype number 2 remaining haplotypes frequencies 10% border collies exhibited haplotype number 1 32% remaining haplotypes occurred frequencies 10% the labrador retriever breed exhibited haplotype number 1 36% german shepherd breed exhibited haplotype 1 16% haplotype number 2 13% remaining haplotypes exhibited frequencies 10% analysis associations snp performed binary response variable logistic regression analysis provided summary genotype frequencies proportions odds ratios 95% confidence intervals we used null hypothesis similar effects studied dog breeds determine significant differences existed among breeds an example locus snp22 shown table 5 snp marker exhibited significant difference control population p 0.0001 case an association effects cyp1a2 gene shown according obtained results following 10 26 snps exhibited significant differences and were associated drug sensitivity snp6 snp12 snp15 snp16 snp20 snp21 snp22 snp24 snp25 cyp1a2 gene snp26 cyp2b11 gene all snps potential candidates studies drug sensitivity breeds the results obtained study revealed three breeds border collie labrador retrievers german shepherd exhibited several candidate alleles useful investigation genes control drug sensitivity the correspondence analysis indicated border collie breed exhibited greater genetic variability reduced inbreeding characteristics -0.52382 fis fis values range -1 1 in contrast uruguayan cimarron breed exhibited less genetic variability fewer inbreeding characteristics -0.27243 fis value also free inbreeding problems snps used markers diagnoses specific features abundant genome genetically stable easily analysed the underlying principle based identifying associations gene genes affect variable e.g. drug resistance snp markers if application statistical programme i.e. snpstat present study reveals significant differences genotypic classes concluded association marker studied characteristic our results indicated border collie labrador retriever german shepherd breeds exhibited significant differences uruguayan cimarron breed 10 snps cyp1a2 cyp2b11 genes different studies examined frequencies several single nucleotide substitutions canine genes associated product resistance drug sensitivity survey purebred populations might genetically risk our results concordant obtained authors examined canine breeds the cyp1a2 gene beagle breed examined using csnp method non functional allele indicated interindividual differences pharmacokinetics cyp1a2 substrates examined breed discovered additionally one snp identified cyp1a2 causes protein deletion deficiencies toxicological evaluations most common diseases involve complex genetic traits multiple genetic environmental components contribute susceptibility it proposed common genetic variants including single nucleotide polymorphisms snps influence susceptibilities common disease this proposal begun tested numerous studies associations genetic variations common dna polymorphisms variations disease susceptibilities our preliminary results indicate several polymorphisms present studied genes polymorphic loci potential candidates investigations drug resistance analysis genetic marker snp candidates might useful subjecting animals drugs medical treatments easily identified methodology simple inexpensive the results presented herein contribute efficient reliable studies drug candidates dogs this technology detect animals might risk verify genotypic characteristics personalise drug therapy individual level rather population level advances provide clear genetic responses identify new drug targets precisely apply drug therapy minimise adverse effects maximise therapeutic benefits academic clinical research efforts pharmacogenetics pharmacogenomics might beneficial entirety veterinary medicine coming years

the fields of pharmacogenetics and pharmacogenomics have become increasingly promising regarding the clinical application of genetic data to aid in prevention of adverse reactions . specific screening tests can predict which animals express modified proteins or genetic sequences responsible for adverse effects associated with a drug . among the genetic variations that have been investigated in dogs , the multidrug resistance gene ( mdr ) is the best studied . however , other genes such as cyp1a2 and cyp2b11 control the protein syntheses involved in the metabolism of many drugs . in the present study , the mdr-1 , cyp1a2 and cyp2b11 genes were examined to identify snp polymorphisms associated with these genes in the following four canine breeds : uruguayan cimarron , border collie , labrador retriever and german shepherd . the results revealed that several snps of the cyp1a2 and cyp2b11 genes are potential targets for drug sensitivity investigations .

emergency department ed known one congested units hospital faces greater pressure terms patient load health care resources compared departments health care system studies across various countries reported quality care decreases ed overcrowded overcrowding result delayed treatment long patient waiting time stay overburdened working staff patient elopement high medical error rate low productivity poor patient outcomes an efficient patient flow system serves critical patient quickly minimizing unnecessary delay treatment hand a patient arriving ed encounters repeated waits progresses different stages may last hours even days waiting time often cited important cause patients dissatisfaction ed cooke cited reduction waits important area improvement ed delays process associated adverse outcome increased violence eds waiting time turn depends multiple factors including volume patients workload existing staff outflow patients ed either means transfer discharge obstructed upstream bottleneck also cause delays treatment the present study conducted assess patient flow system assessing arrival time pattern waiting time distribution patient ed tertiary health care institute india understanding flow trends hospital administrators streamline processes minimize wait times improve efficiency reduce overcrowding emergency patient department eopd turn improve patients satisfaction it short term cross sectional descriptive study conducted may 2011 ed tertiary level medical research health care institution north india the institute caters medical care needs around 370 million populations 7 states india 2010 11 institute catered yearly load around 16,57,200 patients 64,969 inpatients whereas ed institute attended 52,894 patients 32,563 inpatients a data collection tool gather required information developed pilot tested study period the investigator stationed ed 8.00 18.00 h. data regarding waiting time length time patient waiting idle ed delivery service requires inter arrival gap time gap arrivals two consecutive patients ed distribution collected however data regarding arrival time time patient first recognized requesting service ed obtained emergency records the investigator approached patients attendants presented eopd data collection hours asked background characteristics time entry eopd they asked document waiting period service asking question service patient waiting much time consent undertaking study obtained charge ed institute the ed block institute treatment area approximately 23,088 square feet included 2 halls patients medical emergencies known emergency medical opd one hall patients surgical emergencies known emergency surgical opd the ed one main entrance patients two inlets within hospital one main hospital one advance trauma center there cabin enquiry adjoining main entrance two receptionists along one assistant public relation officer respond queries patients 24 7 along assigned works there registration counter manned one medical record technician register patients issued gate passes patients attendants mark payment stamp recommendation form patients checking eligibility a fee clerk receives hospital charges cabin adjoining registration counter the fee counter runs 8.00 20.00 collection fees done registration counter there radiology room attendant waiting hall laboratories chemist shop blood bank toilets male female senior medical officers smo room figure 1 ground floor plan emergency block study institute north india a hall emergency medical patient department emopd b hall b emopd c emergency surgical patient department waiting hall e chemist shop f laboratory g ultrasonography room h x ray room reception j corridors k waiting area patients overall signage system adequate guide patients relatives there designated parking space vehicles patients staff came ed a deputy medical superintendent assisted five smo look ed services present four posts smo lying vacant one smo ad hoc position the administration emergency complex looked smo dealing medico legal cases providing poor free services deserving patients supply life saving medicines consumable items poor patients supervise patient management case disaster eopd medical staff works 3 shifts day viz 8.00 14.00 h 14.00 20.00 h 20.00 8.00 h. it observed majority 70% patients visited eopd male around 28% age group 45 59 years followed 24.7% patients age group 15 29 years majority 85% patients arrived eopd conscious state mind around 64% referred health care facility coming eopd table 1 arrival time pattern patients showed around 26.3% patients came 9.00 12.00 h. peak hour presentation 10.01 11.00 11% total presentations the maximum inter arrival gap 46 min observed 6.01 7.00 h figure 2 background characteristics study population arrival time pattern patients emergency patient department study institute observed maximum patients 29.6% waiting observation preliminary diagnosis physician median waiting time 16 h. next highest category patients 16.4% waiting diagnostics tests results median waiting time 1 h. number patients completed treatment process waiting doctors decision regarding discharge eopd also substantial 14.6% median waiting time 2 h. maximum median waiting time 38 h recorded patients waiting turn operative procedure decision operate observed waiting around 71% patients eopd attributed factors within ed whereas waiting 26% patients attributed reasons outside ed within hospital figure 3 waiting time distribution patients various services emergency patient department study institute waiting time attributed reasons within emergency department ed complex b waiting time attributed reasons outside ed within hospital c waiting time attributed reasons outside hospital with steep increase life style diseases road traffic accidents demand emergency medical health care increasing however resource constraints terms scarce health manpower lead overcrowding hospitals turn compromises quality care most tertiary care hospitals india facing problem patient flow system such problem even apparent ed mostly overcrowded utilized inappropriately staffed often lack coordination care overcrowded eds poorly managed patient flow results excessively long waits patients increase risk inappropriate care poor quality services there steady rise ed attendance institute years absolute terms well daily averages this conformity another study conducted ed tertiary care hospital new delhi 2003 concluded last 3 4 years steady rise ed attendance absolute terms well daily averages the higher use ed tertiary care hospitals also attributed many reasons increase illness chronic diseases changes demographic epidemiological trends diseases increase acute injuries changes people perception emergency need health problems better quality health services rendered growing population etc this supported data accidental deaths shown rapidly increasing trend decade 2000 2010 moreover the patient preference toward tertiary care institute also documented study earlier done institute wherein two third patients satisfied hospital services wish avail secondary level care the provision low cost tertiary level care treatment study institute compared catastrophic cost treatment private hospitals might reason choosing health care service institute lack tertiary care hospitals repute neighboring states also lead high rush referral cases institute present eopd study institute looking around 154 patients time around 5 times original capacity to accommodate heavy load patients additional patients put patient trolleys per guidelines australasian college emergency medicine the area ed study adequate around 29,000 yearly patient load present 53,000 due shortage space even corridors eopd full patients trolleys little space left movement another study conducted institute backs finding patients treated non treatment areas eopd had also concluded frequency medication errors ed increased crowding ed a study goel et al study institute also suggested heavy load patients attendants non treatment areas eds may potential source transmission deadly infections other studies concluded plague infection spread rapidly infected patient patients overcrowded emergency ward tertiary care institute the maximum patients ed study neurosurgery department indicative fact accidents mostly road increasing volume patients ed chan et al study concluded total census major patients ed major reason behind wait ed the present study revealed majority patients using ed services males these results easy attribute culture males exposed hazards due outdoor nature work opposed females a little less one third patients visited ed productive age group 15 29 years closely followed age group 45 59 years somewhat similar using pattern shown another study carried barbados shows around 36% used ed services age group 21 45 years around 26% 50 years age increase number vehicles chandigarh neighboring states resulting steep upward trend number road accidents might lead increase admission males productive age group the peak arrival hours 9.00 12.00 h around one fourth patients arrived eopd the finding present study consonance another study done tertiary care hospital barbados 10% daily census entered ed night another study done turkey also showed ed visit night decreased substantially compare day time visits the results another study conducted saudi arabia contrary finding around 46% patients attended emergency night the reason low arrival patient ed night time study attributed fact public transport facility shut 21.00 h hefty charges made private taxi operators night moreover since call ambulance provided study institute arranging one vehicle might easier option night moreover patients arriving ed study institute belong lower strata society prefer wait morning rather taking patient hospital night time even though highly random arrival patterns observed study yet surprisingly staffing pattern somewhat similar 24 h spans overloading patient due high arrival rate peak hours inappropriate staffing pattern may result clogging patients the guidelines adopted american academy emergency medicine states 1 physician required per 2.5 patients hour nurse patient ratio exceed 1:3 whereas eopd study institute around 10 11 resident doctors 12 14 nurses cater average daily load around 150 patients much lower requirements the volume patients attending ed observed major determinant waiting time resources fixed the resources department need matched workload hour hour basis despite inherent variation workload our study depicts factors influencing patient flow lie solely ed although waiting least three fourths patients explained reasons lying ed complex similar study schull shanks cited unavailability alternative levels care community delay diagnostics patients held ed awaiting admission main causes wait ed derlet richards study concluded important cause ed overcrowding insufficient inpatient capacity ed patients required hospital admission unavailability inpatient beds delay requiring bed create log jam effect leading unavailability space consequently delay patients discharged ed due un specified observation area eopd study area observation chauhan et al study also suggested formulating strong admission discharging policy ed regulate patient turn rate fast decisions life death cases critical ed result doctors face great pressures test treat the fear missing something often leads extra blood tests imaging scans ed complex study institute only routine diagnostic tests x ray hematology biochemistry ultrasonography echocardiogram performed whereas patients visit concerned departments specific tests this finding supported fact around 12.3% patients eopd found waiting specialized tests median waiting time 12 h. eopd blood samples laboratory test taken already overworked resident doctors another study done ed study institute also observed extreme rush laboratories present within premises ed other studies also shown similar trend wherein unnecessary tests conducted laboratory complex increases rush non patients cooke also concluded waiting results tests one four commonest reasons patient waiting ed a review consultants ed tertiary hospitals suggested average response time consultants ranges 30 45 min depending current needs patient study this might due high proportion junior resident doctors ed higher rush patients needed super specialty consultations lack standard operating procedures consultations there ample evidence lack timely consultation coordination emergency team leading cause ed overcrowding poor case management study small fraction 1% patients aware reason wait possibly sketched fact people feel hesitant enquiring treatment care givers frank et al also concluded insufficient information provided waiting time cause people perception waiting time extended one main limitations study small sample size short duration study services availed patients absence investigator investigator rely statement patients attendants time taken service there might possibility recall bias patient may forget certain type service rendered there possibility incorrect data provided patients exaggeration waiting time tackle random inflow patients it recommended experienced staffing ed matched temporal arrival pattern patients further benchmark formulated various services waiting time service time annual audit mechanism a laboratory technician posted eopd area blood sample collection ease already overworked resident doctors the communication patients ed improved waiting inevitable health education promotion introduced one main limitations study small sample size short duration study services availed patients absence investigator investigator rely statement patients attendants time taken service there might possibility recall bias patient may forget certain type service rendered there possibility incorrect data provided patients exaggeration waiting time to tackle random inflow patients recommended experienced staffing ed matched temporal arrival pattern patients further benchmark formulated various services waiting time service time annual audit mechanism a laboratory technician posted eopd area blood sample collection ease already overworked resident doctors the communication patients ed improved waiting inevitable health education promotion introduced based study concluded identifying operational factors influence patient flow ed help hospital administration device suitable strategy improvements functioning ed topic importantthe policy makers faces challenge overcrowded emergency departments eds ill managed patient flow tertiary care institutes especially developing countries result excessively long waits patients study tried analyze patient flow system assessing arrival waiting time distribution patients emergency patient department eopd this study inform hospital administrators policy makers effectively design system reduce waiting times hence overcrowding ed this turn benefit patient increasing satisfaction rate.what study attempt show ? the paper provides insight operational factors solutions easily replicated across countries.what key findings arrival time pattern patients eopd highly random peak hour presentation 10.01 11.00 11% total presentationsthe primary waiting areas patients included patients observation overcrowding resulting poor patient flow excess waits result delayed treatment long patient waiting time stay overburdened working staff patient elopement high medical error rate low throughput poor patient outcomes topic important policy makers faces challenge overcrowded emergency departments eds ill managed patient flow tertiary care institutes especially developing countries result excessively long waits patients through study tried analyze patient flow system assessing arrival waiting time distribution patients emergency patient department eopd this study inform hospital administrators policy makers effectively design system reduce waiting times hence overcrowding ed the paper provides insight operational factors solutions easily replicated across countries arrival time pattern patients eopd highly random peak hour presentation 10.01 11.00 11% total presentationsthe primary waiting areas patients included patients observation arrival time pattern patients eopd highly random peak hour presentation 10.01 11.00 11% total presentations primary waiting areas patients included patients observation 29.6% waiting routine diagnostic tests 16.4% waiting discharge 14.6% around 71% overcrowding resulting poor patient flow excess waits result delayed treatment long patient waiting time stay overburdened working staff patient elopement high medical error rate low throughput poor patient outcomes topic importantthe policy makers faces challenge overcrowded emergency departments eds ill managed patient flow tertiary care institutes especially developing countries result excessively long waits patients study tried analyze patient flow system assessing arrival waiting time distribution patients emergency patient department eopd this study inform hospital administrators policy makers effectively design system reduce waiting times hence overcrowding ed this turn benefit patient increasing satisfaction rate.what study attempt show ? the paper provides insight operational factors solutions easily replicated across countries.what key findings arrival time pattern patients eopd highly random peak hour presentation 10.01 11.00 11% total presentationsthe primary waiting areas patients included patients observation overcrowding resulting poor patient flow excess waits result delayed treatment long patient waiting time stay overburdened working staff patient elopement high medical error rate low throughput poor patient outcomes topic important policy makers faces challenge overcrowded emergency departments eds ill managed patient flow tertiary care institutes especially developing countries result excessively long waits patients through study tried analyze patient flow system assessing arrival waiting time distribution patients emergency patient department eopd this study inform hospital administrators policy makers effectively design system reduce waiting times hence overcrowding ed the paper provides insight operational factors solutions easily replicated across countries arrival time pattern patients eopd highly random peak hour presentation 10.01 11.00 11% total presentationsthe primary waiting areas patients included patients observation arrival time pattern patients eopd highly random peak hour presentation 10.01 11.00 11% total presentations primary waiting areas patients included patients observation overcrowding resulting poor patient flow excess waits result delayed treatment long patient waiting time stay overburdened working staff patient elopement high medical error rate low throughput poor patient outcomes

background : emergency department ( ed ) of tertiary health care institute in india is mostly overcrowded , over utilized and inappropriately staffed . the challenges of overcrowded eds and ill - managed patient flow and admission processes result in excessively long waits for patients.aim:the objective of the present study was to analyze the patient flow system by assessing the arrival and waiting time distribution of patients in an emergency out patient department ( eopd).materials and methods : this short cross - sectional descriptive study was conducted in the eopd of a tertiary level health care institution in north india in the month of may , 2011 . the data was obtained from 591 patients , who were present in the eopd during the month of may , 2011 . the waiting time , inter arrival time between two consecutive patients were calculated in addition to the daily census data ( discharge rate , admission rate and transfer out rates etc . ) of the emergency.results:arrival time pattern of patients in the eopd was highly stochastic with the peak arrival hours to be 9.00 - 12.00 h in which around 26.3% patients arrived in the eopd . the primary waiting areas of patients included patients under observation ( 29.6% ) ; waiting for routine diagnostic tests ( 16.4% ) and waiting for discharge ( 14.6% ) . around 71% patients were waiting due to reasons within emergency complex.conclusion:the patient flow of the ed could only be addressed by multifaceted , multidisciplinary and hospital wide approach .

primary intracystic squamous cell carcinoma scc breast extremely rare neoplasm primary sccs breast quite rare although scc mixed ductal carcinoma common it arise metaplastic epithelium associated primary malignancy breast metastasis primary elsewhere body extension malignancy skin covering a 45-year old female presented lump right breast noticed since three weeks her menstrual cycles regular two children clinical examination revealed firm cystic lump 12 10 cm upper inner central quadrant right breast the skin right breast appeared stretched slight retraction nipple pre operatively patient relegated clinical stage iii t3n0m0 ultrasonographic examination right breast showed irregular shaped hypoechoic lesion measuring 12 6 cm internal anechoic area measuring 6 3 cm suggesting cystic malignant tumor fine needle aspiration lump right breast yielded 10 ml pale yellow serous fluid wet fixed air dried smears prepared centrifuged aspirated fluid stained papanicolaou pap stain may grnwald giemsa mgg stain respectively the smears studied cellular showed malignant squamous cells predominantly singles occasional syncytial groups cells spindle shaped the malignant squamous cells rounded borders hyperchromatic enlarged nucleus coarse irregular granular chromatin cytoplasm squamous cells showed variable degree keratinisation figure 2 cytology smears showing malignant squamous cells singles numerous cyst macrophages mgg 400 cytology smear showing malignant squamous cells singles demonstrating cytoplasmic keratinisation pap 400 exclude metastatic scc search remote primary scc included chest radiograph cystoscopy colposcopy oesophagogastroscopy laryngoscopy cervical pap smear reveal extramammary cancer the criteria include 1 neoplastic element ductal mesenchymal ones present tumor 2 tumor independent adjacent cutaneous structures 3 distant epidermoid tumor exists patient later simple mastectomy axillary clearance performed specimen sent histological examination the cut section mastectomy specimen showed large cystic tumor measuring 8 3 cm containing yellow serous fluid the cystic cavity lined dysplastic squamous epithelium infiltrating tumor showing malignant squamous cells broad sheets groups whorls keratin pearl formation intercellular bridges immunostaining estrogen progesterone receptor her-2/neu oncoprotein negative cytokeratin positive the smears studied cellular showed malignant squamous cells predominantly singles occasional syncytial groups cells spindle shaped the malignant squamous cells rounded borders hyperchromatic enlarged nucleus coarse irregular granular chromatin cytoplasm squamous cells showed variable degree keratinisation figure 2 cytology smears showing malignant squamous cells singles numerous cyst macrophages mgg 400 cytology smear showing malignant squamous cells singles demonstrating cytoplasmic keratinisation pap 400 exclude metastatic scc search remote primary scc included chest radiograph cystoscopy colposcopy oesophagogastroscopy laryngoscopy cervical pap smear reveal extramammary cancer the criteria include 1 neoplastic element ductal mesenchymal ones present tumor 2 tumor independent adjacent cutaneous structures 3 distant epidermoid tumor exists patient later simple mastectomy axillary clearance performed specimen sent histological examination the cut section mastectomy specimen showed large cystic tumor measuring 8 3 cm containing yellow serous fluid multiple sections studied showed cystic breast tumor cystic cavity lined dysplastic squamous epithelium infiltrating tumor showing malignant squamous cells broad sheets groups whorls keratin pearl formation intercellular bridges immunostaining estrogen progesterone receptor her-2/neu oncoprotein negative cytokeratin positive pure scc adenosquamous carcinoma listed metaplastic breast carcinomas world health organization classification the japanese breast cancer society defined scc breast special type cancer malignant cells arranged broad sheets whorls keratin formation intercellular bridge the incidence scc reported western countries 0.13.6% japan 0.17% the age group affected 32 65 years increased tendency left sided involvement the characteristics scc breast generally reported large sized tumor rapidly growing central cyst formed necrosis this unlikely case little necrosis elsewhere tumor the possible mode origin scc epidermoid cyst breast chronic abscess complete metaplasia glandular breast tissue lymph node involvement reported less frequent might expected given larger tumor size the tumor cells negative vimentin estrogen progesterone diffusely positive high molecular weight cytokeratin c erbb-2 squamous cells fine needle aspiration cytology fnac breast lesions found various benign lesions like epidermoid cyst subareolar abscess fibroadenoma infracted papillomas spindle cell metaplasia cystic sarcoma phyllodes pseudosarcoma malignant breast tumors metastatic malignancy benign breast conditions abundant squamous cells may sometimes mimic malignant squamous lesion vice versa general benign squamous cells bland looking often associated anucleated squames tumor cell cannibalism the malignant squamous cells pleomorphic mitotically active dyskeratotic sometimes bizarre shaped cells seen the differential diagnosis malignant squamous cells fnac breast includes primary scc metastatic scc breast careful assessment cytological features squamous cells background appearance appears critical arriving correct diagnosis however case intracystic scc numerous foamy macrophages coexist malignant squamous cells hormonal therapy indicated cases scc negative hormonal receptors scc breast reported resistant radiotherapy standard chemotherapy performed invasive ductal carcinoma case radical mastectomy axillary clearance performed postoperative adjuvant therapy given common types breast cancers there evidence recurrence tumor four years treatment conclusion the presence malignant squamous cells fine needle aspiration breast suggests primary scc metastatic scc the finding pure scc necessitates accurate work exclude skin lesion metastasis the primary scc confused much largely manifested metaplastic change usual breast cancers although presence numerous cyst macrophages background breast fnac smears suggests benign lesion present malignant squamous cells suggest intracystic scc

primary intracystic squamous cell carcinoma ( scc ) of the breast is an extremely rare entity and has a low incidence in comparison with other breast cancers . we report a rare case of primary intracystic scc in a 45-year - old woman who presented with a cystic lump in the right breast . cytological smears of the fluid aspirated from the breast tumor revealed malignant squamous cells dispersed in single and occasional groups along with numerous cyst macrophages , suggesting cystic scc . histological study of the mastectomy specimen confirmed the diagnosis of primary intracystic scc . although the presence of abundant foamy macrophages in the background of fine needle aspiration cytology smears of the breast suggest benign breast lesion , when associated with malignant squamous cells , these suggest cystic primary scc or metastatic scc . the primary scc should not be confused with metaplastic change in other breast carcinomas .

translating empirically validated treatments routine clinical practice essential activity requires multitude factors addressed given disorder some factors include type patients treated setting treatment delivered including funding arrangements costs incurred attitudes perceptions staff treatment psychological physical disorders diseases disabilities may co occur condition treated these various factors mean translation first involve thoughtful adaptation given setting takes account local issues current practices staff attitudes funding arrangements secondly translation involve evaluation effectiveness resulting treatment plus empirical consideration factors believed influence outcome treatment improved 2 3 the present paper reports attempt translate cognitive behavioral program depression inpatient hospital setting there sound evidence supporting cognitive behavioral therapy cbt treatment depression little direction adaptation inpatient settings first people depression require inpatient admissions often time crisis extreme symptom severity aim quickly safely return levels independent function instance inpatient treatment may appropriate risk suicide goal reduce symptoms rapidly need restricted environment removed swiftly within constraints in addition individuals depression require hospitalization may find optimal treatment format brief weekly sessions however individuals hospital logistics organizing time intensive program less especially since patients might otherwise occupied much day hospital staff already present thus intensive day long format cbt becomes viable option also potentially good use time third given constant turnover within hospital harder mount disorder specific treatments patients possess various degrees comorbidity might fit neatly diagnostic categories yet may benefit cbt program reasons perth clinic developed intensive cbt program suitable inpatients it closed group program open various diagnostic groups mainly depression anxiety office hours two week period the first question answer regarded degree outcomes reported published literature regarding group cbt depression would generalize different format second focus present study concerned variables related outcome hospital setting many factors uncontrollable instance degree type comorbidity potentially important moderator treatment success in addition success may moderated frequency depression i.e. whether current presentation single recurrent episode severity depression according beutler prognosis is attenuated patient complexity chronicity absence patient distress facilitating social support enhances likelihood good outcome among patients complex chronic problems therefore assist future treatment planning necessary consider degree patient related variables affected outcome conducting evaluation current program variety constraints imposed context chiefly possible randomly assign patients control condition therefore variety methods used evaluate size treatment effect address alternative explanations pre posttreatment change increase confidence presumption that pre postchanges associated provision treatment outcomes compared inpatients receive cbt program began treatment complete the former provide indication extent improvement without program due nonrandom assignment conditions possible patients poorer outcomes encouraged enroll cbt program this problem partly addressed examining patients enrolled complete cbt program ideally patient groups similar admission hospital patients complete cbt program superior outcomes another way address causal role played cbt program examine specificity treatment changes since cbt aimed changing emotions mood related symptoms change cbt program points inpatient admission finally evaluate size treatment change published treatment data used generate benchmark pre- posttreatment data one study could serve benchmark patients depression reported peterson halstead they examined effectiveness group cbt depressed i.e. major depressive disorder single episode recurrent dysthymic disorder depressive disorder otherwise specified adjustment disorder depressed mood outpatients their study found reduction pretreatment mean 23.1 beck depression inventory bdi 14.4 posttreatment another relevant benchmark derived studies conducted group psychotherapy treat depression used bdi outcome measure reviewed studies found 35 studies calculated overall pretreatment mean bdi 23.9 posttreatment mean 12.3 this review incorporated variety treatments values represent acceptable benchmark studies included versions cbt excluding studies cbt values improved estimate treatment effect somewhat bdi treatment 25.5 11.6 treatment clients mean age 36 mean number 20 therapy hours a final benchmark outpatient evaluation group cbt depressed patients treated within diagnostically heterogenous groups their outcomes bdi revealed decline 26.9 sd 8.9 16.5 sd 11.3 the effect size cohen change large i.e. 1.2 59% patients demonstrating clinically significant change treatment thus aim present study examine effectiveness inpatients depression treated cbt program modified run intensively two week period the total patient population comprised archival dataset 998 consecutive inpatient admissions diagnosed major depressive disorder july 1996 march 1999 spent least one day hospital completed bdi admission hospital patients diagnosed using clinical interview according dsm iv treating psychiatrist the decision admit person inpatient made psychiatrist made apparent required treatment could provided optimally patient community the total patient group mean age 43.2 sd 14.4 73% female of total sample 46% married 26% separated divorced 17% widowed remainder never married the depressive disorders classified single episode among 52% cases recurrent among remaining 48% depression primary diagnosis among 82% patients secondary another diagnosis among remainder for primary diagnosis depression common recorded comorbid disorders anxiety disorders 18% substance use 12% personality disorders 5% 58% patients least one medical condition coded axis iii patients spent average 12.3 days hospital sd 8.9 range 266 days of total patient group 225 began cbt program inpatient stay immediately upon discharge 201 89% completed program patients provided consent data used research evaluation purposes university human research ethics committee approved analysis deidentified data in addition medications managed treating psychiatrist cbt program variety problem focused open group inpatient programs available including acute care program substance abuse program interpersonal therapy program thus patients may exposure treatments cbt program engaged treatment except concurrent psychopharmacotherapy time the type pharmacotherapy admission inpatient stay discharge collected almost inpatients would one type medication perth clinic cbt program closed group eight patients conducted period ten working days 5 10 the program begins problem identification goal setting leading psychoeducation cognitive therapy the cognitive therapy draws upon work beck ellis harper involves self monitoring identification challenging irrational beliefs behavioral interventions anxiety management stress reduction taught e.g. relaxation breathing control etc patients engage pleasant events scheduling behavioral assignments practise newly developed skills latter part treatment focus shifts self esteem assertion communication training concludes relapse prevention module the structure allows sufficient time flexibility discussion group individual issues there also supporters session participant invited attend supporter the aim session increase awareness psychological disorders nature treatment helpful caring behaviors patients could complete cbt program inpatient daypatient immediately following discharge discharged inpatient status point within program the average time admission initiation cbt program 4.9 days sd 7.4 all patients received questionnaires admission discharge applicable beginning end cbt program questionnaires sent patients completed cbt program six week followup staff rated measures administered admission discharge ward staff beginning end cbt program treating therapists clinical psychologists occupational therapists the bdi 21-item self report scale designed measure level depression among clinical non clinical populations widely used research depression in addition patients completed locus control behaviour scale lcb assess sense control lives the rosenberg self esteem scale rses administered measure general self concept consists ten items using four point likert type response format clinic staff rated patients general level psychiatric symptoms using health nation outcome scales honos 16 17 global assessment function gaf the honos 12-item clinician rated scale comprehensive coverage clinically relevant quick administer honos introduced clinic middle 1997 therefore data available one year study first change symptoms admission pre cbt post cbt 6-week followup examined the bdi scores improved somewhat admission 30.3 pre cbt 24.7 f(1,152 60.76 .29 p .001 markedly pre- post cbt 12.2 f(1,152 299.90 .66 p .001 remained stable followup 12.2 f(1,152 0.003 .00 p ns compare effectiveness present form therapy results typically obtained research trials 95% confidence interval placed around difference pre- posttreatment bdi scores compared benchmark values identified introduction thus 95% confidence interval around difference 12.5 pre- posttreatment bdi scores extended 11.1 13.9 mean difference 11.6 studies group psychotherapy depression within confidence interval 13.9 difference studies limited using cbt supporting conclusion difference obtained present investigation comparable found group treatment depression generally another way examine the overall effectiveness examine clinical significance outcomes calculating reliable change index using normative data bdi described robinson et al 60.2% patients demonstrated clinically significant improvement treatment comparing present results depressed outpatient sample apparent inpatients severe admission outpatient population i.e. 30.3 versus 26.9 scores declined less severe i.e. 24.7 start cbt program this consistent treatment model within acutely unwell patients first stabilised gains consolidated cbt program number patients demonstrated clinically significant improvement pre- post cbt i.e. 60.2% similar found outpatient setting i.e. 59% these changes outcome also observable clinician rated self reported measures terms staff rated gaf scores rose admission 49.3 pre cbt 56.9 f(1,120 32.00 .21 p < .001 continued increase pre- post cbt 69.1 f(1,120 219.65 .65 p .001 patients lcb scores increase admission 47.9 pre cbt 47.2 f(1,151 1.24 .01 p ns became substantially post cbt 38.2 f(1,151 144.14 .49 p .001 became marginally external 6-week followup 40.0 f(1,151 6.51 .04 p .05 the patients self esteem scores improve admission 23.5 pre cbt 23.6 f(1,102 0.03 .000 p ns became internal pre post cbt 28.7 f(1,102 106.87 .51 p < .001 remained stable 6-week followup 28.9 f(1,102 0.22 .002 p ns although changes cbt program comparable published studies one possible interpretation present findings patients would improved degree due hospital stay partly address concern the patients completed cbt program compared admission discharge hospital begin cbt program one hand withdrew program there significant differences groups across two time intervals f(2,602 3.74 .01 p .05 follow tests revealed significant differences groups admission patients completed cbt less depressed 13.1 never began cbt 16.6 t(611 2.56 p .05 turn different dropped cbt 16.9 t(516 0.10 p ns thus despite absence differences admission completed cbt program least depressed discharge address concern observed improvement may due general effects hospitalization rather specific effects cbt the profile symptom change admission pre cbt pre- post cbt examined assuming degree symptom specificity treatments cbt focussed symptoms depression anxiety changes domains greater cbt whereas earlier phase inpatient admission focussed acute symptom management examine issues changes admission pre cbt post cbt individual honos items what apparent figure 1 inpatient stay prior entry cbt associated small changes anxiety depression substantial improvements memory orientation activities daily living self harm aggression substance problems whereas cbt program associated greatest changes emotional symptoms thus symptoms targeted cbt change following treatment domains focus treatment shift little cbt program given data showing symptoms depression improved time patients cbt program effect outcome various clinical variables examined of variables examined difference bdi scores patients pre- post cbt depression single episode recurrent episode f(1,146 2.37 .02 p ns depression judged primary secondary another disorder f(1,151 0.15 .001 p ns considering patients depression primary disorder hint total number secondary diagnoses might associated poorer outcome f(1,97 2.98 .03 p .09 indication poorer outcomes patients personality disorders f(1,151 0.19 .001 p ns affective disorders f(1,151 0.15 .001 p ns neurotic disorders f(1,151 0.83 .01 p ns although difference bdi scores cbt patients secondary medical conditions f(6,146 1.67 .06 p ns hint patients secondary substance use problems might worse outcomes f(3,149 2.44 .05 p .07 thus strong evidence present sample presentations major depression complicated axis ii iii resistant cbt program less complicated presentations however one clinical variable strongly related outcome severity depression admission using trecile split divide patients three equal sized groups based admission bdi scores apparent change admission pre cbt f(2,150 7.20 .09 p .01 pre- post cbt f(2,150 16.73 .18 p .001 systematically related severity from figure 2 apparent greatest changes observed severe problems cbt finished patients maintained level symptoms treatment next six weeks f(2,150 0.31 .004 p ns the present study examined effectiveness patients depression cbt program adapted inpatient psychiatric clinic intensive program brought reductions depression ratings compared found literature generally importantly able achieve gains within two week period rather usual 1620 weeks treatment the speed treatment gains important patients require inpatient treatment often distressed may represent threat safety therefore relatively rapid treatment means safer environment provided period time removed typical routines support minimized thus cbt depression adapted intensive program suitable delivery within model care found inpatient settings however within inpatient settings multiple interventions co occur present study this means possible use data conclude cbt program caused symptom changes however empirical issue already answered numerous times controlled efficacy studies present question related effectiveness cbt particular context particular also clear packaging cbt program part inpatient treatment program deliver comparable treatment outcomes spaced treatments achieve outcomes expense longevity treatment gains the gains observed stable six week followup evidence severe patients likely return problematic levels following termination program clinical perspective common issue raised among practitioners treatments developed demonstrated efficacy studies generalize real world clinical settings experience common reasons given lack generalization include greater severity patients outside efficacy studies higher occurrence comorbidity especially substance use personality disorders since patients rarely prohibited receiving treatment way may appropriately excluded efficacy study setting therefore curious see present sample variable associated differential outcomes symptom severity greatest change observed patients depressed given studies found comorbidity type depression impact outcome intriguing present study failed find differences one possible reason lack difference intensity program there may greater opportunity comorbid conditions interfere treatment intervention extends months whereas intense cbt program fills day may less opportunity associated conditions manifest for instance person substance use problem may able maintain harm free use abstinence couple weeks treatment whereas months might difficult without random assignment intensive spaced treatment sessions possible offer speculative suggestion present data would encourage investigation benefits costs alternative forms delivery in considering results number limitations need borne mind first hospital private clinic patients insured therefore extent generalization public sector clear it possible even though comorbid conditions present severity types comorbidity may present public hospitals e.g. greater frequency psychotic disorders may mean particular presentations represented current sample second nature present investigation retrospective examination effectiveness randomized controlled trial therefore variety issues make hard conclude cbt program caused observed effects it possible symptom changes observed cbt program may attributable program due patient selection factors associated hospital care e.g. concurrent pharmacotherapy while ruled attempts made examine degree variables may affected data the difference inpatients began cbt program suggests cbt program providing benefits treatment usual benefits discharge people began cbt finished relative dropped address issue staff selected cbt patients likely improve regardless content treatment dropped different admission discharge never began cbt in addition apparent specificity treatment gains occurred cbt speaks arguments general effect hospital causing reductions symptoms however remains possible treatment cause observed outcomes given cbt demonstrated efficacious treatment depression current treatment effects comparable observed efficacy studies unreasonable presume application empirically validated treatment albeit modified form largely responsible observed improvements third present study set establish efficacy cbt examine effectiveness within clinical setting relied patients self reports judgments treating clinicians although internal validity ratings less carried person blind treatment external validity ratings conducted clinicians spent two weeks patient greater nonetheless possibility bias patients may tried please therapists staff may overestimated change greater degree cbt program hospital generally finally important note contrast efficacy studies hospital context possible exclude patients treatment bdi cut e.g. decision treat particular patient involve consideration self reported severity depression factors also taken account therefore sample contains individuals might meet criteria inclusion efficacy study applying criterion excluding analyses anything strengthens conclusions present study since pre- post cbt scores change 28.4 13.2 increasing absolute treatment difference 12.5 bdi points patients included 15.2 points despite limitations appears reasonable conclude cbt adapted inpatient psychiatric clinic effectiveness program comparable observed homogenous patient samples found efficacy studies however clear present study degree apparent lack comorbidity type depression interfere treatment outcomes associated intensity treatment setting therapy temporal location overall treatment program cbt delivered future research could investigate questions assist clarifying effective way deliver treatment different settings different patient presentations another future research question could relate ability deliver cbt homogeneous patient groups the patients present study diagnosed depression treated within group patients suffered depression the clinic provides cbt program makes available patients might benefit program therefore mixture primary diagnoses although possible evaluate outcomes patients numbers diagnostic group small outcome measures specific conditions sufficient literature disorders measures used benchmark present data clear least people depression treated alongside patients conditions without degradation treatment outcomes the therapy staff observes mixed patient groups rather hindrance assistance therapy since patients assist one another helping different problems the degree clinical observation replicated controlled study possible start isolate factors beneficial treating heterogenous patient groups the present study identify extent incorporating cbt inpatient program reduce overall length hospital stay the length stay present clinic relatively short compared private psychiatric clinics australia treat similar patient groups moira munro personal communication 27 october 2011 different public psychiatric hospitals tend treat proportionally people schizophrenia one possible explanation reliance adjunctive psychotherapy shown elsewhere addition psychotherapy enhances outcomes following inpatient stay however duration hospital stay varies markedly across health care systems countries therefore question probably better addressed using multilevel modelling hospitals nested relevant units e.g. healthcare system nation etc conclusion present study found evidence based treatment depression could modified inpatient treatment setting without substantial loss effectiveness clinically important people often admitted inpatients suicide risk high therefore data consistent view adding cbt treatment available times associated rapid reduction symptoms suicidal patient a reduction symptoms going associated lowered risk harm therefore sooner reduction achieved sooner less intensive treatment regime implemented in addition concerns generalizing efficacy studies clinical effectiveness arise due complexity patients e.g. comorbidity addressed strong evidence found issues compromised outcomes present study

the effectiveness among inpatients with depression of a modified cognitive behavior therapy ( cbt ) program was examined . a group of 300 inpatient admissions with a primary diagnosis of depression attending a private psychiatric clinic were assessed at the beginning and end of a two - week cbt program . the effectiveness of the treatment was demonstrated by improvements on the beck depression inventory ( bdi ) , the health of the nation outcome scales , locus of control of behaviour scale , and the global assessment of function . the changes on the bdi for patients with depression were benchmarked against estimates generated from published studies . the degree of change in a two - week period for inpatients with depression was similar to that observed in efficacy studies of cbt that typically run over a more extended time . implications for integrating cbt with inpatient services are discussed .

retinal vein occlusion rvo obstruction retinal venous system abrupt onset important cause visual morbidity retinal vein occlusions constitute second common cause retinal vascular disease diabetic retinopathy prevalence 1% 2% persons older 40 years age 25 wisconsin beaver dam eye study 12% eyes developed severe visual impairment best corrected visual acuity 20/200 15-year followup due rvo although exact etiology rvo known likely follow thrombotic event possibly caused external compression disease vein wall 1 7 rvo categorized branch retinal vein occlusion brvo obstruction located one branches central vein central retinal vein occlusion crvo located central vein level optic nerve brvo encompasses heterogeneous group disorders different clinical aspects presents dilated tortuous retinal venous system particular quadrant hemisphere retina often associated macular edema crvo presented hemorrhagic changes four quadrants retina dilated tortuous veins brvo crvo cotton wool spots disc edema neovascularization the incidence rvo estimated 0.12%/year adults aged 45 years brvo 0.04%/year adults aged 45 years crvo caucasian populations 6 11 however canadian incidence visual impairment vi due macular edema secondary rvo unknown this study aims determine annual incidence vi characteristics patients secondary brvo crvo real world canadian setting records longitudinal population based database 170,000 patients 53 family practice clinics southwestern ontario canada analyzed january 1 2008 december 31 2009 these records contained chart abstracted information visit diagnosis medications consultation notes order compare characteristics comorbidities patients diabetic macular edema dme general population a control cohort constructed matching age gender patients database 18 years clinic location initial extractions control cohort rvo patients defined retinal thickening within 500 macular center vi defined best corrected visual acuity 20/40 rvo eye accomplished utilizing international classification disease codes icd9/icd10 reviewing patient charts text entries symptoms supported diagnosis rvo concomitant comorbidity reviewing patient treatment records unique rvo including consultation notes hospital discharge summaries data included study comprised patient characteristics demographics cardiovascular comorbidity events medication coverage the swo database recorded patient level data clinical diagnoses visit symptoms corroborating diagnoses clinical data prescribed treatments including lifestyle interventions medications physician visits hospitalizations diagnostic laboratory test results allowing conduct patient level analyses since 2000 data 53 practices participating swo database cohort routinely updated quarterly basis immediate reconciliation point care all practices included swo database part family practice research network involved various audit clinical research activities practices consented centralized accrual clinical data patient record uwo irb 09572 each patient ontario health insurance plan ohip number assigned unique patient identification number swo database protect privacy patient medical information 128-bit ssl certificate installed production swo web server the industry standard data protection method ensures security data transmission across internet validation studies swo database confirming quality completeness recorded data show good agreement estimates prevalence cardiovascular risk factors obtained swo database published estimates 1215 moreover correlation swo database national data i.e. ims brogan pharmastat utilization prescription medication personal communication petrella kamino ims starting index date date first diagnosis rvo study period december 2009 subjects meeting inclusion exclusion criteria analyzed understand demographics treatment patterns care treated patients treatment choices characterized treatment pattern related clinical characteristics patients including type drug coverage public private pocket continuous variables mean standard deviation median minimum maximum values estimated for categorical variables number percentage category within assessment calculated non missing data 73 47,166 patients 40 years age new diagnosis rvo control cohort 76,077 patients extracted analysis please refer table 1 data demographics control rvo cohorts examination recorded episodes observation period gender age revealed following 8% patients new diagnosis rvo time males ages 4059 years compared 43% females age group ninety two percent males new diagnoses rvo age 60 compared 54% females a higher percentage caucasian 81% aboriginal 11% ethnic groups affected rvo compared respective groupings 78% 9% within control cohort please refer table 2 data disease characteristics control rvo cohorts more rvo patients overweight 23% obese 13% compared control cohort more rvo patients hypertension 68% versus 14% dyslipidemia 16% versus 10% control cohort p 0.05 one quarter rvo patients history vascular disease primarily mi stroke fifteen percent patients rvo suffer chronic kidney disease please refer table 3 data incidence type rvo within rvo cohort seventy three patients 40 years old new diagnosis rvo identified 47,166 patients 40 years old fifty three patients brvo 20 patients crvo interpreted consultation notes the annual incidence vi due secondary brvo crvo found 0.056% 95% ci 0.0110.072 0.021% 95% ci 0.0080.081 respectively shown table 4 majority patients public drug coverage macular edema complication rvo lead blindness this first study assess incidence vi due secondary brvo crvo describe disease characteristics patients rvo canadian setting a studies assessed incidence rvo australian blue mountains eye study the 5-year incidence rvo 1.0% 10-year incidence 1.6% us 5-year incidence 0.8% 15-year incidence 2.3% japan 9-year incidence reported 2.0% the incidence brvo appears generally higher incidence crvo studies small number individuals developed incident rvo makes difficult know accuracy ratio incident brvo incident crvo use estimates basis comparison males 92% 60 years versus 54% females diagnosis rvo rvo rarely seen individuals younger 50 may affect 5% individuals age 80as noted laouri et al review literature burden retinal vein occlusion consistent previous literature found patients rvo likely present hypertension dyslipidemia vascular diseases compared general population 4 1719 further findings concur previous studies found risk factors common patients brvo observed lattanzio et al given close association rvo systemic vascular disease new patients rvo evaluated hypertension diabetes lipid abnormalities may presentation significant vascular morbidity case younger patients may otherwise healthy pathogenesis risk factors still poorly understood additional evaluation coagulation disorders history thromboses may necessary 8 20 this retrospective study conducted utilizing icd9/icd10 disease codes reviewing patient charges treatment records including consultation notes hospital discharge summaries brvo crvo easily detected using standard ophthalmological diagnostic tools techniques diagnosis classification rvo valid difficulties interpreting data contained consultation notes well data concerning number episodes requiring consultation treatment observation period may resulted inconsistencies data capture this paper presents description characteristics patients vi due secondary brvo crvo real world canadian setting consistent findings studies rvo patient population associated several vascular comorbidities the annual incidence vi due secondary brvo crvo estimated 0.056% 0.021% respectively

retinal vein occlusion ( rvo ) is an obstruction of the retinal venous system , and macular edema ( me ) is a complication of rvo that can lead to blindness . the canadian incidence of visual impairment ( vi ) due to me secondary to rvo is unknown . this observational , retrospective study used records from the southwestern ontario database to observe the annual incidence , demographics , and comorbidity characteristics of patients with vi due to me secondary to rvo . from 47,166 patients , 73 with rvo ( > 40 years old ) were identified : 53 with branch retinal vein occlusion ( brvo ) , 20 with central retinal vein occlusion ( crvo ) . the annual incidence of vi ( visual acuity < 20/40 in snellen equivalent ) due to me secondary to brvo was ( mean ( 95%ci ) ) 0.056% ( 0.0110.072 ) , and to crvo was 0.021% ( 0.0080.081 ) . furthermore , a greater proportion of rvo patients had hypertension ( 68% versus 14% ) or dyslipidemia ( 16% versus 10% ) , when compared to a healthy control cohort of 76,077 subjects ( p < 0.05 ) . this study presents a description of the characteristics of patients with vi due to me secondary to rvo in a real - world canadian setting . the results demonstrate that brvo was more frequent than crvo , and that rvo in this patient population was associated with several vascular comorbidities .

well documented medical literature diabetes management ramadan fasting poses challenges treating physician pre ramadan diabetes assessment evaluations diabetes education important successful management.1 unplanned diabetes management ramadan fasting may lead hypoglycemia hence past decades efforts made various researchers manage diabetes ramadan fasting without risk hypoglycemia these include alteration reduction dosages oral hypoglycemic agents ohas insulins shifting patients ohas insulins metformin agents dipeptidyl peptidase-4 dpp-4 inhibitors the increase hypoglycemic events month ramadan previously reported literature the epidemiology diabetes ramadan epidiar study reported 7.5-fold increase risk hypoglycemia patients type 2 diabetes.2 however risk reduced extensive diabetes education pre ramadan medication adjustments.1 hence ramadan risk poor education use medications cause hypoglycemia high dosages carries risk hypoglycemia adverse effect quality life obstacle managing diabetes control glycemia associated poor compliance medication treatment it also well documented skipping meals reduced food intake main causes hypoglycemia ramadan fasting hence diabetes education plays central role diabetic patients given education counseling ramadan fasting.1,3,4 general severe hypoglycemia carries risk morbidities major cardiovascular events stroke myocardial ischemia failure ventricular arrhythmias.5 reasons allowing patients fast fasting ramadan without risk hypoglycemia personal patient centered decision a study conducted saudi arabia aziz1 1,046 patients demonstrated ramadan fasting pose risk human metabolism health conversely beneficial health effects physiological parameters eg opportunity lose weight chronic disease prevention this study demonstrated goal achieved optimal pre ramadan assessment diabetes education.1 similar observations reported studies well.68 however 2003 laarijani et al9 demonstrated slight decrease fasting serum glucose among healthy subjects ramadan fasting similar finding also demonstrated aziz1 ramadan study lowest prevalence hypoglycemia 4.58% contrary facts different studies conducted past ramadan fasting demonstrated high prevalence hypoglycemia ramadan fasting 21.7%).1018 however studies mostly observational nature patients selected ramadan extensive diabetes self management education dsme counseling assessment hba1c creatinine alteration therapy hence words concluded general blood glucose levels fall ramadan fasting diabetic nondiabetic subjects prevention hypoglycemia medication adjustments alterations basic strategies manage diabetes ramadan fasting literature background the current review focuses class medications cause hypoglycemia general ramadan fasting one dpp-4 inhibitors drug available market vildagliptin focus pathophysiology type 2 diabetes dpp-4 inhibitors role vildagliptin ramadan fasting dpp-4 inhibitors new oral antidiabetic agents including vildagliptin sitagliptin saxagliptin linagliptin alogliptin agents well extensive research these agents drugs reduce serum glucose concentrations improve glycemic control augmenting effects incretins hence strategy also called incretin based therapy diabetes management normal physiological state gut response meals releases hormones called incretins example glp-1 glucagon like peptide-1 gip gastric inhibitory polypeptide augment biosynthesis secretion insulin known incretin effect well slow gastric emptying well.1924 normally incretin hormones degraded within minutes release enzyme dpp-4 dpp-4 inhibitors contrary normal physiology diabetic patients balance insulin secretion hepatic glucose production dysregulated type 1 diabetic subjects absolute insulin deficiency due autoimmunity -cells destruction cells compared type 2 diabetic subjects exhibit relative insulin deficiency insulin resistance.25,26 furthermore absolute relative hyperglucagonemia due deficiency incretin hormones diabetic state hallmark type 1 type 2 diabetic subjects.27,28 words type 2 diabetic subjects insulin resistance hyperglucagonemia due pathophysiological states lipolysis ketosis may worsen metabolic state leading diabetic ketoacidosis dka prolonged fasting absolute relative insulin deficiency.29,30 insulin resistance reversed metformin hyperglucagonemia incretin based therapy additionally vildagliptin also shown improve -cell function type 2 diabetics apart enhancing incretin effect.31 research studies demonstrated efficacy vildagliptin lower hba1c improve glycemic control well.3236 hence reversion diminished incretin effect also essential manage diabetes effectively general ramadan fasting preventing hypoglycemia time one interesting phenomena dpp-4 inhibitor vildagliptin blood glucose dependent cause hypoglycemia given monotherapy therapeutic advantage dpp-4 inhibitors vildagliptin ramadan fasting in section literature safety efficacy vildagliptin ramadan fasting reviewed sulfonylureas sus oral hypoglycemic agents ohas still widely used general practitioners oral antidiabetic agents general ramadan fasting ability effectively reduce hba1c low cost.37,38 however carry higher risk severe hypoglycemia especially dose reduced ramadan fasting special precautions required together individual considerations especially older age group.1,39 hence highly variable rates hypoglycemia reported published research trials sus ohas 3%40% ramadan fasting however risk significantly reduced vildagliptin prescribed ramadan fasting also recently reported indo pakistani populations uk cohorts uk south asian muslim patients vector vildagliptin experience compared gliclazide observed ramadan study.4042 furthermore general agents also demonstrated safer older age group.43 recent observational study combined metformin vildagliptin therapy together without ohas ramadan fasting demonstrated advantage reduced hypoglycemia incidence.44 shown one case severe hypoglycemia arm treated oha compared vildagliptin group showed hypoglycemia event the hypoglycemia events 12 times group treated oha compared vildagliptin group other studies reported similar results comparing ohas vildagliptin.45 another study conducted vildagliptin sus ohas ramadan fasting reported higher incidence hypoglycemia ramadan fasting group treated su metformin vs vildagliptin plus metformin 26 episodes vs 19 episodes also reported hba1c reduction vildagliptin group however insignificant p values.46 study conducted aziz1 also concluded patient group prescribed dpp-4 inhibitors sitagliptin vildagliptin show episodes hypoglycemia ramadan fasting recent review published switzerland france has studied worldwide role dpp-4 inhibitors including vildagliptin ramadan fasting come conclusion anti diabetic agents dpp-4 inhibitors could safer option managing type-2 diabetes ramadan fasting low risk hypoglycemia.47 another prospective noninterventional study published france assess real life rate hypoglycemia ramadan fasting patients type 2 diabetes ongoing dual therapy metformin vildagliptin metformin sulfonylurea shown hypoglycemia adverse events aes higher su group compared vildagliptin group 17.9% vs 7.5% p=0.025 better compliance seen vildagliptin group.48 virtue vildagliptin experience compared sulphonylureas observed ramadan study recruited 1,333 patients 10 different countries worldwide demonstrated significantly fewer hypoglycemia events compared su therapy 5.4% vs 19.8% respectively p<0.001 additionally good glycemic weight control better tolerance observed vildagliptin treated patients.49 steadfast study evaluating vildagliptin compared gliclazide patients type 2 diabetes fasting ramadan study multicenter double blind randomized trial recruited 557 type 2 diabetic patients demonstrated significantly lower hypoglycemia prevalence compared sus 3.0% vs 7.0% respectively p=0.039).50 similar results reported muslim populations india.51 summary vildagliptin proven effective well tolerated associated low incidence hypoglycemia recent clinical trials this true especially high risk population elderly renal impairment require insulin based therapy metformin dpp-4 inhibitors.44,5256 despite islamic rule exemption diabetic patients essentially fast ramadan fact considered managing diabetes ramadan fasting.1,2 drugs dpp-4 inhibitors vildagliptin selected diabetes medications associated high risk hypoglycemia observed general practitioners limited knowledge diabetes management ramadan fasting furthermore survey results shown 53% patients fasted medical advice.57 however prevented effectively extensive dsme ramadan focused diabetes management ramadan education awareness diabetes read program similar studies demonstrated promising good results terms minimizing hypoglycemia risk ramadan fasting.1,58,59 effective diabetes education patients empowerment motivation self care awareness involves health care professional teams families community religious authorities well.60,61 additionally newer antidiabetic agents vildagliptin dpp-4 inhibitor associated lower risk hypoglycemia considered one safer options managing diabetes ramadan fasting also shown higher treatment adherence compared medications.62

diabetes management during ramadan fasting is challenging to the physician in terms of minimizing the risk of hypoglycemia . as compared to oral hypoglycemic agents ( ohas ) and sulfonylureas ( sus ) , which carry a higher and significant risk of hypoglycemia , newer antidiabetic agents such as dipeptidyl peptidase-4 ( dpp-4 ) inhibitors have demonstrated lower risk of hypoglycemia during ramadan fasting , with better patient compliance . in addition to diabetes education and pre - ramadan assessments , the physician should also consider use of dpp-4 inhibitors ( such as vildagliptin ) during ramadan fasting to minimize the risk of hypoglycemia in type 2 diabetic subjects . severe episodes of hypoglycemia have been demonstrated in recent research and clinical trials with ohas / sus . conversely , these research observations have also demonstrated comparative safety and efficacy with lower risk of hypoglycemia associated with vildagliptin . current research review has collected evidence - based clinical trials and observations for the drug vildagliptin to minimize the risk of hypoglycemia during ramadan fasting , while at the same time focusing the role of diabetes self - management education ( dsme ) , pre - ramadan assessments , and patient care .

pseudoprogression pspd pseudoresponse psr following anticancer therapy major areas controversy management high grade glioma era temozolomide discrimination pspd true progression high grade glioma concurrent radiochemotherapy crt conventional magnetic resonance imaging mri ) novel imaging modalities mr spectroscopy positron emission tomography pet perfusion mri showing promising results yet validated prospective studies we previously reported significance overexpression p53 predicting development pspd10 in addition method interpretation early radiologic deterioration determine also critical issue affects clinician decision making regarding whether switch salvage therapy recurrent disease continue planned adjuvant therapy as pspd termed early necrosis early reports many series determined pspd 4 weeks treatment conversely wen et al.28 proposed new response assessment neuro oncology rano working group criteria high grade glioma an increasing non enhancing component t2-weighted fluid attenuated inversion recovery flair mr images considered progression patients receiving anti angiogenic therapy new criteria they also suggested progression true progression cases recurrence outside radiation field pathologically confirmed cases radiological progression detected within 12 weeks pspd prevalent however validation would necessary suggestion based clinical study demonstrates comparable survival outcome early true progression 4 weeks treatment etpd late true progression progression 4 12 weeks treatment ltpd if survival difference etpd ltpd significant early progressive findings treated based assumption lesion would recurrent tumor psr concept represents temporary radiological improvement using antiangiogenic agent bevacizumab progression effect phase ii trials using bevacizumab cpt-11 recurrent malignant glioma shown high response rate 63% encouraging result lead significantly improved overall survival os)11,27 this phenomenon proposed represent effect bevacizumab normalizing permeability leaky endothelium causes edema showing steroid like feature present study evaluated pspd following radiation therapy combined concurrent temozolomide tmz also assessed psr following anti angiogenic therapy patients recurrent disease using criteria rano working group all data patients diagnosed world health organization grade 3 4 glioma march 2005 february 2011 retrieved archives pathologic reports the clinical pathologic data relevant patients obtained accordance protocol approved institutional review board fifty five sixty two patients high grade glioma received radiotherapy rt concurrent adjuvant tmz included present study gross total resection performed 16 29% patients subtotal near total resection performed 25 45% patients stereotactic biopsy performed 14 26% patients the median dose used radiation therapy 61.2 gy range 59.4 61.2 gy daily fractions 1.8 2.0 gy forty five patients received total dose 61.2 gy 34 fractions nine patients received 60 gy 30 fractions one patient received 59.4 gy 33 fractions the median time surgery initiation crt 26 days range 11 77 days tmz concomitant postoperative rt administered 75 mg day adjuvant tmz 150 200 mg administered daily 5 days every 28 days terms salvage therapy following recurrence the surgical resection performed patients small progressive lesion involving non eloquent area gamma knife surgery reserved cases small lesions medically inoperable condition bevacizumab without cpt-11 considered primary option salvage therapy inoperable cases showing true progression the patients previous episode intracranial hemorrhage able afford bevacizumab received nimustine acnu)/cisplatin cddp procarbazine lomustine vincristine pcv chemotherapy metronomic tmz mri gadolinium enhancement t2/flair performed 1 month crt every 3 months thereafter progressive finding present mri performed 2-month intervals progression defined 25% increase sum products perpendicular diameters macdonald criteria increase non enhancing lesion recurrent cancer bevacizumab15 etpd ltpd defined true progression found first second post treatment mri exception pspd respectively psr scored progressive enhancement non enhancing t2-weighted lesion shown right response bevacizumab discontinuation drug due toxicity cases cerebral blood flow cbf cerebral blood volume cbv values using perfusion mri apparent diffusion coefficient adc maps the kaplan meier method used survival analysis compare os etpd ltpd all data patients diagnosed world health organization grade 3 4 glioma march 2005 february 2011 retrieved archives pathologic reports the clinical pathologic data relevant patients obtained accordance protocol approved institutional review board fifty five sixty two patients high grade glioma received radiotherapy rt concurrent adjuvant tmz included present study gross total resection performed 16 29% patients subtotal near total resection performed 25 45% patients stereotactic biopsy performed 14 26% patients the median dose used radiation therapy 61.2 gy range 59.4 61.2 gy daily fractions 1.8 2.0 gy forty five patients received total dose 61.2 gy 34 fractions nine patients received 60 gy 30 fractions one patient received 59.4 gy 33 fractions the median time surgery initiation crt 26 days range 11 77 days tmz concomitant postoperative rt administered 75 mg day adjuvant tmz 150 200 mg / administered daily 5 days every 28 days terms salvage therapy following recurrence surgical resection performed patients small progressive lesion involving non eloquent area gamma knife surgery reserved cases small lesions medically inoperable condition bevacizumab without cpt-11 considered primary option salvage therapy inoperable cases showing true progression the patients previous episode intracranial hemorrhage able afford bevacizumab received nimustine acnu)/cisplatin cddp procarbazine lomustine vincristine pcv chemotherapy metronomic tmz mri gadolinium enhancement t2/flair performed 1 month crt every 3 months thereafter progressive finding present mri performed 2-month intervals progression defined 25% increase sum products perpendicular diameters macdonald criteria increase non enhancing lesion recurrent cancer bevacizumab15 etpd ltpd defined true progression found first second post treatment mri exception pspd respectively psr scored progressive enhancement non enhancing t2-weighted lesion shown right response bevacizumab discontinuation drug due toxicity cases cerebral blood flow cbf cerebral blood volume cbv values using perfusion mri apparent diffusion coefficient adc maps the kaplan meier method used survival analysis compare os etpd ltpd all patients treated rt combined concurrent adjuvant tmz surgical resection stereotactic biopsy nine 24% thirty seven patients disease progression treatment period 1 case pspd treated bevacizumab without cpt-11 surgical resection gamma knife surgery acnu cddp pcv chemotherapy metronomic tmz also used option salvage therapy 13 55 patients cbv adc pet undertaken insufficient number analyze efficiency techniques the exams provided additional information absolute means diagnosis first follow crt 21 patients 38% showed radiologic progression second post crt mri 16 29% persistently showed progression considered etpd five 9% patients showed progression first mri taken post treatment 4 weeks showed improvement afterwards without specific salvage treatment decreasing stable dose dexamethasone these groups patients finally categorized pspd second post crt mri 7 showed new progression seen previous mri ltpd the follow time defined period surgery including stereotactic biopsy last follow day death the estimated median survival 25.6 months range 3.3 89.7 months the os rates 1 2 years 84.8% 59.2% respectively divided grade iii iv gliomas os 2 years 71.4% 53.3% respectively p=0.125 1 os etpd group vs. ltpd group 57% vs. 86% 1 year oss 30% 51% 2 years respectively we also performed subgroup analysis 33 patients available data o-6 methylguanine dna methyltransferase mgmt methylation status although statistically significant os difference patients methylated mgmt unmethylated mgmt p=0.178 showed trend toward better survival mgmt methylation fig 2 multivariate analysis using covariates recursive partitioning analysis grade p53 status mgmt methylation status extent resection none factors including etpd ltpd related os the median survivals treatment without bevacizumab 10 6 months respectively p=0.835 although 5 50% 10 patients showed radiological response following salvage treatment bevacizumab response durable one case stable state persisted 11 months for one case mri scan showed decreased high t2 signal intensity 1 month bevacizumab cpt-11 lesion showed progression subsequent follow considered case pspd for three cases radiologic improvements shown within 3 months bevacizumab continued thromboembolic events two cases cerebral infarction one case all showed rapid regrowth tumor immediately discontinuation bevacizumab mri fig all patients treated rt combined concurrent adjuvant tmz surgical resection stereotactic biopsy nine 24% thirty seven patients disease progression treatment period 1 case pspd treated bevacizumab without cpt-11 surgical resection gamma knife surgery acnu cddp pcv chemotherapy metronomic tmz also used option salvage therapy 13 55 patients cbv adc pet undertaken insufficient number analyze efficiency techniques at first follow crt 21 patients 38% showed radiologic progression second post crt mri 16 29% persistently showed progression considered etpd five 9% patients showed progression first mri taken post treatment 4 weeks showed improvement afterwards without specific salvage treatment decreasing stable dose dexamethasone these groups patients finally categorized pspd second post crt mri 7 showed new progression seen previous mri ltpd the follow time defined period surgery including stereotactic biopsy last follow day death the estimated median survival 25.6 months range 3.3 89.7 months the os rates 1 2 years 84.8% 59.2% respectively divided grade iii iv gliomas os 2 years 71.4% 53.3% respectively p=0.125 1 os etpd group vs. ltpd group 57% vs. 86% 1 year oss 30% 51% 2 years respectively statistically significant difference found etpd ltpd groups p=0.595 we also performed subgroup analysis 33 patients available data o-6 methylguanine dna methyltransferase mgmt methylation status although statistically significant os difference patients methylated mgmt unmethylated mgmt p=0.178 showed trend toward better survival mgmt methylation fig using covariates recursive partitioning analysis grade p53 status mgmt methylation status extent resection none factors including etpd ltpd related os the median survivals treatment without bevacizumab 10 6 months respectively p=0.835 although 5 50% 10 patients showed radiological response following salvage treatment bevacizumab response durable one case stable state persisted 11 months for one case mri scan showed decreased high t2 signal intensity 1 month bevacizumab cpt-11 lesion showed progression subsequent follow considered case pspd for three cases radiologic improvements shown within 3 months bevacizumab continued thromboembolic events two cases cerebral infarction one case all showed rapid regrowth tumor immediately discontinuation bevacizumab mri fig the primary goal study evaluate survival difference etpd ltpd thus could identify statistically significant survival difference two groups indicating would safe continue planned adjuvant tmz rather treatment recurrence radiologic progression appears first post treatment mri this unique treatment strategy dealing radiologic progression high grade glioma comes absence diagnostic modality providing high accuracy resemblance field recurrence treatment response pspd radiation necrosis the mechanism pspd exclusive phenomenon high grade glioma clarified might related nature tumor blood tumor barrier glioma high expression p glycoprotein 170-kda drug efflux pump protein main component blood brain barrier bbb inhibits penetration23 generally glioblastoma compromised barrier gadolinium penetrate shows enhancement bbb breakage rt well documented mechanism unclear vitro study showed rt induces early transient increase paracellular permeability maintaining tight junction capillary endothelial cells6 an experimental study revealed higher exposures tmz brain tumor also attribute breakdown bbb20 increased permeability blood tumor barrier caused rt tmz would result contrast enhancement due leakage presents pspd rano criteria suggested overcome limitation macdonald criteria drew considerable attentions although provides innovative assessment treatment malignant glioma yet several limitations pointed discriminating true false lesions non enhancing lesions simple tumor size measurement reflecting new imaging techniques14 despite current criteria macdonald rano purely objective judgement treatment response imaging increased cbv cbf decreased adc value imply progressive disease times however decisions made novel imaging modalities confusing cases latest imaging techniques dynamic susceptibility contrast mri perfusion diffusion weighted mri mri pet scans using biomarker thallium single photon emission ct introduced comparison prospective studies performed2,13,29 thus disparity sometimes clinical judgment oncologists radiologists makes treatment planning complex often needs discussed multidisciplinary tumor board for example one patient considered show pspd last study confirmed show true progression surgery case cbv increased tumor demonstrated stabilization 4 weeks second post treatment mri could considered pspd recently vogelbaum et al.26 suggested retrospective assessments serial images distinguish pspd form true progression finding ambiguous response called indeterminate even rano criteria this shows despite latest techniques reorganization criteria guarantee reliable diagnosis appropriate treatment plan therefore validation needed retrospective comparative analysis among various criteria performed placing emphasis nonenhancing tumor8 the major component adopted rano timing decision making well imaging criteria appropriate timing crucial avoiding misdiagnosis would lead discontinuation initiation effective therapy also canadian recommendations pspd diagnosed 12 weeks end rt unless new lesion shows field5 review sanghera et al.22 timing defining pspd varied 2 weeks 6 months among 8 studies considering decisions made 4 weeks 6 months performed studies confirmed observation 12 weeks related decreased os present study as data showed difference etpd ltpd confirmation serial observations would reasonable rather deciding early little information correlation mgmt methylation pspd association improved os reported brandes et al.1 study survival graph comparing mgmt methylated unmethylated group showed difference statistically significant may reflect increased bbb permeability administration bevacizumab misdiagnosis true progression normalizes leaky vasculature therefore may show response"25 it highly persuasive hypothesis needs examination future study present study the regression field recurrence occurred 1 month administration drug remained stable disease 11 months however impression possibilities true progression vs. treatment related change still rule late pspd case counting cases treatment effect may result falsely high response rate anti angiogenic agent perform pathologic confirmation every suspected recurrence pspd psr considered separately regarding psr conflicting views as mentioned earlier strikingly high response rate progression free survival pfs result increased os thus doubt exists regarding whether bevacizumab antitumor effect pooled analysis norden et al.18 ) statistically significant increase found pfs patients receiving bevacizumab however os demonstrated statistically significant difference context bevacizumab causes adverse effects intracranial hemorrhage thromboembolic events proteinuria7 phase ii study bevacizumab cpt-11 recurrent malignant glioma 4 32 13% ) patients thromboembolic event two died toxicity neurologic deterioration27 one possible advantage bevacizumab considered reduction corticosteroid use3 paradoxically cause dependency bevacizumab one expensive drugs additionally bevacizumab cause vegf independent mechanisms progression heavily treated patients18 furthermore glioblastoma known associated high risk thromboembolism anti coagulation would needed prevent severe complication19 however every patient planning administered bevacizumab receive prophylaxis thus positive effect survival may countervailed this life threatening toxicity overlooked costs benefits must considered one limitations current study showing high os reported data may due censored data 9 patients unavailable follow up9 recurrence suspected salvage treatment varied among patients standard chemotherapy regimen management high grade glioma although small sized retrospective nature present study suggests methodology evidence safety postponed treatment recurrent high grade glioma 12 weeks termination crt shows clinical features psr raising question true effect bevacizumab initiating treatment plan 12 weeks recommended rano lead loss overall survival additionally recurrent high grade glioma bevacizumab must administered caution consideration pspd psr however mri gadolinium enhancement alone sufficient characterize tumor response growth in addition clinical correlation adequate duration follow histopathologic validation studies regarding additional physiologic imaging modalities required accurate evaluation tumor response

objectivewe evaluated pseudoprogression ( pspd ) following radiation therapy combined with concurrent temozolomide ( tmz ) , and we assessed pseudoresponse following anti - angiogenic therapy for patients with recurrent disease using the response assessment of the neuro - oncology working group.methodspatients who were pathologically confirmed as having high - grade glioma received radiotherapy with concurrent tmz followed by adjuvant tmz . bevacizumab ( avastin ) with cpt-11 were used as a salvage option for cases of radiologic progression . magnetic resonance imaging ( mri ) was routinely performed 1 month after concurrent radiochemotherapy ( crt ) and every 3 months thereafter . for cases treated with the bevacizumab - containing regimen for progressive disease , mri was performed every 2 months.resultsof 55 patients , 21 ( 38% ) showed radiologic progression within 4 weeks after crt . of these patients , 16 ( 29% ) showed progression at second post - crt mri ( etpd ) and five ( 9% ) showed improvement ( pspd ) . seven of thirty - four initially non - progressed patients showed progression at the second post - crt mri ( ltpd ) . no difference in survival was observed between the etpd and ltpd groups ( p=0.595 ) . five ( 50% ) of ten patients showed a radiological response after salvage bevacizumab therapy . four of those patients exhibited rapid progression immediately after discontinuation of the drug ( drug holiday).conclusiontwelve weeks following treatment could be the optimal timing to determine pspd or true progression . mri with gadolinium enhancement alone is not sufficient to characterize tumor response or growth . clinical correlation with adequate follow - up duration and histopathologic validation may be helpful in discriminating pspd from true progression .

fontan procedure used separate systemic pulmonary circulations patients various forms functionally univentricular hearts fontan circulation systemic venous return sent pulmonary arteries without passage ventricle the fontan procedure result various late complications including central venous hypertension diminished oxygen delivery reduced cardiac output venous thrombosis arrhythmia 1,2 these complications caused central venous hypertension lead parenchymal injury fibrosis cirrhosis liver 2,3 cardiac cirrhosis serious late complication congenital heart disease cause hepatocellular carcinoma hcc 4,5 however prevalence risk factors cirrhotic changes hccs clearly identified furthermore non invasive diagnostic tools hepatic fibrosis management hcc patients undergoing fontan procedure yet clearly established a 29-year old woman history univentricular heart undergone fontan operation 10 years age she followed department pediatric cardiology hospital regularly underwent blood examinations without alpha fetoprotein afp 2- 3-month intervals a routine follow day experienced slight abdominal discomfort received abdominal ultrasonography us b mode conventional us showed 15 mm hypoechoic mass liver segment 4 s4 fig 1a 18 mm hyperechoic mass liver segment 2 s2 fig furthermore liver parenchyma coarsened appearance consistent cirrhosis ascites splenomegaly observed fig b mode conventional ultrasonography showed 15 mm hypoechoic mass liver segment 4 s4 liver parenchyma coarsened appearance consistent cirrhosis ascites splenomegaly observed b c).ceus using sonazoid showed homogeneously enhanced mass arterial dominant phase hypoechoic mass relative adjacent liver parenchyma portal dominant phase e contrast defect clear border postvascular phase f b mode conventional ultrasonography showed 18 mm hyperechoic mass liver segment 2 s2 ceus showed homogeneously enhanced mass arterial dominant phase b isoechoic mass relative adjacent liver parenchyma portal dominant phase c defects postvascular phase we next performed contrast enhanced us ceus using bolus injection 0.015 ml kg sonazoid perfluorobutane daiichi sankyo tokyo japan the mass lesion s4 homogeneously enhanced arterial dominant phase 10 30 seconds fig the lesion became progressively hypoechoic relative adjacent liver parenchyma portal dominant phase 30 120 seconds fig 1e provided contrast defect clear border postvascular phase 10 minutes later fig the mass lesion s2 homogeneously enhanced arterial dominant phase fig the lesion became isoechoic mass relative adjacent liver parenchyma portal dominant phase fig 2c provided defects postvascular phase 10 minutes later fig we subsequently performed magnetic resonance imaging mri conventional t1-and t2-weighted imaging wi contrast media administration including diffusion imaging the contrast media used hepatocyte specific primovist gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid gd eob dtpa bayer schering pharma berlin germany there radiographic evidence liver cirrhosis portal hypertension including nodular surface coarse texture ascites splenomegaly mri fig there radiographic evidence liver cirrhosis portal hypertension including nodular surface coarse texture ascites splenomegaly mri b gd eob dtpa mri showed 15 mm s4 mass moderately low intensity t1-wi c moderately high intensity t2-wi moderately high intensity diffusion e homogeneous arterial enhancement b f complete washout hepatobiliary phase g this imaging revealed mass lesion s4 moderately low intensity t1-wi fig 3b f washout portal phase 70 seconds interstitial phase 180 seconds 20 minutes hepatobiliary phase contrast uptake lesion showed washout fig in addition mri imaging revealed mass lesion s2 moderately high intensity t1-wi fig 4a e isoenhancement relative adjacent liver parenchyma portal interstitial phases high intensity hepatobiliary phase fig gd eob dtpa mri showed 18 mm s2 mass moderately high intensity t1-wi b moderately low intensity t2-wi c moderately low intensity diffusion homogeneous arterial enhancement e high intensity hepatobiliary phase f her liver function well preserved child pugh classification international normalized ratio low patient warfarin atrial arrhythmia other lab findings included high afp level normal less 40 nanograms milliliter ng ml high lens culinaris agglutinin reactive fraction afp afp l3 normal less 10% low platelet count normal greater 150,000/microliter high hyaluronic acid normal less 50 ng ml high type iv collagen 7s normal less 6.0 ng ml the patient significant medical problems risk factors cirrhosis expressed negativity viral autoimmune markers alcoholic consumption hepatotoxic drugs amiodarone metabolic factors diabetes mellitus obesity body mass index 17.7 fatty liver us following reversal warfarin therapy ultrasound guided needle biopsy s2 lesion performed histological findings revealed bridging fibrosis liver section parenchymal cells display cellular structural atypia fig wbc white blood cells rbc red blood cells hb hemoglobin hct hematocrit plt platelets pt prothrombin time inr international normalized ratio tp total protein alb albumin che cholinesterase t.bil total bilirubin d.bil direct bilirubin ast aspartate aminotransferase alt alanine aminotransferase ldh lactate dehydrogenase alp alkaline phosphatase -gtp -glutamyltranspeptidase ztt zinc sulfate turbidity test ttt thymol turbidity test bun blood urea nitrogen cr creatinine crp c reactive protein icg r indocyanine green retention hbsag hepatitis b surface antigen hbsab hepatitis b surface antibody hbcab hepatitis b core antibody hcv hepatitis c virus antibody ana antinuclear antibodies ama m2 anti mitochondrial m2 antibody afp alpha fetoprotein afp l3 lens culinaris agglutinin reactive fraction afp cea carcinoembryonic antigen ca carbohydrate antigen histological specimen obtained ultrasound guided needle biopsy s2 lesion the parenchymal cells display cellular structural atypia hematoxylin eosin staining silver impregnation b low power field the patient underwent curative operation involving partial hepatectomy s4 liver macroscopically sections specimen revealed yellowish white encapsulated solid tumor measuring 15 mm size fig 6a macroscopic appearance resected s4 specimen revealed yellowish white encapsulated solid tumor measuring 15 mm size microscopically growth pattern s4 liver tumor showed expansive growth extracapsular invasions b hematoxylin eosin h&e staining low power field tumor cells increased nuclear cytoplasmic ratio polymorphic chromatin rich nuclei pathological diagnosis poorly differentiated hcc c h&e staining high power field the growth pattern liver tumor showed expansive growth extracapsular invasions fig the tumor cells increased nuclear cytoplasmic ratio polymorphic chromatin rich nuclei pathological diagnosis poorly differentiated hcc fig the non cancerous area resected specimen revealed bridging fibrosis observed without fat deposition patient diagnosed liver cirrhosis fig fibrosis observed portal pericellular areas sinusoidal structure maintained signicant inflammation observed fig the noncancerous area resected specimen revealed bridging fibrosis without fat deposition patient diagnosed liver cirrhosis silver impregnation fibrosis observed portal pericellular areas sinusoidal structure maintained signicant inammation seen silver impregnation b c high power field after discharge hospital patient followed 3-month intervals 1-year follow afp level 2.7 ng ml afp l3 less 0.5% tumor markers remained normal in present case known etiological factors hepatitis viral infection alcoholic liver disease autoimmune liver diseases autoimmune hepatitis primary biliary cirrhosis metabolic liver diseases non alcoholic steatohepatitis nash medication hepatotoxic drugs amiodarone moreover significant hepatic inflammation fat deposition seen result processes hepatitis viral infection nash on histology present case hepatic sinusoidal fibrosis extending centrilobular areas toward portal tract without inflammation observed schwarz et al discussed hepatic fibrosis fontan procedure mixed disease affects portal centrilobular areas liver biopsy autopsy specimens 6,7 sinusoidal fibrosis believed result increase central venous pressure transmitted hepatic cells surround central veins extent cirrhosis strongly correlated elevated hepatic venous pressures low cardiac index patients undergoing fontan procedure 8) after fontan palliation significant liver disease result central venous congestion hypoxia resulting low cardiac output however little known regarding fibrogenic mechanisms independent inflammation mediated pathway congestive liver disease cld cld mechanotransduction associated stretching compression hepatic stellate cells may potent inducer hepatic fibrosis 9,10 using newly characterized murine cld model sinusoidal thrombosis mechanical stretching adjacent hepatic stellate cells caused sinusoidal dilatation was shown induce release fibronectin hepatic stellate cells fibrin stretching stimulated fibronectin fibril assembly 1-integrin actin dependent mechanism 11 in addition hepatic complications fontan procedure associated duration follow 8,12 progression cirrhosis may even observed within 10 years initial fontan procedure 13 34 patients median follow 11.5 years fontan procedure 30% experienced abnormal transaminases 61% abnormal -gtp 32% abnormal bilirubin 58% coagulopathy 14 as compared duration 0 5 years odds ratio hepatic complications 4.4 post fontan duration 11 15 years 9.0 duration 16 20 years respectively 12 liver cirrhosis potential prerequisite hcc however prevalence progression cirrhotic changes fontan population clearly identified non invasive diagnostic tools hepatic fibrosis needed liver biopsy golden standard diagnosing liver cirrhosis difficult perform fontan patients due prophylactic anticoagulation similar present case radiological assessment liver fibrosis using various methods us ct mri may useful as shown table 2 recent pubmed search identified 12 cases hccs among published reports the publications described use surgical resection transcatheter arterial chemoembolization radioembolization local ablation sorafenib therapy 15 22 in previous reports two patients treated surgical resection 20,21 early stage hcc surgical resection provides curative treatment long time survival however hepatectomy rarely performed following fontan procedure difficult detect early stage hccs although present patient receive periodic surveillance hcc us afp fortunate early stage hcc incidentally detected hcc detected onset symptoms poor prognosis 5-year survival rate 0 10% in contrast early stage hccs detected surveillance cured surgical resection liver transplantation 5-year disease free survival rate 50% 23 f female male nd described tace transarterial chemoembolization tae transarterial embolization thus surveillance hcc may necessary patients cld undergo fontan procedure cirrhosis high risk factor hcc however screening interval liver disease fontan procedure yet established surveillance based ultrasound examination recommended screening interval 6 months according american association study liver diseases aasld practice guidelines management hcc 24 ( 25 patients high risk risk factors hcc advised undergo periodic surveillance us laboratory work ups including afp protein induced vitamin k absence antagonists ii pivka ii every 6 months however similar present case pivka ii useful patients fontan procedure prophylactic antiplatelet anticoagulation therapies warfarin administration necessary prevent thromboembolic events one major causes morbidity mortality 26 following fontan procedure patients face risk hcc require lifelong follow pediatric cardiologist also hepatologist experienced care patients liver cirrhosis we herein described patient hcc able safely undergo liver resection following fontan procedure preserved cardiac hepatic function

a 29-year - old woman who underwent the fontan procedure at 10 years of age had an incidental finding of liver masses on abdominal ultrasonography . subsequent gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid magnetic resonance imaging showed a 15 mm hypervascular mass with washout in the hepatobiliary phase in liver segment 4 ( s4 ) , and an 18 mm hypervascular mass without washout in the hepatobiliary phase in liver segment 2 ( s2 ) . the s2 liver mass was pathologically diagnosed to be a regenerative nodule by an ultrasound - guided needle biopsy , and the s4 liver mass was pathologically diagnosed as a poorly differentiated hepatocellular carcinoma after partial hepatectomy .

case describes young non pregnant cat presented uterine prolapse association unusual diffuse polypoid fibrosing perimetritis parametritis no intra abdominal complications seen ultrasound examination 3 months postsurgery time writing uterine prolapse cat relatively rare usually associated periparturient period inflammatory polypoid perimetritis parametritis previously documented cats dogs reported association administration oestrogenic compounds the polypoid inflammation affecting uterus parametrium may contributed increased laxity uterine ligaments predisposed development uterine prolapse uterine prolapse rare queen usually occurring delivery kittens abortion we report uterine prolapse non pregnant queen association unusual diffuse polypoid fibrosis perimetrium parametrium the perimetrium syn uterine tunica serosa equivalent visceral peritoneal lining it composed loose connective tissue containing nerves smooth muscle cells covered mesothelium it blends broad ligaments uterus also covered mesothelium contains core connective tissue parametrium while peritonitis cats frequently associated feline infectious peritonitis specific lesions unique perimetrium documented a stray 6- 7-month old domestic shorthair queen weighing 2.15 kg presented university veterinary clinic uterine prolapse observed several days prior capture examination uterine horns prolapsed everted swollen regions hyperaemia particularly towards tips uterine horns no evidence placental zoning tissue necrosis particular gangrene noted figure 1 prolapsed everted uterus following lavage revealed swollen hyperaemic viable tissue clinical assessment revealed normal appetite demeanour the vital parameters within normal limits temperature 37.8c respiratory rate 40 breaths min heart rate 140 beats min the cat received hydromorphone hydrochloride injection hydromorphone west ward pharmaceutical 0.1 mg kg iv q4h pain management facilitate examination initial haematological blood biochemical evaluations revealed markedly regenerative anaemia packed cell volume 24% reference interval ri 2545% erythrocytes 4.64 10/l ri 510 10/l reticulocytes 375.84 10 /l ri 040 10/l neutrophilic leukocytosis mild toxic changes leukocytes 37.72 10/l ri 5.519.5 10/l neutrophils 25.3 10/l ri 2.512.5 10/l blood biochemistry showed mild hyperkalaemia 6.0 mmol l ri 3.75.8 mmol l mild hypoglycaemia 66 mg dl ri 70150 mg dl oestrogen plasma concentrations retrospectively evaluated 25.7 pg ml ri 1540 pg ml intravenous crystalloid fluid solution plasmalyte travenol laboratories administered correct estimated 8% dehydration deficit maintenance requirement 12 h. antibiotic therapy consisting intravenous cefazolin sodium cefazolin injection west ward pharmaceutical dose rate 22 mg kg iv q8h also administered attempts reducing uterus unsuccessful internal ovariectomy external hysterectomy performed the ovarian pedicles transfixed ligated prior transection manipulating uterine body the everted uterus circumferentially ligated distal cervix approximately 5 cm away vulva transected the remaining uterus returned abdominal cavity within abdominal cavity the serosa uterine body noted diffusely thickened multiple coalescing white villous projections figure 2 formalin fixed perimetrial surface uterine body revealing diffuse coalescing polypoid lesions small amount mildly flocculent abdominal fluid present the cat recovered uneventfully follow examinations 6 month period including ultrasound 3 months postsurgery detected abdominal abnormalities microscopic examination uterine body confirmed presence multiple nodular perimetrial proliferations moderately loose connective tissue forming irregular polyps plaques blending myometrium figures 3 4 most polyps contained multifocal randomly distributed lymphocyte plasma cell infiltrations regions neovascularisation polyps covered combination normal attenuated moderately swollen mesothelial cells sites mesothelial cell loss the stratum vascularis moderately oedematous containing congested medium large size blood vessels dilated lymphatics endometrium uterine epithelium appeared unremarkable low glandular content intact mucosal epithelium minimal inflammatory cell infiltrations the lesion characterised diffuse chronic polypoid fibrotic perimetritis parametritis cross section uterine body u revealing multiple polypoid perimetrial projections p haematoxylin eosin polypoid structures blending myometrium containing foci angiogenesis b regions active fibroplasia haematoxylin eosin ) uterine prolapse rarely documented cats individual case reports citing complete unilateral uterine prolapse without uterine mucosal eversion the majority occurred association periparturient period one proposed associated oestrus furthermore novel lesion inducing extensive perimetrial fibrosis previously reported cats noted could potentially implicated cause however factors suggested including uterine atony excessive abdominal contractions including tenesmus uterus prolapse there requirement laxity stretching rupture uterine ligaments patency cervix laxity uterine ligaments occur due repeated pregnancy changes progesterone oestrogen relaxin concentrations near parturition cervical patency occurs two time points oestrus without ovulation parturition based absence newborn animals no mammary development histopathological features suggest recent pregnancy placental zoning recent uterine haemorrhage inflammation endometrial regeneration unlikely animal advanced pregnancy aborted it possible however establish animal recently oestrus oestrogen levels within normal limits vary significantly short periods time within oestrus cycle uterus large this latter feature could potentially contributed uterine prolapse least two ways namely inducing ligament laxity and/or acting caudal abdominal irritant inducing abdominal contractions tenesmus active lesion extent mature fibrous tissue would suggest presence lesion advance uterine prolapse perimetrial inflammation co exist part peritonitis cats peritonitis frequently associated feline infectious peritonitis primary bacterial septic peritonitis bacteroides species fusobacterium species mainly isolated however lesions manifest polypoid fibrous proliferations similar proliferative fibrotic lesions focused perimetrium described dogs associated experimental stilboestrol synthetic oestrogen administration perimetrial papillae consisting proliferations mesothelial cells subjacent connective tissue documented ovariectomised dogs administered stilboestrol 150350 days similar present case lesions confined genital organ serosal surfaces including broad ligament such perimetrial lesions also noted entire female dogs administered synthetic oestrogens association lesions including oestrogen induced renal fibrosis proposed mechanisms regional fibrous proliferations suggested involvement stromal oestrogen receptors cat develops oestrogen associated pathology similar perimetrial pathology documented association oestrogen the ovaries submitted histopathological evaluation eliminate ovarian pathology definitively cystic follicular disease ovarian neoplasia potential source hyperoestrogenism cats tropics likely cycle continuously recognised unmated cats may appear persistent oestrous owing relatively short interoestrous period thus remain physiological hyperoestrogenic state also unlikely cat access exogenous oestrogenic compounds humans generalised intra abdominal fibrosing lesions peritoneal surfaces intra abdominal fibromatosis described mesenteric retroperitoneal pelvic locations sclerosing peritonitis noted surgery following long term continuous peritoneal dialysis also side effect utilising beta blockers the pathogenesis lesions thought result delayed healing response due loss mesothelial cells continued serosal irritation however appearance described polypoid ranges spectrum gross changes appearance visceral peritoneum opacification serosa disorganised collagen fibres stromal expansion tanned peritoneum hyalinised collagen fibrin vessel sclerosis progress stage viscera palpably stiffened mural fibrosis the unique perimetritis parametritis observed case highlights additional potential cause uterine prolapse warrants cases uterine prolapse non pregnant animals perimetrial parametrial tissue evaluated varying degrees inflammation

case summarythis case describes a young non - pregnant cat that presented with uterine prolapse in association with an unusual diffuse , polypoid , fibrosing perimetritis and parametritis . following ovariohysterectomy the cat recovered fully . no intra - abdominal complications were seen on ultrasound examination 3 months postsurgery . at the time of writing , the cat remains healthy.relevance and novel informationuterine prolapse in the cat is relatively rare and usually associated with the periparturient period . inflammatory polypoid perimetritis and parametritis have not previously been documented in cats , and in dogs have only been reported in association with the administration of oestrogenic compounds . the polypoid inflammation affecting the uterus and parametrium may have contributed to increased laxity of the uterine ligaments and predisposed to the development of uterine prolapse .

previous studies showed a. baumannii already emerging pathogen respiratory tract wounds blood urine 3 since strains a. baumannii usually resistant available drugs except colistin tigecycline would sticky detected patient therefore urgent need study epidemiology resistance surveillance control measures a. baumannii hospital infections warned 4 acinetobacter baumannii could found diverse sources food water soils acinetobacter baumannii strains able survive long time even dry surface could toughly attach human epithelial cells fimbriae side chains 5 these characteristics indicate a. baumannii could easily spread via surface air possible pathways moreover many studies showed certain section hospital one type a. baumannii 1 6 the sources harmful a. baumannii may found analyzing molecular epidemiology strain time however enough studies subject hand although physicians surgeons reported threat a. baumannii raised problems internal medicine icu the differences may caused selective pressure indiscriminate use broad spectrum antibiotics different clinical departments 7 analysis may lead new explanation formation spread carbapenem resistant acinetobacter baumannii crab carbapenem susceptible acinetobacter baumannii csab 8) however researches compared differences distribution different departments the current study aimed analyze distribution certain types a. baumannii clinical departments authors tried figure roles antibiotic resistant genes therapies diseases different departments spread crab csab eighty one patients three wards icu department respiration medicine department burn plastic surgery studied the current study approved medical ethics committee zhejiang university shaoxing china all strains identified vitek32 automatic bacteria identification system corporation us the bacteria cultured mueller hinton mh agar medium oxoid uk lb broth medium shenggong china 2 cultured isolations collected solved boiling double distilled water 10 minutes centrifuged 12000 rpm supernatant collected used template mlst reaction the housekeeping genes ab glta gyrb gdhb gpi rpod selected standard template 9 10 the sequences primers designed according previous studies 3 11 table 2 all primers designed jinsi rui corporation china induced rt pcr kits drr063a takara japan polymerase chain reaction pcr ) was performed follows initial denaturation 95c 5 minutes denaturation 95c 1 minute annealing 56c 30 seconds extension 72c 45 seconds after pcr reaction products gathered purified pcr purification kit axygen ap pcr-250 silicon us the test results used identify strains collected clinical specimens referring ab mlst database 7 9 the evaluation antibiotic resistance induced kirby bauer test k b 10 a disk impregnated antibiotic compounds placed plate concentrations compound highest next disk decreased distance disk increased once compound effective bacteria certain concentration colonies would grow point concentration agar greater effective concentration i. e. inhibition zone pseudomonas aeruginosa atcc27853 escherichia coli atcc25922 used quality control isolates 5 the clinical laboratory standards institute clsi standard selected standard evaluation the drug resistance ability evaluated studying minimum inhibitory concentration mic agar dilution method the antibiotics used study follows imipenem imp aztreonam atm cefepime fep cefoperazone cfp cefotaxime ctx piperacillin pip amikacin amk ciprofloxacin cip 7 the target genes included methicillinase oxa-23 oxa-24 oxa-51 carbapenemase sim imp spm type integrase intl 1 sulfonamides resistance gene qace1-sul1 abbreviations f forward primer r reverse primer data analyzed spss software version17.0 chi square test used compare enumeration data different groups student test measurement data one way analysis variance used analyze dependency drug resistance gene expression eighty one patients three wards icu department respiration medicine department burn plastic surgery studied the current study approved medical ethics committee zhejiang university shaoxing china all strains identified vitek32 automatic bacteria identification system corporation us the bacteria cultured mueller hinton mh agar medium oxoid uk lb broth medium shenggong china 2 cultured isolations collected solved boiling double distilled water 10 minutes centrifuged 12000 rpm supernatant collected used template mlst reaction the housekeeping genes ab glta gyrb gdhb gpi rpod selected standard template 9 10 the sequences primers designed according previous studies 3 11 table 2 all primers designed jinsi rui corporation china induced rt pcr kits drr063a takara japan polymerase chain reaction pcr ) was performed follows initial denaturation 95c 5 minutes denaturation 95c 1 minute annealing 56c 30 seconds extension 72c 45 seconds after pcr reaction products gathered purified pcr purification kit axygen ap pcr-250 silicon us the test results used identify strains collected clinical specimens referring ab mlst database 7 9 the evaluation antibiotic resistance induced kirby bauer test k b 10 a disk impregnated antibiotic compounds placed plate concentrations compound highest next disk decreased distance disk increased once compound effective bacteria certain concentration colonies would grow point concentration agar greater effective concentration i. e. inhibition zone pseudomonas aeruginosa atcc27853 escherichia coli atcc25922 used quality control isolates 5 the clinical laboratory standards institute clsi standard selected standard evaluation the drug resistance ability evaluated studying minimum inhibitory concentration mic agar dilution method the antibiotics used study follows imipenem imp aztreonam atm cefepime fep cefoperazone cfp cefotaxime ctx piperacillin pip amikacin amk ciprofloxacin cip 7 the target genes included methicillinase oxa-23 oxa-24 oxa-51 carbapenemase sim imp spm type integrase intl 1 sulfonamides resistance gene qace1-sul1 data analyzed spss software version17.0 chi square test used compare enumeration data different groups student test measurement data one way analysis variance used analyze dependency drug resistance gene expression results level antibiotic resistance isolates shown table 4 all isolates collected hospital study certain level resistance kinds antibiotics however atm imp still relatively effective antibiotics order evaluate properties isolates group isolates resistant four types antibiotics group ii isolates resistant three four types antibiotics group iii isolates resistant less three types antibiotics it identified among isolates ones isolated blood highest level drug resistance besides among three departments species isolated icu highest level drug resistance table 5 present distribution isolates different clinical departments clinical specimens abbreviations amk amikacin atm aztreonam cfp cefoperazone cip ciprofloxacin ctx cefotaxime fep cefepime mic minimum inhibitory concentration pip piperacillin mic50 minimum inhibitory concentration required inhibit growth 50% organisms mic90 minimum inhibitory concentration required inhibit growth 90% organisms abbreviations bur pla department burn plastic surgery icu intensive care unit res department respiratory medicine percentage refers rate total number example 39 81.25 means 39 cases 48 39/48 81.25% groups groups ii iii refer group classified study group according mlst analysis two types ab strains sorted identified clinical samples st208 st218 the detailed distribution two strains clinical departments specimens listed table 6 the st208 isolates distributed mainly icu respiration medicine st218 burn plastic surgery department no significant correlation found types sources clinical samples p value 0.05 but departments strains came seemed significantly related p value 0.05 according results k p drug sensitive test strains divided two groups carbapenem resistant carbapenem resistant ab crab carbapenem sensitive ab csab 70.21% st208 69.21% st218 included crab strains 15.22% st208 84.78% st218 csab st208 strains isolated icu respiration medicine department mainly crab st218 strains isolated burn plastic surgery departments mainly csab abbreviations bur pla department burn plastic surgery icu department intensive care unit res department respiration medicine number refers number species isolated certain department sample percentage refers rate total number example 40 83.3 means 40 cases 48 40/48 83.3% number group significant difference compared groups p value 0.05 pcr tests revealed prevalence resistance inducing genes follows oxa-23 81.3% oxa-51 61.3% sim 18.8% oxa-24 5.3% imp 12.3% intl1 57.9% qace1-sul1 61.2% results current study showed st208/crab oxa-23 51 main molecular phenotype crab strains hospital drug resistance property isolates related expression oxa-23 51 p value 0.05 meanwhile st208 chances expressions oxa-23 51 compared st218 p value 0.05 abbreviations crab carbapenem resistant ab csab carbapenem susceptible ab there significant differences group crab compared csab p value 0.05 data presented number isolates express gene per number stains results level antibiotic resistance isolates shown table 4 all isolates collected hospital study certain level resistance kinds antibiotics however atm imp still relatively effective antibiotics order evaluate properties isolates group isolates resistant four types antibiotics group ii isolates resistant three four types antibiotics group iii isolates resistant less three types antibiotics it identified among isolates ones isolated blood highest level drug resistance besides among three departments species isolated icu highest level drug resistance table 5 present distribution isolates different clinical departments clinical specimens abbreviations amk amikacin atm aztreonam cfp cefoperazone cip ciprofloxacin ctx cefotaxime fep cefepime mic minimum inhibitory concentration pip piperacillin mic50 minimum inhibitory concentration required inhibit growth 50% organisms mic90 minimum inhibitory concentration required inhibit growth 90% organisms abbreviations bur pla department burn plastic surgery icu intensive care unit res department respiratory medicine percentage refers rate total number example 39 81.25 means 39 cases 48 39/48 81.25% groups groups ii iii refer group classified study group according mlst analysis two types ab strains sorted identified clinical samples st208 st218 the detailed distribution two strains clinical departments specimens listed table 6 the st208 isolates distributed mainly icu respiration medicine st218 burn plastic surgery department no significant correlation found types sources clinical samples p value 0.05 but departments strains came seemed significantly related p value 0.05 according results k p drug sensitive test strains divided two groups carbapenem resistant carbapenem resistant ab crab carbapenem sensitive ab csab 70.21% st208 69.21% st218 included crab strains 15.22% st208 84.78% st218 csab st208 strains isolated icu respiration medicine department mainly crab st218 strains isolated burn plastic surgery departments mainly csab abbreviations bur pla department burn plastic surgery icu department intensive care unit res department respiration medicine number refers number species isolated certain department sample percentage refers rate total number example 40 83.3 means 40 cases 48 40/48 83.3% number group significant difference compared groups p value 0.05 pcr tests revealed prevalence resistance inducing genes follows oxa-23 81.3% oxa-51 61.3% sim 18.8% oxa-24 5.3% imp 12.3% intl1 57.9% qace1-sul1 61.2% results current study showed st208/crab oxa-23 51 main molecular phenotype crab strains hospital drug resistance property isolates related expression oxa-23 51 p value 0.05 meanwhile st208 chances expressions oxa-23 51 compared st218 p value 0.05 abbreviations crab carbapenem resistant ab csab carbapenem susceptible ab there significant differences group crab compared csab p value 0.05 data presented number isolates express gene per number stains acinetobacter baumannii widely distributed environment human body surfaces 12 studies show growing numbers carbapenem resistant pathogenic bacteria crab appearing the prevention treatment crab urgent issue control nosocomial infections 13 15 current study all strains a. baumannii resistant antibiotics different degrees table 4 in fact common phenomenon hospitals china 9 moreover found distribution crab isolates related wards came table 5 isolates icu department respiratory medicine higher levels antibiotic resistance strains surgery department it may due long term use antibiotics former wards 16 18 this finding showed distribution crab may affected therapeutic methods besides authors studied types isolates air hands doctors nurses wards it found icu st208 existed department burn plastic surgery st218 detected considering fact two departments physically separated speculated two different isolates may different origins separated spatial distance epidemic section currently techniques molecular cloning detection widely used study types certain isolates 3 10 13 current study new technique mlst used mlst classified strains according results accurate detection sequences nucleic acid isolates 19 two isolates st208 st218 found hospital one territory this finding showed certain kind bacteria would dominant certain section the existence anti carbapenemase enzymes key factor prevalence multidrug resistant bacteria 20 22 previous reports showed five types anti carbapenemase enzymes oxa-23 51 sim imp spm could detected crab 23 the current study found oxa-23 51 prevalent genes hospital since antibiotic resistance inducing genes expressed plasmid distribution determined gene mutation combined effect antibiotics used certain place thus final results distribution natural selection certain therapeutic milieu hospital identification types genes isolates certain ward would important restrict prevalence moreover although many microbiologists believe nuclear dna related plasmid dna bacteria current study findings indicated st208 chances existence oxa-23 51 table 8) fact revealed might certain connections nuclear gene types appearance plasmid genes may different isolates ab different wards since departments physically separated the levels antibiotic resistance also quite different isolates different locations human body species isolates blood culture highest level antibiotic resistance st208 st218 epidemic isolates ab abilities drug resistance due existence oxa-23 oxa-51 intl 1 qace1-sul 1 hospital managers take measures supervise epidemiology types crab isolates the study molecular epidemiology isolates different wards specimens significantly important besides studies connections plasmid gene expression types isolates required

background : acinetobacter baumannii is the most prevalent strain in hospitals and different clinical departments.objectives:the current study aimed to investigate the genetic characteristics and resistance mechanisms of a. baumannii isolated from clinical samples in shaoxing people s hospital affiliated to zhejiang university , shaoxing , china.patients and methods : acinetobacter baumannii strains were isolated from blood , phlegm and skin of the patients hospitalized in different departments as respiratory medicine , plastic surgery and intensive care unit ( icu ) . multilocus sequence typing ( mlst ) was used to characterize the isolates . kirby - bauer test was used to evaluate antibiotic resistance of the bacteria . the expression of resistance inducing genes was detected by reverse transcription polymerase chain reaction ( rt - pcr ) . the results were analyzed and compared.results:two bacterial types , st208 , and st218 , were identified in all 140 samples . the st208 mainly came from icu and department of respiratory medicine , while st218 from department of plastic surgery ; 70.21% of st208 and 84.78% of st218 were carbapenem - resistant acinetobacter baumannii ( crab ) and carbapenem - susceptible acinetobacter baumannii ( csab ) , respectively . multidrug - resistance genes in crab isolated from the hospital mainly included , oxa-23 , oxa-5 , intl 1 and qace1-sul 1 . besides , the highest and lowest antibiotic resistance was observed in the strains isolated from blood samples and wounds , respectively.conclusions:the distribution of ab varies in different clinical departments and samples . in the hospital under study , the main types of ab were st208 and st218 . the genes which affect the ability of antibiotic - resistance were oxa-23 , oxa-51 , intl 1 and qace1-sul 1 .

high phosphorus p intake known one risk factors impaired bone health several researchers investigated adverse effects high p diet bone metabolism human adults a diet containing p additives increases urinary hydroxyproline excretion bone resorption marker growing male rats elevation parathyroid hormone pth secretion considered one mechanisms high p diet impairs bone metabolism pth stimulates osteoblasts produce mediators osteoclastic bone resorption macrophage colony stimulating factor csf interleukin-6 il-6 receptor activator nf-b ligand rankl 46 we previously reported high p diet increased rankl mrna expression osteoclast number rats it could therefore deduced high p diet induced elevated pth secretion leads increase rankl expression enhances osteoclastic bone resorption overton basu suggested bone loss occurs increasing age rate approximately 1% per year averaged age range 2976 previous mouse study ferguson et al bone mass mechanical properties approached mature levels 12 weeks age age related osteopenia observed 42 weeks age we previously reported effects high p diet mechanical properties femur 4- 12- 24- 80-week old mice the results showed high p diet decreased breaking force femur 80-week old mice stiffness femur 24- 80-week old mice we also found high p diet increased serum pth concentration 12- 24- 80-week old mice 80-week old mice higher serum pth concentration mice ages therefore thought high p diet strongly influences aged mice terms pth response the purpose study clarify mechanism high p diet affects bone metabolism older mice we assessed changes bone metabolism young aged mice fed high p diet measuring mrna expression bone metabolism mediators using real time polymerase chain reaction pcr the mice maintained accordance university guidelines care use laboratory animals the experimental diets based ain-93 g diet table 1 control diet containing 0.3% p high p diet containing 1.2% twenty four 10-week old ddy male mice purchased slc shizuoka japan housed individually stainless cages room maintained 22c 12-hour light dark cycle half mice fed commercial diet ce-2 clea japan tokyo japan 78 weeks age after acclimatization period 12 young 12-week old 12 aged 80-week old mice randomly divided two experimental groups fed control diet high p diet 4 weeks given free access diets distilled water their urine samples collected 5 days prior euthanasia analyses end experimental period mice euthanized anesthesia blood bone duodenum samples collected analyses the serum ca level analyzed atomic absorption spectrophotometry hitachi a-2000 hitachi tokyo japan according method gimblet et al the serum p level analyzed colorimetry using phospha c test wako wako pure chemical industries osaka japan serum pth concentration measured using mouse intact pth enzyme linked immunosorbent assay elisa kit alpco nh usa serum intact osteocalcin oc concentration measured using mouse osteocalcin eia kit biomedical technologies usa urinary c terminal telopeptide type collagen ctx level measured using ratlaps eia kit immunodiagnostic systems boldon uk urinary creatinine level measured using jaffe reaction described lustgarten wenk total rna isolated homogenized femurs duodenum using trizol reagent life technologies ca usa according manufacturer specifications the amount purity rna assessed using nanodrop 2000c thermo fisher scientific usa complementary dna cdna synthesized using high capacity rna cdna kit applied biosystems ca usa real time pcr reaction mixture prepared using taqman gene expression master mix applied biosystems taqman gene expression assays applied biosystems mouse pth receptor assay mm00441046_m1 mouse rankl assay mm00441906_m1 mouse osteoprotegerin opg assay mm01205928_m1 mouse tartrate resistant acid phosphatase trap assay mm00475698_m1 mouse runt related transcription factor 2 runx2 assay mm00501580_m1 mouse osterix assay mm04209856_m1 mouse alkaline phosphatase alp assay mm00475834_m1 mouse osteopontin opn assay mm00436767_m1 mouse oc assay mm03413826_mh mouse type collagen col1a1 assay mm00801666_g1 mouse transient receptor potential vanilloid type 6 trpv6 assay mm00499069_m1 mouse calbindin d9k assay mm00486654_m1 mouse plasma membrane ca atpase 1b pmca1b assay mm01245805_m1 mouse glyceraldehyde-3-phosphate dehydrogenase gapdh assay mm99999915_g1 real time pcr performed using stepone real time pcr system applied biosystems the value young mice fed control diet considered 1.00 after two way analysis variance anova fisher protected least significant difference plsd test used determine significant differences among groups in young aged mice significant difference initial body weight mice fed control high p diets table 2 the initial body weight aged mice significantly higher young mice there significant differences serum ca p concentrations among groups table 2 young aged mice although significant difference serum pth concentration young aged mice fed control diet serum pth concentration significantly higher aged mice young mice fed high p diet young aged mice the high p diet significantly increased serum oc concentration table 2 mice fed both the control high p diets serum oc concentration significantly lower aged mice young mice young aged mice although significant difference urinary excretion ctx young aged mice fed control diet urinary excretion ctx significantly lower aged mice young mice fed high p diet young aged mice high p diet significantly increased mrna expression pth receptor rankl trap runx2 osterix alp opn oc col1a1 figure 1 compared control diet mice fed control high p diets mrna expression pth receptor rankl trap runx2 osterix alp opn oc col1a1 significantly lower aged mice young mice the high p diet significantly increased rankl opg ratio aged mice young mice young aged mice high p diet significantly increased mrna expression trpv6 cabp9k pmca1b figure 2 compared control diet mice fed control high p diets mrna expression trpv6 cabp9k significantly lower aged mice young mice in humans bone loss occurs increasing age rate approximately 1% per year averaged ages 2976 years therefore aging one risk factors bone loss previous mouse study bone mass mechanical properties shown approach mature levels 12 weeks age age related osteopenia observed 42 weeks study investigated bone metabolism measuring markers bone formation resorption serum oc concentration urinary ctx excretion serum oc concentration significantly lower aged mice young mice fed control diet whereas difference urinary excretion ctx young aged mice fed control diet these results showed aged mice present decrease bone formation appears balance bone formation resorption may disrupted aged mice bone formation mediated osteoblasts using gene deficient mouse models runx2 osterix were shown essential transcription factors osteoblast differentiation bone formation 15 16 study runx2 osterix mrna expression significantly lower aged mice young mice these results suggest aging leads reduction osteoblast differentiation runx2 osterix mrna expression changes associated decrease bone formation aged mice consequently decreases mrna expression alp bone matrix proteins opn oc col1a1 also occurred aged mice ikeda et al showed mrna expression opn oc col1a1 decreased cortical trabecular bones aged rats compared young animals in addition cao et al reported alp col1a1 expression declined aged mice compared young mice thus aging results decrease bone formation declined osteoblast related gene expression regard bone resorption study showed rankl trap mrna expression decreased aged mice compared young mice despite unchanging serum pth concentration since pth stimulates rankl result rankl mrna expression seems contradict serum pth concentration however pth receptor mrna expression decreased aged mice compared young mice study this result suggested pth action suppressed decreased rankl mrna expression aged mice though urinary excretion ctx unchanged expression bone resorption rerated genes decreased aged mice compared young mice however generally known age related increase serum pth contributes increase bone resorption therefore results serum pth concentration bone resorption young aged mice study contradictory further studies increase number mice per group needed address discrepancies a decline intestinal ca absorption also one causes age related bone loss the transcellular ca pathway affected 1,25-dihydroxyvitamin 1,25(oh)2d proposed involve ca entry via trpv6 intracellular diffusion ca calbindin d9k basolateral extrusion ca pmca1b study trpv6 calbindin d9k mrna expression duodenum decreased aged mice compared young mice demonstrated plasma 1,25(oh)2d duodenal calbindin protein ca absorption decreased age rats study therefore decrease serum 1,25(oh)2d might reflect results trpv6 calbindin d9k mrna expression many studies reported high p intake induces increase serum pth concentration humans 1 23 animals 2 3 24 study the high p diet increased serum pth concentration young aged mice greater aged mice young mice fed high p diet similar previous study results suggest response high p diet terms pth secretion might different greater aging kidney function decreases age declining kidney function causes increase pth secretion therefore combination aging high p diet might one reasons higher serum pth concentration observed aged mice fed high p diet we previously reported high p diet increased rankl mrna expression bone resorption growing rats therefore suggested elevated pth secretion induced high p diet led increase rankl expression increased bone resorption study the high p diet significantly increased urinary excretion ctx young aged mice regarding mrna expression bone resorption related molecules femora high p diet significantly increased rankl trap mrna expressions young aged mice rankl actions inhibited opg acts decoy receptor blocking rankl binding receptor study the high p diet significantly increased rankl opg ratio aged mice whereas ratio unchanged young mice many reports supported assertion increase rankl opg ratio promotes osteoclastogenesis accelerates bone resorption induces bone loss our previous study showed high p diet decreased breaking force stiffness femur aged mice compared young mice our findings show high p diet aged mice leads increased pth secretion consequent increases rankl opg ratio accelerating osteoclastogenesis previous studies showed pth regulates runx2 osterix mrna expression 30 31 study high p diet significantly increased serum oc concentration mrna expression runx2 osterix alp opn oc col1a1 young aged mice results bone resorption formation markers high bone turnover resorption exceeding formation since high bone turnover risk factor bone fracture osteoporosis high p diet might risk factor bone loss young mice also aged mice pth secretion might reflect decrease ca absorption high p diet group although mechanism underlying high p diet induced decreased ca absorption remains unknown thought formation insoluble ca p salts intestinal lumen important factor our previous study also showed high p diet decreased ca absorption female rats while evaluate ca absorption study possible estimate decrease ca absorption high p diet young aged mice however study showed high p diet significantly increased mrna expression trpv6 cabp9k pmca1b young aged mice previous study demonstrated ca restriction lactation stimulated ca binding protein active ca transport jejunum ileum furthermore ca deficient diet resulted increase duodenal pmca mrna chickens these studies suggested 1,25(oh)2d regulated ca transporters might stimulated low ca status intestinal lumen thus decrease soluble ca induced high p diet might lead trpv6 cabp9k pmca1b mrna expression increases 1,25(oh)2d regulated gene expression seem compensate decrease ca absorption high p diet brief results suggest high p diet accelerates transcellular ca pathway though absorbed amount ca insufficient maintain serum pth concentration it known fibroblast growth factor 23 fgf23 1,25(oh)2d well pth key factors ca p metabolism therefore measuring serum fgf23 1,25(oh)2d important fully elucidate mechanisms high p diet changes ca p metabolism studies needed clarify details in conclusion demonstrated high p diet increased bone metabolism related gene expression young aged mice furthermore high p diet affected pth secretion differently young aged mice leading increase rankl opg mrna ratio aged mice

in this study , the effects of high phosphorus ( p ) diet on bone metabolism - related gene expression were investigated in young and aged mice . twelve- and 80-week - old ddy male mice were divided into two groups , respectively , and fed a control diet containing 0.3% p or a high p diet containing 1.2% p. after 4 weeks of treatment , serum parathyroid hormone ( pth ) concentration was significantly higher in the high p groups than in the control groups in both young and aged mice and was significantly higher in aged mice than in young mice fed the high p diet . high p diet significantly increased receptor activator of nf-b ligand ( rankl ) mrna in the femur of both young and aged mice and significantly increased the rankl / osteoprotegerin ( opg ) mrna ratio only in aged mice . high p diet significantly increased mrna expression of transient receptor potential vanilloid type 6 , calbindin - d9k , and plasma membrane ca2 + -atpase 1b in the duodenum of both young and aged mice . these results suggest that high p diet increased rankl mrna expression in the femur and calcium absorption - related gene expression in the duodenum regardless of age . furthermore , the high p diet - induced increase in pth secretion might increase the rankl / opg mrna ratio in aged mice .

despite advancements modern medical science health management industry incidence cerebral palsy cp continues rise1 the common features cp decreased muscle strength abnormal muscle tone2 cp sufferers lack theability generate enough force maintain antigravity postural control result abnormal postures3 the development movement posture may altered non progressive damage brain subsequent neurological impairments spasticity muscle weakness co contractions visual impairment)5 studies indicate children adults mild severe forms cp postural impairments6,7,8 the emergence sitting postural control early infancy changes way infants interact world sitting position looking reaching interacting become functional allow exploration supports learning development motor skills therefore independent sitting defined needing support caregiver pillow sitting one first developmental goals every child individual differences present children characteristic signs developmental disorders infancy relatively unspecific therefore specific child able achieve sitting postural control always clear one method examining postural control adults children measure center pressure cop base support using force platform task remaining upright cop frequently used investigate postural control standing young children healthy cp9 10 the purpose study investigate differences pressure distributions sitting postures typical developmental td children children cp twelve cp childrens recruited outpatient rehabilitation clinic hemiparetic mmse k score 2411 could maintain independent sitting posture without support two cp children subsequently excluded refused participate experiment all parents enrolled participants provided written informed consent children participation prior experiment accordance ethical principles established declaration helsinki result exclusion this study used two school chairs mounted force platform assess quiet sitting pressure distribution subjects fsa seating assessment canada ) the acquisition frequency set 5 hz stated working range device 0200 mmhg resolution 1 mmhg the system also calibrated assign absolute pressure values digital output converter connected sensing pad this done applying pressure distribution similar actual conditions possible then thus subjects sat one two school chairs according height the chairs generally used school research purposes study used two basic school chair students spend long time day day sitting one chair 40 cm floor seat height 35 cm seat depth 32 cm seat width designed 122.4133.5 cm height subjects the another 35 cm floor seat height 38 cm seat depth 35 cm seat width designed 133.6152.7 cm height subjects it used posture symmetry study13 statistical analyses performed using pasw 18.0 descriptive statistics calculated frequency mean standard deviation range the mann whitney u tests wilcoxon signed rank tests used analyze differences groups differences lesion side respectively the si age matched td group employed normal criteria table 1table 1.general characteristics subjectscerebral palsygroup n=10)typicaldevelopmentalgroup n=10)age years)8.040.827.840.94gender male female)5/54/6lesion side right left)4/6symmetry index meansd)5.041.342.302.76 shows general characteristics participants the si td group significantly closer zero cp group children cp right hemiparetic cp respectively 4.96 2.24 5.12 0.83 significant difference hemiparetic sides the purpose present study investigate differences sitting posture children cp sat school chairs it known dynamic postural control sitting reliably assessed using cop data infants developing typically infants risk cp14 the principal finding study cp children sitting posture asymmetrical leaning less paretic side we thought infants developing typically develop ability sit exhibiting optimal range movement variability whereas cp may present either much little variability leading rigid narrow unpredictable set movement solutions achieve independent sitting the finding study agreement previous studies dissimilarities cop patterns infants cp td obviously demonstrated14 the results generalized cp children sample limited ten children one stage developmental process future studies assess relationship symmetry sitting posture functional activities

[ purpose ] the purpose of this study was to investigate the differences in symmetry of sitting posture between typical developmental ( td ) children and hemi - cerebral palsy ( cp ) children . [ subjects and methods ] a school chair mounted on a force platform was used to assess the quiet - sitting pressure distribution of 10 td and 10 cp children . [ results ] the symmetry index of the td children was significantly closer to zero than that of the cp children irrespective of the latter group s hemiparetic side . [ conclusions ] sitting posture on school chairs of cp children was more asymmetrical than that of td children .

bioethanol one employed liquid biofuels due easy adaptability fuel existing engines cleaner fuel higher octane rating gasoline ethanol market grew less billion liters 1975 39 billion liters 2006 expected reach 100 billion liters 2015 among widely used substrates ethanol production are molasses wastes byproduct sugar industries sugarcane sugar beet this cheap raw materials readily available ready conversion limited pretreatments compared starchy cellulosic materials sugars present readily fermentable form ongoing research development seeking improve methods minimizing numbers experiments provide information direct additive effects study variables interaction effects using design experiment methods recently statistical technique successfully applied many fields 58 response surface methodology rsm combination mathematical statistical techniques used modeling analysis problems response interest influenced several variables objective optimize response the popular rsm design central composite design ccd analysis experimental data the ccd efficient flexible providing sufficient information effects variables overall experimental error minimum number experiments center points ccd design usually repeated 46 times get good estimate experimental error pure error five center points created default factor alpha negative zero positive values 1 0 1 study alpha value taken one resulting 3 levels lowest 1 middle 0 highest 1 specifically known central composite face centered design ccfd attempt optimize variables incubation period initial ph incubation temperature molasses concentration affect bioethanol production the main goal present work maximize bioethanol production sugarcane molasses batch fermentation process using previously isolated saccharomyces cerevisiae y-39 the application ccfd rsm assisted designing modeling optimizing fermentation process performing series controlled laboratory experiments sugarcane molasses purchased sugars integrated industries egyptian distillation plants hawamdeia city giza egypt stored 4c use wickerham wh medium prepared according wickerham used maintenance inoculum preparation medium fermentation experiments prepared follows 2 g kh2po4 10 g nh4)2so4 1 g mgso4 7h2o 2 g yeast extract dissolved 1 l distilled water molasses concentration ph adjusted according experimental conditions sterilization 121c 20 min avoid contamination a yeast strain saccharomyces cerevisiae y-39 previously isolated ability produce bioethanol different saccharides used study active cultures fermentation experiments prepared growing y-39 wh medium 48 h 30c shaking incubator 150 rpm harvested cells washed twice sterile saline 8.5 g nacl per 1 l distilled water resuspended sterile saline used fresh pure stock inoculation the types sugars molasses determined high performance liquid chromatography hplc according method reported madian et al ashes quantified gravimetric analysis burning samples 550c 3 h minerals concentrations determined atomic absorption spectrophotometer central analytical lab egyptian petroleum research institute all chemical characterizations molasses done agricultural research center giza egypt ethanol yield measured gas chromatography model 6890 agilent equipped flame ionization detector nominal capillary column 60 530 5.00 all experiments carried triplicates listed results average batch fermentations done 100 ml erlenmeyer flasks fitted rubber stoppers containing 50 ml culture media inoculated 5 ml fresh yeast inoculum stock 10 mg fresh yeast ml incubation performed shaking incubator 100 rpm set temperatures according required experimental conditions samples analyses taken beginning end fermentation prescribed incubation periods response surface methodology rsm used optimize bioethanol production process sugarcane molasses investigate influence different fermentation process variables bioethanol yield central composite face the experimental runs carried according 2 full factorial design four identified design independent variables namely incubation period h x1 initial ph x2 incubation temperature c x3 molasses concentration wt.% x4 low 1 high 1 levels the total number experiments runs given simple formula 30 2 2k 6 k number independent variables k 4 includes 16 factorial points 24 full factorial ccfd augmented 6 replicates center point assess pure error the design factors variables low 1 high 1 levels namely x1 24 72 h x2 5 7 x3 20 40c x4 15 25 wt.% central values zero level chosen experimental design 48 h 6 30c 20% x1 x2 x3 x4 respectively table 1 once experiments preformed next step perform response surface experiment produce prediction model determine curvature detect interactions among design factors independent variables optimize process determine local optimum independent variables maximum yield bioethanol the model used study estimate response surface quadratic polynomial represented following equation 1)y=o+i=14ixi+i=13j i+14ijxixj+i=14iixi2 bioethanol yield g l value fixed response center point design ij ii linear interactive quadratic coefficients respectively xi xj independent variables factors study statistical software design expert 6.0.7 stat ease inc minneapolis usa used design experiments regression graphical analyses data obtained statistical analysis model evaluate analysis variance anova sugarcane molasses dark viscous fluid ph value 5 rich nutrients required microorganisms carbon nitrogen phosphorus sodium potassium contents 64 6 0.3 0.33 5.5 wt.% respectively non nitrogenous compounds e.g. citric acid oxalic acid represent 28% wt.% molasses found rich calcium 0.7% contains significant quantities trace minerals copper 2.2 ppm zinc 3.91 ppm manganese 4.74 ppm iron 78.37 ppm magnesium 1370 ppm sugarcane molasses rich fermentable sugars 55% wt% non fermentable sugars recorded 5% wt% most chemical parameters determined study close agreement reported chen chou found molasses containing 4555% total sugars 2025% reducing sugars 2535% sucrose 1016% ash 0.40.8% calcium 0.10.4% sodium 1.55% potassium ph 55.5 soil climate variety maturity cane processing conditions factory influence molasses composition consequently considerable variation may found nutrient content flavor color viscosity molasses based ccfd experimental results rsm used optimize fermentation process design factors independent variables the statistical combinations variables coded actual values along predicted experimental responses presented table 2 regression equation characterizing influence different considered variables process yield obtained 2)y=177.4 6.34x10.41x23.33x32.39x4 33.32x1x2 28.51x1x322.11x1x4 27.60x2x3 30.38x2x416.39x3x4 0.56x1292.58x22 44.08x3266.47x42 positive sign front terms indicate synergetic effect whereas negative sign indicates antagonistic effect pareto chart figure 1 used work make much easier visualize main interaction effects factors response variable bioethanol yield the model identified within studied range experiments quadratic effect incubation temperature interactive effect initial ph molasses concentration incubation period temperature highly significant positive influence bioethanol yield that increment incubation period temperature bioethanol yield increases occurred increment ph molasses concentration but incubation period relatively low significant positive effect bioethanol yield quadratic effect initial ph molasses concentration interactive effect incubation period initial ph well interactive effect incubation temperature initial ph highly significant inverse effect bioethanol yield the interactive effect incubation period molasses concentration incubation temperature molasses concentration significant negative impact bioethanol yield initial ph incubation temperature molasses concentration quadratic effect incubation period seem negative impact bioethanol yield thus bioethanol yield decreases increase initial ph incubation period incubation temperature initial ph incubation period molasses concentration incubation temperature molasses concentration the validity fitted model evaluated statistical significance controlled f test the analysis variance anova response surface full quadratic model given table 3 it indicated model highly statistically significant 95% confidence level f value 82.14 low probability p value 0.0001 the values determination coefficients r radj measure model fitting reliability model 2 calculated 0.9871 0.9751 respectively this suggests approximately 98.71% variance attributed variables indicated high significance model thus 0.0129 total variations explained model ensures good adjustment model experimental data confirmation adequacy regression model reflected also good agreement experimental predicted values response variables shown table 2 the lack fit test performed comparing viability current model residuals variability observations replicate settings factors the lack fit statistically significant f value 53.24 p value 0.0002 a significant lack fit suggests may systematic variation unaccounted hypothesized model this may due exact replicate values independent variable model provide estimate pure error the relationship predicted experimental values bioethanol yield shown figure 2 it seen high correlation r 0.9853 predicted experimental values indicating predicted experimental values reasonable agreement it means data fit well model give convincingly good estimate response system experimental range studied figure 3 shows normal probability plots standardized residuals bioethanol production efficiency a normal probability plot indicates residuals follow normal distribution case points follow straight line since scattering expected even normal data shown figure 3 assumed data normally distributed thus obtained normal probability plot indicates good validity approximation quadratic regression model , points observed runs scattered randomly within constant range residuals across graph that model adequate reason suspect violation independence constant variance assumption runs the standardized residuals versus run plot represented figure 4(b shows randomly scattered points ranged 2.8 errors normally distributed insignificant the perturbation plot figure 5 shows comparative effects independent variables bioethanol yield the sharp curvature three factors initial ph incubation temperature molasses concentration shows response bioethanol yield sensitive three variables the comparatively semiflat incubation period curve shows less sensitivity bioethanol yield towards incubation period thus incubation period studied range experiments major function fermentation process compared three factors the empirical predicted quadratic model response bioethanol yield terms process variables incubation period initial ph incubation temperature molasses concentration plotted three dimensional diagrams figure 6 investigate interaction among variables determine optimum condition factor maximum bioethanol yield figure 6(a represents effects varying incubation period initial ph bioethanol yield constant incubation temperature 38c molasses concentration 18 wt% however increase bioethanol yield occurred increase incubation period ph range 5.5 6.5 further increase initial ph would decrease bioethanol yield according interaction effects a significant positive impact detectable bioethanol yield increased increase incubation temperature time figure 6(c shows interactive effect incubation period h molasses concentration wt.% bioethanol production constant initial ph 5.6 incubation temperature 38c it obvious increase molasses concentration 20 wt% bioethanol yield decreases g l obtained higher incubation period 72 h 20 wt.% molasses concentration figure 6(d shows cooperative effect incubation temperature initial ph bioethanol yield constant incubation period 72 h molasses concentration 18 wt% shown figure 6(d low high ph values bioethanol yield decreases maximum bioethanol yield 257 g l obtained high incubation temperature 40c initial ph 6 figure 6(e illustrates interactive effect initial ph molasses concentration bioethanol yield constant incubation period 72 h temperature 40c where bioethanol yield low low high molasses concentrations initial ph but bioethanol yield increases within ph range 5.56.5 molasses concentration 18%22% maximum yield 257 g l ph molasses concentration 6 20% respectively figure 6(f represents interactive effect incubation temperature molasses concentration bioethanol yield constant incubation period 72 h initial ph 5.6 it obvious increase temperature bioethanol yield increases reaching maximum 40c low high molasses concentrations yield decreases increased within 18%22% g l obtained molasses concentration incubation temperature 20% 40c the optimization process carried determine optimum value bioethanol production efficiency using design expert 6.0.7 software according software optimization step desired goal operational condition x1 incubation period x2 initial ph x3 incubation temperature x4 molasses concentration chosen within studied range the response bioethanol production defined maximum achieve highest performance the program combines individual desirability single number searches optimize function based response goal accordingly optimum working conditions respective bioethanol production established results presented table 4 additional experiment performed confirm optimum results shown maximum bioethanol production 255 g l incubation period 71 h initial ph 5.6 incubation temperature 38c molasses concentration 18% recording average 1.08 0.87% respectively indicating process optimization ccfd capable reliable optimize bioethanol production sugarcane molasses using saccharomyces cerevisiae y-39 rsm ccfd proved reliable powerful tool modeling optimizing studying interactive effects four process variables incubation period initial ph incubation temperature molasses concentration bioethanol production batch fermentation sugarcane molasses using locally isolated saccharomyces cerevisiae y-39 a highly significant r 0.9871 p 0.0001 regression quadratic model equation obtained analyzing data 2 factorial design maximum predicted actual bioethanol yields 253 255

a statistical model was developed in this study to describe bioethanol production through a batch fermentation process of sugarcane molasses by locally isolated saccharomyces cerevisiae y-39 . response surface methodology rsm based on central composite face centered design ccfd was employed to statistically evaluate and optimize the conditions for maximum bioethanol production and study the significance and interaction of incubation period , initial ph , incubation temperature , and molasses concentration on bioethanol yield . with the use of the developed quadratic model equation , a maximum ethanol production of 255 g / l was obtained in a batch fermentation process at optimum operating conditions of approximately 71 h , ph 5.6 , 38c , molasses concentration 18% wt.% , and 100 rpm .

1989 caffe et al published method organ culture neuroretina placed photoreceptor layer facing downward rafts made nitrocellulose filters polyamide gauze grids since mammalian retinal organ cultures commonly used research retinal physiology pathobiology retinal cell dynamics shown organotypic models comparable vivo conditions especially outer retina thus retinal cultures still used electrophysiological studies monitor drug effects retinal cell functionality evaluate neurotrophic factors physical tension retinal cells study molecular basis potential therapies photoreceptor death our group previously used neuroretina explant cultures evaluate modifications induced exogenous cells blood derived macrophages cytokines tumor necrosis factor alpha tnf- implicated pathobiology proliferative vitreoretinopathy we also established mixed coculture model composed three cellular layers neuroretina rpe adipose tissue derived stem cells evaluate neuroprotective effects stem cells there obvious limitations culture systems axotomy ganglion cells part dissection procedure absence blood supply thus degenerative changes retinal cells especially inner retina could differ vivo conditions the rpe monolayer pigmented cuboidal epithelial cells closely associated photoreceptor outer segments the important functions rpe synthesis maintenance interphotoreceptor matrix photoreceptor membrane turnover retinoid metabolism rpe association supported fact neuroretinas adjoined rpe monolayer cultures better preserved tissue architecture culture studies the rpe also considered key element development retinal diseases importantly physiopathology retinal detachment central serous chorioretinopathy pathologies neuroretina rpe furthermore rpe secretes across apical membrane neuroprotective factors helps maintain retinal homeostasis thus adequate ex vivo tool studying retinal degeneration importance molecular signaling across subretinal space physical separation neuroretina rpe would inherent overcome complexities inherent vivo studies study developed characterized novel organotypic coculture system central cone dominated porcine neuroretina cocultured rpe cells maintained physically separated neuroretina culture medium porous membrane this arrangement prevented physical contact cocultured cells allowed signal molecules pass this model could useful studying vitro interactions neuroretina rpe lose natural contact happens several retinal diseases retinal detachment central serous chorioretinopathy further ex vivo model allow study role rpe secreted factors evaluation potential therapies reducing progression neuroretina degeneration fifteen porcine eyes animals aged 68 months obtained local slaughterhouse processed within 2 h death immediately enucleation eyes immersed ice cold transport medium composed dulbecco modified eagle medium dmem supplemented 10% antibiotic antimycotic mixture containing penicillin streptomycin amphotericin b gibco invitrogen paisley uk transported ice laboratory aseptic conditions laminar airflow hood eyes dissected free periocular tissue full immersion eyeballs 70% ethanol 2 min eyes washed clean dmem supplemented 10% antibiotic antimycotic room temperature briefly eyes dissected ora serrata exclude iris lens the vitreous removed posterior eyecup cotton swabs neuroretina detached discarded the remaining eye cup covered 0.05% trypsin tetrasodium ethylene diamine tetra acetate trypsin edta gibco 30 min 37 c rpe cells removed filling eye cup dmem supplemented 10% fetal bovine serum fbs gibco plus 1% antibiotic antimycotic mixture complete dmem swabbing gently after resuspension complete dmem cells plated 25 cm flasks nunc roskilde denmark the rpe cells maintained complete dmem standard culture conditions 37 c atmosphere 5% co2 95% humidity rpe cell morphology evaluated nikon eclipse ts100 inverted phase contrast microscope nikon instruments inc louis mo used determine viability cell numbers reaching 90% confluence the cells trypsinized 0.05% trypsin edta washed resuspended pbs gibco passage 2 rpe cells seeded 30,000 cells cm bottom transwell culture plates corning inc corning ny grown 24 h complete dmem allow cellular adhesion coculturing neuroretina explants the porcine cone enriched visual streak identified described hendrickson hicks two 55 mm adjacent explants eye obtained castroviejo corneal scissors john weiss son ltd milton keynes uk temporal area 1 mm superior optic disc figure 1 the neuroretina explants laid transwell membranes 24-mm diameter 0.4-m pore polycarbonate membrane insert product 3412 corning inc photoreceptor layer facing membrane nine neuroretina explants cocultured rpe cells physically separated culture medium overlying rpe cells transwell membrane figure 2 cultures maintained 1:1 neurobasal dmem supplemented 10% fetal bovine serum 2% b-27 gibco 1% l glutamine sigma aldrich st louis mo 1% antibiotic antimycotic mixture maintained standard culture conditions culture medium level 1.5 ml suggested manufacturer maintained contact support membrane beneath explants changed freshly prepared warmed medium every day the explants harvested analysis 9 days culture previously described group cut two halves subsequent processing two fresh central neuroretina specimens used culture day 0 samples processed parallel two neuroretina explants 55 mm obtained eye porcine cone enriched visual streak superotemporal optic disc schematic view coculture model porcine neuroretina explant isolated rpe cells the neuroretina explant placed cell culture membrane rpe cells placed bottom cell culture insert cocultured together well configuration the cell culture membrane physically separated signal molecules could pass porous membrane rpe cells cocultured neuroretina explants 9 days fixed transwell culture plates ice cold methanol panreac quimica s.a.u barcelona spain 15 min 4 c primary antibodies table 1 zonula occludens protein 1 zo-1 rpe specific 65 kda protein rpe65 cellular retinaldehyde binding protein cralbp diluted pbs containing 0.5% triton x-100 10% goat serum 1% bovine serum albumin bsa sigma aldrich the antibodies applied directly cultures incubated overnight 4 c washing pbs the corresponding species specific secondary antibodies immunoglobulin gamma conjugated alexa fluor 488 568 green red molecular probes eugene applied 1:200 dilution 1 h. nuclei stained 10 g ml 4,6-diamino-2-phenylindole dihydrochloride dapi molecular probes finally cells washed pbs mounted fluorescent mounting medium dakocytomation inc carpinteria ca coverslipped rpe retinal pigment epithelium cells nr neuroretina explant neuroretina samples fixed overnight 1% paraformaldehyde 1% glutaraldehyde panreac quimica s.a.u phosphate buffer pb gibco after gradual dehydration ethanol series pieces embedded low viscosity epoxy resin spurr taab aldermaston uk semithin sections 1 obtained ultramicrotome lkb bromma 8800 ultratome iii freiburg germany stained 1% toluidine blue 3% sodium tetraborate panreac quimica s.a.u immunohistochemistry the half samples fixed 4% paraformaldehyde pb ph 7.4 2 h subjected sucrose panreac quimica s.a.u ) sections 5 cut cryostat leica instruments nussloch germany mounted glass slides superfrost plus menzel glser braunschweig germany the neuroretinas immunostained phenotype specific markers table 1 rhodopsin rho calbindin d-28k cb glial fibrillary acidic protein gfap cralbp specific combinations antibodies diluted pbs containing 0.5% triton x-100 incubated overnight 4 c thereafter corresponding species specific secondary antibodies conjugated alexa fluor 488 and/or 568 applied 1:200 dilution 1 h. nuclei stained 10 g ml dapi finally sections washed pbs mounted fluorescent mounting medium coverslipped the primary antibodies used work used previous studies well characterized group authors regarding specific cell type immunostaining whole fixed porcine eyes porcine neuroretina samples 6 8,16 furthermore control samples primary antibodies omitted processed parallel immunoreactivity found case samples analyzed leica dm4000b light microscope leica microsystems wetzlar germany equipped epifluorescence leica hcx pl fluotar ph2 20x/0.50 40x/0.75 leica n plan 63x/0.8 objectives leica microsystems used images obtained leica dfc490 digital camera leica microsystems brightness contrast minimally adjusted final figures composed pixelmator 3.4 twist apple cupertino ca the thickness neuroretina thickness outer nuclear layer onl inner nuclear layer inl measured toluidine blue histological images image j 1.47v nih image national institute health bethesda md neuroretina thickness determined outer inner limiting membranes case six measurements performed 20x images neuroretina explants n=20 statistical analysis performed using r statistical software version 3.1.0 foundation statistical computing vienna austria a kruskal one way anova anova used compare mean thicknesses freshly isolated neuroretinas cultured 9 days alone 9 days coculture rpe cells the homogeneity variance assumption checked robust brown forsythe levene type test using group medians implemented r lawstat package when homogeneity variance validated welch test used comparison mean values pair wise comparisons performed student test bonferroni correction multiple testing rpe cells porcine eyes n=5 obtained previously described briefly eyes dissected ora serrata exclude iris lens the vitreous removed posterior eyecup cotton swabs neuroretina detached discarded the remaining eye cup covered 0.05% trypsin tetrasodium ethylene diamine tetra acetate trypsin edta gibco 30 min 37 c rpe cells removed filling eye cup dmem supplemented 10% fetal bovine serum fbs gibco plus 1% antibiotic antimycotic mixture complete dmem swabbing gently after resuspension complete dmem cells plated 25 cm flasks nunc roskilde denmark the rpe cells maintained complete dmem standard culture conditions 37 c atmosphere 5% co2 95% humidity rpe cell morphology evaluated nikon eclipse ts100 inverted phase contrast microscope nikon instruments inc louis mo used determine viability cell numbers reaching 90% confluence the cells trypsinized 0.05% trypsin edta washed resuspended pbs gibco passage 2 rpe cells seeded 30,000 cells cm bottom transwell culture plates corning inc corning ny grown 24 h complete dmem allow cellular adhesion coculturing neuroretina explants the porcine cone enriched visual streak identified described hendrickson hicks two 55 mm adjacent explants eye obtained castroviejo corneal scissors john weiss son ltd milton keynes uk temporal area 1 mm superior optic disc figure 1 the neuroretina explants laid transwell membranes 24-mm diameter 0.4-m pore polycarbonate membrane insert product 3412 corning inc photoreceptor layer facing membrane nine neuroretina explants cocultured rpe cells physically separated culture medium overlying rpe cells transwell membrane figure 2 cultures maintained 1:1 neurobasal dmem supplemented 10% fetal bovine serum 2% b-27 gibco 1% l glutamine sigma aldrich st louis mo 1% antibiotic antimycotic mixture maintained standard culture conditions culture medium level 1.5 ml suggested manufacturer ) was maintained contact support membrane beneath explants changed freshly prepared warmed medium every day the explants harvested analysis 9 days culture previously described group cut two halves subsequent processing two fresh central neuroretina specimens used culture day 0 samples processed parallel were obtained eye porcine cone enriched visual streak superotemporal optic disc schematic view coculture model porcine neuroretina explant isolated rpe cells the neuroretina explant placed cell culture membrane rpe cells placed bottom cell culture insert cocultured together well configuration cell culture membrane physically separated signal molecules could pass porous membrane rpe cells cocultured neuroretina explants 9 days fixed transwell culture plates ice cold methanol panreac quimica s.a.u primary antibodies table 1 zonula occludens protein 1 zo-1 rpe specific 65 kda protein rpe65 cellular retinaldehyde binding protein cralbp diluted pbs containing 0.5% triton x-100 10% goat serum 1% bovine serum albumin bsa sigma aldrich the antibodies applied directly cultures incubated overnight 4 c washing pbs the corresponding species specific secondary antibodies immunoglobulin gamma conjugated alexa fluor 488 568 green red molecular probes eugene applied 1:200 dilution 1 h. nuclei stained 10 g ml 4,6-diamino-2-phenylindole dihydrochloride dapi molecular probes finally cells washed pbs mounted fluorescent mounting medium dakocytomation inc carpinteria ca coverslipped rpe retinal pigment epithelium cells nr neuroretina explant neuroretina samples fixed overnight 1% paraformaldehyde 1% glutaraldehyde panreac quimica s.a.u phosphate buffer pb gibco after gradual dehydration ethanol series pieces embedded low viscosity epoxy resin spurr taab aldermaston uk semithin sections 1 obtained ultramicrotome lkb bromma 8800 ultratome iii freiburg germany stained 1% toluidine blue 3% sodium tetraborate panreac quimica s.a.u for immunohistochemistry half samples fixed 4% paraformaldehyde pb ph 7.4 2 h subjected sucrose panreac quimica s.a.u ) sections 5 cut cryostat leica instruments nussloch germany mounted glass slides superfrost plus menzel glser braunschweig germany the neuroretinas immunostained phenotype specific markers table 1 rhodopsin rho calbindin d-28k cb glial fibrillary acidic protein gfap cralbp specific combinations antibodies diluted pbs containing 0.5% triton x-100 incubated overnight 4 c thereafter corresponding species specific secondary antibodies conjugated alexa fluor 488 and/or 568 applied 1:200 dilution 1 h. nuclei stained 10 g ml dapi finally sections washed pbs mounted fluorescent mounting medium coverslipped the primary antibodies used work used previous studies well characterized group authors regarding specific cell type immunostaining whole fixed porcine eyes porcine neuroretina samples 6 8,16 furthermore control samples primary antibodies omitted processed parallel immunoreactivity found case samples analyzed leica dm4000b light microscope leica microsystems wetzlar germany equipped epifluorescence leica hcx pl fluotar ph2 20x/0.50 40x/0.75 leica n plan 63x/0.8 objectives leica microsystems used images obtained leica dfc490 digital camera leica microsystems brightness contrast minimally adjusted final figures composed pixelmator 3.4 twist apple cupertino ca the thickness neuroretina thickness outer nuclear layer onl inner nuclear layer inl measured toluidine blue histological images image j 1.47v nih image national institute health bethesda md neuroretina thickness determined outer inner limiting membranes case six measurements performed 20x images neuroretina explants n=20 statistical analysis performed using r statistical software version 3.1.0 foundation statistical computing vienna austria a kruskal one way anova anova used compare mean thicknesses freshly isolated neuroretinas cultured 9 days alone 9 days coculture rpe cells the homogeneity variance assumption checked robust brown forsythe levene type test using group medians implemented r lawstat package when homogeneity variance validated welch test used comparison mean values pair wise comparisons performed student test bonferroni correction multiple testing isolated porcine rpe cells started adhere culture day 1 form clusters cells day 4 flasks figure 3a these cells polygonal contained pigment day 7 culture flasks rpe cells almost confluent monostratified epithelium polygonal shape pigmented cytoplasmic granules figure 3b however cells dedifferentiated morphology increased size reduced pigment content figure 3b arrowheads cellular characteristics maintained passage 2 rpe cells cocultured neuroretina explants coculture day 9 figure 3c rpe cells formed confluent monolayer bottom cell culture inserts retained polygonal shape still pigment content figure 3c insert cells looked dedifferentiated figure 3c arrowheads cellular morphology immunochemistry rpe cells culture coculture period day 4 culture flasks rpe cells formed clusters pigmented polygonal cells day 7 flasks b rpe cells reached confluence maintained morphological characteristics dedifferentiated cells present b arrowheads 9 days coculture neuroretina explants c rpe cells cell culture inserts confluent monolayered pigmented polygonal shaped c insert dedifferentiated cells still present c arrowheads 9 days coculture rpe cells cell culture inserts showed variable zo-1 immunoexpression cell periphery green nuclei blue maintained cytoplasmic rpe65 e red nuclei blue cralbp detectable f scale bars=100 c 20 f assess preservation rpe morphology phenotype coculture neuroretina explants the rpe cells examined antibodies zo-1 peripheral adaptor protein tight junction structure rpe cells the expression rpe65 protein located cytoplasm involved production 11-cis retinal visual pigment regeneration cralbp retinoid binding protein implicated vitamin metabolism found rpe apical microvilli also detected immunohistochemistry nine days coculture neuroretina explants rpe cells showed variable zo-1 immunoexpression polygonal cell periphery figure 3d maintained expression cytoplasmic rpe65 figure 3e neuroretina explants stained toluidine blue showed general overview layers porcine retina figure 4 freshly isolated neuroretina explants figure 4a d characteristic neuroretina architecture apparent notable presence cone photoreceptors corresponding porcine visual streak the delicate structures photoreceptor outer inner segments os well preserved culturing absence cocultured rpe cells the retina structure 9 days culture figure 4b e f disorganized cellular vacuolization present throughout retina layers photoreceptor os lost remaining appeared shorter swollen compared freshly isolated neuroretina explants onl photoreceptor cell bodies oriented form degenerative rosette like structures figure 4b e asterisks the mller cells hypertrophied nuclei translocated onl cytoplasmic pigmented granules present figure 4f open arrow open arrowheads furthermore cellular extensions mller cells covered photoreceptor figure 4b f arrows 9 days coculture rpe cells figure 4c g the cellular architecture neuroretina explants better preserved retina layers easily discerned neuroretina explants cultured alone photoreceptor condensed swollen figure 4 g arrowheads rosette like formation evident cellular extensions photoreceptors present figure 4c g arrows neuroretina morphology culture coculture period freshly isolated retina explants ) porcine central retina showed characteristic highly organized layered structure 9 days culture alone b e f photoreceptor os lost cell bodies formed degenerative rosette like structures b e asterisks mller cells hypertrophied nuclei translocated outer nuclear layer f open arrow cell cytoplasm pigmented granules f open arrowheads cellular extensions photoreceptors also present b f arrows 9 days coculture rpe cells c g the layered retinal morphology preserved condensed photoreceptor still present g arrowheads os outer segments inner segments olm outer limiting membrane onl outer nuclear layer opl outer plexiform layer inl inner nuclear layer ipl inner plexiform layer gcl ganglion cell layer neuroretina explants examined antibodies rho opsin protein present rod os cb calcium binding protein present cone photoreceptors additionally assess degree glial cell activation neuroretina explants immunostained antibodies gfap intermediate filament protein present glial cells cralbp found mller cells freshly fixed neuroretina explants the rods maintained normal long straight os morphology expressing rho figure 5a 9 days culture without rpe partial displacement rho immunoexpression detected swollen rod figure 5b additionally photoreceptor nuclei displaced region figure 5b asterisks after 9 days coculture rpe rho expression figure 5c evident onl figure 5d arrows displaced shorter swollen rod neuroretina immunohistochemistry culture coculture period freshly isolated retina explants ) rho immunoreactivity green showed normal appearance rod outer segments os 9 days neuroretina explants culture alone b rho scarcely detected inner segments discernable photoreceptor nuclei appeared region b arrowhead 9 days coculture rpe cells c rho displaced short swollen rod outer nuclear layer arrows freshly isolated retina explants cb red showed cone photoreceptors normal morphology 9 days neuroretina culture alone e ) cone morphology markedly altered revealed cb immunostaining 9 days coculture rpe f cone morphology underwent degeneration freshly isolated retina explants g cralbp green showed mller cells normal morphology reduced immunostaining gfap red observed 9 days neuroretina culture alone h gfap upregulated cytoplasm glial cells gfap positive extensions formed layered like structure outside retinal tissue arrows 9 days coculture rpe cralbp scarcely detected gfap reduced compared explants cultured alone os outer segments inner segments onl outer nuclear layer inl inner nuclear layer gcl ganglion cell layer freshly fixed neuroretina explants cones immunoreactive cb normal morphology figure 5d 9 days culture without rpe the cb immunostained cones showed markedly altered morphology figure 5e 9 days coculture rpe cones underwent morphological degenerative changes figure 5f the freshly fixed neuroretina explants cralbp immunoexpression revealed mller cells normal morphology figure 5 g gfap immunostaining limited innermost layers neuroretinal tissue 9 days culture without rpe gfap clearly upregulated cytoplasm glial cells figure 5h gfap cellular processes extended outside retinal tissue formed layered structures figure 5h arrows 9 days coculture rpe cralbp scarcely present glial cell immunoexpression gfap reduced compared cultures without rpe figure 5i gfap processes also present outer limiting membrane thickness cellular extensions reduced figure 5i arrows the thickness neuroretina inl onl changed time culture period figure 6 the thickness freshly isolated neuroretinas n=2 135.2917.02 9 days culture alone thickness neuroretinas n=9 92.2814.94 rpe cocultured neuroretinas n=9 109.354.25 p<0.0001 the onl thickness freshly isolated samples 34.641.43 9 days culture alone onl thickness 21.73.57 rpe cocultured neuroretinas 30.823.16 p<0.0001 the inl thickness freshly isolated samples 35.541.81 9 days culture alone inl thickness 21.892.68 rpe cocultured neuroretinas 27.882.35 p<0.0001 porcine neuroretina thickness culture coculture period neuroretina tissue thickness day 9 culture day 9 coculture rpe reduced compared freshly isolated tissue the thickness neuroretina cultures alone day 9 significantly less neuroretinas cocultured rpe onl b inl c thickness day 9 culture day 9 coculture rpe reduced compared freshly isolated tissue the onl b inl c thickness neuroretina cultures alone day 9 significantly less neuroretinas cocultured rpe isolated porcine rpe cells started adhere culture day 1 form clusters cells day 4 flasks figure 3a these cells polygonal contained pigment day 7 culture flasks rpe cells almost confluent monostratified epithelium polygonal shape pigmented cytoplasmic granules figure 3b however cells dedifferentiated morphology increased size reduced pigment content figure 3b arrowheads cellular characteristics maintained passage 2 rpe cells cocultured neuroretina explants coculture day 9 figure 3c rpe cells formed confluent monolayer bottom cell culture inserts retained polygonal shape still pigment content figure 3c insert cells looked dedifferentiated figure 3c arrowheads cellular morphology immunochemistry rpe cells culture coculture period day 4 culture flasks rpe cells formed clusters pigmented polygonal cells day 7 flasks b rpe cells reached confluence maintained morphological characteristics dedifferentiated cells present b arrowheads 9 days coculture neuroretina explants c rpe cells cell culture inserts confluent monolayered pigmented polygonal shaped c insert dedifferentiated cells still present c arrowheads 9 days coculture rpe cells cell culture inserts showed variable zo-1 immunoexpression cell periphery green nuclei blue maintained cytoplasmic rpe65 e red nuclei blue cralbp detectable f scale bars=100 c 20 f assess preservation rpe morphology phenotype coculture neuroretina explants the rpe cells examined antibodies zo-1 peripheral adaptor protein tight junction structure rpe cells the expression rpe65 protein located cytoplasm involved production 11-cis retinal visual pigment regeneration cralbp retinoid binding protein implicated vitamin metabolism found rpe apical microvilli also detected immunohistochemistry nine days coculture neuroretina explants rpe cells showed variable zo-1 immunoexpression polygonal cell periphery figure 3d maintained expression cytoplasmic rpe65 figure 3e neuroretina explants stained toluidine blue showed general overview layers porcine retina figure 4 freshly isolated neuroretina explants figure 4a d characteristic neuroretina architecture apparent notable presence cone photoreceptors corresponding porcine visual streak the delicate structures photoreceptor outer inner segments os well preserved culturing absence cocultured rpe cells the retina structure 9 days culture figure 4b e f disorganized cellular vacuolization present throughout retina layers photoreceptor os lost remaining appeared shorter swollen compared freshly isolated neuroretina explants onl photoreceptor cell bodies oriented form degenerative rosette like structures figure 4b e asterisks the mller cells hypertrophied nuclei translocated onl cytoplasmic pigmented granules present figure 4f open arrow open arrowheads furthermore cellular extensions mller cells covered photoreceptor figure 4b f arrows 9 days coculture rpe cells figure 4c g the cellular architecture neuroretina explants better preserved retina layers easily discerned neuroretina explants cultured alone photoreceptor condensed swollen figure 4 g arrowheads rosette like formation evident cellular extensions photoreceptors present figure 4c g arrows ( porcine central retina showed characteristic highly organized layered structure 9 days culture alone b photoreceptor os lost cell bodies formed degenerative rosette like structures b e asterisks mller cells hypertrophied nuclei translocated outer nuclear layer f open arrow cell cytoplasm pigmented granules f open arrowheads cellular extensions photoreceptors also present b f arrows 9 days coculture rpe cells c g the layered retinal morphology preserved condensed photoreceptor still present g arrowheads os outer segments inner segments olm outer limiting membrane onl outer nuclear layer opl outer plexiform layer inl inner nuclear layer ipl inner plexiform layer gcl ganglion cell layer neuroretina explants examined antibodies rho opsin protein present rod os cb calcium binding protein present cone photoreceptors additionally assess degree glial cell activation neuroretina explants immunostained antibodies gfap intermediate filament protein present glial cells cralbp found mller cells freshly fixed neuroretina explants the rods maintained normal long straight os morphology expressing rho figure 5a 9 days culture without rpe partial displacement rho immunoexpression detected swollen rod figure 5b additionally photoreceptor nuclei displaced region figure 5b asterisks 9 days coculture rpe rho expression figure 5c evident onl figure 5d arrows displaced shorter swollen rod neuroretina immunohistochemistry culture coculture period freshly isolated retina explants rho immunoreactivity green showed normal appearance rod outer segments os 9 days neuroretina explants culture alone b rho scarcely detected inner segments discernable photoreceptor nuclei appeared region b arrowhead 9 days coculture rpe cells c rho displaced short swollen rod outer nuclear layer arrows freshly isolated retina explants cb red showed cone photoreceptors normal morphology 9 days neuroretina culture alone e cone morphology markedly altered revealed cb immunostaining 9 days coculture rpe f reduced immunostaining gfap red observed 9 days neuroretina culture alone h gfap upregulated cytoplasm glial cells gfap positive extensions formed layered like structure outside retinal tissue arrows 9 days coculture rpe ) cralbp scarcely detected gfap reduced compared explants cultured alone os outer segments inner segments onl outer nuclear layer inl inner nuclear layer gcl ganglion cell layer cones immunoreactive cb normal morphology figure 5d 9 days culture without rpe the cb immunostained cones showed markedly altered morphology figure 5e 9 days coculture rpe cones underwent morphological degenerative changes figure 5f freshly fixed neuroretina explants gfap immunostaining limited innermost layers neuroretinal tissue 9 days culture without rpe gfap clearly upregulated cytoplasm glial cells figure 5h gfap cellular processes extended outside retinal tissue formed layered structures figure 5h arrows 9 days coculture rpe cralbp scarcely present glial cell immunoexpression gfap reduced compared cultures without rpe figure 5i gfap processes also present outer limiting membrane thickness cellular extensions reduced figure 5i arrows the thickness neuroretina inl onl changed time culture period figure 6 the thickness freshly isolated neuroretinas n=2 135.2917.02 9 days culture alone thickness neuroretinas n=9 92.2814.94 rpe cocultured neuroretinas n=9 109.354.25 p<0.0001 the onl thickness freshly isolated samples 34.641.43 9 days culture alone onl thickness 21.73.57 rpe cocultured neuroretinas 30.823.16 p<0.0001 the inl thickness freshly isolated samples 35.541.81 9 days culture alone inl thickness 21.892.68 rpe cocultured neuroretinas 27.882.35 p<0.0001 neuroretina tissue thickness day 9 culture day 9 coculture rpe reduced compared freshly isolated tissue the thickness neuroretina cultures alone day 9 significantly less neuroretinas cocultured rpe onl b inl c thickness day 9 culture day 9 coculture rpe reduced compared freshly isolated tissue the onl b inl c thickness neuroretina cultures alone day 9 significantly less neuroretinas cocultured rpe * p<0.02 n=20 p<0.0001 n=20 onl outer nuclear layer inl inner nuclear layer the present study described development characterization novel coculture model cone dominated porcine central retina rpe cells closely simulate ex vivo subretinal space microenvironment retinal degeneration common finding many retinal diseases cases retinal detachment central serous chorioretinopathy part consequence physical separation neuroretina rpe this separation starts cascade events result cellular changes throughout retina these events part responsible poor functional results occur even successful reattachment surgery currently research retinal degeneration largely limited due difficulty obtaining specimens freshly detached human retinas limitations experimental animal models sense importantly closely simulate vivo retinal cellular molecular dynamics source improved knowledge retinal physiopathology recent decades nevertheless previously reported culture models lack influence rpe cells necessary maintaining viability functionality outermost retinal layers furthermore rpe cells secrete various growth trophic factors act neuroretina choroidal endothelium the rpe also plays important role retinal degeneration process considered key element retinal detachment physiopathology there obvious limitations culture systems absence choroidal retinal blood flow lack vitreous axotomy ganglion cells may considerably limit study inner retina modifications however neuroretina organotypic cultures still considered adequate tool improving knowledge retinal physiopathology other authors previously described models rpe monolayers cocultured contact neuroretina those models complex develop adequately mimic retinal pathology related separation neuroretina rpe our purpose study characterize differences neuroretinas cultured alone cocultured rpe cells physically separated this novel model could basis studying interaction neuroretina rpe cells separated vivo studies currently exist model neuroretina explants cultured cell culture membranes physically separated rpe cells cultured bottom cell culture inserts thus porous cell culture membranes inhibit rpe cell migration retinal tissue direct contact neuroretina cells molecular exchange among different cell types occur scenario rpe secreted factors diffuse cell culture membrane pores influence neuroretina cell dynamics thus recreating subretinal space milieu the porcine retina possesses characteristics make species particularly useful retinal research retinal extent ultrastructure lack tapetum the retinal parenchyma quite similar humans double circulation system central zone visual streak this zone broad horizontal retinal streak optic disc high cone density without direct vascularization unlike human fovea the porcine retina rod density similar humans cellular dynamics neuroretina rpe separation comparable species especially key cells mller rpe cells therefore in coculture model developed used explants porcine cone dominated area centralis similarity human central retina as shown phase contrast microscopy rpe cells largely maintained morphological characteristics throughout coculture period furthermore cells maintained expression zo-1 outlining polygonal shape rpe cells within monolayer rpe65 supporting preservation rpe phenotype without dedifferentiation form cell types undergoing epithelial mesenchymal transition as previously described primary rpe cell cultures clusters cells became partially dedifferentiated cralbp immunoexpression observed rpe cells cocultured neuroretinas probably due loss normal cell polarization especially seen disappearance apical microvilli the ability rpe culture secrete neurotrophic factors described authors cultures present study neuroretina explants without rpe showed degenerative alterations tissue retina cells previously described however presence degenerative rosette like structures reported observed group previous work central retina used our immunohistochemical studies revealed retinal cell degenerative modifications others previously described organotypic porcine retina cultures although rho cralbp immunoexpression better preserved lower levels gfap detected neuroretinas cocultured rpe cells quantify gfap cralbp labeling part future experiments however clear related cellular swelling better cellular viability sense useful explore cell death survival future studies case neuroretina explants cocultured rpe cells maintained better preserved tissue cellular characteristics significantly better conserved tissue nuclear layer thicknesses these findings consistent complete 5-mm neuroretina explants observed experiments performed based morphological immunohistochemical results found study neuroretina preservation cocultures rpe cells may partially linked neuroprotective factors secreted rpe cells the secretion neurotrophic neuroprotective factors rpe cells stimulated retinal damage our group developed standardized novel coculture model central cone dominated porcine neuroretina supplemented rpe cells maintained separately within culture wells this coculture system mimics ex vivo subretinal space develops retinal detachment rd disease conditions retina compared neuroretina explant cultures alone presence cocultured rpe provided improved neuroretina architecture thickness better preservation rho cralbp immunoexpression lower levels gfap these data suggest cocultured rpe direct contact neuroretina explant neuroprotective role effect may linked beneficial effects neurotrophic factors secreted induced rpe cells coculture furthermore proposed model useful better study interactions rpe neuroretina test neuroprotective and/or anti inflammatory drugs retinal degenerative diseases our group developed standardized novel coculture model central cone dominated porcine neuroretina supplemented rpe cells maintained separately within culture wells this coculture system mimics ex vivo subretinal space develops retinal detachment rd disease conditions retina compared neuroretina explant cultures alone presence cocultured rpe provided improved neuroretina architecture thickness better preservation rho cralbp immunoexpression lower levels gfap these data suggest cocultured rpe direct contact neuroretina explant neuroprotective role effect may linked beneficial effects neurotrophic factors secreted induced rpe cells coculture furthermore proposed model useful better study interactions rpe neuroretina test neuroprotective and/or anti inflammatory drugs retinal degenerative diseases

purposeto develop and standardize a novel organ culture model using porcine central neuroretina explants and rpe cells separated by a cell culture membrane.methodsrpe cells were isolated from porcine eyes , expanded , and seeded on the bottom of cell culture inserts . neuroretina explants were obtained from the area centralis and cultured alone ( controls ) on cell culture membranes or supplemented with rpe cells in the same wells but physically separated by the culture membrane . finally , cellular and tissue specimens were processed for phase contrast , cyto-/histological , and immunochemical evaluation . neuroretina thickness was also determined.resultscompared to the neuroretinas cultured alone , the neuroretinas cocultured with rpe cells maintained better tissue structure and cellular organization , displayed better preservation of photoreceptors containing rhodopsin , lower levels of glial fibrillary acidic protein immunoexpression , and preservation of cellular retinaldehyde binding protein both markers of reactive gliosis . neuroretina thickness was significantly greater in the cocultures.conclusionsa coculture model of central porcine neuroretina and rpe cells was successfully developed and standardized . this model mimics a subretinal space and will be useful in studying interactions between the rpe and the neuroretina and to preclinically test potential therapies .

ectopic kidney relatively rare renal anomaly however endourologist encounter stone disease ectopic kidney occasionally factors anomalous blood vessels tortuous ureter high insertion lead poor drainage predisposition formation renal calculi patients the common management option stones laparoscopy ultrasound guided percutaneous nephrolithotomy pcnl we report experience use recently described micro pcnl microperc two cases two male patients aged 57 60 years respectively presented us calculi ectopic pelvic kidneys the first patient undergone flexible ureterorenoscopy elsewhere stone could reached due difficult angulation inflamed tissue leading poor vision computed tomography urogram revealed ectopic malrotated left kidney lying sacrum there 13 mm 11 mm sized calculus renal pelvis figure 1a b second patient computed tomography urogram revealed normal right kidney ectopic malrotated left kidney lying sacrum there 18 mm 17 mm sized calculus 1232 hu pelvis 5 mm 4 mm sized calculus 423 hu lower calyx ectopic kidney figure 1c ( x ray kub showing renal calculus pelvis pelvic ectopic kidney double j stent situ b computed tomography urogram showing pelvicalyceal system anatomy c x ray kub showing renal calculus yellow arrow ectopic kidney computed tomography urogram showing pelvicalyceal system anatomy location pelvic stone yellow arrow location lower calyceal stone red arrow procedures performed general anesthesia lithotomy position ureteric catheterization carried cystoscopic guidance using 7 fr ureteric catheters 0.035 inch glidewire terumo tokyo japan the position changed supine oblique sandbag ipsilateral hemipelvis move overlying bowel away kidney an ultrasound probe pressed anterior abdominal wall displace bowel away line access further colour doppler used rule significant blood vessel path needle puncture three way connector attached needle allowing saline irrigation passage 0.9 mm flexible microperc telescope 272 holmium yttrium aluminum garnet ho yag laser fiber figure 2 the stones completely fragmented dust laser x ray ultrasound was obtained first post operative day document stone clearance rule fluid collection the urethral catheter removed first post operative day patient discharged ( surface view patient position ultrasound guided percutaneous renal access b intraoperative ultrasonography picture showing entry puncture needle yellow arrows lower calyx containing calculus red arrow c confirmation access lower calyx antegrade contrast study delineate pelvicalyceal system intraoperative surface view showing microperc instruments e puncture site end procedure yellow arrow f post operative x ray kub showing complete clearance operating times 30 35 min respectively in first patient ureteric catheter left situ 1 day second patient ureteric catheter replaced double j stent end procedure visual analog pain scores 1 10 scale first post operative day two three respectively first post operative day 1 month follow x ray ultrasound kub revealed complete stone clearance evidence fluid collection abdomen options managing small renal calculi pelvic ectopic kidney shock wave lithotripsy swl retrograde intrarenal surgery rirs ultrasound laparoscopy guided pcnl since kidneys surrounded bowel bone moreover clearance fragmented stones also impaired due high insertion ureter impaired pyeloureteral motility due surrounding fibrous bands rirs technically demanding due abnormal tortuous course ureter pelvic kidney this exemplified first patient stone could reached flexible ureteroscope procedure abandoned the potential hazards percutaneous access ectopic kidney 1 risk injury surrounding bowel particularly track dilatation carried bowel 2 abnormal vasculature resulting bleeding tract dilatation 3 spillage fluid peritoneal cavity microperc minimally invasive form pcnl percutaneous renal access stone fragmentation achieved single step using 16 g needle since dilatation performed potential hazards associated avoided fluid collection less likely puncture end procedure since needle puncture site closes quickly both patients rapid post operative recovery probably due lack fluid spillage hence paralytic ileus though laparoscopic guidance advocated percutaneous access ectopic kidney experienced hands ultrasound guidance provide safe entry appropriate calyx appropriate patient positioning move bowel away kidney placing sand bag push kidney towards anterior abdominal wall compression ultrasound transducer allow safe access kidney further use colour doppler rules significant blood vessel along path needle puncture hemoglobin drop patients minimal supporting safety procedure laparoscopy guided pcnl abdominal drain may need left situ prolonged period persistent urinary leakage the average hospital stay series 15 patients underwent laparoscopy guided pcnl 4.8 days 4 11 days the patients series sequential removal nephrostomy tube urethral catheter abdominal drain lead prolonged post operative stay thus ultrasound guided microperc safe efficient technique management small renal calculi ectopic kidneys minimally invasive procedure short hospital stay

management of stone disease in an ectopic kidney is challenging . laparoscopy or ultrasound guided percutaneous nephrolithotomy and retrograde intra - renal surgery are the preferred techniques for these stones . we performed ultrasound guided microperc using a 16 g needle for the management of renal calculi in pelvic ectopic kidneys in two patients . there was no intraoperative or post - operative complication . both patients had complete stone clearance and were discharged on the first post - operative day . ultrasound guided microperc is a safe and effective option for the management of small renal calculi in pelvic ectopic kidneys .

although use prefabricated posts gained popularity several years custom cast dowel core used retain restorations clinical evidence success one important advantage post system dowel fit flared irregularly shaped canal closely prefabricated post systems however although endodontic therapy shown high success rate adverse situations require endodontic retreatment rare when signs symptoms radiographic images suggest failure endodontic treatment atraumatic efficient post removal essential optimal non surgical endodontic management drills extractors exert high force root may result root fractures ultrasonic energy transmitted post causing cracks cement thus facilitating removal recent evidence suggests ultrasonic vibration safe fast method post removal however several vitro studies evaluated post removal ultrasonic devices using roots extract teeth included resin cylinders procedure used order facilitate handling test application however clinically root enveloped periodontal ligament pdl presents higher resiliency resins used root inclusion this difference resiliencies enables pdl dissipate ultrasonic energy easily root included resin cylinder thus facility found vitro studies post removal ultrasound activation may consistent clinical reality the simulation pdl tests would important bring laboratory findings clinical application the aim vitro study evaluate effect simulated periodontal ligament spdl custom cast dowel core removal using ultrasonic device the null hypothesis spdl influence action ultrasonic device used remove cast dowel core root canal thirty two human maxillary canines mature apices un pronounced flattening roots curves single canal selected study crowns removed order obtain 15-mm long root remainder endodontic treatment root canals were prepared according crown technique using stainless steel k files 2 4 gates glidden drills dentsply maillefer ballaigues switzerland all enlargement procedures followed irrigation 2.5% sodium hypochlorite solution instrumented root canals obturated gutta percha cones sealer-26 resin sealer dentsply petrpolis rj brazil using lateral condensation technique the filled roots stored relative humidity least 72 h allow resin sealer set the specimens randomly allocated according presence spdl application ultrasonic vibration half roots included directly self cured acrylic resin cylinders jet clssico paulo sp brazil without spdl the external surfaces root remainders dipped melted wax epoxiglass diadema sp brazil resulting 0.2 0.3-mm thick wax layer after resin polymerization roots removed cylinder wax removed root surface creating space resin cylinder the polyether impression material impregum f 3m espe seefeld germany mixed placed space created resin cylinder the tooth inserted cylinder excess material removed scalpel blade order allow tensile testing without root becoming dislodged roots included resin cylinder spdl fixed cylinders two stainless steel wires 1 mm diameter figure 2 note presence two wires order prevent root dislodgement testing post holes prepared standardizing length 8 mm preparation performed size 6 largo drill dentsply maillefer this drill used low speed handpiece attached parallelometer the root canal impressions made self cured resin acrylic duralay reliance dental worth il usa the dowel cores cast nickel chromium alloy wironia bego bremen germany all custom cast dowel cores luted dual cured resin cement panavia f kuraray osaka japan accordance manufacturer instructions after storage period half specimens inclusion type without spdl submitted ultrasonic vibration this applied calibrated operator using piezoelectric ultrasonic device enac osada electric co ltd tokyo japan st 09 tip osada electric co. ltd maximum power water cooling the vibration applied 1 min buccal mesial lingual distal incisal surfaces successively total application time 4 min sample figure 3 samples positioned universal testing machine model 4411 instron corp canton usa ring core attached load cell 500 n tensile load applied crosshead speed 0.5 mm min cast dowel core dislodged figure 4 the factors evaluated presence spdl ultrasonic vibration application post hoc tests calculated using tukey`s multiple comparison test =0.05 application ultrasonic vibration application tensile load samples dislodgment custom cast dowel core a b without simulated periodontal ligament c simulated periodontal ligament there statistically significant effect factors presence spdl p<0.01 ultrasonic vibration application p<0.01 interaction factors p<0.05 the means tensile strength values kgf sd necessary dislodge cast post cores results tukey test shown table 1 when ultrasonic vibration applied samples spdl presented lowest tensile bond strength values no significant differences observed presence absence spdl samples submitted ultrasonic vibration ultrasonic vibration application led lowest values pdl simulated effect samples spdl tensile strength means values kgf sd necessary dislodge cast post cores the influence pdl often omitted vitro tests evaluate post removal using ultrasonic devices opposed studies evaluate vitro fracture resistance restored teeth the pdl important structure distributing stress generated load application teeth based elastomeric materials used reproduce pdl several studies polyether impression material adequate purpose ease use consistency deformation limit one difficulty using spdl tensile tests possibility dislodged load application present study roots fixed resin cylinder two stainless steel wires prevent dislodgment root allow testing this methodology enabled tensile load application samples included spdl influence tensile bond strength values samples included cylinder presence spdl presented lower bond strength values directly included resin ultrasonic vibrations applied one possible explanation may related forces resulting tensile load application test deformation wires used fix root allowed slight dislodgement root thus tensile load concentrated interface resin cement dowel samples included spdl this results need lower loads remove cast dowel cores despite influence load distribution the simulation pdl also intervened effectiveness ultrasonic vibration reduce dowel retention the tensile bond strength samples included resin cylinders spdl altered use ultrasound device opposed samples included without spdl the reduction cast dowel core retention application ultrasonic energy demonstrated several studies the ultrasonic device used study piezoelectric transducer transforms electricity ultrasonic vibrations quartz crystals within transducer vibrated electricity flowing applying alternating electrical field across crystal these ultrasonic vibrations transmitted dowel core fracturing cement interposed dowel root canal walls facilitate removal several studies reported type luting agent influence post retention removal procedure posts cemented resin cements usually require greater force removal compared cemented zinc phosphate glass ionomer cements study cast dowel cores luted resin cement panavia f. cement contains resin monomer 10-mdp 10-methacryloyloxydecyl di hydrogen phosphate composition bonds metal oxides considering essential vibrations reach resin cement order facilitate dowel core removal root inclusion method may affect efficacy ultrasonic device the high rigidity acrylic resin used sample inclusion allow root movement ultrasound application thus approximately energy dispensed tip ultrasonic device transmitted cast dowel core reach resin cement hand root mobility permitted impression material may reduce energy reaches resin cement thus resin cement submitted lower strain sufficient reduce retention cast dowel core the latter situation closer clinical reality directly including root resin cylinders therefore within limitations study demonstrated pdl simulation significant effect custom cast dowel core removal ultrasonic vibrations this means several vitro studies evaluated ultrasonic devices dowel core removal may overestimated efficacy the ultrasonic vibration effect cast dowel core retention pdl simulated the present outcomes demonstrate importance simulation vitro evaluation avoid overestimating efficacy ultrasonic vibration used cast dowel core removal

objectivethe aim of this study was to evaluate the effect of simulated periodontal ligament ( spdl ) on custom cast dowel and core removal by ultrasonic vibration.material and methodsthirty - two human maxillary canines were included in resin cylinders with or without spdl made from polyether impression material . in order to allow tensile testing , the roots included in resin cylinders with spdl were fixed to cylinders with two stainless steel wires . post - holes were prepared by standardizing the length at 8 mm and root canal impressions were made with self - cured resin acrylic . cast dowel and core sets were fabricated and luted with panavia f resin cement . half of the samples were submitted to ultrasonic vibration before the tensile test . data were analyzed statistically by two - way anova and tukey 's post - hoc tests ( p<0.05).resultsthe ultrasonic vibration reduced the tensile strength of the samples directly included in resin cylinders . there was no difference between the values , whether or not ultrasonic vibration was used , when the pdl was simulated . however , the presence of spdl affected the tensile strength values even when no ultrasonic vibration was applied.conclusionsimulation of pdl has an effect on both ultrasonic vibration and tensile testing .

human metapneumovirus hmpv rna virus pneumovirinae subfamily paramyxoviridae family first isolated netherlands 2001 subsequently identified worldwide it implicated significant cause hospitalization young children second respiratory syncytial virus rsv infants hospitalized acute respiratory infections aris hmpv detected 1.543.0% patients aris circulates predominantly winter seroprevalence studies shown almost children five years age evidence past infection clinical syndromes associated hmpv infection similar rsv infection ranging mild upper respiratory tract infections wheezing severe lower respiratory tract infections requiring mechanical ventilation rare cases fatalities associated hmpv implicated handful cases encephalitis although hmpv infections diagnosed adults greatest impact occurs children a significant association hmpv wheezing seen young children hmpv linked apparent life threatening events infants hmpv associated aris super infections result staphylococcus aureus streptococcus pneumoniae begin understand impact hmpv institution analyzed children admitted tertiary care center southeast michigan respiratory symptoms respiratory season 20062007 observational retrospective study the primary purposes study establish utility testing hmpv children admitted hospital respiratory virus season compare impact hmpv rsv healthcare system we identified convenience sample 256 nasopharyngeal np specimens children younger 18 years age admitted respiratory symptoms november 1 2006 may 31 2007 the specimens obtained np wash swab based admitting physician discretion routine testing direct fluorescent antibody dfa and/or culture rsv parainfluenza viruses 13 influenza viruses b adenovirus rhinovirus the np specimens frozen 70c later subjected nucleic acid extraction using easymag system biomrieux durham nc usa following manufacturer protocol slight modifications two hundred microliters specimen pre treated 20 units dnase new england biolabs uk 37c 45 min extraction extracts used template detection hmpv using nuclisense real time analyte specific reagent asr assay performed easyq instrument biomrieux a proprietary internal control containing primer binding sites hmpv target unique internal sequences targeted separate molecular beacon probe spiked specimen extraction monitor amplification integrity laboratory personnel blinded clinical data clinical investigator blinded laboratory results first specimen virus identified per admission considered the -test used analysis categorical variables comparing hmpv positive patients rsv positive patients well patients negative hmpv rsv the student test used analysis compared continu ous variables of 256 specimens rsv identified 52 20.3% hmpv 18 7.0% influenza 9 3.5% rhinovirus 5 2.0% parainfluenza 8 3.1% adenovirus 4 1.6% three specimens co infections hmpv rsv two rsv influenza one hmpv detected primarily specimens collected january march 83.4% rsv uniformly detected november february decreased may no hmpv detected specimens obtained april may figure 1 primary goal study compare patients hmpv rsv and because number patients isolated viruses hmpv rsv low cases viruses hmpv rsv specific virus isolated combined separate group additional analyses detailed following section while several studies shown hmpv occurs older children rsv reports showed difference age predilection gender predominance presence underlying medical disorders study majority hmpv positive patients aged 1324 months n=7 43.8% whereas rsv positive patients n=35 71.4% younger 12 months age p<0.01 underlying medical disorders considered prematurity chronic lung disease bronchopulmonary dysplasia asthma congenital heart disease congestive heart failure immunosuppression immunodeficiencies hematological solid organ malignancies diabetes renal failure we also found children attending daycare school increased risk positive hmpv rsv respiratory illness associated either viruses table 1 table 1demographic data.hmpv+(% n=16rsv+(% n=49negative hmpv rsv n=188age 012 months4 25.0)*35 71.4)77 41.0 1324 months7 43.8)9 18.4)38 20.2 25 months5 31.2)5 10.2)73 38.8)male7 43.8)26 53.1)98 52.1)daily activities home11 68.8)37 75.5)118 62.8 school daycare5 31.2)12 24.5)70 37.2)presence underlying medical condition11 68.8)32 65.3)140 74.5)p values listed 0.05 thus deemed statistically significant;*p<0.01 p values listed 0.05 thus deemed statistically significant hmpv previously reported rare cause community acquired pneumonia we found hmpv positive patients likely diagnosed pneumonia 37.5% two groups 14% p=0.04 rsv positive p=0.02 negative hmpv rsv in addition hmpv positive patients equally likely diagnosed bronchiolitis rsv positive children approximately 30% less likely children respiratory symptoms negative hmpv rsv 11% p=0.02 study the rate abnormal chest radiographs comparable three groups peribronchial cuffing likely present hmpv positive patients two groups table 2 table 2clinical features.featurehmpv+(% n=16rsv+(% n=49negative hmpv rsv n=188pneumonia6 37.5)*,**7 14.3)*27 14.4)**bronchiolitis5 31.2)14 29.2)21 11.2)abnormal chest radiograph14 87.5)35 77.8)123 71.9)peribronchial cuffing6 37.5),5 11.1)18 10.4)oxygen supplementation12 75.0)38 77.6)104 55.3)antibiotic use13 81.2)25 51.0)136 72.3)antiviral use0 0)1 2.0)4 2.1)steroid use8 50.0)15 30.6)66 35.1)mechanical ventilation2 12.5)2 4.1)26 13.8)icu admission6 37.5)6 12.2)50 26.6)pvalues listed 0.05 thus deemed statistically significant;*p=0.04;**p=0.02;p=0.02;p<0.01;p=0.03;p=0.02 pvalues listed 0.05 thus deemed statistically significant order compare severity illness across three patient populations examined hospital course mean duration hospital stay 6 days range 137 hmpv positive patients 6 days range 1112 rsv positive patients 12 days range 1117 patients negative hmpv rsv p=0.83 hmpv vs. rsv p=0.06 hmpv vs. negative hmpv rsv hmpv positive patients likely treated antibiotics rsv positive patients 81.2% vs. 51.0% p=0.03 likely admitted intensive care unit icu 37.5% vs. 12.2% p=0.02 hmpv positive patients likely require oxygen supplementation mechanical ventilation steroid use rsv positive patients table 2 infections hmpv rsv reported clinically indistinguishable subtle differences identified study the common presenting symptom hmpv positive patients fever occurred often hmpv positive patients two patient populations hmpv positive patients also likely patients negative hmpv rsv decreased urine output table 3 43.8% vs. 14.4% p<0.01 physical examination hmpv positive patients likely rsv positive patients exhibit focal decreased breath sounds 18.8% vs. 2.0% p=0.01 likely patients negative hmpv rsv exhibit signs otitis media 18.8% vs. 4.8% p=0.02 otherwise differences signs symptoms noted hmpv rsv positive patients table 4 table 3clinical symptoms.hmpv+(% n=16rsv+(% n=49negative hmpv rsv n=188fatigue6 37.5)9 18.4)153 81.4)fever15 93.8)*,**30 61.2)*102 54.3)**rash1 6.2)1 2.0)10 5.3)vomiting5 31.2)19 38.8)67 35.6)diarrhea4 25.0)11 22.4)31 16.5)poor feeding9 56.2)30 61.2)80 42.6)decreased urine output7 43.8)14 28.6)27 14.4)watery eyes1 6.2)1 2.0)7 3.7)red eyes0 0)1 2.0)4 2.1)rhinorrhea6 37.5)30 61.2)74 39.4)congestion8 50.0)25 51.0)61 32.4)ear pain1 6.2)2 4.1)7 3.7)sore throat1 6.2)1 2.0)15 8.0)cough13 81.2)44 89.8)128 68.1)rapid breathing4 25.0)12 24.5)30 16.0)difficulty breathing9 56.2)29 59.2)80 42.6)apnea2 12.5)1 2.0)12 6.4)pvalues listed 0.05 thus deemed statistically significant;*p=0.01;**p=<0.01;p<0.01 pvalues listed 0.05 thus deemed statistically significant table 4clinical signs.hmpv+(% n=16rsv+(% n=49negative hmpv rsv n=188respiratory distress6 37.5)24 49.0)63 33.5)respiratory failure1 6.2)0 0)15 8.0)tachypnea5 31.2)16 32.7)40 21.3)retractions4 25.0)*30 61.2)*48 25.5)crackles4 25.0)10 20.4)28 14.9)rhonchi2 12.5)6 12.2)17 9.0)wheezing6 37.5)23 46.9)48 25.5)rales0 0)2 4.1)7 3.7)focal decreased breath sounds3 18.8)1 2.0)13 6.9)tachycardia5 31.2)18 36.7)46 24.5)poor perfusion1 6.2)4 8.2)13 6.9)conjunctivitis1 6.2)0 0)5 2.7)phayrngitis2 12.5)1 2.0)10 5.3)signs otitis media3 18.8)8 16.3)9 4.8)lymphadenopathy head neck region1 6.2)0 0)11 5.9)pvalues listed 0.05 thus deemed statistically significant;*p=0.01;p=0.02;p=0.02 hmpv second commonly identified respiratory virus respiratory season 20062007 study our results confirm previous findings children symptomatic hmpv infection older infected rsv possibly differences upper respiratory tract lung anatomy older children allow hmpv acquisition study although previous report found hmpv rare cause community acquired pneumonia among hospitalized patients 4.9% 37.5% hmpv positive patients admitted diagnosis pneumonia this difference likely requirement three independent radiologists interpretations radiographs earlier study compared diagnosis one admitting physician study hmpv positive patients likely two groups peribronchial cuffing chest radiographs evidence suggesting patients interstitial edema likely result disruption respiratory epithelial structure inflammation hmpv shown cause animal studies hmpv may stronger predilection respiratory epithelial cells rsv corroborated reports hmpv positive patients frequently exhibited signs otitis media further research needed pathogenesis hmpv humans specifically regarding ability hmpv infect human respiratory epithelial cells compared rsv viruses the present study demonstrates previously reported hmpv positive patients likely severely ill rsv positive patients in fact study hmpv positive patients likely rsv positive patients admitted icu even though likely underlying medical illness in addition hmpv positive patients likely rsv positive patients receive antibiotics study time period institution routinely test specimens hmpv physicians may used antibiotics often patients retrospectively shown hmpv positive aware specific virus contributing patients illnesses the finding increased antibiotic use children aris negative viral cultures corroborates suggestion it also possible increased diagnosis pneumonia hmpv positive patients contributed increased use antibiotics this study however shows may subtle differences clinical features hmpv rsv infections previously reported fever common presenting symptom hmpv positive patients common hmpv positive children two groups hmpv positive patients also likely experience decreased urinary output patients negative hmpv rsv more information needed compare level inflammation produced hmpv viruses our study limited extraction data convenience sample fact one respiratory viral season studied in addition signs symptoms associated studied viruses may exaggerated hospitalized patients included lastly acknowledge li mi tations associated use different methods detecting different viruses study the real time asr assay used detect hmpv likely sensitive dfa- culture based techniques used detect rsv viruses as possible children infected rsv assigned group patients infected hmpv rsv even potential drawback however data strongly suggest hmpv common patient population included study furthermore as molecular methods gain widespread use detection many respiratory pathogens continued studies assessing correlation laboratory clinical information warranted our study confirms hmpv significant pathogen particularly young children frequently associated respiratory symptoms resulting hospitalization general clinical manifestations hmpv- rsv associated infections children similar although data suggest subtle differences illness presentation severity our data also suggest underidentification children hmpv may lead inappropriate use antibiotics therefore considering high prevalence hmpv severity hmpv illness ease accuracy detection routine diagnostic testing hmpv implemented increasingly hmpv recognized significant cause disease populations elderly patients immunocompromised cases highlighting groups would likely benefit routine testing hmpv future studies expanded patient populations help determine identifying cases hmpv associated disease allow clinicians anticipate patient clinical course identify cohort patients appropriately decrease use unnecessary antibiotics

human metapneumovirus ( hmpv ) is a recently discovered virus that causes respiratory illness in children that can lead to hospitalization . our study was undertaken to further understand hmpv - associated illness , compare clinical characteristics of hmpv and respiratory syncytial virus ( rsv ) , and establish the utility of routine screening for hmpv . we retrospectively identified hmpv - associated illnesses described among children with respiratory symptoms admitted to a tertiary care center in southeast michigan during the 20062007 respiratory viral season . a convenience sample of 256 nasopharyngeal specimens was subjected to nucleic acid extraction and amplification to identify those specimens positive for hmpv . a medical record review was undertaken to retrieve demographic and clinical data of patients with hmpv , comparing them to rsv - positive patients and patients evaluated for respiratory symptoms who were negative for hmpv and rsv . we found that hmpv was the second most commonly identified virus after rsv . hmpv - positive patients were older than rsv - positive patients . among hmpv - positive patients , pneumonia was diagnosed in 37.5% and bronchiolitis in 31.2% , peribronchial cuffing was present on chest radiographs of 37.5% , antibiotic treatment was used in 81.2% , and admission to the icu was seen in 37.5% . finally , hmpv - positive patients were more likely to have fever than rsv - positive patients or patients negative for hmpv and rsv . we concluded that hmpv is a major pathogen associated with hospitalization of children and with the same severity of illness as rsv but in a slightly older population . because of the apparent prevalence and severity of illness , routine screening should be implemented .

recent whole transcriptome studies revealed three quarters human genome capable transcribed protein coding regions account 2% genome 13 therefore vast majority transcribed sequences encode proteins called non coding rna ncrna accumulating evidence shows non coding rnas play key roles various biological processes imprinting control circuitry controlling pluripotency differentiation immune responses chromosome dynamics 4 ncrnas important protein coding genes cellular functions 5,6 notably growing number long ncrnas lncrnas considered 200 nt length often multiexonic 7 implicated disease etiology 810 it therefore great importance collect lncrna information store information one stop knowledge gateway lncrnas noncode database the development high throughput sequencing methodologies reduced cost rna sequencing result explosive rise number newly identified lncrnas established consensus set 384 066 predicted transcripts 7256 rna seq libraries designated mitranscriptome assembly 11 since revolutionary advancement sequencing methods single molecule long read techniques leads us closer real lncrna transcriptome given sufficient material amplification free sequencing full length cdna molecules noncode collected data literature published since last update includes latest versions several public databases ensembl 13 refseq(14 lncrnadb 15 gencode 16 after removal false redundant lncrnas noncode contains total 527,336 transcripts in addition identification new lncrnas data genetics biochemical properties lncrnas accumulated rapidly papers retrieved pubmed lncrnas found vast majority studied lncrna function especially relationship lncrnas disease 810 in large scale searches single base differences diseased healthy individuals 40% disease related differences show genomic regions outside protein coding genes this implicates non coding regions vital genetic risk factors disease 2 order enable systematic compilation integration information the sources annotations derived literature mining differential lncrna analysis utilizing public rna seq data microarray data mutation analysis public genome wide association study gwas data along ever increasing number lncrnas amount functional study data genome wide conservation information required biologists study mechanisms lncrna actions order explore conservation information lncrnas noncode collected six new mammalian species chimpanzee gorilla orangutan rhesus macaque opossum platypus 17 users browse conserved counterparts human lncrna gene species phylogenetic tree layout similar former iterations noncode 1821 source noncode 2016 includes previous versions noncode collated literature public databases noncoding non coding code non code lncrna lincrna found 6532 new articles since 1 june 2013 last collection date noncode we retrieved newly identified lncrnas annotations supplementary material web site articles together newest data ensembl refseq lncrnadb gencode old versions noncode data literature data processed standard pipeline species all input data processed bed gtf formats based one assembly version example hg38 human mm10 mouse.combination all normalized data files combined together using cuffcompare program cufinks suite 22 eliminating redundancy every new transcript accompanying resources extracted.filtering protein coding rna firstly rna compared coding rna refseq ensembl c transcripts excluded secondly rna filtered coding non coding index cnci 23 program rnas considered non coding cnci kept.information retrieval we assigned transcript name according criterion noncode v4 extracted basic information location 24 exons length assembly sequence source etc.advanced annotation advanced annotations included expression profiles predicted functions conservation disease information etc human expression profiles collected 16 tissues human bodymap 2.0 data ena archive erp000546 eight cell lines geo accession gse30554 mouse data collected six different tissues ena archive erp000591 functions lncrna genes predicted lnc gfp 25 coding non coding co expression network 26,27 based global function predictor.web presence more annotation information added user friendly interface introduced all input data processed bed gtf formats based one assembly version example hg38 human mm10 mouse all normalized data files combined together using cuffcompare program cufinks suite 22 eliminating redundancy firstly rna compared coding rna refseq ensembl c transcripts excluded secondly rna filtered coding non coding index cnci 23 program rnas considered non coding cnci kept we assigned transcript name according criterion noncode v4 extracted basic information location 24 exons length assembly sequence source etc advanced annotations included expression profiles predicted functions conservation disease information etc human expression profiles collected 16 tissues human bodymap 2.0 data ena archive erp000546 eight cell lines geo accession gse30554 mouse data collected six different tissues ena archive erp000591 functions lncrna genes predicted lnc gfp 25 coding non coding co expression network 26,27 based global function predictor more annotation information added user friendly interface introduced noncode contains 527,336 lncrna transcripts 16 species human mouse cow rat chimpanzee gorilla orangutan rhesus macaque opossum platypus chicken zebrafish fruitfly caenorhabditiselegans yeast arabidopsis according definition lncrna genes 18 a total 101,700 86,935 genes generated 167,150 130,558 lncrnas human mouse shown table 1 respectively following nomenclature noncode v4 18 lncrna transcripts genes designated systematically non+ three characters representing species transcript g gene six sequential numbers noncode annotated expression profiles human mouse transcripts genes large number genes annotated predicted functions definitive evidence proven transcription non coding genome produced functional rnas 1 particular lncrnas implicated biological developmental pathological processes acted mechanisms chromatin reprogramming cis regulation enhancers post transcriptional regulation mrna processing 28 lncrnas therefore considered important regulators tissue physiology disease processes including cancer 11 although collected functional interactions ncrnas biomolecules npinter 2931 think also necessary include disease information noncode recent published papers explored proven associations noncode transcripts diseases integrated latest version the noncode assembly also assessed overlaps transcripts unique disease associated single nucleotide polymorphisms snps catalog gwass 32 snp database dbsnp 33 there also lot relational data lncrnas diseases analyzed rna seq microarray data after collecting basic data compared lncrnas noncode retained data overlapped noncode lncrnas noncode 2016 contains 1110 lncrnas related 284 diseases among associations disease related data acquisition pipeline lncrna gene description pages users retrieve related diseases entry also get source information pmid(s reference paper(s there also mutational information retrieved literature gwass dbsnp database compared protein coding genes small rnas e.g. mirnas snornas several reports suggested lncrnas modestly conserved 11 most lncrnas less conserved sequence 34 still many lncrnas conserved genomic loci exonic sequences promoter regions 35 these preserved across multiple species attesting important functional potentials 36 benefiting next generation sequencing technologies ncrnas easily identied via transcript sequencing noncode added six new species mainly multi species rna seq data 37,38 an evolutionary tree 12 commonly studied species human mouse cow rat chicken zebrafish chimpanzee gorilla orangutan rhesus macaque opossum platypus constructed using methods introduced phylononcode 39 each human lncrna gene counterpart listed species retrieved browsing evolutionary tree shown figure 2 the counterpart lncrna computed using ucsc liftover tool 40 brief liftover utilized blastz 41 independent implementation gapped blast algorithm specifically designed aligning two long genomic sequences core algorithm detect homologous regions genomes mapping second species however proper method silico transcript reconstruction ongoing challenge according assessment paul bertone group 40% known transcripts the complexity higher eukaryotic genomes imposes severe limitations transcript recall splice product discrimination likely remain limiting factors analysis current generation rna seq data 42 furthermore multiple amplification steps library preparation complicate quantification expression levels extent given sufficient material amplification free fragmentation free sequencing full length cdna molecules provides direct view rna molecules 12 noncode contacted authors third generation single molecule long read survey human transcriptome paper 12 after analysis single molecule lncrna transcripts included noncode meet quality demands researchers users choose subset considered high quality quality controls include source data literature support database support long read sequencing method support the controls also include selection exon numbers lengths transcripts prediction tools support the web interface return subset according conditions users chose allow users download data noncode 2016 contains 527,336 lncrnas 16 different species compares favorably lncrna databases for example lncipedia human contains 111 685 transcripts 43 lncrnator human mouse fly zebrafish worm yeast contains 34 605 transcripts 44 lncrnawiki human contains 105 255 transcripts 45 mentioned technical limitations imposed short read sequencing lead number computational challenges transcript reconstruction quantification many transcripts automated methods failed identify constituent exons cases exons reported protocols tested often failed assemble exons complete isoforms 42 for example although obtained data mitranscriptome 11 contains 384 066 human lncrnas 7256 rna seq libraries detection precise refseq splicing patterns mitranscriptome 31% fraction annotated genes within entire mitranscriptome 46% although reasonable assume unannotated transcription unique specific lineages low refseq detection rate unusual we therefore made decision noncode would include mitranscriptome data current version future attempt make clear real reason(s perhaps comprehensive construction tool required answer question national high technology research development program 863 program china 2014aa021103 2014aa021502 training program major research plan national natural science foundation china national natural science foundation china 31371320 31401119 chinese academy science strategic project leading science technology xda01020402 funding open access charge national high technology research development program 863 program china 2014aa021103

noncode ( http://www.bioinfo.org/noncode/ ) is an interactive database that aims to present the most complete collection and annotation of non - coding rnas , especially long non - coding rnas ( lncrnas ) . the recently reduced cost of rna sequencing has produced an explosion of newly identified data . revolutionary third - generation sequencing methods have also contributed to more accurate annotations . accumulative experimental data also provides more comprehensive knowledge of lncrna functions . in this update , noncode has added six new species , bringing the total to 16 species altogether . the lncrnas in noncode have increased from 210 831 to 527,336 . for human and mouse , the lncrna numbers are 167,150 and 130,558 , respectively . noncode 2016 has also introduced three important new features : ( i ) conservation annotation ; ( ii ) the relationships between lncrnas and diseases ; and ( iii ) an interface to choose high - quality datasets through predicted scores , literature support and long - read sequencing method support . noncode is also accessible through http://www.noncode.org/.

chemical modification proteins established tool studying structure function regulation class biopolymer moreover recent years great deal effort directed toward modification proteins therapeutic applications traditionally protein conjugation chemistries exploited reactivity surface exposed nucleophilic amino acids cysteine lysine however methods typically result heterogeneous mixtures products complicate biological studies efficacious medicinal applications address concern several strategies developed site specific modification proteins ranging total chemical synthesis usually via native chemical ligation ncl genetic incorporation unnatural amino acids bio orthogonal functional groups two extremes the widely used protein semisynthesis technique extension ncl termed expressed protein ligation epl recombinant protein -thioester building block ligated synthetic molecule equipped 1,2-aminothiol moiety commonly n terminal cys containing peptide formation native peptide bond scheme 1 since inception epl applied wide variety proteins including enzymes ion channels transcription factors transmembrane receptors antibodies reviews see refs 15 16 one basic requirements epl thioester group c terminus recombinant protein this reactive handle introduced exploiting process known protein splicing scheme 1 mediated autoprocessing domain called intein protein splicing typically takes place formation one protein thioester intermediates ultimately resolve form native peptide bond sequences flanking intein referred n- c exteins using appropriate intein mutants it possible intercept intermediates exogenous thiols resulting n extein choice cleaved mutant intein reactive -thioester derivative suitable chemical ligation int int represent n- c intein fragments respectively despite many successes epl approach often suffers low overall efficiency due complications associated generation protein -thioesters in particular fusions inteins varying extents susceptible premature extein cleavage vivo initial purification cell lysates reduces isolated yield intein fusion needed subsequent thiolysis step importantly cleaved extein side product unreactive toward epl separation desired -thioester ligation product often difficult large proteins antibodies compound matters the thiolysis reaction slow inefficient strengthening need develop customized purification regimes involving multiple chromatographic steps isolate desired product complex mixtures collectively technical issues mean considerable investment time resources usually required semisynthetic protein obtained useful quantities ( schematic procedure isolation -thioester derivative protein interest poi using engineered split intein fragments int int epl performed one pot fashion thiolysis split intein immediately elution without need purification ( b sequences npu wt npuaa mutant used split intein column catalytic residues mutated npuaa shown bold linker sequence added immobilization onto solid support underlined sequences numbered according intein sequence alignment shown figure s2 overcome various drawbacks associated intein thiolysis process central epl envisioned alternative strategy based naturally occurring split inteins unlike inteins used standard epl contiguous polypeptides catalyze protein splicing cis split inteins consist two discrete polypeptides herein termed int int upon association catalyze protein splicing trans protein trans splicing pts scheme 1 split inteins two important properties make attractive improved epl strategy first cognate int int pairs often bind tightly one another dissociation constants low nanomolar range reflecting extremely fast rates reported members naturally split dnae inteins cyanobacteria this ability split inteins self associate recently exploited lu et al part traceless protein purification system case using artificially split intein pair the potential utility split inteins epl enhanced remarkable splicing efficiency members family for instance many split dnae inteins half lives splicing reaction less minute compared several hours case cis splicing inteins commonly used epl moreover recent mechanistic investigations indicate ultrafast dnae inteins highly activated n terminal splice junction making superior reagents protein -thioester generation given unique properties split inteins particular ultrafast split dnae inteins conceived integrated protein modification system shown figure 1 split intein association employed purify desired protein complex biological mixtures trigger generation desired protein -thioester epl principle complementation system address major issues attendant standard epl protocol including premature cleavage intein occur case split intein fragment absent cognate partner to implement system designed mutant ultrafast nostoc punctiforme npu split dnae intein suitable efficient -thioester generation specifically mutated catalytic c terminal residue int fragment asn137 first residue c extein cys+1 ala allow efficient build desired splicing intermediates upon exposure n extein npu fusion figure 1 preliminary studies showed mixing n extein npu fusions n extein corresponded various model proteins mutant npu npuaa led highly efficient n extein -thioester formation thiol dependent manner figures s3 s4 importantly low levels intein cleavage i.e. unwanted hydrolysis observed absence thiols thereby fulfilling requirement integrated strategy encouraged results adapted system one pot purification generation c terminally modified proteins taking full advantage strong specific interaction split intein fragments accordingly npuaa peptide immobilized solid support unique cys residue engineered within c extein region figure s5 resulting npuaa column hereafter referred int column evaluated affinity modification resin three test proteins maltose binding protein mbp ubiquitin ub protein histidine phosphatase type 1 phpt1 genetically fused npu expressed escherichia coli case cells lysed soluble fraction loaded onto int column allow binding npu tagged protein immobilized npuaa after brief incubation 5 min rt column extensively washed remove contaminants thiolysis triggered addition buffer containing thiol mercaptoethansulfonate mes three cases desired -thioester protein eluted int column high recovery 7595% high purity 95% determined rp hplc mass spectrometry figure 2 total isolated yields purified protein -thioesters varied one protein another ranged 2.5 mg per l bacterial culture ub mes 40 mg mbp mes the calculated loading capacity int column used experiments 36 mg protein per ml beads 0.12 mols ml higher lower loadings could easily achieved modifying amount npu aa immobilized solid support furthermore showed int column could regenerated reused least 5 times minimal loss capacity figures s11 s12 the utility -thioester derivatives ub mbp phpt1 obtained column demonstrated ligating n terminal cys containing fluorescent peptide cgk(fl give corresponding semisynthetic products excellent yield figure s10 importantly one pot thiolysis ligation reactions could carried allowed us obtain site specifically modified protein directly cell lysates without isolating intermediate thioester figure 3 ( mbp b phpt1 c ub mercaptoethansulfonate mes -thioesters purified one step e. coli cell lysates using int column the purifications monitored coomassie stained sds page analysis top inp input ft column flow w1 3 washes e1 4 elutions bds resin beads rp hplc detection 214 nm esi tof ms analysis eluted fractions bottom left right respectively confirmed identity protein -thioesters indicated high purity one pot purification ligation ubiquitin h cgk(fluorescein)-nh2 peptide cgk(fl ub npu e. coli cell lysates bound int column shown figure 2 removal contaminants extensive washes intein cleavage ligation triggered addition 200 mm mes 1 mm cgk(fl peptide coomassie stained sds page analysis gel fluorescence purification ligation left rp hplc detection 214 440 nm esi tof ms right eluted fractions confirm desired ligated protein obtained one step directly cell lysates ligation yield close 95% quantified rp hplc an attractive feature epl allow preparation site specifically modified proteins virtually traceless manner this contingent ability efficiently generate recombinant protein -thioesters 20 proteinogenic amino acids present c terminus protein the activity split inteins known sensitive identity amino acids immediately flanking splice junction thus eager test generality strategy asked whether could generate -thioesters 20 amino acids using ubiquitin n extein template twenty ub npu fusion proteins individually expressed e. coli purified int column thiol induced cleavage yields solid support determined sds page analysis eluted resin beads fractions levels competing side reactions mainly hydrolysis measured rp hplc esi tof ms the results clearly show amino acids 60% bound proteins recovered mes treatment furthermore 8095% eluted material desired -thioester product the exceptions pro glu recovery 49 50% respectively figure 4 the asn mutant displayed high levels cleavage split intein almost -thioester could isolated due side chain cyclization form c terminal succinimide a second problematic residue asp observed premature cleavage initial binding int resin moreover rp hplc analysis eluted fractions upon thiolysis revealed two species molecular weight desired -thioester these results wholly unexpected asp known cleave prematurely contiguous inteins side chain cyclization -thioesters reported migrate side chain carboxylate yielding mixtures -isomers these minor constraints aside clear studies streamlined epl system compatible majority amino acids present last residue protein interest the 20 mutants protein ub x npu expressed e. coli varying identity c terminal amino acid ub x wt gly proteinogenic amino acids thiol induced cleavage yields int column calculated sds page analysis eluted fractions left resin beads ratios -thioester vs side products determined rp hplc esi tof ms analysis eluted fractions the major competing reaction amino acids hydrolysis exception asn succinimide form isolated instead * protein semisynthesis frequently requires preparation protein fragments often poorly behaved need purified presence strong chaotropic agents we first confirmed model protein ub npu could bind int column presence 2 4 urea corresponding ub -thioester could generated similar yields obtained native conditions figure s14 this consistent previous study demonstrated npu dnae could splice presence high concentration denaturants we turned challenging target namely fragment histone h2b residues 1116 polypeptide prone aggregation difficult generate -thioester derivative using standard epl procedures we expressed human histone h2b(1116 fused npu e. coli extracted inclusion bodies 6 urea diluted 2 urea prior loading int intein column the incubation performed 3 h ph 6.0 maximize binding avoiding premature cleavage hydrolysis the ph raised 7.2 thiolysis carried 36 h rt note presence denaturant slows thiolysis rate using conditions hh2b(1116)-mes obtained excellent purity 90% rp hplc isolated yield 20 mg per l culture this represents significant improvement previous protocols afford less protein 4 mg per l culture require use multiple chromatographic purification steps including rp hplc importantly hh2b(1116)-mes thioester obtained int column could directly used epl reactions without purification accordingly successfully ligated protein hh2b(117125 peptide containing acetylated lys position 120 yield semisynthetic hh2b k120ac figure 5 coomassie stained sds page analysis hh2b(1116 -thioester generation presence 2 urea sup cell lysate supernatant trit 1% triton wash inclusion bodies inp solubilized inclusion bodies used input int column e1e3 collected 18 h incubation mes e4e6 additional 18 h. e1e6 pooled concentrated 150 ligated peptide h cvtk(ac)ytsak oh 1 mm 3 h rt ( b rp hplc left ligation reaction mixture ms right ligated hh2b k120ac product finally tested streamlined epl methodology modification monoclonal antibody the site specific modification antibodies become highly desirable area biopharmaceuticals diagnostics currently commercially utilized methods conjugate cargo antibodies relatively nonspecific result polydisperse mixtures may vary batch batch since heterogeneity adversely affect efficacy safety conjugate indeed protein semisynthesis via standard epl pts recently used generate monoclonal antibody conjugates full activity given eager see whether streamlined epl process could used facile generation antibody conjugates as model immunoglobulin igg studies used antibody dec205 receptor c type lectin found predominantly dendritic cells accordingly designed construct npu fused c terminus heavy chain antibody dec205-npu initial expression tests dec205-npu 293 cells resulted low levels antibody secreted figure 6a we observed previously identity int effect expression levels fusions consequently asked whether could obtain higher levels secreted dec205-int varying identity intein n fragment several new dec205-int constructs generated int corresponded n fragment series ultrafast split dnae inteins namely ava csp cra cwa mcht oli ter also tested npu mutant c noncatalytic cysteines cys28 cys59 mutated ser determine whether residues influence secretion maturation igg tetramer figure 6a importantly int fragments set cross react c fragment npu without significant loss splicing efficiency thus int column already hand compatible dec205-int fusions the contiguous mxe gyra intein also fused dec205 test whether use n intein fragments negatively affected expression levels compared full length intein an expression screen dec205-int library performed 293 cells revealing ava csp fusions reproducibly exhibited higher expression levels n inteins former best indeed expression levels dec ava construct least good dec gyra construct based the dec205-ava fusion chosen purified int column analogous manner soluble proteins described figure 6b elutions column contained thiolyzed dec205 subsequently ligated cgk(fl peptide figure 6c size exclusion chromatography sec coupled multiple angle light scattering mals analysis confirmed antibody retained tetrameric folded state thiolysis ligation figure 6d we also performed ms analysis deglycosylated fully reduced antibody confirmed formation stable nonreducible amide bond dec205 heavy chain fluorescent peptide 75% yield figure 6e importantly demonstrated semisynthetic dec cgk(fl retains ability bind dec205 receptor extent control dec205 previously shown fully functional vivo(46 figure 7 binding dec cgk(fl dec205 receptor could monitored flow cytometry using antimouse igg secondary antibody figure 7 also site specifically incorporated fluorescein figure s20 purification monoclonal antibody -thioester using split intein site specific modification ( test expression dec205 genetically fused contiguous mxe gyra intein different split dnae inteins c terminus heavy chain hc western blot 293 cell supernatants several dec205-int fusions using antibody mouse igg ( c elution fractions containing dec205-mes concentrated 20 ligated cgk(fl fluorescent peptide 1 mm 48 h rt ( sec mals analysis ligated antibody showing retains tetrameric structure thiolysis ligation mw 151 kda mw calcd 148 kda ( e esi tof ms analysis degycosylated fully reduced hc ligation showing 75% hc labeled ( dose dependent binding dec205-cgk(fl cho cells expressing mouse dec205 receptor monitored flow cytometry using pe labeled -mouse igg binding control cho neo cells express receptor shown gray ( b using control -dec205 antibody we shown split dnae inteins engineered efficient generation isolation protein -thioesters furthermore strategy seamlessly integrated epl one pot purification ligations performed without isolation -thioester intermediates we note however ligation step i.e. ncl still requires use high concentrations high n terminal cysteine peptide efficient reactions strong specific interaction two intein fragments facilitates purification protein -thioesters variety conditions including strong denaturants isolation products directly cell lysates proceeds significantly faster via many mainstream epl strategies often take multiple days several intermediate enrichment purification steps additionally split intein fusions usually associated low levels expression show certain dnae int fragments express levels commonly used contiguous inteins fused monoclonal ab moreover absence premature cleavage events allowed us generate semisynthetic proteins h2b k120ac far superior yields previously established protocols importantly shown utility methodology modification complex biomolecules igg thus streamlined epl via split dnae intein column provide efficient route site specifically modified proteins basic biochemical research well translational applications npu aa cys ome peptide 0.5 mols per ml resin dissolved 2 column volumes cv coupling buffer 50 mm trishcl ph 8.5 250 l 125 l resin treated 25 mm tcep 1 stock 15 min the peptide solution added 1 cv agarose sulfolink resin pierce loading 18.4 mol iodoacetyl groups ml resin small fritted column incubated 15 min nutator followed 30 min standing rt the column flow collected column washed twice 2 cv coupling buffer unreacted iodoacetyl groups resin blocked treatment 50 mm h cys ome coupling buffer 15 min nutator followed 30 min standing rt column washed twice 1 cv coupling buffer 2 cv 1 nacl finally 2 cv water int columns stored storage buffer 100 mm phosphate 150 mm nacl 1 mm edta 0.05% nan3 ph 7.2 4 c 2 weeks e. coli bl21(de3 cells transformed desired poi npu plasmid grown 1 l lb containing 100 g ml ampicillin 37 c od600 0.6 after harvesting cells centrifugation 10 500 rcf 30 min cell pellets transferred 50 ml conical tubes 20 ml high salt binding buffer 100 mm phosphate 500 mm nacl 1 mm edta 1 mm tcep ph 7.2 complete protease inhibitor cocktail roche stored 80 c resuspended cells lysed sonication soluble fraction recovered centrifugation 17 000 rcf 10 min onto 62.5 l int column 300 l soluble fraction loaded incubated rt 5 min after incubation flow collected column washed 300 l high salt binding buffer 300 l wash buffer 100 mm phosphate 300 mm nacl 1 mm edta 1 mm tcep ph 7.2 300 l binding buffer 100 mm phosphate 150 mm nacl 1 mm edta 1 mm tcep ph 7.2 column capped incubated 150 l elution buffer 100 mm phosphate 150 mm nacl 200 mm mes 10 mm tcep 1 mm edta ph 7.2 18 h. flow collected column washed three times 150 l elution buffer onto 62.5 l int column 300 l cell lysate containing ub npu loaded incubated described the column washed 300 l high salt binding buffer 300 l wash buffer 300 l binding buffer the column capped incubated 75 l epl buffer 100 mm phosphate 150 mm nacl 200 mm mes 50 mm mpaa 1 mm edta 10 mm tcep ph 7.9 containing 1 mm cgk(fl peptide 18 h. flow collected column washed three times 75 l elution buffer gels first imaged using green fluorescence channel ge imagequant las 4010 imager coomassie stained t cells transiently cotransfected antimouse dec205-lc antimouse dec205-hc ava using lipofectamine 2000 invitrogen according manufacturer instructions after 4 days incubation 37 c 5% co2 cell supernatants harvested spun 2000 rcf 20 min 4 c filtered 0.22 filter supplemented complete protease inhbitors for typical purification 50 ml dec205-ava transfected cell supernatants concentrated final volume 5 ml exchanged binding buffer resulting ab solution input ) was applied int column 300 l beads loading 1.8 mol npuc peptide ml incubated rt 30 min column flow collected column washed three times 3 cv wash buffer 3 cv binding buffer the column capped incubated 3 cv ab elution buffer 100 mm phosphate 150 mm nacl 200 mm mes 1 mm tcep 1 mm edta ph 7.2 20 h. column flow collected column washed three times 3 cv ab elution buffer ligation initiated addition 1 mm cgk(fl peptide 1 mm tcep adjusting ph 7.58.0 the reaction incubated dark rt 48 h monitored sds page imaged using fluorescence scanner coomassie staining once reaction completed ligated ab diluted 200500 l dialyzed 100 mm phosphate 150 mm nacl 1 mm edta 1 mm tcep ph 7.2

chemically modified proteins are invaluable tools for studying the molecular details of biological processes , and they also hold great potential as new therapeutic agents . several methods have been developed for the site - specific modification of proteins , one of the most widely used being expressed protein ligation ( epl ) in which a recombinant -thioester is ligated to an n - terminal cys - containing peptide . despite the widespread use of epl , the generation and isolation of the required recombinant protein -thioesters remain challenging . we describe here a new method for the preparation and purification of recombinant protein -thioesters using engineered versions of naturally split dnae inteins . this family of autoprocessing enzymes is closely related to the inteins currently used for protein -thioester generation , but they feature faster kinetics and are split into two inactive polypeptides that need to associate to become active . taking advantage of the strong affinity between the two split intein fragments , we devised a streamlined procedure for the purification and generation of protein -thioesters from cell lysates and applied this strategy for the semisynthesis of a variety of proteins including an acetylated histone and a site - specifically modified monoclonal antibody .

ameloblastoma benign locally aggressive neoplasm odontogenic epithelium.1 accounting 11% odontogenic tumors ameloblastoma common odontogenic neoplasm affecting jaws yet accounts 1% tumors maxilla mandible.24 average age patients presenting ameloblastoma 36 years men women equally affected.5 ameloblastomas classified solid multicystic intraosseous unicystic peripheral subtypes.6,7 radiographic appearance ameloblastoma dentigerous cysts odontogenic keratocysts similar reason biopsy recommended obtain precise diagnosis ameloblastoma.8 although benign tumor ameloblastoma aggressive myeline nature growth conservative treatment associated high rate recurrence 50%90% primary resection benign tumor therefore considered predictable curative form therapy.711 unfortunately treatment leaves patient defect affected region jaw a multi disciplinary approach needed patient complete rehabilitation including bone grafting planned placement implant prosthetic work sometimes orthodontist role multidisciplinary approach create optimum occlusal relationship sufficient space allow successful reconstruction affected region jaw a 31-year old caucasian female referred graduate orthodontic clinic aristotle university thessaloniki following mandibulectomy immediate replacement removed bone autologous calvarial bone graft purposes evaluation treatment occlusal disturbances the patient medical dental history referred tumor histologically classified follicular ameloblastoma according history the tumor located posterior right mandibular region encompassing three teeth specifically first second premolars first molar the first author performed partial mandibular resection immediate reconstruction utilizing bone graft taken patient calvaria extraoral examination revealed orthognathic profile without facial asymmetry lips competent rest figure 2 intraoral examination revealed amalgam restorations maxillary mandibular second molars left first maxillary premolar left mandibular first molar clinical examination indicated satisfactory dental hygiene periodontal condition absence bleeding probing significant pocket depths an analysis dental casts revealed class malocclusion characterized 3 mm overjet 3 mm overbite moderate crowding anterior region dental arches 3 mm crowding maxillary dental arch 6 mm mandibular one respectively labially positioned mandibular right canine 2 mm deviation lower dental arch midline right moderate bolton tooth size discrepancy mandibular anterior relatively larger maxillary ones edentulous space due aforementioned surgical procedure figure 2 analysis cephalometric radiograph indicated harmonious sagittal vertical skeletal relationships sna 83 snb 80 facial angle 90 anb 3 individual anb 3.5 figure 3 the problem list patient included presence class malocclusion characterized condition moderate crowding anterior region dental arches labially positioned mandibular right canine mild deviation mandibular dental arch midline right moderate tooth size discrepancy edentulous space right posterior area mandible due extractions right premolars first molar the patient chief complaint referred need optimum prosthetic rehabilitation desire improvement dental esthetics a comprehensive orthodontic treatment aiming correct mentioned occlusal problems proposed accepted patient the placement two dental implants took place first author prior orthodontic treatment according orthodontic treatment plan a setup patient dental casts made facilitate accurate determination exact positions two implants following placement lingual arch banded lower dental arch anchorage space preservation fixed appliances .018 x .022 roth bonded maxillary teeth order level align dental arch following placement bracket mandibular right canine uprighted brought dental arch means loop then fixed appliances bonded remaining mandibular teeth reproximation took place lower incisors the fixed appliances removed alignment leveling dental arches crowding alleviation correction dental arch midlines essix maxillary mandibular splints used retention placement prostheses figure 4 new essix mandibular retainer delivered following end prosthetic work due patient advanced stage pregnancy 9 month relevant inconvenience stage possible place bonded lingual retainer lower anterior teeth originally planned superimposition initial final tracings lateral cephalometric x rays indicated slight labial proclination upper lower incisors occurred post treatment figure 5 prosthodontic rehabilitation partially edentulous right mandibular dental arch region achieved placement two implants two crowns respectively figure 6 a 31-year old caucasian female referred graduate orthodontic clinic aristotle university thessaloniki following mandibulectomy immediate replacement removed bone autologous calvarial bone graft purposes evaluation treatment occlusal disturbances the patient medical dental history referred tumor histologically classified follicular ameloblastoma according history the tumor located posterior right mandibular region encompassing three teeth specifically first second premolars first molar the first author performed partial mandibular resection immediate reconstruction utilizing bone graft taken patient calvaria extraoral examination revealed orthognathic profile without facial asymmetry lips competent rest figure 2 intraoral examination revealed amalgam restorations maxillary mandibular second molars left first maxillary premolar left mandibular first molar clinical examination indicated satisfactory dental hygiene periodontal condition absence bleeding probing significant pocket depths an analysis dental casts revealed class malocclusion characterized 3 mm overjet 3 mm overbite moderate crowding anterior region dental arches 3 mm crowding maxillary dental arch 6 mm mandibular one respectively labially positioned mandibular right canine 2 mm deviation lower dental arch midline right moderate bolton tooth size discrepancy mandibular anterior relatively larger maxillary ones edentulous space due aforementioned surgical procedure figure 2 analysis cephalometric radiograph indicated harmonious sagittal vertical skeletal relationships sna 83 snb 80 facial angle 90 anb 3 individual anb 3.5 figure 3 the problem list patient included presence class malocclusion characterized condition moderate crowding anterior region dental arches labially positioned mandibular right canine mild deviation mandibular dental arch midline right moderate tooth size discrepancy edentulous space right posterior area mandible due extractions right premolars first molar the patient chief complaint referred need optimum prosthetic rehabilitation desire improvement dental esthetics a comprehensive orthodontic treatment aiming correct mentioned occlusal problems proposed accepted patient the placement two dental implants took place first author prior orthodontic treatment according orthodontic treatment plan a setup patient dental casts made facilitate accurate determination exact positions two implants following placement lingual arch fixed appliances .018 x .022 roth bonded maxillary teeth order level align dental arch following placement bracket mandibular right canine uprighted brought dental arch means loop then fixed appliances bonded remaining mandibular teeth reproximation took place lower incisors the fixed appliances removed alignment leveling dental arches crowding alleviation correction dental arch midlines essix maxillary mandibular splints used retention placement prostheses figure 4 new essix mandibular retainer delivered following end prosthetic work due patient advanced stage pregnancy 9 month relevant inconvenience stage possible place bonded lingual retainer lower anterior teeth originally planned superimposition initial final tracings lateral cephalometric x rays indicated slight labial proclination upper lower incisors occurred post treatment figure 5 prosthodontic rehabilitation partially edentulous right mandibular dental arch region achieved placement two implants two crowns respectively figure 6 ameloblastoma benign odontogenic tumor arising residual epithelial components tooth development it slow growing locally aggressive tumor capable causing facial deformity high recurrence rate due capacity infiltrate trabecular bone bone grafts replace surgically removed bone autologous bone grafting desirable it typically harvested intraoral sources e.g. chin extraoral sources e.g. iliac crest fibula calvarial bone the commonly used graft material alveolar ridge reconstruction free autogenous iliac bone.12 case however autologous calvarial bone grafts used reconstruct missing mandibular bone following surgical resection tumor removal three teeth region the advantages calvarial bone grafting include good integration absence pain donor site visible scar these advantages however applicable case thin calvaria bone thickness less 5 mm.12 recent reports use calvarial bone grafting reconstruction subsequent placement dental implants presented good clinical outcomes low rates graft resorption high implant survival rates.1316 results studies showed calvarial bone grafting appears less prone resorption iliac grafts case this goal could achieved placement two implants bridge replacing three teeth however treatment plan would addressed patient chief complaint would result optimum functionality esthetics accordingly placement two implants decided relation orthodontic treatment plan aiming optimum result the two implants placed posterior region edentulous area hence replacing two missing teeth extra space used correct crowding improve dental occlusion the outcome interdisciplinary approach satisfactory restoration occlusal function esthetics a multidisciplinary approach including oral surgery orthodontics prosthodontics able provide patient diagnosed follicular ameloblastoma right mandibular region satisfactory occlusion following partial mandibular resection removal adjacent teeth affected site

this case report describes the combined surgical , orthodontic and prosthodontic rehabilitation of an adult female patient with a previous history of follicular ameloblastoma , which was treated through partial mandibulectomy and an immediate replacement of missing bone with an autologous calvarial bone graft . orthodontic treatment was undertaken in order to restore occlusal disturbances and obtain sufficient space for two dental implants and an optimum prosthodontic rehabilitation .

laparoscopic removal cervical stump following supra cervical subtotal hysterectomy first described nezhat et al concluded cervical stump could removed laparoscopically experienced surgeon the advantages laparoscopic approach included possible stump adhesiolysis providing adequate postoperative vault support assessment pelvic lymph nodes the 43-year old presented history persistent p v discharge occasional post coital bleeding she undergone subtotal hysterectomy 1994 due postpartum hemorrhage following normal delivery a colposcopic biopsy done january 2009 reported severe dysplasia cervix human papillomavirus hpv effect crypt extension there strong family history cancer cervix mother succumbed disease on general examination fair general condition well built well nourished adequate hydration the hemoglobin 13.3 g dl blood sugar 5.3 mmols l urea electrolytes normal an initial diagnosis abnormal pap smear entertained patient opted laparoscopic trachelectomy option laparotomy discussing options there dense adhesions pouch douglas involving bowel cervical stump the pelvic lymph nodes clearly visualized intracervical methylene blue injection appear enlarged gentle adhesiolysis undertaken using sharp dissection bipolar cautery harmonic scalpel the vaginal vault subsequently opened ceramic cup clermont ferrand elevator a cystoscopy retrograde ureteral catheterization confirm integrity bladder ureters undertaken the cervical stump laparoscopic trachelectomy one week follow patient well a postoperative intravenous urogram ivu confirmed ureters bladder intact subtotal hysterectomy developed procedure 1990s regarded safe option total abdominal hysterectomy management benign uterine conditions obstetrics due severe postpartum hemorrhage okaro et al assessment long term outcomes laparoscopic supracervical hysterectomy analyzed case records 70 consecutive women undergoing procedure of 24.3% 17 cases reported symptoms related cervical stump within 14 months original surgery in series 14 patients underwent laparoscopic trachelectomy one laparoscopic adhesiolysis two underwent laparotomy trachelectomy due dense bowel adhesions cervical stump histologically stumps showed endometriosis 23.5% mild dysplasia 7.6% patients case patient presented persistent p v discharge occasional post coital bleeding the subsequent pap smears abnormal retrospective 41 patients undergoing laparoscopic subtotal hysterectomy van der stege et al noted 98% patients satisfied procedure 10% monthly spotting they concluded although laparoscopic hysterectomy benign diseases satisfactory procedure special attention paid careful management cervical stump hilger et al reviewed indications 310 trachelectomies performed mayo clinic 1974 2003 they included stump prolapse 4% fibroid mass 1% cervical dysplasia 6% carcinoma situ 5% irregular bleeding 2% cervicitis 53% the complications following vaginal trachelectomies encountered 80% procedures 37% abdominal procedure report cervical stump confirmed carcinoma situ

a 43-year - old , who underwent a subtotal hysterectomy for postpartum hemorrhage following a normal delivery , 10 years ago , presented with a history of persistent vaginal discharge and post - coital bleeding . a pap smear reported moderate dysplasia , and a subsequent colposcopic biopsy reported severe dysplasia with crypt extension . the patient underwent a laparoscopic trachelectomy , and histology of the stump reported cervical squamous carcinoma in situ , with no microinvasion .

biological membranes mainly composed phospholipids sphingolipids cholesterol membrane associated proteins molecules nonhomogeneously distributed membranes rearrange leading formation membrane domains highly differentiated molecular compositions supramolecular architectures stabilized lateral interactions among membrane components although glycosphingolipids gsls originally thought structural components plasma membranes several experiments suggested gsls involved regulation numerous cellular functions the membrane lipid bilayer stable structure constituting physical boundary intra- extracellular environments gsls expressed surface cellular membranes based physicochemical properties especially many hydroxyl acetamide groups act hydrogen bond donors acceptors gsls form clusters cis interactions there general consensus roles played ceramide moiety gsls promoting formation stabilization membrane lipid domains in addition ceramide also shown involved gsl mediated functions several biological activities 3 4 we recently showed long fatty acid chains ceramide c24:0 c24:1 responsible direct connection lactosylceramide laccer cdw17 palmitoylated signal transducer molecules moreover phosphorylated product sphingosine sphingosine-1-phosphate s1p shown important immunological especially inflammatory reactions 4 6 this review describes role fatty acid chains ceramide gsl mediated outside signaling promoting gsl enriched domain mediated cellular functions well activities s1p inflammatory reactions keratinocytes human the studied gsl enriched domains membrane lipid microdomains called lipid rafts defined gsl- cholesterol rich nature enrichment gpi anchored proteins membrane anchored signaling molecules cytoskeletal association 7 8 as shown artificial membrane models gsls tend form clusters cluster formation confirmed intact cells immunoelectron microscopy 1012 the gsl enriched microdomains plasma membranes diameter 50100 nm include signal transducer molecules src family kinases 11 12 gsls contain saturated fatty acid chains higher transition temperatures show ordered less fluid liquid phase cholesterol composed highly hydrophobic sterol ring system 3-hydroxy moiety hydrophilic part molecule small cholesterol sterol ring system ceramide moiety sphingolipids thought interact via hydrogen bonds hydrophobic van der waal interactions in addition hydrophilic interactions sugar moieties gsls promote lateral association gsls cholesterol phospholipids tend loosely packaged bilayers resulting formation liquid disordered membranes allow rapid lateral rotational movement lipids these interactions result separation gsl- cholesterol enriched lipid microdomains phospholipids cell membrane formation distinct domains electron microscopy using labeled anti gsl antibodies revealed gsl clusters surface glycosphingolipid phosphatidylcholine pc liposomes even absence sphingomyelin sm cholesterol the anti laccer mabs t5a7 huly m13 recognized laccer human neutrophils t5a7 recognized laccer mouse neutrophils interestingly huly m13 t5a7 used immunoprecipitation suggesting difference binding and/or cluster formation huly m13 t5a7 laccer enriched microdomains indeed stimulated emission depletion sted superresolution microscopy showed t5a7 huly m13 bind different regions laccer dioleoylphosphatidylcholine dopc liposomes figure 1 laccer enriched microdomains composed laccer sm phospholipids cholesterol surface plasmon resonance analysis showed reduction laccer content dopc cholesterol laccer sm lipid layer markedly decreased huly m13 t5a7 binding laccer suggesting content laccer laccer enriched microdomains affects binding avidity huly m13 laccer in contrast molecular species pc including dopc dipalmitoylphosphatidylcholine dppc palmitoyl oleoyl phosphatidylcholine popc affect binding avidity huly m13 laccer coated plastic wells lactose inhibited binding huly m13 laccer dopc liposome coated dopc laccer mixture coated plastic wells suggesting huly m13 binds laccer clusters laccer enriched microdomains in contrast binding avidity t5a7 laccer coated plastic wells much weaker binding avidity dopc laccer- popc laccer- dppc laccer mixture coated wells suggesting binding t5a7 laccer affected pc ability lactose inhibit binding t5a7 dopc laccer liposome coated plastic wells similar ability inhibit binding huly m13 in contrast lactose inhibition t5a7 binding dopc laccer mixture coated plastic wells significantly lower inhibition huly m13 binding suggesting t5a7 recognizes pc enhanced three dimensional structure laccer clusters thus huly m13 may bind core region lactose clusters laccer enriched domains t5a7 binds dispersed laccer clusters phase boundary regions microdomains these findings suggest specificities antibodies gsls dependent organizations gsls molecules surrounding gsl enriched domains disorders degradation gsls sometimes cause human diseases 18 19 degradation molecules constitutively degraded suitable catabolic enzymes activities lysosomal enzymes impaired degradation able proceed normally undegraded molecules accumulate organelle intracellular membranes causing several metabolism diseases for instance genetic disorder glucocerebrosidase gba ec 3.2.1.45 gaucher disease results accumulation glccer deacetylated form glucosylsphingosine caused abnormality gba gaucher disease multisystem disorder whose features include peripheral blood cytopenias hepatosplenomegaly bone disease neurological manifestations cases the form intravenous enzyme replacement therapy 1990s developed resulted dramatic improvements haematological visceral disease recognition complications including multiple myeloma parkinson disease challenged traditional macrophage centric view pathophysiology disorder however pathways enzyme deficiency results clinical manifestations disorder remain obscure spinal cords amyotrophic lateral sclerosis als patients levels gm1 gm3 laccer glccer galcer ceramide significantly elevated furthermore glucocerebrosidase-1 glucocerebrosidase-2 hexosaminidase galactosylceramidase -galactosidase -galactosidase activities also elevated patients inhibition glucosylceramide synthesis accelerated disease course als model mice whereas infusion exogenous gm3 significantly slowed onset paralysis increased survival these observations suggest gsls metabolism likely important participants pathogenesis als further studies gsl metabolism pathways gsl related disease serve advance understanding associated disorders over last 30 years many studies indicated gsls expressed cell surface may act binding sites microorganisms the binding avidities microorganisms several types gsl 2427 suggest gsls involved host pathogen interactions indeed microorganisms shown recognize enter host cells via gsl enriched membrane microdomains cells among gsls laccer well described bind several kinds microorganisms including viruses fungi for instance candida albicans specifically bind laccer though binding -1,6-long glucosyl side chain branched -1,3-glucan laccer enriched domains 26 29 it also well known microorganisms derived toxins shiga toxin specifically bind gsls 3032 furthermore sphingolipid metabolite ceramide demonstrated play crucial role pulmonary infection inflammation .ceramide degraded product gsls sphingomyelin reported form ceramide rich membrane platforms involve uptake several microorganisms including pseudomonas aeruginosa abnormal amounts enzymes involved synthesis ceramide demonstrated emphysematic smokers patients severe sepsis gsls reported interact membrane proteins modulate properties proteins 2 25 in addition certain proteins including glycosylphosphatidylinositol- gpi- anchored palmitoylated proteins tend enter gsl enriched membrane microdomains however mechanism gsls interact proteins mediate outside signaling unclear the ceramide moiety consists long chain base linked fatty acid chain sphingosine containing c18 carbons 2s,3r,4e)-2-amino-1,3-dihydroxy octadecene generally main structure mammals structure containing 20 carbons relatively abundant neurons ceramide synthesized ceramide synthases cers 16 uses restricted subset fatty acyl coas n acylation sphingoid long chain base the expression levels genes encoding cers tissue specific suggesting molecular varieties expression patterns gsls associated functions cells although gsl enriched microdomains implicated number important membrane events 2 38 39 molecular mechanisms responsible gsl mediated cell functions still unclear one main issues centers around association gsls signal transducer molecules localized cytosolic side laccer along src family kinase lyn forms lipid microdomains plasma membranes human neutrophils involved several cellular functions including chemotaxis phagocytosis superoxide generation highly dependent lyn 16 29 38 hl-60 cells differentiated neutrophilic lineage cells dmso hl-60 found acquire superoxide generating activity laccer despite expression laccer plasma membranes most laccer lyn recovered microdomain fractions neutrophils hl-60 cells lipidomics analysis revealed laccer neutrophil plasma membrane mainly composed molecular species containing c16:0 c24:1 c24:0 fatty acid chains whereas 70% laccer plasma membranes hl-60 cells contained c16:0 fatty acid chains 14% c24:1 c24:0 lyn immunoprecipitated anti laccer antibody neutrophils hl-60 cells importantly lyn coimmunoprecipitated anti laccer antibody detergent resistant membrane drm fraction plasma membranes c24:0 c24:1 c16:0 c22:0 laccer loaded hl-60 cells anti laccer antibody induced superoxide generation hl-60 cells loaded c24:0-laccer c16:0-laccer lyn colocalized laccer enriched domains hl-60 cells loaded c24:0-laccer c16:0-laccer these results suggested c24 fatty acid chain laccer indispensable connecting lyn laccer enriched microdomains knockdown lyn molecules human lyn specific short interfering rna sirna hl-60 cells completely abolished effects c24:1-laccer loading function suggesting lyn crucial c24-laccer mediated neutrophil function experiments using azide photoactivatable tritium labeled c24- c16-laccer revealed c24- c16-laccer directly associated lyn heterotrimeric g protein subunit gi these results confirm specific direct interaction c24-laccer signal transduction molecules lyn gi associated cytoplasmic layer via palmitic acid chains figure 2 laccer species long fatty acids indispensable lyn coupled laccer enriched membrane microdomain mediated neutrophil functions gpi anchored proteins composed glycerol phospholipids c24 fatty acid chains suggesting gpi anchored proteins able form large clusters directly connect signal transduction molecules fatty acid chains mediate cell functions gpi anchored proteins require signal transduction molecule coupled transmembrane proteins gsl enriched domains laccer enriched domains the epidermis consists single layer proliferating undifferentiated keratinocytes stratum basale several superficial layers stratum spinosum stratum granulosum sg form stratum corneum sc the sc acts air liquid interface barrier avoid drying tissues contact air ceramide main component sc important water retention permeability barrier functions sc sphingosine phosphorylated sphingosine kinase form s1p molecule involved wide range cellular functions including growth differentiation survival chemotaxis angiogenesis embryogenesis various types cells 44 45 s1p shown inhibit keratinocyte proliferation promote corneocyte differentiation chemoattract keratinocytes roles s1p skin immunological functions demonstrated mouse models 45 4751 mice good experimental tool choice majority immunologists study immune responses mice provided considerable insight human immune system function however significant differences immunological reactions mice human little known however role ceramide metabolites immunological functions differentiating keratinocytes a neutral cdase pseudomonas aeruginosa an17 pacdase isolated patient atopic dermatitis ad shown require detergents hydrolyze ceramide staphylococcus aureus derived lipids consist primarily cardiolipin phosphatidylglycerol enhanced pacdase hydrolysis normal ceramide human skin specific omega hydroxyacyl ceramide absence detergents a three dimensionally cultured human primary keratinocyte 3d keratinocyte culture system utilized simulate epidermal differentiation air liquid interface resulting generation basal spinous granular layers sc latter displaying permeability barrier functions treatment 3d keratinocytes pacdase water soluble stimulants keratinocytes including trypsin dermatophagoides pteronyssinus class 1 allergen der p1 dermatophagoides farinae allergen der f1 effect expression genes dna microarray analysis indicating sc 3d keratinocyte culture acts permeability barrier triton x-100 detergent reduces permeability barrier functions thereby moderately increasing transepidermal water loss production erythema human skin presence 0.1% triton x-100 pacdase markedly enhanced tnf- mrna expression 3d keratinocytes increase observed cells treated triton x-100 alone tnf- mrna expression enhanced heat inactivated mutant pacdase suggesting ceramide metabolites induce tnf- mrna expression keratinocytes tnf- critical cytokine several dermatological diseases secreted keratinocytes shown involved progression atopic dermatitis ad among metabolites ceramide s1p synthesized sphingosine sphingosine kinase sphk stimulates 3d keratinocytes specific receptors both specific sphk inhibitor cas 1177741 83 1 s1p receptor antagonist vpc 23019 suppressed pacdase induced expression tnf- mrna 3d keratinocytes s1p generally considered stimulate cells plasma membrane g protein coupled receptors example s1p1s1p5 however vpc2301 competitive antagonist s1p1 s1p3 receptors inhibited pacdase enhanced gene expression tnf- also endothelin-1 il-8 thus s1p induced production inflammatory mediators mediated s1p receptors human primary keratinocytes 3d culture system cdna microarray analysis showed s1p strongly upregulated expression endothelin-1 cxcl1 tnf- -defensin 5 il-8 cxcl2 interferon regulatory factor 1 gadd45 gamma il-23 subunit mrnas il-8 cxcl1 cxcl2 reported upregulated lesional skin patients ad psoriasis s1p also enhanced expression claudin-4 mrna observed layers psoriatic normal epidermis tnf- induce production endothelin-1 il-8 human keratinocytes 66 67 infliximab chimeric igg1 monoclonal antibody human tnf- inhibits tnf--mediated production il-8 keratinocytes the nf-b inhibitor curcumin inhibited pacdase induced expression il-8 endothelin-1 mrnas tnf- mrna therefore likely s1p induces tnf- production release 3d keratinocytes via s1p receptors resulting tnf- induction cytokine production nf-b mediated signal transduction figure 3 tnf- critical cytokine psoriatic immunopathology development effective strategy required counteract effects infliximab used treat patients plaque psoriasis psoriatic arthritis pustular psoriasis excluding localized type psoriatic erythroderma 71 72 downregulates antiapoptotic proteins regressing psoriatic skin the effects infliximab also evaluated inflammatory dermatoses systemic diseases involving skin pityriasis rubra pilaris pyoderma gangrenosum cutaneous sarcoidosis ad characterized marked reduction ceramides sc lesional nonlesional forearms 74 75 increased activities enzymes ceramidase cdase the metabolic conversion ceramide s1p found protect keratinocytes uvb induced ceramide mediated apoptosis histamine h1-receptor blockers used treat types itch resulting serious skin diseases ad well renal liver diseases however often lack efficacy chronic itch profound clinical problem decreases quality life nerve density epidermis partly involved itch sensitization pruritic skin diseases ad endothelin-1 shown elicit itch humans 8082 the molecular pathways contribute transduction itch responses endothelin-1 require either plc3 trpv1 neurons mediate histamine- serotonin induced itch responses respectively several reports described roles ceramide metabolites immunological inflammatory diseases 8488 however physiological roles gsl enriched microdomains largely undetermined although much known organization functions laccer enriched microdomains 24 89 90 analogous patterns gsls motifs pamps result generation autoantibodies gsls inducing severe autoimmune inflammatory diseases antibodies neuronal tissues involved immune mediated neurological disorders expression several antibodies found correlate pathophysiology diseases 92 93 therefore elucidation organization structural specificities based interactions gsls surrounding molecules important understanding physiological functions gls enriched microdomains related diseases

glycosphingolipids ( gsls ) are composed of hydrophobic ceramide and hydrophilic sugar chains . gsls cluster to form membrane microdomains ( lipid rafts ) on plasma membranes , along with several kinds of transducer molecules , including src family kinases and small g proteins . however , gsl - mediated biological functions remain unclear . lactosylceramide ( laccer , cdw17 ) is highly expressed on the plasma membranes of human phagocytes and mediates several immunological and inflammatory reactions , including phagocytosis , chemotaxis , and superoxide generation . laccer forms membrane microdomains with the src family tyrosine kinase lyn and the gi subunit of heterotrimeric g proteins . the very long fatty acids c24:0 and c24:1 are the main ceramide components of laccer in neutrophil plasma membranes and are directly connected with the fatty acids of lyn and gi . these observations suggest that the very long fatty acid chains of ceramide are critical for gsl - mediated outside - in signaling . sphingosine is another component of ceramide , with the hydrolysis of ceramide by ceramidase producing sphingosine and fatty acids . sphingosine is phosphorylated by sphingosine kinase to sphingosine-1-phosphate , which is involved in a wide range of cellular functions , including growth , differentiation , survival , chemotaxis , angiogenesis , and embryogenesis , in various types of cells . this review describes the role of ceramide moiety of gsls and its metabolites in immunological and inflammatory reactions in human .

stimulated promise mechanically interlocked molecular architectures potential employment future nanotechnological applications development molecular machines switches interest shown construction ever increasing rotaxane catenane species also however designed function selective host systems whereby topologically unique interlocked three dimensional cavities exploited selectively recognise specific guest species previous work utilised anion templation construct range rotaxanes catenanes upon removal template bind anions strongly selectively competitive solvent mixtures furthermore selective anion binding exploited bring triggered motion within interlocked supramolecular architectures underlining possible application systems preparation molecular switches sense monitor anion binding and/or binding triggered motion desirable integrate redox- photoactive reporter group proximity interlocked anion binding site examples interlocked hosts capability sensing anions electrochemical optical[3b 4 6 methods rare herein report preparation first anion templated rotaxane incorporating optically- electro active osmium(ii tris(bipyridine reporter group removal anion template selective anion binding investigated monitoring optical electronic output osmium(ii tris(bipyridine reporter moiety develop fabricate prototype molecular sensory system surface association removes complications associated brownian motion typically increases conformational rigidity underpin potential applications molecular architectures ultimately device integration.[1b date limited number surface bound interlocked structures reported.[5b c 7 hence additionally report anion templated assembly electrochemically active osmium(ii bipyridyl rotaxane structures gold substrates template removal specific reporting anion recruitment solution figure 1 schematic representation recognition sensing anions surface bound rotaxane axle blue macrocycle green anion binding functionalities shown red design system target design mechanically bonded host features incorporation osmium(ii tris(bipyridine reporter group macrocyclic component rotaxane molecular framework containing convergent hydrogen bond donor anion binding interlocked cavity the macrocycle contains 4,4-bis(amide)-2,2-bipyridyl motif coordination osmium(ii metal centre electron rich hydroquinone units facilitate supplementary secondary aromatic donor acceptor interactions electron deficient positively charged pyridinium axle.[3a choice osmium(ii bipyridyl reporter group comes established electro- photochemical properties making system attractive probe sense anion binding event synthesis rotaxanes three distinct osmium(ii bipyridyl bipy rotaxanes 13 prepared clipping stoppering anion templated synthetic methodologies comprising macrocycle component different axle lengths scheme 1 rotaxane 1 obtained two steps chloride anion templated clipping reaction axle[3a components bis(amine 8 4,4-bis(chlorocarbonyl)-2,2-bipyridine 9 presence et3n dry dichloromethane scheme 2 scheme s1 supporting information the crude metal free rotaxane intermediate treated os(bipy)2cl2 16 etoh h2o mixture reflux give anion exchange using 0.1 aqueous nh4pf6 rotaxane 1 6 overall yield macrocycle 19 synthesised 37 yield condensation bis(amine 8 bis(acid chloride 9 presence et3n template 17 dry dichloromethane reaction macrocycle 19 16 etoh h2o mixture reflux afforded anion exchange macrocycle 4 74 yield the reaction carboxy terphenyl amide pyridine derivative 10[3f oxalyl chloride produced corresponding acid chloride upon condensation 3-bromopropylamine hydrobromide presence et3n dry dichloromethane gave bis(amide 11 this converted azide derivative 12 reaction sodium azide dimethylformamide reaction compound 12 methyl iodide reflux followed anion exchange using 0.1 aqueous nh4pf6 gave desired axle precursor 13 an analogous condensation reaction acyl chloride derivative 10 4-(azidomethyl)biphenyl-4-yl methanamine followed methylation anion exchange produced axle precursor 14 scheme 2 rotaxanes 2 3 obtained 16 72 yield respectively mono stoppering reactions followed washing nh4pf6/h2o remove chloride template see supporting information scheme s1 axle precursors 13 14 added macrocycle 4 leading chloride anion templated pseudo rotaxane assembly alkyne cycloaddition cuaac reactions dry dichloromethane using alkyne stopper 15 copper(i tetrakis(acetonitrile hexafluorophosphate tris(benzyltriazolylmethyl)amine tbta diisopropylethylamine afforded desired rotaxanes 2 3 rotaxanes 13 characterised nmr spectroscopy h c f p mass spectrometry maldi tof h nmr spectra rotaxanes 13 figure 2 reveal splitting upfield shift hydroquinone protons macrocycle component owing aromatic donor acceptor interactions electron rich hydroquinone groups electron deficient pyridinium moiety axle characteristic interlocked structure h nmr spectra 500 mhz d6]acetone d2o 7:3 293 k rotaxanes 1 2 c 3 e chloride complexes 1 cl b 2 cl 3 cl f addition one equivalent chloride anion binding studies solution anion binding probed using nmr luminescence electrochemical methods assorted solvents dictated various factors these techniques dramatically different limits detection different requirements system case nmr titration initial assessments acetonitrile revealed strong association anions compounded low solubility generated complexes fact necessitated use competitive aqueous solvent mixture d6]acetone d2o 7:3 luminescence spectroscopy offering much lower detection limits presents quantum yield weighted average consequence response observed luminescence anion concentrations varies solvents this particularly true case osmium tris(bipy complexes solvation excited state plays important role determining form spectrum small quantities water added acetonitrile mixtures found addition 3 water produced optimal reproducible changes osmium emission spectrum case electrochemistry need large potential window resolve bipy ligand based voltammetry use relatively concentrated electrolytes dictated use acetonitrile solvent system h nmr investigations preliminary h nmr anion titration experiments rotaxanes 1 2 3 various anions cl aco h2po4 performed competitive the addition one equivalent tetrabutylammonium tba chloride resulted downfield shift inner protons 0.13 0.05 0.29 ppm c 0.10 0.05 0.34 ppm rotaxanes 1 2 3 respectively indicative halide binding within rotaxane interlocked binding cavity figure 2 in addition modest upfield perturbations macrocyclic hydroquinone protons respective rotaxane observed g 0.02 0.02 0.06 ppm h 0.03 0.02 0.03 ppm 1 2 3 respectively oxoanions acetate dihydrogen phosphate added small perturbation observed inner protons case rotaxane 3 protons move upfield indication oxoanion binding periphery rotaxane wineqnmr2 analysis binding isotherms chloride acetate anions obtained monitoring chemical shift perturbation proton axle component respective rotaxane versus equivalent anion figure 3 determined 1:1 stoichiometric association constants table 1 it proved impossible obtain quantitative binding data dihydrogen phosphate titration experiments indicative complex equilibrium binding process chemical shifts observed small association constants 100 sensibly fitted within error a chemical shift perturbation proton upon addition cl aco h2po4 tba salt solution rotaxane 1 unfilled 2 half filled 3 filled 7:3 d6]acetone d2o 293 k. b chemical shift perturbation proton c upon addition cl tba salt solution macrocycle 4 7:3 d6]acetone d2o 9:1 d6]acetone d2o 293 k. symbols represent experimental data continuous lines represent calculated curves association constants rotaxanes 1 2 3 7:3 acetone d2o macrocycle 4 9:1 acetone d2o cl aco tba salts 293 k obtained monitoring proton error 15 values b determined monitoring proton c. error 10 c 9:1 acetone d2o the results indicate selectivity towards cl weak peripheral association aco h2po4 the oxoanions large penetrate interlocked binding domain whereas chloride anions complementary size shape leading strong binding even competitive 30 aqueous solvent mixture comparing strength chloride anion binding three rotaxanes interlocked host 3 containing rigid biphenyl axle linkage exhibits significantly stronger binding this may reflect optimal degree preorganisation interlocked binding domain respective rotaxane determined nature axle component the relatively preorganised binding sites rotaxanes 1 3 compared flexible propyl linked axle component present 2 potentially enhances chloride binding steric congestion could responsible 1 relatively inferior halide complexing reagent comparison 3 the binding chloride anions macrocycle 4 also investigated table 1 figure 3 b as expected binding weak 7:3 acetone water shown small downfield shift protons c 0.03 ppm addition one equivalent chloride the h nmr titration experiment repeated 9:1 acetone water less competitive solvent mixture enabled 1:1 stoichiometric association constant 210 determined analogous titrations acetate anions revealed small perturbations macrocycle proton indicative weak binding these observations serve highlight rotaxanes potency chloride anion recognition consequence three dimensional binding cavities capable encapsulation guest macrocycle 4 rotaxanes 13 display two emission bands near ir region centred approximately 800 925 nm figure 4 following excitation metal ligand charge transfer mlct absorption bands 430 nm lifetimes two bands determined 44 35 ns 800 950 nm respectively a titration rotaxane 1 tbacl 97:3 acetonitrile water showing emission spectra uncorrected detector sensitivity increasing chloride anion concentration b binding isotherms fits titration following two emission bands these observations consistent nozaki co workers assigned presence two bands emission spectra osmium bipyridyl complexes subtle variations osmium environment arising solvation induced polarisation triplet state upon titration tbacl 97:3 acetonitrile water an increase emission intensity observed four species increases pronounced higher energy bands given conclusions nozaki regarding solvent induced distortions triplet state structure plausible anion binding also influence local structure differing degrees representative spectra shown figure 4 along corresponding binding isotherms fits association constants obtained dynafit software using 1:1 binding model table 2 association constants determined luminescence titration macrocycle 4 rotaxanes 1 2 3 97:3 acetonitrile water chloride dihydrogen phosphate acetate anions tba salts 99 confidence intervals given square brackets 1:1 binding model acetate dihydrogen phosphate tba salts also titrated rotaxane 1 macrocycle 4 comparison although latter phosphate anion binding could determined owing precipitation the data indicate binding chloride anions rotaxanes two orders magnitude greater macrocycle the chloride anion association constants obtained luminescence measurements three rotaxanes confirm observations nmr spectroscopy strong binding chloride ions rotaxane 3 it noted change solvent system also reflected differences binding owing different solvation host relatively uncompetitive solvent system likely many anions associate cationic receptors indeed studies dihydrogen phosphate acetate bear hypothesis it important point likely optical titrations notably less reflective binding selectivity report anion association cavity confined peripheral cyclic voltammetric scans figure 5 acetonitrile solution containing macrocycle 4 revealed electrochemical redox system 0.605 v vs. ag ag assigned reversible os(+2/+3 redox couple in addition three bipyridyl ligand centred redox systems observed 1.375 1.715 1.975 v vs. ag ag labelled x z respectively consistent previous reports tris(bipy ruthenium moiety these assignments confirmed equivalent integration charges associated os(+2/+3 redox system bipyridyl ligand centred redox events expected one electron redox systems species accordance previous report least cathodic bipy redox couple x figure 5 ) is assigned amide substituted bipyridyl located next anion binding site light electron withdrawing nature carbonyl amide moieties cyclic voltammetric scans 0.2 mm macrocycle 4 0.15 tbapf6/ch3cn scan rate=0.1 vs. insert electrochemical titrations 4 tbacl monitoring bipy couples x z. titrations macrocycle 4 tbacl tbaoac carried determine ability ligand- metal centre based redox potentials report anion association shown figure 6 cathodic perturbations approximately 20 mv 40 mv metal centred osmium based 2/+3 couple observed upon addition 5 molar equivalents tbacl tbaoac respectively the bipy ligand centred responses scale accordance vicinity anion association site x showing strongest perturbations z weakest presence 5-fold excess cl aco see insert figure 5 schemes s2 s3 supporting information these observations fully consistent anionic species bound vicinity bis(amide bipy cavity dynafit modelling analysis simultaneous fit metal centred os(+2/+3 couple least cathodic ligand centred x potential perturbations function anion concentration gave 1:1 stoichiometric association constants 2.510 2.22.710 1.510 1.12.110 chloride acetate anions respectively the larger association constant potential shifts observed addition tbaoac compared tbacl attributed stronger association aco amide based binding site cl owing relative basicities anions e1/2 os(+2/+3 b bipy couple versus equivalents tbacl tbaaco added we assign higher luminescence electrochemically defined association constants comparison obtained nmr spectroscopy competitive aqueous solvent system used latter acknowledge also optical redox transitions may exactly equivalent response specific anion association indeed optical transition energies likely sensitive broad electrostatic dielectric change vicinity relevant chromophore orbitals redox signatures specifically sensitive electron density bipyridyl osmium centres hence may reflective specific anion association resolved fundamental electrochemical characteristics rotaxane hosts similar macrocycle reported supporting information page 4 unfortunately combination sluggish diffusion strong physical adsorption based electrode fouling precluded attainment reliable electrochemical anion binding data rotaxanes surface immobilisation rotaxane cui catalysed huisgen cycloaddition axles 13 14 prepared alkyne terminated molecular films gold see experimental section presence templating chloride anions 5-fold excess macrocycle 4 generates surface confined rotaxane assemblies scheme 3 accompanying ellipsometry defined increases film thickness 1.30.1 nm consistent axle surface orientations normal close normal control surface analyses absence cui catalyst confirm surface assembly specifically click chemistry driven the associated template locking osmium bipy macrocycle surface process confirmed resolved osmium based 2/+3 electrochemical signatures figure 7 an integration signals resolves surface macrocycle concentrations 1.05 2.510 mol cm axles 13 14 respectively corresponding 20 45 densely packed monolayer based macrocycle footprint 3.0 nm surface association stable ultrasonic washing observed chloride anion template specifically involved importantly macrocycle osmium based 2/+3 electrochemical response observed pf6 salts 13 14 used the linear scaling os(+2/+3 redox peak current voltage scan rate confirmatory surface entrapment the surface density differences axle rotaxane films additionally resolvable electrode access solution phase faradaic redox probe surface capacitance analysis axle film significantly densely packed 10 f cm rotaxane film mushroom shaped component 25 15 f cm surfaces prepared 13 14 respectively scheme s4 supporting information estimated surface concentration 50 theoretical maximum calculated molecular footprint macrocycle 3.0 nm distance faradaic exchange estimated approximately 2 nm based localisation macrocycle pyridinium station perpendicular orientation axle underlying gold surface insert potential shifts surface bound os(+2/+3 couple immersion rotaxane interface tbaaco ch3cn tbacl ch3cn solutions following washing nh4pf6/h2o remove cl template it notable axle 14 generates significantly higher 200 macrocycle capture surface observed axle 13 consistent increased axle rigidity higher associated chloride anion template association constant table 1 these films constitute addition surface confined tethered interlocked systems best knowledge also first incorporating redox active osmium(ii bipy reporter motif we also investigated possibility surface presented vacant interlocked cavities generated using axle 14 provide means selectively recruiting electrochemically sensing chloride anions the chloride anion template initially removed washing copious amounts 0.1 nh4pf6/h2o pleasingly subsequent halide anion recruitment detectable selective exhibiting cathodic perturbation upon immersion 50 solution acetate anions observed 2 mv shift less 3 mv error reference electrode 14 mv cathodic shift upon immersion chloride anion containing solution concentration(with solutions formed tba salt anhydrous ch3cn figure 7 no potential shift observed cavity depopulated prior immersion chloride solution expected cavity exhibiting 1:1 binding stoichiometry these results consistent presented table 1 confirm selectivity three dimensional cavity towards chloride anion binding furthermore smaller potential shift observed upon binding chloride anions comparison observed macrocycle attributed electron withdrawing nature positively charged pyridinium motif axle component mitigating donation electron density osmium centre indicative interlocked surface bound structure interlocked structures engineered bind specific guests within topologically constrained three dimensional cavities created template driven syntheses this binding ability coupled signal transduction capabilities associated appended reporter groups make catenanes rotaxanes highly promising candidates development molecular sensors we reported herein solution surface synthesis number anion templated rotaxanes incorporating photo- electroactive tris(bipy osmium moiety upon removal chloride anion template h nmr titration solution rotaxanes number anions chosen basis contrasting size shape basicity showed selectivity chloride anions acetate anions dihydrogen phosphate oxoanions observations broadly supported associated perturbations osmium luminescence redox character rotaxane immobilisation onto alkyne modified gold electrode substrate copper(i)-catalysed huisgen cycloaddition produced molecular films capable responding electrochemically selectively chloride anions this work clearly demonstrates successful application self assembled monolayers electrochemical sensing halide ions general procedure commercially available solvents chemicals used without purification unless otherwise stated where dry solvents used degassed nitrogen dried passing mbraun mpsp-800 column used immediately h c f p nmr spectra recorded varian mercury vx 300 bruker avii500 spectrometer synthesis macrocycle 19,[6b axle 5,[3a bis(amine 8 thread 17,[3a axle 14 synthesized according reported procedures the syntheses 11 12 13 along electrochemical surface analysis luminescence anion titration details given supporting information rotaxane 1 50 ml round bottom flask 2,2-bipyridine-4,4-dicarboxylic acid 60 mg 245 mol 1.2 equiv suspended 10 ml thionyl chloride drop dmf added solution heated reflux n2 16 h. removal solvent residue dissolved 10 ml dry dichloromethane added dropwise 50 ml dry dichloromethane solution containing bis(amine 8 86 mg 204 mol 1 equiv axle 5 220 mg 204 mol 1 equiv triethylamine 71 l 510 mol 2.5 equiv the reaction mixture stirred room temperature 213 c 2 h washed 10 hcl(aq ) after removal solvent residue dissolved 10 ml acetonitrile filtered solvent removed vacuo give 121 mg crude mixture containing rotaxane macrocycle 19 crude material 121 mg 71 mol 1 equiv suspended 20 ml 4:1 etoh h2o mixture os(bipy)2cl2 16 82 mg 142 mol 2 equiv added after removal solvent crude product purified preparative tlc sio2 ch3cn h2o kno3(sat rotaxane 1 30 mg 6 obtained brown solid anion exchange hexafluorophosphate salt washing dichloromethane solution rotaxane 0.1 nh4pf6(aq ) h nmr 500 mhz d6]acetone d2o 7:3 =9.40 2 h py 9.33 1 h py 9.29 2 h arhc 8.75 2 h j=8.4 hz bipy 8.71 2 h j=8.4 hz bipy 8.05 2 h j=6.2 hz arha 8.02 2 h j=8.1 hz bipy 7.90 2 h j=8.1 hz bipy 7.78 2 h j=5.6 hz bipy 7.71 2 h j=5.6 hz bipy 7.66 2 h dd j=6.1.4 hz j=1.7 hz arhb 7.59 4 h j=8.9 hz arh 7.46 2 h j=6.7 hz bipy 7.067.27 28 h arhstopper bipy 6.50 4 h j=8.9 hz arhg 6.39 4 h j=9.1 hz arhh 4.61 3 h h 3.92 4 h ch2 3.86 4 h ch2 3.79 4 h ch2 3.77 4 h ch2 3.54 4 h ch2 1.18 ppm 36 h tbuh c nmr 125 mhz d6]acetone =164.0 160.7 159.7 159.5 152.4 151.7 149.5 147.9 144.7 138.9 138.8 132.3 131.7 131.4 129.4 129.3 128.4 127.4 126.9 125.7 125.7 125.3 123.2 121.1 116.5 115.4 71.4 70.9 67.5 64.7 40.4 34.9 31.7 ppm f nmr 282.5 mhz d6]acetone =72.4 ppm j=707 hz pf6 p nmr 121.6 mhz d6]acetone =144.3 ppm sept j=707 hz pf6 ms maldi tof z calcd c128h130f18n11o10osp3 mh3(pf6 1085.98 found 1085.98 rotaxane 2 50 ml round bottom flask osmium macrocycle 4 100 mg 70 mol 2 equiv thread 13 26 mg 35 mol 1 equiv slowly stirred 1 h 25 ml dry dichloromethane after removal solvent residue dissolved 25 ml dry degassed dichloromethane stopper 15 19 mg 35 mol 1 equiv cu(ch3cn)4pf6 2.6 mg 7 mol 0.2 equiv tbta 3.7 mg 7 mol 0.2 equiv diisopropylethylamine dipea 12 l 70 mol 2 equiv successively added reaction mixture stirred room temperature 213 c 3 d. 10 ml kno3(sat after removal solvent crude product purified preparative tlc sio2 ch3cn h2o kno3(sat rotaxane 2 16 mg 16 obtained brown solid anion exchange hexafluorophosphate salt washing chloroform solution rotaxane 0.1 nh4pf6(aq ) h nmr 500 mhz d6]acetone =10.06 1 h nhd 9.42 1 h pyg 9.36 3 h pyb b arhc arhc 9.20 1 h pyb b 8.82 2 h j=7.5 hz bipy 8.77 2 h j=8.2 hz bipy 8.74 2 h j=8.2 hz bipy 8.36 1 h nhd 8.20 2 h arha 8.04 1 h hc 7.968.03 8 h bipy 7.78 2 h j=6.1 hz arhbj 7.74 2 h j=5.8 hz arhe 7.51 4 h bipy 7.147.39 29 h arhstopper 6.90 2 h j=8.8 hz arhe 6.63 4 h arhg 6.47 4 h arhh 5.03 2 h hl 4.65 3 h ha 4.44 2 h j=6.8 hz hnj 4.04 4 h och2 3.99 4 h och2 3.91 8 h nch2 och2 3.63 4 h och2 3.48 2 h hr 2.19 2 h hk 1.29 18 h tbuh 1.27 ppm 27 h tbuh c nmr 125 mhz d6]acetone =164.4 160.8 159.8 159.7 159.6 157.4 153.3 152.5 152.4 152.0 151.7 149.6 149.3 148.0 145.3 144.8 144.2 142.3 140.8 138.9 132.9 132.4 131.8 131.5 131.4 129.6 129.4 128.5 127.2 126.9 125.7 125.4 125.2 125.2 sic 125.1 123.4 121.0 120.9 116.6 115.5 115.5 sic 114.3 71.5 70.9 68.5 67.5 67.4 64.8 64.0 62.2 55.0 50.2 48.5 40.3 36.2 34.9 31.7 ppm f nmr 282.5 mhz d6]acetone =72.5 ppm j=708 hz pf6 p nmr 121.6 mhz d6]acetone =144.3 ppm sept j=709 hz pf6 ms maldi tof z calcd c138h147f18n14o11osp3 m(pf6 2658.03 found 2658.26 rotaxane 3 50 ml round bottom flask osmium macrocycle 4 100 mg 70 mol 2 equiv thread 14 31 mg 35 mol 1 equiv slowly stirred 1 h 25 ml dry dichloromethane after removal solvent residue dissolved 25 ml dry degassed dichloromethane stopper 15 19 mg 35 mol 1 equiv cu(ch3cn)4pf6 2.6 mg 7 mol 0.2 equiv tbta 3.7 mg 7 mol 0.2 equiv dipea 12 l 70 mol 2 equiv successively added reaction mixture stirred room temperature 213 c 3 d. 10 ml kno3(sat after removal solvent crude product purified preparative tlc sio2 ch3cn h2o kno3(sat rotaxane 3 74 mg 72 obtained brown solid anion exchange hexafluorophosphate salt washing chloroform solution rotaxane 0.1 nh4pf6(aq 815 ml h2o 215 ml h nmr 500 mhz d6]acetone =9.92 1 h nh 9.44 1 h py 9.34 1 h py/ 9.27 1 h arhc c 9.23 1 h py/ 9.12 1 h arhc c 8.78 4 h j=8.5 hz bipy 8.36 1 h nh 8.18 2 h j=6.1 hz arha 8.17 1 h h 8.03 4 h bipy 7.87 4 h bipy 7.80 2 h arhb 7.70 2 h arh 7.48 4 h bipy 7.097.42 37 h arhstopper arhbiphenyl 6.92 2 h j=9.0 hz arh 6.61 4 h arhg 6.41 4 h arhh 5.67 2 h h 5.16 2 h h 4.67 3 h h 4.08 4 h och2 4.04 4 h och2 3.94 4 h och2 3.87 8 h nch2 och2 3.71 2 h h 1.30 18 h tbuh 1.28 ppm 27 h tbuh c nmr 125 mhz d6]acetone =163.8 162.4 160.3 159.4 159.1 157.1 152.8 152.2 151.9 151.4 151.3 151.2 149.1 148.8 147.6 146.4 144.9 144.3 141.8 140.3 138.5 137.6 135.8 132.4 132.0 131.3 131.1 131.0 129.2 129.1 129.0 128.7 128.1 127.6 127.5 127.0 126.5 125.3 125.0 124.7 123.2 122.7 120.5 116.3 116.2 115.0 113.9 71.0 70.4 68.0 67.2 67.0 64.4 63.4 61.9 54.6 53.6 49.7 39.8 35.8 34.6 34.5 31.3 ppm;f nmr 282.5 mhz d6]acetone =72.5 ppm j=708 hz pf6 p nmr 121.6 mhz d6]acetone =144.3 ppm sept j=709 hz pf6 ms maldi tof z calcd c149h153f18n14o11osp3 m(pf6 2796.07 found 2796.10 macrocycle 4 250 ml round bottom flask macrocycle 19 168 mg 270 mol 1 equiv suspended 100 ml 4:1 etoh h2o mixture os(bipy)2cl2 16 153 mg 142 mg 2 equiv added the brown solution left cool room temperature 213 c filtered celite bed solvent removed vacuo the brown residue redissolved 20 ml h2o nh4pf6(s added solution precipitation ceased the precipitate collected vacuum filtration dried vacuum give macrocycle 4 black solid 282 mg 198 mol 74 h nmr 300 mhz d6]acetone =9.14 2 h arhc 8.82 4 h bipy 8.51 2 h j=5.3 hz nhd 8.19 2 h j=5.6 hz arha 8.05 4 h j=7.9 hz bipy 7.97 4 h j=6.0 hz bipy 7.80 2 h dd j=6.1 hz j=1.7 hz arhb 7.53 2 h j=6.8 hz bipy 7.47 2 h j=6.8 hz bipy 6.88 8 h arhg h 4.14 4 h j=5.3 hz ch2 4.06 4 h j=4.5 hz ch2 3.733.85 8 h ch2 3.67 ppm 4 h ch2 c nmr 75.5 mhz d6]acetone =163.6 160.8 159.7 153.9 152.4 152.2 151.7 142.4 138.8 129.4 127.5 125.6 122.7 116.5 116.3 71.4 70.4 68.9 67.4 40.5 ppm f nmr 282.5 mhz d6]acetone =72.5 ppm j=708 hz pf6 p nmr 121.6 mhz d6]acetone =144.3 sept j=709 hz pf6 ms maldi z calcd c54h52f12n8o8osp2 m(pf6)2 1132.35 found 1132.38 as service authors readers journal provides supporting information supplied authors such materials peer reviewed may organized online delivery copy edited typeset technical support issues arising supporting information missing files addressed authors

we report the preparation of [ 2]rotaxanes containing an electrochemically and optically active osmium(ii ) bipyridyl macrocyclic component mechanically bonded with cationic pyridinium axles . such interlocked host systems are demonstrated to recognise and sense anionic guest species as shown by 1h nmr , luminescence and electrochemical studies . the rotaxanes can be surface assembled on to gold electrodes through anion templation under click copper(i)-catalysed huisgen cycloaddition conditions to form rotaxane molecular films , which , after template removal , respond electrochemically and selectively to chloride .

fatty acids fas components phospholipids organelle cellular membranes play important biological roles maintaining processing membrane protein function fluidity.1 addition fas modulate vascular inflammation key mechanism atherosclerosis cerebral small vessel pathologies stroke altering intracellular signal transduction controlling lipid mediators prostaglandins thromboxanes leukotrienes.2 among fas 3-polyunsaturated fas 3-pufas eicosapentaenoic acid epa docosahexaenoic acid dha potent anti inflammatory molecules epa dha decrease expression receptors chemoattractants blood inflammatory cells prohibit migration neutrophils monocytes therefore 3-pufas may protect atherosclerotic changes.3,4 several clinical studies emphasized role fas risk occurrence stroke5 cardiovascular disease6 however effects due composition fas stroke cardiovascular disease remain controversial high dose 3-pufas reported beneficial effects cardiac sudden death.7 high levels plasma 3-pufas decrease risk myocardial infarction.8 terms stroke low levels circulating 3-pufas blood risk factor ischemic hemorrhagic stroke.9 decreased proportion linoleic acid also associated ischemic stroke.10 compared normal controls stroke patients moderate severe intracranial arterial stenosis occlusion decreased levels dha.11 hand recent meta analysis revealed evidence beneficial effects 3-pufas insufficient adults peripheral arterial disease associated poor cardiovascular outcome.12 date little information available relationship composition fas prognosis stroke therefore investigated whether composition fas associated stroke severity hospital admission functional outcomes 3-months follow patients acute non cardiogenic ischemic stroke between september 2007 may 2010 prospectively enrolled patients diagnosed first episode ischemic stroke admitted hospital within 7 days onset symptoms the patient demographic information well past medical medication familial history brain imaging studies computerized tomography ct and/or magnetic resonance imaging mri vascular imaging studies digital subtraction angiography ct angiography mr angiography chest radiography 12-lead electrocardiography electrocardiography monitoring median time period 3 days intensive stroke care unit transthoracic echocardiography routine blood test data collected.13 patients preferentially excluded agree provide blood samples study taking lipid lowering agents statins niacin fenofibrate 3-pufas components health foods supplements.11 total 401 subjects enrolled however basis trial org 10,172 acute stroke treatment classification system,14 moderate high risk cardiac sources embolism n=127 excluded patients undetermined stroke subtype n=45 negative evaluation n=19 two causes identified rare causes stroke subtype n=10 moyamoya disease arterial dissection venous thrombosis also excluded study moreover patients received incomplete vascular imaging work n=2 transient ischemic attacks negative diffusion weighted images n=42 enrolled for extracranial arteries degree arterial stenosis measured according published method used north american symptomatic carotid endarterectomy trial.15 intracranial arteries degree arterial stenosis measured based methods used warfarin aspirin symptomatic intracranial disease study.16 vascular images evaluated two independent vascular neurologists blinded clinical information inter observer agreement presence 50% stenosis and/or occlusion excellent kappa=0.95 the severity neurologic deficits determined using national institutes health stroke scale nihss admission.17 recurrent ischemic stroke considered presence acute onset focal neurological signs 24 hours duration evidence new ischemic lesion ct mri scan new lesions absent clinical syndrome consistent stroke admission 3 months index stroke functional outcomes assessed using modified rankin scale mrs 3 months index stroke blood samples lipid profiles collected patients within 24 hours admission fasting state 12 hours they centrifuged separate plasma serum whole blood stored -70 analysis could performed the methods measuring fa composition described previously.11 briefly plasma total lipids extracted according folch method18 phospholipid fraction isolated thin layer chromatography using development solvent composed hexane diethyl ether acetic acid 80:20:2 the phospholipid fractions methylated fa methyl esters fames lepage roy method.19 fames individual fas phospholipids separated gas chromatography using model 6890 apparatus agilent technologies palo alto ca usa 30 omegawaz tm 250 capillary column supelco bellefonte pa usa).20 peak retention times obtained comparison known standards 37 component fame mix pufa-2 supelco glc37 nucheck prep elysian mn usa analyzed chemstation software agilent technologies the average duplicate measurements sample calculated.11,21 plasma phospholipid fas expressed percentage total fas the sum 16:0 palmitic acid 18:0 stearic acid 20:0 arachidonic acid 22:0 behenic acid defined saturated fatty acids 16:1 palmitoleic acid 18:1 9 oleic acid 22:1 erucic acid defined monounsaturated fatty acids 18:2 6 linolenic acid 18:3 6 -linolenic acid 20:3 6 dihomo--linolenic acid 20:4 6 arachidonic acid defined 6-pufas 18:3 3 -linolenic acid 20:3 3 5 8 11-eicosatrienoic acid 20:5 3 epa 22:6 3 dha reported 3-pufas hypertension defined resting systolic blood pressure 140 mmhg diastolic blood pressure 90 mmhg repeated measurements receiving treatment anti hypertensive medications diabetes mellitus diagnosed patient fasting blood glucose level 7.0 mmol l treated oral hypoglycemic agents insulin patients defined smokers smokers stroke event stopped smoking within 1 year stroke event body mass index estimated dividing body weight height kg the presence coronary artery disease defined patient history unstable angina myocardial infarction angiographically confirmed coronary artery disease all statistical analyses performed using windows spss software package version 18.0 chicago il usa independent test mann whitney u test one way analysis variance anova bonferroni post hoc analysis kruskal wallis test used compare continuous variables categorical variables compared using chi square test fisher exact test continuous variables expressed meansstandard deviations sd medians interquartile ranges iqr univariate multivariate linear regression analyses performed determine factors associated nihss admission functional outcome dichotomized good outcome mrs <3 poor outcome mrs 3 univariate multivariate binary logistic regression analyses performed determine predictive factors functional outcome assess the goodness fit logistic regression model cox snell r square calculated hosmer lemeshow test performed between september 2007 may 2010 prospectively enrolled patients diagnosed first episode ischemic stroke admitted hospital within 7 days onset symptoms the patient demographic information well past medical medication familial history brain imaging studies computerized tomography ct and/or magnetic resonance imaging mri vascular imaging studies digital subtraction angiography ct angiography mr angiography chest radiography 12-lead electrocardiography electrocardiography monitoring median time period 3 days intensive stroke care unit transthoracic echocardiography routine blood test data collected.13 patients preferentially excluded agree provide blood samples study taking lipid lowering agents statins niacin fenofibrate 3-pufas components health foods supplements.11 total 401 subjects enrolled however basis trial org 10,172 acute stroke treatment classification system,14 moderate high risk cardiac sources embolism n=127 excluded patients undetermined stroke subtype n=45 negative evaluation n=19 two causes identified rare causes stroke subtype n=10 moyamoya disease arterial dissection venous thrombosis also excluded study moreover patients received incomplete vascular imaging work n=2 transient ischemic attacks negative diffusion weighted images n=42 enrolled for extracranial arteries degree arterial stenosis measured according published method used north american symptomatic carotid endarterectomy trial.15 intracranial arteries degree arterial stenosis measured based methods used warfarin aspirin symptomatic intracranial disease study.16 vascular images evaluated two independent vascular neurologists blinded clinical information inter observer agreement presence 50% stenosis and/or occlusion excellent kappa=0.95 the severity neurologic deficits determined using national institutes health stroke scale nihss admission.17 recurrent ischemic stroke considered presence acute onset focal neurological signs 24 hours duration evidence new ischemic lesion ct mri scan new lesions absent clinical syndrome consistent stroke admission 3 months index stroke functional outcomes assessed using modified rankin scale mrs 3 months index stroke blood samples lipid profiles collected patients within 24 hours admission fasting state 12 hours they centrifuged separate plasma serum whole blood stored -70 analysis could performed the methods measuring fa composition described previously.11 briefly plasma total lipids extracted according folch method18 phospholipid fraction isolated thin layer chromatography using development solvent composed hexane diethyl ether acetic acid 80:20:2 the phospholipid fractions methylated fa methyl esters fames lepage roy method.19 fames individual fas phospholipids separated gas chromatography using model 6890 apparatus agilent technologies palo alto ca usa 30 omegawaz tm 250 capillary column supelco bellefonte pa usa).20 peak retention times obtained comparison known standards 37 component fame mix pufa-2 supelco glc37 nucheck prep elysian mn usa analyzed chemstation software agilent technologies the average duplicate measurements sample calculated.11,21 plasma phospholipid fas expressed percentage total fas the sum 16:0 palmitic acid 18:0 stearic acid 20:0 arachidonic acid 22:0 behenic acid defined saturated fatty acids 16:1 palmitoleic acid 18:1 9 oleic acid 22:1 erucic acid defined monounsaturated fatty acids 18:2 6 linolenic acid 18:3 6 -linolenic acid 20:3 6 dihomo--linolenic acid 20:4 6 arachidonic acid defined 6-pufas 18:3 3 -linolenic acid 20:3 3 5 8 11-eicosatrienoic acid 20:5 3 epa 22:6 3 dha reported 3-pufas hypertension defined resting systolic blood pressure 140 mmhg diastolic blood pressure 90 mmhg repeated measurements receiving treatment anti hypertensive medications diabetes mellitus diagnosed patient fasting blood glucose level 7.0 mmol l treated oral hypoglycemic agents insulin patients defined smokers smokers stroke event stopped smoking within 1 year stroke event body mass index estimated dividing body weight height kg the presence coronary artery disease defined patient history unstable angina myocardial infarction angiographically confirmed coronary artery disease all statistical analyses performed using windows spss software package version 18.0 chicago il usa independent test mann whitney u test one way analysis variance anova bonferroni post hoc analysis kruskal wallis test used compare continuous variables categorical variables compared using chi square test fisher exact test continuous variables expressed meansstandard deviations sd medians interquartile ranges iqr univariate multivariate linear regression analyses performed determine factors associated nihss admission functional outcome dichotomized good outcome mrs <3 poor outcome mrs 3 univariate multivariate binary logistic regression analyses performed determine predictive factors functional outcome assess the goodness fit logistic regression model cox snell r square calculated hosmer lemeshow test performed the demographic data study subjects comparative analysis according functional outcome 3 months index stroke summarized table 1 of patients 60 38.5% patients large artery atherosclerosis stroke subtype 96 61.5% patients small vessel occlusions the meanssd proportions epa dha 2.00.7 8.91.4 respectively case 3-pufa considering stroke subtypes difference large artery atherosclerosis small vessel occlusion stroke subtypes terms proportion epa dha 3-pufas supplementary table 1 of 156 patients 122 78.2% patients good functional outcome meanssd epa dha proportions 2.10.7 9.11.3 respectively remaining 34 21.8% patients poor outcome relatively smaller proportion epa 1.80.6 p=0.032 dha 8.11.3 p=0.001 the proportion 3-pufa patients poor outcome significantly lower patients good outcome 10.81.6 vs. 12.21.9 p=0.001 table 1 after adjusting factors including age sex variables p<0.1 univariate analysis stroke subtypes hemoglobin high density lipoprotein high sensitivity c reactive protein fasting glucose 16:0 palmitic acid saturated fatty acids lower proportion epa dha independently associated stroke severity admission -0.751 standard error se 0.376 p=0.048 epa -0.610 se 0.215 p=0.005 dha moreover the 3-pufa significantly associated stroke severity admission -0.462 se 0.156 p=0.004 table 2 fig 1 considering stroke subtypes dha 3-pufa correlated stroke severity admission large artery atherosclerosis even though showed tendency 3-pufa p=0.065 small vessel occlusion however epa supplementary table 2 appear correlated multivariate linear regression analysis there six recurrent stroke cases events epa relatively lower recurrent group compared non recurrent group 1.60.2 vs. 2.00.7 p=0.006 however proportions dha 3-pufas different two groups supplementary table 3 regarding functional outcome three months index stroke lower proportion dha odds ratio 0.20 95% confidence interval ci 0.04 0.88 p=0.033 3-pufa 0.22 95% ci 0.05 0.84 p=0.028 showed significant relationship poor functional outcome however epa independently associated poor functional outcome 0.60 95% ci 0.12 -2.93 p=0.533 multivariate analysis adjusting age sex smoking status nihss score stroke subtypes 16:0 palmitic acid table 3 considering stroke subtypes lower proportion dha 3-pufas independently associated poor functional outcome large artery atherosclerosis subtype 0.62 95% ci 0.42 0.93 p=0.023 dha 0.65 95% ci 0.47 0.90 p=0.011 3-pufa small vessel occlusion subtype 0.49 95% ci 0.28 0.85 p=0.012 dha 0.64 95% ci 0.42 0.98 p=0.044 3-pufa supplementary table 4 the demographic data study subjects comparative analysis according functional outcome 3 months index stroke summarized table 1 of patients 60 38.5% patients large artery atherosclerosis stroke subtype 96 61.5% patients small vessel occlusions the meanssd proportions epa dha 2.00.7 8.91.4 respectively case 3-pufa considering stroke subtypes difference large artery atherosclerosis small vessel occlusion stroke subtypes terms proportion epa dha 3-pufas supplementary table 1 of 156 patients 122 78.2% patients good functional outcome meanssd epa dha proportions 2.10.7 9.11.3 respectively remaining 34 21.8% patients poor outcome relatively smaller proportion epa 1.80.6 p=0.032 dha 8.11.3 p=0.001 the proportion 3-pufa patients poor outcome significantly lower patients good outcome 10.81.6 vs. 12.21.9 p=0.001 table 1 after adjusting factors including age sex variables p<0.1 univariate analysis stroke subtypes hemoglobin high density lipoprotein high sensitivity c reactive protein fasting glucose 16:0 palmitic acid saturated fatty acids lower proportion epa dha independently associated stroke severity admission -0.751 standard error se 0.376 p=0.048 epa -0.610 se 0.215 p=0.005 dha moreover the 3-pufa significantly associated stroke severity admission -0.462 se 0.156 p=0.004 table 2 fig 1 considering stroke subtypes dha 3-pufa correlated stroke severity admission large artery atherosclerosis even though showed tendency 3-pufa p=0.065 small vessel occlusion however epa supplementary table 2 appear correlated multivariate linear regression analysis six recurrent stroke cases events epa relatively lower recurrent group compared non recurrent group 1.60.2 vs. 2.00.7 p=0.006 however proportions dha 3-pufas different two groups supplementary table 3 regarding functional outcome three months index stroke lower proportion dha odds ratio 0.20 95% confidence interval ci 0.04 0.88 p=0.033 3-pufa 0.22 95% ci 0.05 0.84 p=0.028 showed significant relationship poor functional outcome however epa independently associated poor functional outcome 0.60 95% ci 0.12 -2.93 p=0.533 multivariate analysis adjusting age sex smoking status nihss score stroke subtypes 16:0 palmitic acid table 3 considering stroke subtypes lower proportion dha 3-pufas independently associated poor functional outcome large artery atherosclerosis subtype 0.62 95% ci 0.42 0.93 p=0.023 dha 0.65 95% ci 0.47 0.90 p=0.011 3-pufa small vessel occlusion subtype 0.49 95% ci 0.28 0.85 p=0.012 dha 0.64 95% ci 0.42 0.98 p=0.044 3-pufa supplementary table 4 our study revealed 3-pufas especially dha associated stroke severity hospital admission poor functional outcome even adjusting nihss score considered strong predictive factor stroke outcome for example treatment dha albumin complex animal studies decreased brain injury transient permanent focal cerebral ischaemia.22 dha showed anti inflammatory neuroprotective effects decreasing oxidative stress via activation nuclear factor e2-related factor 2 heme oxygenase-1 expression attenuating c jun phosphorylation activating protein-1 signaling pathway.23 consistently studies humans produced similar results one previous study 281 japanese patients acute ischemic stroke diagnosed within 24 hours onset showed epa arachidonic acid aa ratio dha aa ratio epa+dha aa ratio independently negatively associated early neurological deterioration.24 furthermore population based study u.s.a included 2,692 elderly adults without prevalence stroke cardiovascular disease revealed higher plasma 3-polyunsaturated fas epa dha total 3 fas associated lower mortalities.25 overall previous studies showed 3-pufas especially epa dha related vascular outcome line study results however reason epa independently associated poor functional outcome study population remains elucidated dha exerts vasodilating properties stimulating nitric oxide release vascular endothelium hence may also responsible decreasing heart rate variability potentially dyslipidemia improvement effects observed epa.26 loss nitric oxide control heart rate variation dyslipidemia may related poor vascular disease outcome,27 study results explained correlation observed dha epa functional outcome in addition difference race staple food study population could represent another possible explanation results demonstrated relationship 3-pufas stroke severity hospital admission poor functional outcomes large artery atherosclerosis small vessel occlusion stroke subtypes 3-pufas give rise anti inflammatory molecules resolvins protectins lipoxygenase cyclo oxygenase pathways.28,29 resolvins protectins play important role resolution inflammation decreases atherosclerotic changes tissue injuries because cerebral small vessel pathologies lacunar infarction white matter changes cerebral microbleeds linked inflammatory reactions30 increase arterial stiffness31 caused progressive atherosclerosis anti inflammatory anti atherogenic effects 3-pufas could explain results observed study moreover because progressive cerebral atherosclerosis associated poor stroke outcome,32 finding relative association 3-pufas stroke prognosis may valid in addition atherosclerotic plaque stabilization effect 3-pufas may important mechanism previous study performed patients awaiting carotid endarterectomy showed plaques patients treated fish oil 1.4 g 3-polyunsaturated fas per day well formed thick fibrous cap i.e. less vulnerable atheroma compared patients placebo group.33 pathologically infiltration macrophages less severe patients treated fish oil.33 furthermore another prospective study confirmed patients treated fish oil lower plaque inflammation instability.34 vulnerability atherosclerotic plaque important determinant thrombosis related stroke well degree arterial stenosis results relative relationship stroke outcome 3-pufas within expectation lastly recent study revealed 3-pufas enhanced cerebral angiogenesis animal models,35 increased angiogenesis could augment brain repair improve long term functional recovery cerebral infarction findings may support study.35 one limitation study blood samples obtained acute stroke patients admission therefore fatty acids composition serially assessed time course stroke moreover even though prospectively enrolled study subjects study design mainly cross sectional in addition short term observation small sample size another limitation study further studies long term follow larger population size needed finally study design randomized control study furthermore stroke severity admission correlated lower proportion 3-pufas functional outcome 3 months could bias due interaction severity admission 3-pufas that may possibility functional outcome 3 months weakly associated 3-pufas our results demonstrate 3-pufa levels correlate stroke severity admission functional outcomes 3 months 3-pufas may considered potential blood biomarkers prognosis acute non cardiogenic ischemic stroke patients comparison proportions 3-pufas according stroke subtypes relationship fatty acids composition stroke severity admission according stroke subtypes comparison functional outcome based occurrence recurrent stroke relationship fatty acids composition poor functional outcome according stroke subtype

background and purposealterations in blood fatty acid ( fa ) composition are associated with cardiovascular diseases . we investigated whether plasma fa composition was related to stroke severity and functional outcome in acute ischemic stroke patients.methodswe prospectively enrolled 156 patients with first - episode cerebral infarction , within 7 days of symptom onset . the proportion of fas was analyzed using gas chromatography , and the summation of the omega-3 polyunsaturated fatty acids ( 3-pufa ) , 18:3 3 -linolenic acid , 20:3 3 eicosatrienoic acid , 20:5 3 eicosapentaenoic acid ( epa ) , and 22:6 3 docosahexaenoic acid ( dha ) was reported as 3-pufas . stroke severity was assessed using the national institutes of health stroke scale ( nihss ) score on admission . poor functional outcome was defined by modified rankin scale ( mrs ) 3 at three months after the index stroke.resultslower proportions of epa ( =-0.751 ) , dha ( =-0.610 ) , and 3-pufas ( =-0.462 ) were independently associated with higher nihss score , after adjusting for stroke subtype , hemoglobin , high density lipoprotein , high sensitivity c - reactive protein , fasting glucose , 16:0 palmitic acid , and saturated fatty acids . moreover , a lower proportion of dha ( odds ratio [ or ] : 0.20 , 95% confidence interval [ ci ] : 0.04 - 0.88 ) , and 3-pufas ( or : 0.22 , 95% ci : 0.05 - 0.84 ) showed an independent relationship with poor functional outcome after adjusting for age , sex , smoking status , nihss score , stroke subtype , and 16:0 palmitic acid.conclusionsour results demonstrate that 3-pufas correlated with stroke severity on admission and functional outcomes at 3 months . 3-pufas are potential blood biomarkers for prognosis of acute non - cardiogenic ischemic stroke patients .

different methods introduced predicting mgp assessing symphyseal morphology one ricketts others stated morphology symphysis may used predict direction mandibular growth he found forward inclination condylar head associated forward mandibular rotation along greater curvature mandibular canal compared mandibular contour a tendency toward backward mandibular rotation associated pronounced apposition symphysis overall concavity inferior mandibular border jarabak cephalometric analysis predicted direction mandibular growth facial polygon including saddle angle n ar articular angle ar go gonial angle ar go sum three angles greater 396 predictive posterior mgp sum less 396 associated anterior mgp also ratio posterior go anterior face height n 56 62% indicated posterior mgp whereas ratio 65 80% indicated anterior growth tendency although many cephalometric measurements introduced still difficult accurately predict direction mandibular growth although skieller four variables accounted 86% variability changes direction mandibular growth mandibular inclination intermolar angle shape inferior border mandible inclination symphysis however accounted 8% lee study thus concluded predicting direction mandibular growth perplexing problematic assessed symphyseal height depth ratio h angle study morphology symphysis found associated direction mandibular growth a mandible anterior growth direction associated small height large depth small ratio large angle symphysis conversely posterior growth direction associated large height small depth large ratio small angle symphysis the purpose study evaluate symphyseal morphology height depth height depth patients aged 9 14 years different sagittal vertical mgps lateral cephalometric radiographs charts adolescents aged 9 14 years used study the first part study conducted 60 normal cephalograms taken normal individuals according normal occlusion defined moyer appropriate facial proportions approved two orthodontists the samples history orthodontic treatment history systemic developmental diseases find normal value vertical growth pattern mandible vertical indices sum bijork angles gonial angle sn man angle used wylie analysis also used assessing sagittal growth pattern mandible included mandibular length lower mandibular length the second part study define case groups included cl ii vertical cl iii vertical cl ii horizontal cl iii horizontal patients normal maxillae the four case groups range classified horizontal vertical growing patterns according vertical indices cl ii cl iii skeletal growing patterns according wylie indices there 15 samples group matched normal groups according sex cervical maturation stage cvms baccetti method evaluation cervical maturation stage symphyseal measurement included height depth ratio h measured according aki et al linear measurements used tangent drawn point b parallel perpendicular lines drawn tangent the method measuring height depth symphysis shown figure 2 cephalometric measurements used quantify symphysis morphology radiographs taken panoramic radiographic device planmeca proline x helsinki finland all scanned digitized dpi 300 measurements made using dolphin imaging software version 10 patterson dental supply usa symphyseal parameters real size tracings printed dolphin imaging software measured using grid normal data distribution evaluated one sample k test levene test performed indicate variance equality groups after confirmation normality groups equation variances two way analysis variance anova model assessed adjusted chi square analysis data p < 0.001 comparison cases normal group performed using dunnett method for evaluation intraobserver reliability intraclass correlation coefficient icc assessed reliability approved icc coefficiency 0.0726 0.0871 normal data distribution evaluated one sample k test levene test performed indicate variance equality groups after confirmation normality groups equation variances two way analysis variance anova model assessed adjusted chi square analysis data p 0.001 comparison cases normal group performed using dunnett method evaluation intraobserver reliability intraclass correlation coefficient icc assessed reliability approved icc coefficiency 0.0726 0.0871 evaluation 60 normal radiographs baccetti method showed 21 stage iii 29 stage ii remaining stage i. case groups also selected patients stage iii matched 15 samples normal radiographs stage mean standard deviation sagittal vertical parameters shown table 1 mean standard deviation mandibular parameters sagittal vertical analysis table 2 lists height depth symphysis based sex growth pattern study groups comparison case group normal group showed statistically significant differences normal group cl ii horizontal cl iii vertical groups height p 0.001 mean standard deviation symphyseal height depth normal group four subgroups based gender comparison case group normal group showed statistically significant difference cl iii horizontal cl ii vertical groups depth p 0.001 significant difference relevant sex table 3 shows symphysis ratio relevant sex growth pattern mean standard deviation symphyseal ratio normal group four subgroups based gender intraobserver reliability assessed icc reliability evaluation 60 normal radiographs baccetti method showed 21 stage iii 29 stage ii remaining stage i. case groups also selected patients stage iii matched 15 samples normal radiographs stage mean standard deviation sagittal vertical parameters shown table 1 table 2 lists height depth symphysis based sex growth pattern study groups comparison case group normal group showed statistically significant differences normal group cl ii horizontal cl iii vertical groups height p 0.001 mean standard deviation symphyseal height depth normal group four subgroups based gender comparison case group normal group showed statistically significant difference cl iii horizontal cl ii vertical groups depth p 0.001 significant difference relevant sex mean standard deviation symphyseal ratio normal group four subgroups based gender intraobserver reliability assessed icc reliability quantification skeletal data shown effective reliable method demonstrating variation human growth well monitoring interpreting growth various skeletal elements living morphological changes mandibular body studied previous researches found gonial angle became significantly smaller third molar eruption also posterior mandibular body length linear correlation gonial angle is symphysis morphology due ease selection landmarks evaluating symphysis morphology cephalograms the characteristics normal subjects mentioned studies despite wide variations size shape human face head body significant relationships found measures mandibular incisor crowding basal bone dimensions female subjects except vestibular part cancellous bone thickness all mandibular incisor bone measurements greater male subjects female subjects we matched study groups according baccetti skeletal age order lower developmental age effect mandibular growth the samples also matched based gender way confounding effect gender skeletal age minimized mandibular growth it also found sexual dimorphism morphology symphysis mean symphyseal ratio higher female samples male samples this difference indicated studies mandibular growth bone deposition pogonion region x linked trait it noticed horizontal growth patterns mandible whether cl ii cl iii symphyseal ratio higher normal group cases vertical growth patterns the height symphysis greatest cl iii vertical least clii horizontal groups significant difference subgroups regarding depth symphysis greatest depth measured cl iii horizontal least indicated cl ii vertical group the size shape mandibular symphysis important consideration evaluation orthodontic patients prominent symphysis more protrusion incisors esthetically acceptable therefore greater chance nonextraction approach treatment may considered conversely patients greater symphyseal height small chin extraction approach preferred compensation arch length discrepancies this concept confirmed results study measured deepest depth horizontal growth patterns therefore use nonextraction approach individuals furthermore vertical growth patterns better extract teeth decreased symphyseal depth did study determine whether symphysis morphology could used predictor direction mandibular growth assess growth changes symphysis they determined direction mandibular growth based vertical parameters divided anterior posterior growth patterns consequently subdivided size symphysis large small medium symphysis morphology found associated direction mandibular growth especially male subjects symphysis ratio strongest relationship adults a mandible anterior growth direction associated small height large depth small ratio large angle symphysis conversely posterior growth direction associated large height small depth large ratio small angle symphysis symphyseal dimensions continued change adulthood male subjects greater later change compared female subjects in addition ratio low horizontal growth anterior growth direction depth greater it seems based studies symphysis morphology different various vertical growth patterns mandible in summary conclude female adolescents greater symphyseal ratio comparison male adolescents also symphyseal depth differed significantly vertical horizontal growth patterns comparison normal groups symphysis ratio height depth strongly related vertical pattern mandibular growth genders

background : this study sought to assess symphyseal morphology in adolescents with different mandibular growth patterns ( mgps ) in order to see if a relation exists.materials and methods : in this study the symphyseal parameters ( height , depth , and ratio ) of normal subjects were compared with four groups with malocclusion ( cl iii vertical , cl ii vertical , cl iii horizontal , and cl ii horizontal ) . these groups ( 15 samples each ) were matched ( for sex and cervical maturation stage [ cvms ] ) based on their cephalograms and patient charts . growth patterns were differentiated by seven vertical parameters and the wylie analysis . after confirmation of normality of the groups and similarity of their variances the two - way analysis of variance ( anova ) was used for analysis of data assessed by adjusted chi - square ( p < 0.001 ) . the comparison of cases with the normal group was performed by the dunnett method . intraclass correlation coefficient ( icc ) was used for evaluation of intraobserver reliability.results:we found the symphyseal ratio to have a significant correlation with the mgp ( p < 0.001 ) . the symphyseal ratio ( height / depth ) was small in a mandible with vertical growth pattern cl ii or cl iii . conversely , a horizontal growth pattern of a cl ii or cl iii mandible was associated with a larger ratio of the symphysis in comparison with the normal group . the symphyseal ratio was also found to be greater in females.conclusion:the symphyseal ratio was found to be strongly associated with the mgp .

established 2003 erste foundation history stretching back 190 years year 1819 founded erste sterreichische spar casse vienna rooted history social enterprise financial service provider erste foundation recognises contemporary society faces huge challenges new united europe work integration crucial means uniting economic cultural social capital region we develop ideas concepts increase social participation ensure one left whatever circumstances turn creates stable effective fairer societies we want work prejudice nationalism integrate thinking living across borders make experiences accessible particularly young generation we intend play active role giving people opportunities increase understanding therefore erste foundation supported research projects concern effects societal transformation processes particular looking effects demographic changes long term care needs elderly people central eastern europe this intend develop policy recommendations decision makers civil society economy politics

introductionestablished in 2003 , erste foundation has a history stretching back 190 years to the year 1819 when it was founded as the erste sterreichische spar casse in vienna . rooted in our history as a social enterprise and financial service provider , erste foundation recognises that contemporary society faces huge challenges and that for a new and united europe to work , integration is crucial and that means uniting the economic , cultural and social capital of our region.descriptionwe develop ideas and concepts to increase social participation and to ensure that no - one is left out , whatever their circumstances which in turn creates stable , effective and fairer societies.european integration is important to us . we want to work against prejudice and nationalism , to integrate thinking and living across borders , and make these experiences accessible , particularly to the young generation.projectswe intend to play an active role in giving people opportunities to increase their understanding of each other . therefore , erste foundation has supported research projects that concern themselves with the effects of societal transformation processes : in particular we have been looking at the effects that demographic changes will have on the long - term care needs of elderly people in central and eastern europe . from this , we intend to develop policy recommendations for decision makers in civil society , economy and politics .

modern nursing nurses expected deliver holistic care taking account patients biological psychosocial spiritual needs however formal nursing programs iran follow biomedical approach focuses mainly medical problems.[35 formal education programs focus transfer knowledge mastery techniques related patient physical care however students also learn informally hidden curricula nursing education direct observation situations birth death loss suffering results emotional learning additionally students gain experience interactions teachers senior professionals known peer learning studies found life long effects students the limited scope formal nursing education iran may result informal learning experience nursing students differs countries if nursing education recognizes informal learning achievements may able take advantages opportunities improve students competency informal indirect learning occur function observing retaining replicating behaviors educational experiences the aim qualitative study explore perceived informal learning achievements undergraduate nursing programs iranian student perspective this study designed answer question undergraduate nursing student perceptions informal learning occurs nursing studies ? this study conducted 2-year period 2009 2010 using qualitative content analysis the participants 14 nursing students fourth year study x university recruited purposeful sampling method they final stage clinical apprenticeship passed major part education could share achievements learning experiences including informal ones sampling done according maximum variant approach terms gender age marital status geographical location local local student work experience level academic achievement e.g. good average superior each interview began open question apart theoretical practical knowledge gained four years nursing study changes think experienced study ? answers explored deeply relevant probing statements questions please tell make feel ? please describe actual experience help understand meant interviews lasted average 70 min usually single session each interview transcribed verbatim converted rich text format use maxqda software facilitated data management data analyzed constant comparison analysis method using strauss corbin coding techniques develop categories data analyzed initially main author emerging codes revised independently 2 members research team throughout coding category development the analysis process accuracy codes categories discussed checked regular sessions discrepancies resolved continuing discussion analysis lincoln guba 1985 criteria used address trustworthiness p.296 dependability gained precise documentation data methods decisions research credibility established participant confirmation understanding prolonged engagements participants peer check external check throughout study data managed maxqda analysis precisely documented maintain audit trail student participation voluntary written information purpose study confidentiality management delivered participants written consent obtained study including permission recording entire interview transcribing the 14 participants consisted 3 male 11 female nursing students age 21 23 years last semester studies based students experiences 5 categories extracted personal maturity emotional growth social development closeness god alterations value systems ethical professional commitments table 1 although participants described process leading internal changes challenging difficult almost stated facilitated growth whole person students achievements informal learning nursing study category consisted 2 subcategories personal maturity consisting self awareness self discovery accelerated mental maturity open mindedness emotional growth consisting greater patience appreciation kindness compassion altruism the participants viewed 4 years nursing school opportunity gaining deeper self awareness self knowledge they believed duties nursing practice frequent personal interactions development relationships great influence deep reflection humanity well personal beliefs in contrast importance rigorous assessment throughout nursing studies resulted regular self assessment daily lives these helped obtain realistic knowledge personal strengths weaknesses talents some participants described awareness self discovery saying learned based taught us humans program i understood university setting eyes friends criticized another significant change reported students increasing maturity mind resulted rational behavior the frequent exposure life death situations nursing helped promote maturity one student said personality developed school first behaved childishly there series realities wholly ignorant acquired school also attributed increase maturity multiple factors including interactions instructors senior students people various personalities well engagement difficult situations one participant said maturity partly influenced interactions professional instructors encounters classmates well senior students i improved little little experiencing critical situations seeing darker side life troubles pains people unaware previous relaxed lifestyle significant experience reported participants increasing open mindedness respect principles fundamental nursing classes learned focus patient human regardless nationality ethnicity religious beliefs financial sociocultural status as result students reported able accept people adopting nonjudgmental manner one student caring people various religious beliefs described primary concern encountering clients different beliefs challenging experience explained successfully overcame conflicts overlooking religious prejudices she said first thought treated differently well one patient muslim held another belief however realized ultimately human job judge belief system people then differences longer serious problematic found adapting easily getting closer i grateful able communicate well various religious minorities despite many differences some said learned patience patients suffering chronic illnesses when saw patients worked manage disease disabling consequences realized triviality problems i patients underwent dialysis every day yet quite happy spoke joy laughed induced others good mood comparison could concern minor issues good health physically emotionally ? i felt embarrassed saw struggle steadfastly pain illness deprivation other participants believed patience resulting nature discipline made stronger handle difficulties one participant stated nowadays problems exhaust spirit since nursing practice you see pains troubles people series problems longer considered problems perhaps experiences abilities improved you patient order help people need hope invested they said seeing patients suffer made realize importance gifts took granted especially gift health now see patients problems realize gifts life nt appreciate also explained nursing made similar mothers kind ready help show compassion now feel stronger sense love concern humankind well ability love care others one indicated studying nursing field gives sense mother they teach us must able establish good communication understand others support one participant stated generally morale personality really like people it seems traits developed inner satisfaction affection induce others the second category derived interviews related becoming social expert interpersonal relationships participants especially limited communication skills entering program acknowledged nursing studies turning point improved social relationships they asserted gaining communication skills patients clinical settings fundamental improving interpersonal proficiency saying nursing improved communication others i habit building fences around forced closed space period my communication skills got much stronger since required necessary skills nursing encouraging developing communication patients required students exploit strategies developing cultural ideological common ground respecting others interests wishes adapting others gradually noticed applying advanced communication skills personal life now look subject point view i take initiative try begin point welcomed party another factor contributed students social development existing social environment larger high school involved interaction heterogeneous population various towns ethnic groups cultures regarded expanding friends circle participants i nt limit making friends people type always search new thoughts new ideas experiences nonlocal participants resided student dormitory studies explained significant influence living dormitory social growth development when become college student live dormitory students coming different places cultures various upbringings obliged encounter automatically learn adapt fact flexible since participants muslim belief god one fundamental characteristic however stated experienced different type connection god nursing studies some attributed new belief gaining deeper knowledge intricacies human body human beings general nursing program i understood complexities perfect system human body accurately designed many problems caused small deviation we told patients nt respond treatment saw eyes effect prayers person accompanying patient offered depths heart worked i touched god power presence others stated felt closer god nursing practice satisfying god wishes caring vulnerable this optimism significantly improved quality prayers replaced traditional ceremonies enthusiastic worship inner dialog god this type close connection god led pleasant feeling dependency made calmer confronting difficulties feel god looking interest caring people i tell since serve god people sincerely certainly would also pay attention ... noticed whenever troubles resolved i sense resolution interpret god promise help someone god help return participants believed confrontation light well dark side life nursing expanded world view change focus living superficial life life concerned serious substantive matters beyond calendar age one participant explained nursing see beginning life termination faced certain facts make issues trivial insignificant life still important peers challenging confrontations acted awakening experiences allowing students obtain new perspective life death stimulated changes value systems result they described alterations achieving deeper realistic point view life objectives future changed principle whenever see patients days live longer waste time child like view life bowl cherries worries know life hardships considered part life moreover asserted feel chosen god profession appreciated opportunity growth transcendence i sensed invited god take right path changed destiny forever i began taking steps road supposed begin interpreted special attention god part ... believe summoned god nurses invited kind heart sympathy ability provide relaxation patients if knew beginning appreciated invited experience along way would become important valuable participants admitted feel strong commitment work try conscientious care patients they believed inner commitment stronger influence rules external controls some attributed inner commitment increased closeness god consequent sense observed constantly god they delighted connection attention tried sustain perceived attention working ethically conscientiously even though work unsterile patient may know notice god however ethical commitment forces work better respecting patient rights important anything else i always tell imagine patient one dear siblings i always try hold thought work according feelings best addition participants attributed commitment reasons respecting patient rights dignity enjoy humanistic proper care human responding inner desire best nurse trying improve nursing image society one participant related sense ethical professional commitment satisfaction inner philanthropic desire matter whether someone see appreciate efforts ethically working best firstly human secondly nurse satisfies so enough follow inner voice desire work well help needy patient make condition better this study designed answer question undergraduate nursing student perceptions informal learning occurs nursing studies ? this study conducted 2-year period 2009 2010 using qualitative content analysis the participants 14 nursing students fourth year study x university recruited purposeful sampling method they final stage clinical apprenticeship passed major part education could share achievements learning experiences including informal ones sampling done according maximum variant approach terms gender age marital status geographical location local local student work experience level academic achievement e.g. good average superior each interview began open question apart theoretical practical knowledge gained four years nursing study changes think experienced study ? answers explored deeply relevant probing statements questions please tell make feel ? please describe actual experience help understand meant each interview transcribed verbatim converted rich text format use maxqda software facilitated data management data analyzed constant comparison analysis method using strauss corbin coding techniques develop categories data analyzed initially main author emerging codes revised independently 2 members research team throughout coding category development the analysis process accuracy codes categories discussed checked regular sessions discrepancies resolved continuing discussion analysis lincoln guba 1985 criteria used address trustworthiness p.296 dependability gained precise documentation data methods decisions research credibility established participant confirmation understanding prolonged engagements participants peer check external check throughout study data managed maxqda analysis precisely documented maintain audit trail student participation voluntary written information purpose study confidentiality management delivered participants written consent obtained study including permission recording entire interview transcribing the 14 participants consisted 3 male 11 female nursing students age 21 23 years last semester studies based students experiences 5 categories extracted personal maturity emotional growth social development closeness god alterations value systems ethical professional commitments table 1 although participants described process leading internal changes challenging difficult almost stated facilitated growth whole person this category consisted 2 subcategories personal maturity consisting self awareness self discovery accelerated mental maturity open mindedness emotional growth consisting greater patience appreciation kindness compassion altruism the participants viewed 4 years nursing school opportunity gaining deeper self awareness self knowledge they believed duties nursing practice frequent personal interactions development relationships great influence deep reflection humanity well personal beliefs in contrast importance rigorous assessment throughout nursing studies resulted regular self assessment daily lives these helped obtain realistic knowledge personal strengths weaknesses talents some participants described awareness self discovery saying learned based taught us humans program i understood university setting eyes friends criticized another significant change reported students increasing maturity mind resulted rational behavior the frequent exposure life death situations nursing helped promote maturity one student said personality developed school first behaved childishly there series realities wholly ignorant acquired school also attributed increase maturity multiple factors including interactions instructors senior students people various personalities well engagement difficult situations one participant said maturity partly influenced interactions professional instructors encounters classmates well senior students i improved little little experiencing critical situations seeing darker side life troubles pains people unaware previous relaxed lifestyle significant experience reported participants increasing open mindedness respect principles fundamental nursing classes learned focus patient human regardless nationality ethnicity religious beliefs financial sociocultural status as result students reported able accept people adopting nonjudgmental manner one student caring people various religious beliefs described primary concern encountering clients different beliefs challenging experience explained successfully overcame conflicts overlooking religious prejudices she said first thought treated differently well one patient muslim held another belief however realized ultimately human job judge belief system people then differences longer serious problematic found adapting easily getting closer i grateful able communicate well various religious minorities despite many differences almost participants indicated increasing patience significant change some said learned patience patients suffering chronic illnesses when saw patients worked manage disease disabling consequences realized triviality problems i patients underwent dialysis every day yet quite happy spoke joy laughed induced others good mood comparison how could concern minor issues good health physically emotionally ? i felt embarrassed saw struggle steadfastly pain illness deprivation other participants believed patience resulting nature discipline made stronger handle difficulties since nursing practice see pains troubles people series problems longer considered problems perhaps experiences abilities improved you patient order help people need hope invested they said seeing patients suffer made realize importance gifts took granted especially gift health now see patients problems realize gifts life nt appreciate also explained nursing made similar mothers kind ready help show compassion now feel stronger sense love concern humankind well ability love care others one indicated studying nursing field gives sense mother they teach us must able establish good communication understand others support one participant stated generally morale personality really like people it seems traits developed inner satisfaction affection induce others the second category derived interviews related becoming social expert interpersonal relationships some participants especially limited communication skills entering program acknowledged nursing studies turning point improved social relationships they asserted gaining communication skills patients clinical settings fundamental improving interpersonal proficiency saying nursing improved communication others i habit building fences around forced closed space period communication skills got much stronger since required necessary skills nursing encouraging developing communication patients required students exploit strategies developing cultural ideological common ground respecting others interests wishes adapting others gradually noticed applying advanced communication skills personal life now look subject point view i take initiative try begin point welcomed party i moved away partial limited understanding others feel growing regards social relations general another factor contributed students social development existing social environment larger high school involved interaction heterogeneous population various towns ethnic groups cultures regarded expanding friends circle participants tried experience group types i nt limit making friends people type always search new thoughts new ideas experiences nonlocal participants resided student dormitory studies explained significant influence living dormitory social growth development when become college student live dormitory students coming different places cultures various upbringings obliged encounter automatically learn adapt fact flexible since participants muslim belief god one fundamental characteristic however stated experienced different type connection god nursing studies some attributed new belief gaining deeper knowledge intricacies human body human beings general nursing program i understood complexities perfect system human body accurately designed many problems caused small deviation we told patients nt respond treatment saw eyes effect prayers person accompanying patient offered depths heart worked i touched god power presence others stated felt closer god nursing practice satisfying god wishes caring vulnerable this optimism significantly improved quality prayers replaced traditional ceremonies enthusiastic worship inner dialog god this type close connection god led pleasant feeling dependency made calmer confronting difficulties feel god looking interest caring people i tell since serve god people sincerely certainly would also pay attention ... noticed whenever troubles resolved i sense resolution interpret god promise help someone god help return participants believed confrontation light well dark side life nursing expanded world view change focus living superficial life life concerned serious substantive matters beyond calendar age one participant explained nursing see beginning life termination faced certain facts make issues trivial insignificant life still important peers these challenging confrontations acted awakening experiences allowing students obtain new perspective life death stimulated changes value systems result they described alterations achieving deeper realistic point view life objectives future changed principle whenever see patients days live longer waste time child like view life bowl cherries worries know life hardships considered part life moreover asserted feel chosen god profession appreciated opportunity growth transcendence i sensed invited god take right path changed destiny forever i began taking steps road supposed begin interpreted special attention god part ... believe summoned god nurses invited kind heart sympathy ability provide relaxation patients if knew beginning appreciated invited experience along way would become important valuable most participants admitted feel strong commitment work try conscientious care patients they believed inner commitment stronger influence rules external controls some attributed inner commitment increased closeness god consequent sense observed constantly god they delighted connection attention tried sustain perceived attention working ethically conscientiously even though work unsterile patient may know notice god however ethical commitment forces work better respecting patient rights important anything else i always tell imagine patient one dear siblings i always try hold thought work according feelings best addition participants attributed commitment reasons respecting patient rights dignity enjoy humanistic proper care human responding inner desire best nurse trying improve nursing image society one participant related sense ethical professional commitment satisfaction inner philanthropic desire matter whether someone see appreciate efforts ethically working best firstly human secondly nurse satisfies so enough follow inner voice desire work well help needy patient make condition better since students felt personal growth eager participate study possible students experiences ignored unintentionally researchers according results study undergraduate nursing students achieved knowledge insights provided formal biomedical centered nursing program these included following personal maturity emotional development social development greater closeness god alteration value systems ethical professional commitment these nontechnical professional competencies important goals nursing curriculum although included specific lesson beyond theoretical knowledge technical skills could considered soul nursing education the results indicate nursing students develop important qualities hidden curriculum help fulfill mission nursing education creating ethical spiritual legal professional values study one important categories personal growth included self awareness self knowledge self discovery due frequent interactions patients people reflection experiences relationships the meaning category compatible previous study indicating self awareness theme related experiences presence participants previous study also experienced nurse patient interactions resulted self awareness the participants increased self awareness reflection found meaning nurse chiu outcome university courses limited professional development also promoted personal growth nurses being nurse requires compassion respect humanity present study participants described nursing opportunity gain empathy humanistic feelings they appreciated characteristics patience thankfulness kindness compassion concern humankind could help achieve emotional growth emotional growth means increasing capacity recognize one emotions allow mature wilson stated nursing students develop emotional competence critical reflection classroom sessions clinical experiences the participants present study also indicated reflection occurred outside formal sessions classroom nursing profession involving difficult situations nursing students need preparation mature enough successfully handle professional role especially crisis studies reveal nurses younger 30 years experience greater burnout older counterparts such findings support idea emotional issues addressed nursing curricula nurture healthier competent nursing workforce the participants present study also emphasized improvement interpersonal communication skills result nursing studies they believed nursing taught put patient place see eyes increased awareness patients feelings perceptions needs these critical abilities referred social growth qualities gained interactions professors classmates roommates patients classroom although participants muslims must maintain respectful distance members opposite sex interfere nursing tasks findings indicated even strongly religious female participants problems caring male patients another aspect nursing training affected students study relationship god islamic perspective additionally nursing considered divine profession iran ideologic context almost study participants appreciated nursing call god toward transcendence ravari et al also reported perception nursing opportunity worship god common belief among iranian nurses provided career transcendental meaning the study participants reported changes value systems finding deeper meaning beyond superficial activities everyday life lasting goals values university studies provided awakening experience inspired students evaluate redefine personal values since universities expected help students achieve broader perspective nursing education help promote student receptivity human values compatible professional mission indicated empathizing patients caring feelings initiates ethical reflection process nursing students may eventually lead development set personal values the last category revealed although participants starting career gained sense professional ethical commitment similar study rahimaghaee et al participant views commitment went beyond work commitment include inner commitment related belief god supervision lives including careers additionally le duc kotzer found even novice nurses hold professional values indicating nursing education initiate sense professional commitment students according nursing theorists including watson nursing schools acknowledge hidden learning occurs outside classroom foster facilitate development students whole human beings professional nurses according watson curriculum development combine integrate cognitive education beauty art ethics intuition esthetics spirituality human human interactions within nursing nursing discipline needs incorporate type curriculum improve nursing student personal development education this aspect nursing reported paterson zedrad 2007 humanistic nursing theory the findings study revealed nursing education especially clinical training patient student interactions provides excellent opportunity student personal growth less likely majors studies nursing students gain expertise theoretical technical matters alone also fulfill potential whole person nursing education prepare students deliver holistic care consider aspects students humans the ability provide holistic care patients requires treating nursing student whole person formal informal education this idea agrees theories student development consider college education involving whole person process involving interaction intellectual development interpersonal competence therefore recognizing encouraging covert aspects nursing education well overt ones provide opportunities student development cognitive technical domains also affective ones this help bring nursing education closer ideal comprehensiveness enables nurses care holistically patients

background : nursing education is both formal and informal . formal education represents only a small part of all the learning involved ; and many students learn more effectively through informal processes . there is little information about nursing student informal education and how it affects their character and practice.materials and methods : this qualitative study explores undergraduate nursing student perceptions of informal learning during nursing studies . data were gathered through semi - structured interviews with a sample of undergraduate nursing students ( n = 14 ) . strauss and corbin s constant comparison analysis approach was used for data analysis.results:the categories that emerged included personal maturity and emotional development , social development , closeness to god , alterations in value systems , and ethical and professional commitment.conclusion:findings reveal that nursing education could take advantage of informal learning opportunities to develop students nontechnical skills and produce more competent students . implications for nursing education are discussed .

vitro ageing human diploid fibroblasts hdfs first described hayflick moorhead become classical experimental model study cellular ageing hdfs limited ability divide cultured vitro normally 50 cell divisions hdfs enter state irreversible proliferative arrest termed replicative senescence cellular senescence cells less 10 passages considered young cells high proliferative ability cultures 1020 passages entered intermediate state pre senescence cultures 25 passages detectable doubling cell numbers 2 weeks considered senescent cells senescent cells shown accumulate age human tissues thus suggested contribute organismal ageing reactive oxygen species ros implicated replicative senescence ageing physiological metabolism endogenous ros include superoxide anion hydrogen peroxide hydroxyl radicals singlet oxygen accumulation oxidatively damaged cellular macromolecules suggested account free radical theory ageing upon entering state senescence the cell size volume increased accumulation cellular debris intracellular vesicles many lysosomes reported senescent fibroblasts became flattened irregular shape increased expression senescence marker senescence associated -galactosidase sa--gal both vitro vivo studies shown increase percentage cells positive sa--gal cumulative population doublings cpds age nevertheless mechanisms responsible continuous cell growth increased sa--gal expression senescent cells well elucidated cell cycle checkpoints sense damage dna structure elicit complex cellular repair response the checkpoints maintain cell cycle arrest repair takes place followed cell cycle progression repair completed cells undergo permanent cell cycle arrest apoptosis dna repaired adequately a permanent cell cycle arrest occurs cells remaining g0/g1 phase senescence besides oxidative dna damage accumulation telomere shortening widely considered another important mechanism trigger replicative senescence human fibroblasts significant telomere shortening occurred consequence prolonged h2o2 treatment acute treatment showed telomere shortening largely dependent interplay oxidative stress antioxidant defence rather cell divisions a comparison telomere shortening rates different hdfs different conditions revealed ratio oxidative damage antioxidative defence quantitatively important determinant telomere shortening prevent progressive dna loss subsequent rounds dna replication many cells maintain telomeres action telomerase specialized rna protein complex uses rna component template extension telomere reverse transcriptase subunit absence adequate repair system accumulated dna damage caused mutagenic alterations resulting cancer ageing abnormalities nervous system accumulating evidence demonstrated vitamin e potent lipid peroxyl radical scavenger significantly reduced free radical induced chromosomal damage vitamin e shown reduce h2o2-induced oh generation subsequent dna base pair modification human oral epithelial cells h2o2-induced dna strand breaks human skin cell line vh10 measurement dna repair ability tested lymphocytes indicated vitamin e increased removal rate damaged dna compared cells treated vitamin e natural vitamin e comprises eight different isomers -tocopherols -tocotrienols based lipophilicity vitamin e considered major chain breaking antioxidant preventing propagation oxidative stress especially biological membranes vitamin e specifically -tocopherol reported able modulate signal transduction gene expression via antioxidant non antioxidant properties recently different isomers tocotrienol gained increasing scientific interest due eminent antioxidant effects non antioxidant activity profile differs tocopherols tocotrienols found abundance rice bran palm oil oat barley tocotrienol rich fraction consists -tocopherol four isomers tocotrienols potent membrane soluble antioxidants differences properties tocotrienol suggested protective effects diseases cardiovascular disease cancer experimental animals animal model ageing tocotrienol extended lifespan 19% reducing protein carbonylation particularly toxic oxidation process indicative ageing since oxidants contribute ageing process ageing related diseases many species possible protection ageing antioxidants tocotrienol tocopherol suggested even though beneficial effects vitamin e established ageing ongoing studies done determine mechanisms involved actual response different stages cellular ageing view study designed evaluate molecular mechanism tocotrienol rich fraction possibly modulating different stages cellular ageing hdfs determining changes molecular markers cell ageing sa--gal dna damage telomere shortening cell cycle progression young presenescent senescent hdfs this research approved ethics committee national university malaysia approval project code ff-218 2008 primary hdfs derived foreskins three 9- 12-year old boys circumcision the samples aseptically collected washed several times 75% alcohol phosphate buffered saline pbs containing 1% antibiotic antimycotic solution paa austria removing epidermis pure dermis cut small pieces transferred falcon tube containing 0.03% collagenase type solution worthington biochemical corporation usa pure dermis digested incubator shaker 37c 612 h. cells rinsed pbs cultured dulbecco modified eagle medium dmem containing 10% fetal bovine serum fbs paa austria 1% antibiotic antimycotic solution 37c 5% co2 humidified incubator after 56 days cultured hdfs harvested trypsinization culture expand new t25 culture flasks nunc denmark expansion degree 1 4 when subcultures reached 8090% confluence serial passaging done trypsinization number population doublings pds monitored hdfs reached senescence subsequent experiments cells used either passage 4 young cell population doubling pd 12 passage 15 presenescent cell 30 pd 40 passage 30 senescent cell pd 55 cell viability study performed using mts assay briefly stock solutions trf gold tri e 50 golden hope bioganic sdn bhd malaysia freshly prepared 100% ethanol 1 1 kept 20c one month serial dilutions trf concentrations 0.1 0.2 0.3 0.4 0.5 mg ml prepared culture medium mixed 50% ethanol 1 1 medium replaced new medium containing various concentrations trf incubated 24 h 37c 5% co2 after incubation 20 l mts added cells incubated 2 h. absorbance mts formazan formed measured 490 nm microtiter plate reader veramax molecular devices usa the viability assay performed obtain optimum dose trf treatment subsequent experiments subsequent experiments treated hdfs incubated 0.5 mg ml trf 24 h untreated cells cultured dulbecco modified eagle medium dmem containing 10% fetal bovine serum fbs paa austria sa--gal staining performed senescent cell staining kit sigma usa according manufacturer instructions a total 1 10 cells trf treated control groups seeded six well plates incubated fixation buffer 2% formaldehyde/0.2% glutaraldehyde 67 mins room temperature cells rinsed three times pbs incubated 5-bromo-4-chloro-3-indolyl -d galactopyranoside 1 mg ml buffer containing 40 mm citric acid phosphate ph 6.0 5 mm k3fecn6 5 mm k4fecn6 150 mm nacl 2 mm mgcl2 4 h 37c absence co2 blue staining visible incubation percentage blue cells observed 100 cells light microscope calculated telomere assay performed using telotaggg telomere length assay kit roche usa determined telomere length using terminal restriction fragment principle three g dna digested 20 units hinfi rsai 2 h 37c fractionated dna fragments transferred nylon membranes hybond n amersham uk alkaline transfer technique using capillary blotting the blotted dna fragments hybridized digoxigenin dig)-labeled probe specific telomeric repeats ttaggg 3 h 42c gentle agitation dig specific antibody covalently coupled alkaline phosphatase finally immobilized telomere probe visualized alkaline phosphatase metabolizing cdp star highly sensitive chemiluminescence substrate the average terminal restriction fragment length determined comparing signals relative nuclear weight standard x ray film the average terminal restriction fragment length sample obtained scanning exposed x ray film quantifying using imagemaster total lab software detection telomerase activity using telomeric repeat amplification protocol trap cultured cells involves addition ttaggg repeats telomerase oligonucleotide ts subsequent pcr amplification extension products forward ts reverse cx primers the trapeze telomerase detection kit chemicon usa used recommended manufacturer minor modifications the lysis buffer contained 1% nonidet p-40 0.25 mm sodium deoxycholate increase efficiency extraction cells lysed left ice 30 min centrifuged 14,000 g 20 min 4c the extracted protein concentration determined bovine serum albumin bsa standard plot od595 versus g bsa based bradford method pcr reaction the total counts sample added 48 l reaction mixture 2 units taq dna polymerase promega usa after incubation room temperature 30 min telomerase extension reaction samples heated 92c 3 min inactivate telomerase followed pcr amplification pcr products electrophoresed 10% polyacrylamide gel gel analysed quantitated using imagemaster total lab software telomerase activity calculated ratio intensity telomerase ladders intensity 36-bp internal standard dna damage assessed using single cell gel electrophoresis assay scge comet assay the alkaline version comet assay performed according method described singh et al cells embedded 65 l 0.6% low melting point agarose layer boehringer mannheim germany dnase free rnase free agarose coated frosted slides the cells lysed buffer containing 2.5 nacl 100 mm edta 10 mm tris 1% n laurylsarcosine 1% triton 2% dimethylsulphoxide ph 10 1 h 4c thereafter cells exposed strong alkaline solution 300 mm naoh 1 mm edta ph 13 25 min electrophoresis chamber electrophoresis performed 20 min 25 v 200 the slides neutralized 0.4 tris ph 7.5 stained ethidium bromide 20 g ml comets analysed fluorescence microscopy carl zeiss germany visual inspection tail length nuclei the cell nuclei classified five categories 0 undamaged nuclei without comet tail 1 low damaged nuclei comet tails two fold longer nucleus diameter 2 damaged nuclei comet tail two three fold longer nucleus diameter 3 highly damaged nuclei comet tails three fold longer nucleus diameter 4 severely damaged cell nuclei almost visible long dispersed comet tails at least 300 cells per slide counted two slides prepared treatment a total damage score determined multiplying number cells assigned grade damage numeric value grade according methods describe heaton et al total dna damage score calculated follows 1)total dna damage=[(0n0)+(1n1)+(2n2 3n3)+(4n4 n0 cells score 0 n1 cells score 1 n2 cells score 2 n3 cells score 3 n4 cells score 4 untreated control trf treated hdfs subcultured 10 cm tissue culture dishes 24 h incubation cells harvested prepared using cycletest plus dna reagent kit becton dickinson usa according manufacturer instruction cell cycle status analyzed facs caliber flow cytometer becton dickinson usa using propidium iodide pi specific fluorescent dye probe each experiment carried triplicates least 3 independent cultures comparable results comparison groups made anova student test two tailed figure 1 shows percentage viable fibroblast cells incubated trf various concentrations 0.10.5 mg ml 24 h. percentage viable cells significantly increased p .05 trf treatment 0.5 mg ml figure 1(a young hdfs presenescent hdfs percentage viable cells significantly increased p .05 trf incubation concentrations 0.3 mg ml 0.4 mg ml 0.5 mg ml figure 1(b percentage viable cells senescent hdfs significantly increased p .05 trf treatment concentrations figure 1(c therefore trf concentration 0.5 g ml used subsequent experiments types hdfs however senescence original fibroblastic shape lost hdfs became larger flattened accumulation cytoplasmic granular inclusions figures 2(a)2(c the morphology trf treated hdfs resembled young cells spindle shaped cells present figures 2(d)2(f positive blue stain sa--gal appeared mainly hdfs passage 30 suggesting presence senescent cells figure 3 quantitative analysis showed percentage cells positive sa--gal staining increased p .05 senescent cells compared young presenescent hdfs incubation senescent cells 0.5 mg ml trf significantly decreased p .05 percentage positive sa--gal stained cells compared untreated control figure 4 damaged dna higher senescent hdfs compared young presenescent cells p .05 decreased trf treatment p < cell cycle progression analysis showed cell population phase lower p .05 senescent hdfs compared young hdfs treatment trf significantly increased p .05 cells phase g2/m phase stages cellular senescence hdfs in contrast cell populations g0/g1 phase decreased significantly p .05 trf treatment young presenescent senescent hdfs figure 6 figure 7(a shows representative southern blot analysis hdfs various passages trf treatment telomere length senescent hdfs significantly decreased compared untreated young hdfs p .05 similar trf treatment effect telomere length young hdfs figure 7(b figure 7(c shows representative pcr analysis telomerase activity hdfs various passages trf treatment treatment trf significantly increased telomerase activity senescent hdfs p .05 whereby effects observed young hdfs figure 7(d the present study evaluated effects tocotrienol rich fraction trf possibly modulating cellular ageing hdfs our results showed hdfs reached senescence clear changes cell morphology decreased cell proliferation increased senescence associated -galactosidase activity typical morphology senescent cells observed study also reported multiple passages cells granular cytoplasmic vacuoles accumulation common features senescent cells accounting large cells senescent cells also filled large numerous lysosomes contain hydrolytic enzymes function ingesting digesting organelles aged damaged the increased sa--gal positive cells observed senescence hdfs attributed increased lysosomal content increased autophagy may associated increase lysosomal mass sa--gal vitro ageing addition possible find correlation increase sa--gal appearance senescent morphotypes increase sa--gal activity senescent hdfs stress induced premature senescence sips irreversible process upon subculture suggesting cellular ageing results irreversible nonmitotic growth however incubation trf shown decrease percentage cells positive sa--gal suggesting reversal cellular ageing hdfs this effect possibly due antioxidant property trf consists -tocopherols isomers tocotrienols previous study shown tocotrienols high antioxidant activity uptake tocotrienols culture medium cultured cells efficient tocopherols our data cells viability study showed trf treatment increased percentage viable cells increasing concentrations trf the percentage viable cells highest maximum concentration studied 0.5 mg ml indicating trf promoted cells propagation viability stages cellular ageing hdfs oxidative stress caused damage dna increased significantly senescent cells especially postmitotic tissues levels correlated ageing age related diseases reported age related increased levels oxidative dna base damage significant contributor many age related pathological diseases if accumulation damaged dna exceeds elimination dna repair mechanism cellular senescence apoptosis takes place contributes ageing process comparison damaged dna different treatment groups hdfs present study showed total dna damage significantly increased senescent cells less damaged dna observed young presenescent hdfs ros caused chronic persistent damage dna causing double strand breaks remain unrepaired tend accumulate senescent cells indicating possible cause ageing mammals increased ros induced dna damage reported correlated cell cycle arrest similarly data showed significant decreased phase cells increased damaged dna senescent hdfs indicating less cell proliferation greater dna damage cells aged it highly probable protection dna damage trf treatment senescent hdfs could attributed either prevention oxidative stress induced dna damage enhancement dna repair mechanism vitamin e particularly -tocopherol suggested inhibit activation endonuclease triggered intracellular oxidative stress enhanced dna repair increasing rate removal damaged dna previous study reported vitamin e reduced h2o2-induced ho generation subsequent dna base pair modification human oral epithelial cells besides decreasing h2o2-induced dna strand breaks human skin cell line vh10 cellular senescence response dna damage degradation would make cells progeny nonviable might indicate decline renewal capability cells reach irreversible growth arrest our data showed significant difference g0/g1 g2/m phase cellular ageing hdfs phase cells decreased significantly the reduction phase cells senescence attributed slow rate proliferation cells undergoing ageing the decreased increased cells g0/g1 phase phase respectively observed trf treatment various stages cellular ageing indicating inhibition growth arrest enhancement cell replication this observation indicated trf able promote cell progression increased cells replicative capacity decreased level total dna damage increased percentage cells phase trf treated senescent cells indicated potential protective role trf dna strand breaks cell cycle arrest it clearly demonstrated cell replication caused telomere shortening due inability dna polymerase completely replicate 3-ends lagging strands majority normal human somatic cells lack telomerase activity our data present study suggested telomere shortening decreased telomerase activity might contributing factors cellular ageing it reported due inherent limitations mechanics dna replication telomeres shorten cell division absence telomerase telomere shortening reaches critical limit cells susceptible chromosomal aberrations end end fusion aneuploidy situation treatment trf however protected telomere shortening senescent hdfs concomitant increased telomerase activity our previous study shown -tocotrienol -tocopherol protected oxidative stress induced telomere shortening hdfs derived differently aged individuals the restoration telomerase activity induced -tocopherol caused significant increased telomere length especially skin fibroblast obtained old donor 26 41 summary trf treatment exerted better protection senescent hdfs compared young presenescent hdfs possibly higher requirement antioxidants indicated senescent hdfs uptake trf utilization senescent hdfs enhanced protection ros related damage therefore concluded tocotrienol rich fraction delays prevents cellular ageing particularly senescent hdfs shown elongated telomere length decreased levels damaged dna progression cell cycle phase besides increased cell propagation stages cellular ageing hdfs trf treatment

this study determined the molecular mechanisms of tocotrienol - rich fraction ( trf ) in preventing cellular senescence of human diploid fibroblasts ( hdfs ) . primary culture of hdfs at various passages were incubated with 0.5 mg / ml trf for 24 h. telomere shortening with decreased telomerase activity was observed in senescent hdfs while the levels of damaged dna and number of cells in g0/g1 phase were increased and s phase cells were decreased . incubation with trf reversed the morphology of senescent hdfs to resemble that of young cells with decreased activity of sa--gal , damaged dna , and cells in g0/g1 phase while cells in the s phase were increased . elongated telomere length and restoration of telomerase activity were observed in trf - treated senescent hdfs . these findings confirmed the ability of tocotrienol - rich fraction in preventing hdfs cellular ageing by restoring telomere length and telomerase activity , reducing damaged dna , and reversing cell cycle arrest associated with senescence .

young healthy organisms strive maintain proteome functional state tight control rates protein synthesis folding degradation extensive quality control systems set throughout cell prevent manage protein damage as organism ages control mechanisms become less efficient leading disruption protein homeostasis balch et al 2008 david 2012 aging main risk factor variety neurodegenerative diseases specific proteins accumulate pathological aggregates recently considerable interest investigating widespread protein aggregation absence disease multiple studies demonstrated several hundred proteins become highly detergent insoluble aged animals ayyadevara et al 2016 david 2012 david et al 2010 demontis perrimon 2010 reis rodrigues et al 2012 2016 walther et al 2015 computational analysis insoluble proteome indicates overrepresentation proteins functional structural similarities david et al 2010 the examination proteins vivo reveals assembly large the discovery endogenous age dependent protein aggregation model organisms gives us unprecedented opportunity dissect intrinsic cellular machineries responsible preventing protein aggregation without using ectopically expressed human disease associated proteins time little known concerning regulation widespread protein insolubility age consequences health organism interestingly several studies show protein insolubility modified long lived animals reduced insulin insulin growth factor igf)-1 daf-2 signaling remains unclear extent david et al 2010 demontis perrimon 2010 walther et al 2015 a growing number familial sporadic forms neurodegenerative diseases show pathological inclusions caused abnormal aggregation rna binding proteins rbps the first rbps identified inclusions tar dna binding protein 43 kda tdp-43 fused sarcoma fus associated amyotrophic lateral sclerosis als frontotemporal lobar degeneration ftld arai et al 2006 neumann et al 2009 neumann et al 2006 since additional rbps taf15 ewsr1 hnrnpa2b1 hnrnpa1 hnrnpa3 all known rbps associated dementia contain low complexity lc prion like domain enriched glycines uncharged polar amino acids similar sequences driving yeast prion aggregation alberti et al lc prion like domains also present key rbps mediate assembly rna granules liquid liquid phase separation lin et al 2015 significantly small proportion liquid droplets made rbps transform solid aggregates time vitro lin et al 2015 2015 murakami et al 2015 patel et al 2015 clarity use term aggregation referring formation non dynamic rbp aggregates an important question whether special assembly properties rbps puts risk aggregating aging multicellular organism context disease interestingly several rbps lc prion like domains identified insoluble proteome aged animals david et al 2010 overall imperative know causes consequences wild type rbp aggregation aging order fully understand rbp aggregation neurodegenerative diseases furthermore likely organism evolved specific mechanisms control liquid droplet protein aggregation current study chose focus key rbps responsible stress granule formation stress granules specific type rna granule protect cell sequestering mrna translational machinery periods stress importantly stress granule proteins often found co localize pathological inclusions tdp-43 fus bentmann et al whether stress granule proteins innocent bystanders transiently interacting tdp-43 fus whether co aggregate accelerate disease associated rbp aggregation remains intensely debated bentmann et al 2013 li et al 2013 we show key stress granule related rbps sgrbps accumulate aberrant stress granule like puncta large solid aggregates aged c. elegans proteomic analysis revealed long lived animals reduced daf-2 signaling preferentially abrogate insolubility rna granule components importantly sgrbp aggregates associated reduced animal size motility lifespan we show sgrbp aggregation triggered earlier age co aggregation misfolded proteins process prevented daf-16 daf-2 mutants in addition proteostasis network established heat shock transcription factor 1 hsf-1 development required maintain dynamic stress granule proteins throughout animal life to identify quantify changes aggregation prone proteins animals reduced daf-2 signaling performed depth proteomic analysis insoluble proteome control long lived animals figure 1a table s1 protein misfolding aggregation highly abundant aged c. elegans gonads masks changes somatic tissues david et al 2010 goudeau aguilaniu 2010 zimmerman et al 2015 used gonad less mutant focus analysis protein insolubility non reproductive tissues we isolated large aggregates pelleted low centrifugal forces 20,000 g insoluble 0.5% sds in three biological replicates identified 260 insoluble proteins 186 highly prone aggregate age control animals figure 1b the strong correlations control replicates r 0.77 r 0.85 r 0.67 long lived replicates r 0.87 r 0.86 and r 0.82 attest quality quantification experimental reproducibility figures s1a s1b none insoluble proteins prone aggregate age long lived animals compared controls these results consistent previous observations david et al 2010 demontis perrimon 2010 surprisingly recent proteomic study showed endogenous protein insolubility higher daf-2 mutants wild type animals walther et al 2015 to account procedural differences performed extraction following less stringent extraction protocol walther et al ( 2015 omitting sds using ultracentrifugation 500,000 g walther et al analyzed highly insoluble large aggregates study well smaller soluble aggregates however observe general change action daf-2 signaling protein insolubility age using less stringent extraction protocol figure s1c next asked whether inconsistencies studies could related protein aggregation gonad indeed found long lived animals gonads proportionally insoluble proteins compared wild type animals gonads figure s1d these results suggest aggregation gonad masks protective effect reduced daf-2 signaling somatic tissues importantly confirmed protective action reduced daf-2 signaling several candidates see taken together data demonstrate reduced daf-2 signaling promotes protein solubility tissues age investigate specifically changes insolubility regulated reduced daf-2 signaling sorted proteins prone aggregate age control animals 2-fold fold change insolubility age long lived conditions we restricted analysis 87 proteins aggregated less age 0.83-fold 52 proteins continued aggregate age 1.2-fold long lived animals figure 1b table s1 previous bioinformatics analysis aggregation prone proteins revealed enrichment -sheet propensity aliphatic amino acids david et al 2010 a similar enrichment whole insoluble proteome identified study figures s1e s1f intriguingly examining two groups insoluble proteins differentially regulated reduced daf-2 signaling found segregation two properties proteins abrogated aggregation daf-2 rnai treatment highly enriched aliphatic amino acids particular alanine glycine valines sheets figures 1c 1d s1 g conversely proteins still aggregate daf-2( condition significant propensity form sheets modestly enriched aliphatic amino acids figures 1c 1d next searched functional differences two groups differentially regulated reduced daf-2 signaling strikingly ribosomal proteins rna granule components including stress granule p granule rbps highly overrepresented among proteins prevented aggregating age long lived animals tables s2a s3 conversely chaperones vitellogenin yolk proteins overrepresented among proteins still prone aggregate age daf-2( conditions table s2b among rbps four are predicted lc prion like domains alberti et al 2009 : pab-1 fib-1 hrp-1 car-1 figure s2a western blot confirmed reduced daf-2 signaling abrogated aggregation age figures 1e s2b in addition evaluated two proteins without rna binding lc prion like domains par-5 daf-21 quantified mass spectrometry insoluble age control long lived animals we confirmed par-5 daf-21 continued aggregate age long lived animals 7-fold 10-fold respectively albeit reduced extent compared controls figure s2c note changes aggregation correlated changes total protein levels figure s3 a previous study found chemical b isox exclusively causes rna granules precipitate whole cell lysates inducing assembly hydrogel han et al proteins precipitated b isox also enriched aliphatic amino acids particular glycine lesser extent alanine valine propensity form sheets figures 1c 1d s1 g we checked proteins common study found significant overlap proteins precipitated b isox proteins longer aggregating long lived conditions figure 1f table s4 together results indicate different types rna granule components including key rbps responsible assembly become insoluble age long lived animals reduced daf-2 signaling highly successful preventing aggregation interestingly higher levels aliphatic amino acids rna granule components could help assembly and/or drive age dependent aggregation to investigate aggregation key rbps lc prion like domains understand mechanisms involved generated c. elegans strains expressing pab-1 tiar-2 pharyngeal muscles fused fluorescent tags pab-1 tiar-2 c. elegans homologs human polyadenylate binding protein 1 pabp-1 cell restricted intracellular antigen-1 tia-1 two prominent rbps localize stress granules also minor components pathological inclusions occur als fltd bentmann et al 2012 both rbps harbor lc prion like domains figure s2a alberti et al 2009 exposing cells stressors heat induces rbps lc prion like domains form liquid droplets molliex et al 2015 patel et al 2015 similarly heat shock c. elegans caused pab-1 tiar-2 form stress granules figures 2a 2c figure s4a murakami et al 2012 rousakis et al 2014 co expressed pab-1 tiar-2 localized stress granules figure s4b consistent dynamic nature stress granules puncta longer observed 24 hr heat shock figures 2a 2c using antibodies observed similar change pattern endogenous pab-1 indicating effect merely due overexpression fluorescent tag figure s4c as additional control showed kinase kin-19 rbp lacks lc prion like domain localize stress granules upon heat shock figure s4d age observed striking change distribution pattern key stress granule rbps whereas majority pab-1 tiar-2 proteins diffusely localized non stressed young animals found stress granule components accumulated distinct puncta aged animals figures 2b 2d figure s4f ) this change specific pab-1 tiar-2 fluorescent tags alone venus tagrfp remained diffuse age figure s4e david et al 2010 the results simply caused overexpression endogenous pab-1 formed similar puncta age different head regions natively expressed figure s4 g for pab-1 tiar-2 observed significant increase age number worms large puncta visible low magnification microscopy figures 2e 2f figure s4h interestingly imaging high magnification revealed tiar-2 aged animals localized predominantly small puncta highly reminiscent stress granules assembled heat shock whereas pab-1 accumulated greater extent large puncta normally observed upon heat shock young animals figures 2b 2d single plane insets 3a 3c 3d quantification confirmed stress granules induced heat shock aberrant tiar-2 stress granule like puncta formed aging similar sizes figure 3b remarkably co expressed pab-1 preferentially co localized within tiar-2-positive age dependent stress granule like puncta figure 2 g suggesting interactions rbps change aggregation patterns hallmark protein aggregation associated disease immobility proteins within aggregates to evaluate aspect monitored fluorescence recovery photobleaching frap large pab-1 tiar-2 puncta figures 3e3i all large tiar-2 puncta half large pab-1 puncta aged animals showed fluorescence recovery demonstrating solid aggregates figures 3e3i because small size age dependent stress granule like puncta possible assess mobility tiar-2 pab-1 structures consequently used large puncta readout sgrbp aggregation subsequent experiments the consequences age dependent protein aggregation animal health poorly understood here evaluated impact aggregation key stress granule component aging surprisingly pab-1 overexpression protective animals grown 15c 20c 25c life lived longer non transgenic animals table s5 however effect likely due higher levels functional pab-1 related protein aggregation pab-1 aggregate animals 15c figure s5a order distinguish effects specifically related pab-1 aggregation separated pab-1 transgenics day 7 three groups depending aggregation levels we found animals pab-1 aggregation significantly smaller size compared animals without pab-1 aggregation figure 4a figure s5b in addition smaller animals visibly less motile movie s1 importantly mildly stressed animals highest levels aggregation died earlier figure 4b table s5 our proteomic study revealed reduced daf-2 signaling efficiently prevents insolubility stress granule components age we confirmed long lived daf-2 mutants greatly delay formation stress granule like structures large aggregates pab-1 tiar-2 figures 5a 5b figures s6a s6b to gain insight mechanisms controlling protein aggregation investigated role transcription factor hsf-1 activated reduced daf-2 signaling hsu et al 2003 hsp40 modulate stress granule dynamics saccharomyces cerevisiae and/or cell culture cherkasov et al because chaperone expression controlled hsf-1 c. elegans speculated hsf-1 may regulate sgrbp aggregation indeed impairing hsf-1 activity daf-2( wild type backgrounds caused severe pab-1 aggregation already young adults well aged individuals effect reversed overexpressing hsf-1 figures 5c5e figures s6c s6d interestingly hsf-1 daf-2 mutants control aggregation kinase kin-19 previously shown misfold form solid aggregates age figure 5 g david et al 2010 these results suggest hsf-1 regulates different types endogenous protein aggregation age different extents it previously shown assure daf-2( longevity hsf-1 highly expressed acts mainly development volovik et al 2012 we observed impairing hsf-1 activity rnai adulthood effect pab-1 aggregation figure 5f figure s6e conversely reducing hsf-1 activity rnai development caused pab-1 aggregation young adults albeit mainly anterior bulb figure 5f the chaperones discovered regulate stress granule dynamics model systems could also play role preventing sgrbp aggregation yeast hsp40 proteins sis1 ydj1 shown co localize stress granules play role stress granule disassembly walters et al 2015 we evaluated worm strains overexpressing yellow fluorescent protein yfp)-tagged dnj-13 dnj-19 worm orthologs sis1 ydj1 together tagrfp::pab-1 we observed occasional co localization chaperones heat induced pab-1 stress granules age dependent large stress granule like pab-1 puncta figures s6 g s6h these findings confirmed single tagrfp::pab-1 transgenics using antibodies dnj-13 dnj-19 data shown however overexpression dnj-19 dnj-13 significantly reduce pab-1 aggregation age figures s6f s6i indicating chaperones may modulate sgrbp aggregation however observed co staining either pab-1 stress granules induced heat shock pab-1 puncta formed aging therefore possible hsp110 modulate stress granule dynamics sgrbp aggregation c. elegans finally investigated role hsp70 sgrbp aggregation found inhibition hsp70 rnai alter pab-1 aggregation table s6 note later results could caused redundancy chaperone functions collectively results reveal hsf-1 important regulator sgrbp aggregation throughout life contributes maintaining dynamic stress granule proteins long lived daf-2 mutants next examined role transcription factor daf-16 activated reduced daf-2 signaling lin et al 1997 daf-2 mutants daf-16 protected pab-1 aggregation aged animals figures 5c 5d ) importantly daf-2 mutants use daf-16 control different types age dependent protein aggregation delayed aggregation kinase kin-19 also dependent daf-16 figure 5 g recent work performed s. cerevisiae drosophila reveals stress granules dynamically interact misfolded proteins cherkasov et al 2013 kroschwald et al 2015 we speculated widespread protein misfolding aggregation occurring age could promote sgrbp aggregation support hypothesis observed co localization pab-1 kin-19 large immobile aggregates double transgenics figures 6a 6b next evaluated rate pab-1 kin-19 aggregation double transgenics relative aggregation rates single transgenics figures 6c 6d single transgenics importantly pab-1 kin-19 co expressed presence misfolded kin-19 triggered abundant pab-1 aggregation earlier age figure 6c conversely kin-19 aggregation slightly impeded pab-1 overexpression figure 6d indicating sgrbp aggregation cross seed kin-19 aggregation notably pab-1 aggregation accelerated co expression fluorescent tag alone figure 6e furthermore observe general induction pab-1 stress granules young double transgenic animals expressing kin-19 pab-1 figure s6j these data strongly suggest pab-1 aggregation simply consequence generalized cellular stress induced kin-19 overexpression rather co localization kin-19 pab-1 aggregates well earlier accelerated aggregation pab-1 consistent seeding mechanism related kin-19 aggregation taken together interpret findings imply accumulation misfolded proteins age acts seed sgrbp aggregation therefore overall reduction widespread protein aggregation long lived daf-2( conditions evidenced proteomic analysis least part increased daf-16 activity could effective strategy prevent sgrbp aggregation several experimental manipulations shown extend lifespan c. elegans protect proteotoxicity kenyon 2010 taylor dillin 2011 we found dietary restriction mimicked eat-2 mutant animals well inhibition mitochondrial function achieved targeting cyc-1 rnai strongly limited pab-1 aggregation age figures 7a 7b we show wide variety rna granule components become highly insoluble age c. elegans together previous vitro cell culture results our findings demonstrate capacity rbps cycle assembled disassembled states become liability aging organisms already young animals maintaining sgrbp dynamics necessitates active control system established hsf-1 the accumulation misfolded proteins age acts seed aggregation key sgrbps significantly one main outcomes longevity program initiated reduced daf-2 signaling responding widespread protein aggregation preservation rbp solubility age study examined detail aggregation pattern pab-1 tiar-2 two key rbps lc prion like domains important formation stress granules aging process proteins spontaneously assembled small puncta similar liquid droplets induced stress larger aggregates significantly upon co expression pab-1 tiar-2 co localized age related stress granule like structures proteomic analysis revealed number stress granule components insoluble proteome likely secondary stress granule proteins also incorporated therefore attractive hypothesis small puncta represent stress granules formed response stress related aging the inherent aggregation propensity sgrbps would induce least droplets undergo irreversible transition solid state stabilized stress granules could grow large aggregates observed pab-1 simply accumulate age seen tiar-2 an important question remains inherent propensity rna granule components aggregate age could influence pathogenesis neurodegenerative diseases one possibility inherent rbp aggregation impacts cellular health thereby indirectly accelerates pathology we observed reduced lifespan striking decrease size mobility animals higher levels pab-1 aggregation as yet remains unclear whether reduced fitness cause consequence pab-1 aggregation support gain function related sgrbp aggregation two rare diseases caused mutated pabpn1 tia-1 human homologs pab-1 tiar-2 accumulate pathological aggregates brais et al our proteomic analysis aging c. elegans highlighted three rbps lc prion like domains highly prone aggregate age hrp-1 fib-1 car-1 the aggregation human homologs hrp-1 hnrnp a1 hnrnp a3 recently discovered cause multisystem proteinopathy als ftld kim et al indeed loss nucleolar protein fib-1 function caused aggregation age could impair ribosomal biogenesis tollervey et al the mammalian lsm14b lsm14a homologs car-1 localize p bodies eulalio et al 2007 aggregation key p body components could impair non sense mediated decay therefore important investigate consequences fib-1 car-1 aggregation cellular health apart accelerating pathology indirectly reducing cellular health the presence stress granule proteins pathological protein aggregates emerging common denominator different types neurodegenerative diseases including als ftld alzheimer disease huntington disease aulas vande velde 2015 bentmann et al the inherent aggregation propensity stress granule proteins demonstrates unlikely transient interacting partners pathological aggregates it remains determined whether age related sgrbp aggregation acts seed disease associated protein aggregation overall role stress granules neurodegenerative diseases clearly highly complex evidence supporting recruitment disease associated proteins stress granules vice versa interestingly recent cell culture data show assembly stress granules caused disease associated protein aggregation turn promotes pathological aggregation vanderweyde et al 2016 the age dependent aggregation sgrbps prevalence stress granule components neurodegenerative diseases underline relevance therapeutic targets one successful strategy would prevent initial assembly stress granules kim et al 2014 important understand longevity pathways relying dietary restriction defective mitochondrial respiration efficiently prevent sgrbp aggregation case reduced daf-2 signaling sgrbp aggregation suppressed least two mechanisms daf-16 activation prevents cross seeding delaying accumulation misfolded aggregation prone proteins increased activity hsf-1 development assures enhanced sgrbp proteostasis throughout adulthood to date cross seeding rbp aggregation observed huntingtin protein containing expanded polyglutamine repeat region furukawa et al 2009 it important investigate means endogenous aggregation prone proteins lacking motifs similar lc prion like domain would cross seed rbps aggregation overall strategies used restore dynamic nature stress granule proteins could also directly prevent disease associated rbp aggregation a list strains strain maintenance rnai treatment lifespan assays described supplemental experimental procedures worms either fixed imaging analysis directly heat shock allowed recover normal growth ng plates kept 20c fixation images taken synchronized live c. elegans using leica fluorescence microscope m165 fc planapo 2.0 objective leica dfc310 fx camera body length determined worm using fiji software schindelin et al 2012 significance evaluated unpaired two tailed test using graphpad prism 6 worms examined leica sp8 confocal microscope fixation immunostaining protocol including imaging parameters representative confocal images displayed maximum z stack projection unless mentioned otherwise population of synchronized live c. elegans aggregation levels determined using leica fluorescence microscope m165 fc planapo 2.0 objective animals overexpressing pmyo-2::venus::tiar-2 divided two categories animals 10 low aggregation 10 venus::tiar-2 puncta intermediate aggregation anterior posterior pharyngeal bulb animals overexpressing pmyo-2:tagrfp pab-1 divided three categories animals 10 low aggregation 10 intermediate aggregation tagrfp::pab-1 puncta posterior bulb 10 high aggregation tagrfp pab-1 puncta anterior bulb animals expressing pkin-19::kin-19::meos pkin-19::kin-19::venus divided less 10 puncta low aggregation 10 100 puncta intermediate aggregation 100 puncta anterior bulb high aggregation compare aggregation levels kin-19::venus tagrfp::pab-1 evaluated tagrfp::pab-1 puncta formation anterior bulb counting scheme kin-19::venus when comparing different strains counting done blind fashion statistics two tailed fisher exact test significance high intermediate low aggregation levels calculated unless indicated otherwise frap analysis performed previously described david et al 2010 using leica sp8 confocal microscope harmonic compound plan apochromatic hc pl apo cs2 63 1.30 glycerol objective photomultiplier tube pmt detector further experimental details described supplemental experimental procedures obtain large synchronized populations aged animals quantify protein aggregation somatic tissues used temperature induced sterile gon-2 mutants previously described david et al 2010 to induce longevity subjected animals daf-2 rnai control animals gfp rnai last larval stage l4 onward animals aged 25c collected day 3 adulthood young half control animals died days 14 18 aged isolation large sds insoluble aggregates immunoblot mass spectrometry analysis performed previously described david et al 2010 the mass spectrometry proteomics data deposited proteomexchange consortium http://proteomecentral.proteomexchange.org via pride partner repository vizcano et al 2014 dataset identifier pxd003451 aliphatic amino acid residues defined g l v. secondary structure content predicted using psipred v2.6 jones 1999 the p values calculated using unequal variance test compared background set proteins detected mass spectrometry n 5,637 additional details functional analysis described supplemental experimental procedures m.c.l n.g j.c.t d.c.d designed performed experiments

summarylow - complexity prion - like domains in key rna - binding proteins ( rbps ) mediate the reversible assembly of rna granules . individual rbps harboring these domains have been linked to specific neurodegenerative diseases . although their aggregation in neurodegeneration has been extensively characterized , it remains unknown how the process of aging disturbs rbp dynamics . we show that a wide variety of rna granule components , including stress granule proteins , become highly insoluble with age in c. elegans and that reduced insulin / insulin - like growth factor 1 ( igf-1 ) daf-2 receptor signaling efficiently prevents their aggregation . importantly , stress - granule - related rbp aggregates are associated with reduced fitness . we show that heat shock transcription factor 1 ( hsf-1 ) is a main regulator of stress - granule - related rbp aggregation in both young and aged animals . during aging , increasing daf-16 activity restores dynamic stress - granule - related rbps , partly by decreasing the buildup of other misfolded proteins that seed rbp aggregation . longevity - associated mechanisms found to maintain dynamic rbps during aging could be relevant for neurodegenerative diseases .

frequency region essential good speech understanding complex listening environments particularly noise.4 5 individuals substantial bilateral highfrequency hearing loss experience hearing difficulties aspects life home phone work social situations highly frustrated existing hearing aid technology overcome problems reduced word understanding quiet noise.6 7 8 due communication problems may become isolated withdrawing family colleagues friends severe hearing loss areas minimal nonfunctioning hair cells auditory neurons often present resulting cochlear dead regions vibrations basilar membrane detected via inner hair cells neurons region frequencies falling dead region detected via apical basal spread vibrations cochlear places therefore hearing loss given frequency may greater indicated audiometric threshold.9 typically acoustic amplification dead regions improve speech understanding may worsen it.10 11 individuals hearing loss profile may candidates electric plus acoustic stimulation ear treatment options individuals bilateral severe skislope hearing loss limited stateofthe art amplification including frequency lowering,12 effort improve speech intelligibility these attempts often end rejection hearing aids due lack benefit leaving individual alternatives studies shown implant shorter electrode array provides beneficial electric stimulation high frequencies preserving acoustic lowfrequency hearing resulting improved speech understanding.13 14 recently lenarz et al described results european multicenter study using cochlear ltd sydney australia nucleus hybrid l24 implant.15 report results clinical trial leading u.s food drug administration approval firstofitskind combined electric acoustic hybrid implant system address substantial hearing difficulties individuals benefitting amplification eligible standard cochlear implant ci this prospective singlearm multicenter trial determine safety effectiveness hybrid system subjects implanted 10 clinical sites united states served controls test conditions the protocol approved us food drug administration relevant institutional review boards participants gave written informed consent the ear selected implantation severe 75 db hl averaged 2000 3000 4000 hz highfrequency sensorineural hearing loss relatively good lowfrequency hearing 60 db hl 125 250 500 hz in addition aided consonantnucleusconsonant cnc monosyllabic word score 10% 60% using appropriately fit hearing aid required aided word recognition contralateral ear required similar better ear treated better 80% those durations severe profound hearing loss greater 30 years and/or onset hearing loss less 2 years excluded the protocol included acoustic thresholds measured ear preoperatively postoperatively device activation 3 6 12 months postactivation consonantnucleusconsonant words presented 60 dba azbio sentences noise presented 60 dba 10talker babble noise 5 db signaltonoise ratio evaluate effectiveness hybrid system used routinely speech perception outcomes analyzed everyday listening condition listening hybrid system combination acoustic hearing opposite unimplanted ear gain insight into how hearing impacts quality life validated speech spatial qualities hearing questionnaire ssq)16 administered selfassessment hearing within three domains hearing speech various environments spatial hearing sound qualities a device use questionnaire administered addressed overall satisfaction hybrid system relative hearing aids surgery hybrid l24 implant cochlear modification standard cis similar description gantz et al.13 details provided nucleus hybrid l24 implant surgeon guide cochlear).17 postauricular incision surgeon creates well bed skull posterior mastoid opens facial recess posterior tympanotomy widely provide good visibility round window niche middle ear although hybrid implant electrode may inserted round window cochleostomy trial electrodes inserted small cochleostomy created inferior round window performing cochleostomy surgeon opens endosteum cochlea pick prior inserting electrode array the array slowly inserted 16 mm scala tympani instead 19 24 mm typical standard cis the 16mm straight electrode thin 22 halfband modiolarfacing electrode contacts stimulate basal region cochlea intent maintain apical cochlear structures responsible lowfrequency hearing coprimary efficacy hypotheses outcomes cnc words 100 recorded words administered)18 azbio sentences noise 40 recorded sentences administered)19 presented hybrid implant system cochlear would significantly better 6 months postimplantation preoperative performance using hearing aid the sample size 50 subjects exceeded minimum requirement 90% statistical power ensuring adequate power mean differences subjects cnc word azbio sentence recognition scores preoperatively 6month endpoint analyzed using paired tests if evidence assumptions hold wilcoxon signed rank test used secondary efficacy objectives compared individual preoperative performance hearing aid performance 6month endpoint cnc words phonemes azbio sentences although formal hypothesis test conducted endpoints success would achieved 75% subjects showed equal better performance preoperative postoperative scores using binomial model.20 primary safety objective describe safety implantation hybrid system the primary safety endpoint defined surgical and/or devicerelated event reported number proportion individuals experiencing adverse event coprimary efficacy hypotheses outcomes cnc words 100 recorded words administered)18 azbio sentences noise 40 recorded sentences administered)19 presented hybrid implant system cochlear would significantly better 6 months postimplantation preoperative performance using hearing aid the sample size 50 subjects exceeded minimum requirement 90% statistical power ensuring adequate power mean differences subjects cnc word azbio sentence recognition scores preoperatively 6month endpoint analyzed using paired tests if evidence assumptions hold wilcoxon signed rank test used secondary efficacy objectives compared individual preoperative performance hearing aid performance 6month endpoint cnc words phonemes azbio sentences although formal hypothesis test conducted endpoints success would achieved 75% subjects showed equal better performance preoperative postoperative scores using binomial model.20 primary safety objective describe safety implantation hybrid system the primary safety endpoint defined surgical and/or devicerelated event reported number proportion individuals experiencing adverse event mean age 64.1 years standard deviation sd 14.7 years ranging 23 86.2 years implantation there 50/50 split gender 52% right ears implanted mean duration overall hearing loss 28.1 years mean duration severetoprofound highfrequency loss 13.1 years hearing loss etiologies unknown 50% noise exposure 22% familial 20% individual cases 8% related ototoxic drugs autoimmune ear disease high fever infection noise exposure viral hl hearing loss lf low frequency pta pure tone average sd standard deviation table 2 provides summary primary outcomes cnc words azbio sentences noise implanted ear testing implanted ear contralateral ear plugged mitigate contribution speech scores cncs subjects experienced significant p 0.001 improvement 35.8 sd 27.7 percentage points hybrid device hearing aid preoperatively similarly azbio sentences experienced significant p 0.001 improvement 32.0 sd 29.4 percentage points one subject missed 6month assessments data imputed based 3month evaluation < 0.001 one subject missed 6month assessments data imputed based 3month evaluation table 3 presents secondary objective outcomes based binomial comparisons preoperative postoperative changes cnc words azbio sentences implanted ear 6month endpoint the secondary endpoint objectives met 75% subjects demonstrated equal improved performance cnc words phonemes azbio sentences hybrid implant relative performance hearing aid specifically 96% 92% subjects performed equal better cnc words phonemes respectively 90% azbio sentences furthermore 82% 86% showed improved performance cnc words phonemes respectively 74% improved sentences the consistency primary endpoints treated ear examined across subject subgroups defined baseline characteristics gender age duration hearing loss duration severetoprofound highfrequency hearing loss etiology baseline speech perception scores results indicated baseline characteristics gender age duration hearing loss main factors terms speech perception outcomes this case duration severetoprofound highfrequency hearing loss etiology baseline speech scores mean benefit scores i.e. improvement females significantly greater males cnc words females 48.8% males 25.7% azbio tests females 42.6% males 23.5% p 0.002 0.02 respectively ) subjects median implantation age 68 years showed significantly greater benefit cncs 68 years 46.6% 68 years 27.8% p 0.01 azbio sentences 68 years 41.0% 68 years 25.0% ) the mean benefit subjects median hearing loss duration 23.5 years significantly better p 0.01 hearing loss durations 23.5 years cncs 23.5 years 46.2% 23.5 years 27.5% azbio sentences 23.5 years 40.7% 23.5 years 24.7% p 0.05 although trend favored shorter durations mean differences cnc words azbio sentences noise 6months postactivation using implant contralateral hearing aid preoperatively compared bilateral amplification for cncs subjects n 49 showed significant p 0.001 improvement 34.7 percentage points sd 17.4 compared bilateral amplification for azbio sentences subjects n 49 showed significant p 0.001 improvement 33.0 percentage points sd 23.5 compared bilateral amplification no subject showed significant decrement preoperatively postoperatively either measure 6month endpoint subjects performed equal better preoperatively bilateral amplification hearing aids fortyeight subjects completed ssq preoperatively using hearing aids 6 months using hybrid system everyday listening condition speech hearing scale subjects improved significantly p 0.001 showing mean change score 2.2 sd 1.8 spatial hearing scale significant p 0.003 mean change score .9 sd 2.0 sound quality scale subjects experienced significantly p 0.001 improved mean change 1.3 sd 2.0 of 48 subjects completed device use survey four 8% the type frequency events consistent reported cochlear implantation e.g. electrode open short circuits postoperative dizziness changes tinnitus mastoid operations unanticipated adverse events reported fifty events medical surgical nature included instances increased tinnitus vertigo symptoms associated mastoidectomy facial recess approach used cochlear implantation noted nine adverse events reporting dizziness imbalance vertigo likely reported patients nine separate patients one could symptoms dizziness imbalance vertigo unlike prior ci trials first quantify changes residual hearing changes preoperative postoperative hearing sensitivity measured throughout study period changes resulting profound 90 db hl hearing loss reported anticipated adverse events 6months postactivation 66% subjects 33 50 retained functional acoustic sensitivity determined 5frequency pure tone average 125 250 500 750 1000 hz severe degree better 90 db hl the degree hearing loss number subjects hearing loss category postintervention outcomes depicted figure 2a b. addition amount residual hearing number subjects category postintervention outcomes depicted figure 3a 3b subjects aidable residual hearing performed better without aidable residual hearing however even subjects residual aidable hearing performed better ci electriconly condition preoperatively hearing aids regarding 17 subjects maintain functional acoustic hearing five chose hybrid implant explanted replaced standard ci improved speech perception varying degrees observed compared obtained preoperatively hearing aid recent hybrid evaluation prior revision surgery ( cnc word scores subjects 10 1020 2030 30 db hearing loss 6 months postcochelar implant activation ( b azbio 5 db signaltonoise ratio scores subjects 10 1020 2030 30 db hearing loss 6 months postcochlear implant activation number subjects category hearing loss shown color figure viewed online issue available www.laryngoscope.com ( cnc word scores subjects category lowfrequency hearing loss ( b azbio 5 db signal noise ratio scores subjects category lowfrequency hearing loss color figure viewed online issue available www.laryngoscope.com apart cases profound hearing loss two events one sound quality issue one decreased performance resolved database closure association baseline characteristics adverse events including profound hearing loss examined univariate cox proportional hazards regression models baseline characteristics evaluated included age implantation hearing loss duration severetoprofound hearing loss duration etiology preoperative speech perception none found significantly associated either outcome adverse event profound hearing loss mean age 64.1 years standard deviation sd 14.7 years ranging 23 86.2 years implantation there 50/50 split gender 52% right ears implanted mean duration overall hearing loss 28.1 years mean duration severetoprofound highfrequency loss 13.1 years hearing loss etiologies unknown 50% noise exposure 22% familial 20% individual cases 8% related ototoxic drugs autoimmune ear disease high fever infection noise exposure viral hl hearing loss lf low frequency pta pure tone average sd standard deviation table 2 provides summary primary outcomes cnc words azbio sentences noise implanted ear testing implanted ear contralateral ear plugged mitigate contribution speech scores cncs subjects experienced significant p 0.001 improvement 35.8 sd 27.7 percentage points hybrid device hearing aid preoperatively similarly azbio sentences experienced significant p 0.001 improvement 32.0 sd 29.4 percentage points one subject missed 6month assessments data imputed based 3month evaluation < 0.001 one subject missed 6month assessments data imputed based 3month evaluation table 3 presents secondary objective outcomes based binomial comparisons preoperative postoperative changes cnc words azbio sentences implanted ear 6month endpoint the secondary endpoint objectives met 75% subjects demonstrated equal improved performance cnc words phonemes azbio sentences hybrid implant relative performance hearing aid specifically 96% 92% subjects performed equal better cnc words phonemes respectively 90% azbio sentences furthermore 82% 86% showed improved performance cnc words phonemes respectively 74% improved sentences the consistency primary endpoints treated ear examined across subject subgroups defined baseline characteristics gender age duration hearing loss duration severetoprofound highfrequency hearing loss etiology baseline speech perception scores results indicated baseline characteristics gender age duration hearing loss main factors terms speech perception outcomes this case duration severetoprofound highfrequency hearing loss etiology baseline speech scores mean benefit scores i.e. improvement females significantly greater males cnc words females 48.8% males 25.7% azbio tests females 42.6% males 23.5% p 0.002 0.02 respectively ) subjects median implantation age 68 years showed significantly greater benefit cncs 68 years 46.6% 68 years 27.8% p 0.01 azbio sentences 68 years 41.0% 68 years 25.0% p 0.05 although trend favored younger subjects the mean benefit subjects median hearing loss duration 23.5 years significantly better p 0.01 hearing loss durations 23.5 years cncs 23.5 years 46.2% 23.5 years 27.5% azbio sentences 23.5 years 40.7% 23.5 years 24.7% p 0.05 although trend favored shorter durations mean differences cnc words azbio sentences noise 6months postactivation using implant contralateral hearing aid preoperatively compared bilateral amplification cncs subjects n 49 showed significant p 0.001 improvement 34.7 percentage points sd 17.4 compared bilateral amplification azbio sentences subjects n 49 showed significant p 0.001 improvement 33.0 percentage points sd 23.5 compared bilateral amplification no subject showed significant decrement preoperatively postoperatively either measure 6month endpoint subjects performed equal better preoperatively bilateral amplification hearing aids fortyeight subjects completed ssq preoperatively using hearing aids 6 months using hybrid system everyday listening condition for speech hearing scale subjects improved significantly p 0.001 showing mean change score 2.2 sd 1.8 spatial hearing scale significant p 0.003 mean change score .9 sd 2.0 sound quality scale subjects experienced significantly p 0.001 improved mean change 1.3 sd 2.0 48 subjects completed device use survey four 8% the type frequency events consistent reported cochlear implantation e.g. electrode open short circuits postoperative dizziness changes tinnitus mastoid operations unanticipated adverse events reported fifty events medical surgical nature included instances increased tinnitus vertigo symptoms associated mastoidectomy facial recess approach used cochlear implantation noted nine adverse events reporting dizziness imbalance vertigo likely reported patients nine separate patients one could symptoms dizziness imbalance vertigo this trial specified implanting subjects functional lowfrequency acoustic hearing unlike prior ci trials this first quantify changes residual hearing changes preoperative postoperative hearing sensitivity measured throughout study period changes resulting profound 90 db hl hearing loss reported anticipated adverse events 6months postactivation 66% subjects 33 50 retained functional acoustic sensitivity determined 5frequency pure tone average 125 250 500 750 1000 hz severe degree better 90 db hl the degree hearing loss number subjects hearing loss category postintervention outcomes depicted figure 2a b. addition amount residual hearing number subjects category postintervention outcomes depicted figure 3a 3b subjects aidable residual hearing performed better without aidable residual hearing however even subjects residual aidable hearing performed better ci electriconly condition preoperatively hearing aids regarding 17 subjects maintain functional acoustic hearing five chose hybrid implant explanted replaced standard ci improved speech perception varying degrees observed compared obtained preoperatively hearing aid recent hybrid evaluation prior revision surgery ( cnc word scores subjects 10 1020 2030 30 db hearing loss 6 months postcochelar implant activation number subjects category hearing loss shown ( b azbio 5 db signaltonoise ratio scores subjects 10 1020 2030 30 db hearing loss 6 months postcochlear implant activation number subjects category hearing loss shown color figure viewed online issue available www.laryngoscope.com cnc word scores subjects category lowfrequency hearing loss ( b azbio 5 db signal noise ratio scores subjects category lowfrequency hearing loss color figure viewed online issue available www.laryngoscope.com apart cases profound hearing loss two events one sound quality issue one decreased performance resolved database closure association baseline characteristics adverse events including profound hearing loss examined univariate cox proportional hazards regression models baseline characteristics evaluated included age implantation hearing loss duration severetoprofound hearing loss duration etiology preoperative speech perception none found significantly associated either outcome adverse event profound hearing loss results study support conclusion nucleus hybrid system cochlear delivers significantly improved speech understanding quiet noise compared hearing aid individuals bilateral severe highfrequency hearing loss ninety percent subjects achieved better performance speech perception measures listening hybrid system when using ears subjects performed equal better preoperatively measures the ssq selfassessment supported speech intelligibility results significant improvement scales greatest improvement hearing speech scale overall listening satisfaction the number individuals satisfied increased 8% preoperatively amplification 79% hybrid system this system delivers important highfrequency information electrical stimulation opportunity combine beneficial lowfrequency residual hearing one ears outcomes five subjects undergoing revision surgery suggest standard ci remains viable treatment hybrid implantation meet expectations current hearing aid technology often provide audible clear highfrequency sound individuals type hearing loss individuals substantial highfrequency losses frequently nonfunctional inner outer hair cells therefore amplification effective individuals precipitously sloping losses predictably frustrated due significant communication struggles regularly reject amplification leaving alternative treatments prior availability hybrid system limitations study include nonrandomized design limited sample size duration followup using subjects control enables clinically meaningful comparisons account patient heterogeneity use standardized objective measures hearing helps ensure validity the effect sample size large enough produce statistically significant improvements 6 months followup additional longer term followup safety study device larger diverse subgroups important typically accessible amplification individuals bilateral severe highfrequency hearing loss beneficial aidable lowfrequency hearing this system new effective treatment provides clinically significant improvements speech understanding integrated electric acoustic stimulation implanted ear additional benefit listening using ears thus fulfilling need individuals date treatment options

objectives / hypothesisto evaluate the safety and efficacy of acoustic and electric sound processing for individuals with significant residual lowfrequency hearing and severetoprofound highfrequency sensorineural hearing loss.study designprospective , singlearm repeated measures , singlesubject design.methodsfifty individuals , 18 years old , with lowfrequency hearing and severe highfrequency loss were implanted with the cochlear nucleus hybrid l24 implant at 10 investigational sites . preoperatively , subjects demonstrated consonantnucleusconsonant word scores of 10% through 60% in the ear to be implanted . subjects were assessed prospectively , preoperatively , and postoperatively on coprimary endpoints of consonantnucleusconsonant words , azbio sentences in noise , and selfassessment measures.resultssignificant mean improvements were observed for coprimary endpoints : consonantnucleusconsonant words ( 35.8 percentage points ) and azbio sentences in noise ( 32.0 percentage points ) , both at p < 0.001 . ninetysix percent of subjects performed equal or better on speech in quiet and 90% in noise . eightytwo percent of subjects showed improved performance on speech in quiet and 74% in noise . selfassessments were positive , corroborating speech perception results.conclusionthe nucleus hybrid system provides significant improvements in speech intelligibility in quiet and noise for individuals with severe highfrequency loss and some lowfrequency hearing . this device expands indications to hearingimpaired individuals who perform poorly with amplification due to bilateral highfrequency hearing loss and who previously were not implant candidates.level of evidence2b . laryngoscope , 126:175181 , 2016

zoonotic endemic disease mediterranean area asia latin america 2 3 iran the prevalence cvl various parts iran different correlated weather condition humidity 4 it endemic ardebil east azerbaijan fars bushehr provinces 5 domestic dogs canis familiaris main reservoir hosts human visceral leishmaniasis iran 6 prevalence vl human distinct area associated amount cvl dogs 4 foci canine leishmaniasis symptomatic disease low due systemic nature disease clinical manifestations variable 68 it seemed among different control strategies disease vaccination efficient vaccine accessible best possible way eradication disease dogs 6 immunization naked dna latest method promote cd4- cd8-mediated responses 9 10 past years studies focused antigens gp63 cp tsa gp64 lmsti1 leif p8 p4 lack 11 among antigens lack gene one best candidates strains pathogen 12 lack leishmania homologue receptors activated c kinase 36 kda protein localized cytosol external surface membrane 13 it expressed promastigote amastigote forms parasite 14 the protective effect lack vaccine mediated il-12-dependent ifn g production 15 the objective study investigate quality lack protein expression iran strain l. infantum leishmania infantum iran strain mcan ir/07/moheb gh provided school public health tehran university medical sciences promastigotes grown rpmi 1640 medium gibco germany supplemented 5% fetal calf serum 200iu /ml penicillin g crystalizeh dna extracted dna extraction kit mbst iran electrophoresed 1% agarose gel the extracted dna used template amplify lack gene pcr the reaction performed 100l solution containing 1l template dna 2l dntp 100 mol 0.5l taq dna polymerase 5u/l 10l 10x pcr buffer 2 l mgcl2 50 mmol 79.5l distilled water 2l primers 10 pmol/l).we designed pair primer based lack gene sequence accession number u49695 bamhi hindii restriction enzymes 5 forward reverse primer lack f 5- ggatcc tga act acg ag g gtc acc -3 reverse primer introduced hindiii pcr product purified purification kit mbst iran cloned ptz57r vector fermentase vector cat / lack digeted bamhi released lack gene purified sub cloned pcdna3.1 shuttel vector briefly reaction performed 10 l solution containing 5 l purified lack gene 2 l 5x buffer 0.5 l t4 dna ligase 5u/l fermentase 1.5 l d.w distilled water 4c overnight transformed competent cell previously described 16 analysis recombinant colony done three methods colony pcr reaction restriction analysis sequencing the universal primers used detection t7promoter taa tac gac tca cta tag gc bgh rev cta gaa ggc aca gtc gag gc a single colony pc lack cultured od reach 0.6 induced 0.1 molar iptg electrophoresed proteins transferred nitrocellulose membrane western blot analysis done specific protein detected leishmania antibody positive dog serum anti dog conjugate anti igg dog sigma for dilution serum western blot best antigen antibody reaction found dot blot leishmania infantum iran strain mcan ir/07/moheb gh provided school public health tehran university medical sciences promastigotes grown rpmi 1640 medium gibco germany supplemented 5% fetal calf serum 200iu /ml penicillin g crystalizeh dna extracted dna extraction kit mbst iran electrophoresed 1% agarose gel the extracted dna used template amplify lack gene pcr the reaction performed 100l solution containing 1l template dna 2l dntp 100 mol 0.5l taq dna polymerase 5u/l 10l 10x pcr buffer 2 l mgcl2 50 mmol 79.5l distilled water 2l primers 10 pmol/l).we designed pair primer based lack gene sequence accession number u49695 bamhi hindii restriction enzymes 5 forward reverse primer lack f 5- ggatcc tga act acg ag g gtc acc -3 reverse primer introduced hindiii pcr product purified purification kit mbst iran cloned ptz57r vector fermentase vector cat recombinant ptz57r lack digeted bamhi released lack gene purified sub cloned pcdna3.1 shuttel vector briefly reaction performed 10 l solution containing 5 l purified lack gene 2 l 5x buffer 0.5 l t4 dna ligase 5u/l fermentase 1.5 l d.w distilled water 4c overnight transformed competent cell previously described 16 analysis recombinant colony done three methods colony pcr reaction restriction analysis sequencing the universal primers used detection t7promoter taa tac gac tca cta tag gc bgh rev cta gaa ggc aca gtc gag gc a single colony pc lack cultured od reach 0.6 induced 0.1 molar iptg electrophoresed proteins transferred nitrocellulose membrane western blot analysis done specific protein detected leishmania antibody positive dog serum anti dog conjugate anti igg dog sigma specific binding revealed diaminobenzidine dab dako denmark dilution serum western blot leishmania infantum genomic dna extracted lack gene amplified pcr reaction gene cloned ptz57r vector confirmation plasmid colony pcr restriction analysis recombinant plasmid digested bamhi sub cloned pcdna3.1 expressing vector fig 3 electrophoresis lack pc- lack recombinant plasmids pcdna3 plasmid loaded 1% agarose gel./ column1 lack recombinant plasmid column2 pc- lack recombinant plasmid column3 band pcdna3 electrophoresis pcr amplification lack gene pc lack./ column 1 dna ladder column 27 pcr amplification lack gene pc lack column 8 control negative electrophoresis extracted pc lack digestion enzyme/ column 1 dna ladder column 2 pclack digested bamh1 recombinant pcdna3.1 plasmid purified sequenced dideoxy chain termination method compared lack gene accession u49695.1 www.ncbi.nlm.nih.gov/blast-showed high homology 98 fig 4 sequencing lack gene universal primers pc dna3.1 36 kd band recognized leishmania antibody positive polyclonal dog sera protein extracts cells transfected pc lack western blot lack protein ( fig 5 6 sds page analysis expressed gene product column1:dh5 expressing iptg control)column2 pc dna3.1 expressing iptg control)column3 pc lack expressing iptg control)/column4 pre stain protein laddercolumn5 pc lack 1h expressing iptgcolumn6 pc lack 2h expressing iptgcolumn7 pc lack 3h expressing iptgcolumn8 pc lack 4h expressing iptgcolumn9 pc lack 5h expressing iptg column1:dh5 expressing iptg control column2 pc dna3.1 expressing iptg control column3 pc lack expressing iptg control)/ column4 pre stain protein ladder column5 pc lack 1h expressing iptg column6 pc lack 2h expressing iptg column7 pc lack 3h expressing iptg column8 pc lack 4h expressing iptg column9 pc lack 5h expressing iptg western blot analysis expressed gene product column1:dh5 expressing iptg control)column2 pc dna3.1 expressing iptg control)column3 pc lack expressing iptg control)/column4 pre stain protein laddercolumn5 pc lack 1h expressing iptgcolumn6 pc lack 2h expressing iptgcolumn7 pc lack 3h expressing iptgcolumn8 pc lack 4h expressing iptgcolumn9 pc lack 5h expressing iptg column1:dh5 expressing iptg control column2 pc dna3.1 expressing iptg control column3 pc lack expressing iptg control)/ column4 pre stain protein ladder column5 pc lack 1h expressing iptg column6 pc lack 2h expressing iptg column7 pc lack 3h expressing iptg column8 pc lack 4h expressing iptg column9 pc lack 5h expressing iptg leishmania infantum genomic dna extracted lack gene amplified pcr reaction gene cloned ptz57r vector confirmation plasmid colony pcr restriction analysis recombinant plasmid digested bamhi sub cloned pcdna3.1 expressing vector fig 3 electrophoresis lack pc- lack recombinant plasmids pcdna3 plasmid loaded 1% agarose gel./ column1 lack recombinant plasmid column2 pc- lack recombinant plasmid column3 band pcdna3 electrophoresis pcr amplification lack gene pc lack./ column 1 dna ladder column 27 pcr amplification lack gene pc lack column 8 control negative electrophoresis extracted pc lack digestion enzyme/ column 1 dna ladder column 2 pclack digested bamh1 recombinant pcdna3.1 plasmid purified sequenced dideoxy chain termination method compared lack gene accession u49695.1 www.ncbi.nlm.nih.gov/blast-showed high homology 98 fig a 36 kd band recognized leishmania antibody positive polyclonal dog sera protein extracts cells transfected pc lack western blot lack protein ( fig 5 6 sds page analysis expressed gene product column1:dh5 expressing iptg control)column2 pc dna3.1 expressing iptg control)column3 pc lack expressing iptg control)/column4 pre stain protein laddercolumn5 pc lack 1h expressing iptgcolumn6 pc lack 2h expressing iptgcolumn7 pc lack 3h expressing iptgcolumn8 pc lack 4h expressing iptgcolumn9 pc lack 5h expressing iptg column1:dh5 expressing iptg control column2 pc dna3.1 expressing iptg control column3 pc lack expressing iptg control)/ column4 pre stain protein ladder column5 pc lack 1h expressing iptg column6 pc lack 2h expressing iptg column7 pc lack 3h expressing iptg column8 pc lack 4h expressing iptg column9 pc lack 5h expressing iptg western blot analysis expressed gene product column1:dh5 expressing iptg control)column2 pc dna3.1 expressing iptg control)column3 pc lack expressing iptg control)/column4 pre stain protein laddercolumn5 pc lack 1h expressing iptgcolumn6 pc lack 2h expressing iptgcolumn7 pc lack 3h expressing iptgcolumn8 pc lack 4h expressing iptgcolumn9 pc lack 5h expressing iptg column1:dh5 expressing iptg control column2 pc dna3.1 expressing iptg control column3 pc lack expressing iptg control)/ column4 pre stain protein ladder column5 pc lack 1h expressing iptg column6 pc lack 2h expressing iptg column7 pc lack 3h expressing iptg column8 pc lack 4h expressing iptg column9 pc lack 5h expressing iptg canine family particularly domestic dogs considered main reservoir zoonotic transmission 17 dogs usually develop systemic form infection highly variable clinical appearance may involve organ tissue body fluid manifested non specific clinical signs both symptomatic asymptomatic leishmania infected dogs act source parasites vl transmission 17 although anti leishmania drugs successfully used human vl therapy show low efficacy canines 9 18 thus recent studies tended cvl control instead treating control methods variable reservoir control vector control insecticide impregnated materials culling none useful 8 9 19 due simple nature parasite fact recovery resistance results reinfection leishmaniasis vaccination vl feasible 13 20 studies protective vaccine candidate advanced recent years several vaccination methods several antigens tested immunization naked dna dna vaccination new approach promote cd4 cd8 mediated responses helped inducing protective response infection 10 15 21 22 found lack required parasite viability potential drug target leishmaniasis 20 lack might bind enhance plasminogen activation vivo promoting formation plasmin might contribute invasiveness parasite 13 studies for example study combination dna vectors expressing il-12 il-18 booster vaccinia virus recombinant expressing lack mice revealed combined prime booster immunization regime efficient approach protect leishmaniasis 14 heterologous prime boost regime using dna recombinant vaccinia virus vectors expressing lack tested studies relative protection achieved 2225 lack dna vaccine induced robust parasite specific th1 immune response ifn- protective cutaneous systemic l. donovani challenge 15 cloning expression lack gene l. major iranian strain immunology study production recombinant vaccine done 26 27 according prevalence importance visceral leishmaniasis iran necessity production efficient vaccine study the results blast gene bank western blot analyze showed gene correctly cloned vector active immunological aspect therefore study start design recombinant vaccine canine visceral leishmaniasis future

background : there are several methods , such as vaccination , to control visceral leishmaniasis . although there is no efficient vaccine , it seem dna vaccination with stimulates both cellular and humoral immunity apparently is the best way . the aim of this study was cloning and expression of lack gene , a 36kd protein , as a candidate protein for vaccination against iranian l. infantum.methods:iranian strain of l. infantum [ mcan / ir/07/moheb - gh ] was used as a template for pcr to amplify lack gene . the lack gene was cloned in ptz57r / t vector and after confirmation it was digested by restriction enzymes ( bamh1 ) and cloned in pcdna3.1 expression vector . recombinant plasmid was extracted and analyzed by sequencing , restriction digestion analysis and pcr reaction . the pc- lack recombinant plasmid was purified from transformed e.coli ( dh5 ) and its expression was analyzed by sds - page and western blot.results:the results of sequencing , restriction digestion analysis and pcr reaction revealed that lack gene was cloned correctly in pcdna3.1 vector and the results of sds page and western blot emphasized that lack protein of iranian l. infantum is a well - expressed protein.conclusion:we amplified , cloned and expressed iranian l. infantum lack gene successfully .

arterial stiffness increased patients type 2 diabetes mellitus t2 dm potentially increase risk morbidity mortality associated cardiovascular diseases cvd the brachial ankle pulse wave velocity bapwv non invasive technique often used clinically assessment arterial wall stiffness evaluation state atherosclerosis prediction cvd t2 dm patients 2 3 previous studies demonstrated conventional risk factors cvd age gender body mass index bmi duration t2 dm glycemic control dyslipidemia systolic blood pressure sbp estimated glomerular filtration rate egfr uric acid albuminuria associated increased arterial stiffness patients t2 dm 1 4 6 however life style related problems fully taken consideration studies although risk cvd patients t2 dm correlates numerous lifestyle problems 7 8 in fact recently demonstrated poor sleep quality patients t2 dm correlates increased arterial stiffness however study take conventional cardiovascular risk factors full consideration another study showed low physical activity associated increased arterial stiffness newly diagnosed patients t2 dm hand the association lifestyle habits energy intake morningness eveningness sleep duration depressive state bapwv remains largely unknown patients t2 dm aim present study identify lifestyle habits associated bapwv t2 dm patients free apparent cvd using model adjusted numerous conventional cardiovascular risk factors lifestyle habits the subjects cohort study recruited three institutions diabetes outpatient clinic juntendo university tokyo japan naka memorial clinic naka japan secomedic hospital funabashi japan 7 9 10 the inclusion criteria follows 1 male female t2 dm patients 2 age 25 70 years 3 signing consent form participation study the following exclusion criteria also applied 1 type 1 secondary diabetes 2 presence severe infection recent surgery severe trauma 3 history myocardial infarction angina pectoris stroke 4 chronic renal failure requiring hemodialysis 5 liver cirrhosis 6 moderate severe heart failure nyha new york heart association stage iii higher 7 active malignancy 8) pregnancy lactation possible pregnancy 9 patients judged ineligible clinical investigators total 1,032 consecutive subjects screened june 2013 january 2014 906 1,032 patients met eligibility criteria were enrolled study among 906 patients 736 patients agreed participate study the self administered questionnaire survey evaluate sleep quality used present study pittsburgh sleep quality index psqi 9 11 based total psqi score patients divided three groups good sleep quality group psqi score 5 average sleep quality group psqi 6 8 poor sleep quality group psqi 9 we also used morning evening questionnaire meq evaluate morningness eveningness individuals the remaining five questions allowed choice time scales scored 1 5 the sum scores converted three point meq scales follows scores 16 52 represented evening type scores 53 64 represented neither type scores 65 86 considered morning type reported previously the participating patients also completed beck depression inventory bdi)-ii 21-item questionnaire assesses hopelessness irritability cognition guilt fatigue weight loss sexual interest representing depression related symptoms dietary habits preceding month also assessed self administered diet history questionnaire bdhq briefly four page structured bdhq includes questions selected foods designed estimate dietary consumption 56 food beverage items physical activity level assessed four question international physical activity questionnaire ipaq results expressed metabolic equivalent scores metsh week questionnaires the subjects also divided non smokers former smokers current smokers blood samples collected outpatient clinic overnight fast liver renal function tests lipid profile hba1c national glycohemoglobin standardization program measured standard techniques urinary albumin excretion uae measured latex agglutination assay using spot urine sample the egfr calculated formula egfr ml min/1.73 194 age serum creatinine 0.739 females bapwv measured using automatic waveform analyzer bp-203rpe colin medical technology komaki japan described previously 7 9 10 briefly recording performed patients supine position 5-min rest occlusion monitoring cuffs placed snugly around areas upper lower extremities the pressure waveforms recorded simultaneously brachial arteries oscillometric method all scans automatically conducted well trained investigators blinded clinical information a resting ankle brachial index 0.90 considered reflect presence peripheral artery disease the diagnosis peripheral artery disease based ankle brachial index confirmed computed tomographic angiography magnetic resonance angiography catheter angiography based data obtained procedures six patients arterial lumen narrowing 50% excluded analysis results presented mean sd median interquartile range 25 75% continuous variables number proportion patients categorical variables multiple linear regression analysis performed investigate whether possible cardiovascular risk factors evaluated clinical biochemical metabolic tests lifestyle habits associated bapwv patients t2 dm free apparent cvd conventional atherosclerotic risk factors based clinical judgment certain life styles included model all analyses performed using sas software version 9.3 sas institute cary nc the subjects cohort study recruited three institutions diabetes outpatient clinic juntendo university tokyo japan naka memorial clinic naka japan secomedic hospital funabashi japan 7 9 10 the inclusion criteria follows 1 male female t2 dm patients 2 age 25 70 years 3 signing consent form participation study the following exclusion criteria also applied 1 type 1 secondary diabetes 2 presence severe infection recent surgery severe trauma 3 history myocardial infarction angina pectoris stroke 4 chronic renal failure requiring hemodialysis 5 liver cirrhosis 6 moderate severe heart failure nyha new york heart association stage iii higher 7 active malignancy 8) pregnancy lactation possible pregnancy 9 patients judged ineligible clinical investigators total 1,032 consecutive subjects screened june 2013 january 2014 906 1,032 patients met eligibility criteria were enrolled study among 906 patients 736 patients agreed participate study the self administered questionnaire survey evaluate sleep quality used present study pittsburgh sleep quality index psqi 9 11 based total psqi score patients divided three groups good sleep quality group psqi score 5 average sleep quality group psqi 6 8 poor sleep quality group psqi 9 we also used morning evening questionnaire meq evaluate morningness eveningness individuals briefly 11 questions allowed choice scored 1 4 two questions allowed choice scored 0 2 4 6 the remaining five questions allowed choice time scales scored 1 5 the sum scores converted three point meq scales follows scores 16 52 represented evening type scores 53 64 represented neither type scores 65 86 considered morning type reported previously the participating patients also completed beck depression inventory bdi)-ii 21-item questionnaire assesses hopelessness irritability cognition guilt fatigue weight loss sexual interest representing depression related symptoms dietary habits preceding month also assessed self administered diet history questionnaire bdhq briefly four page structured bdhq includes questions selected foods designed estimate dietary consumption 56 food beverage items physical activity level assessed four question international physical activity questionnaire ipaq results expressed metabolic equivalent scores metsh week questionnaires the subjects also divided non smokers former smokers current smokers blood samples collected outpatient clinic overnight fast liver renal function tests lipid profile hba1c national glycohemoglobin standardization program measured standard techniques urinary albumin excretion uae measured latex agglutination assay using spot urine sample the egfr calculated formula egfr ml min/1.73 194 age serum creatinine 0.739 females bapwv measured using automatic waveform analyzer bp-203rpe colin medical technology komaki japan described previously 7 9 10 briefly recording performed patients supine position 5-min rest occlusion monitoring cuffs placed snugly around areas upper lower extremities the pressure waveforms recorded simultaneously brachial arteries oscillometric method all scans automatically conducted well trained investigators blinded clinical information a resting ankle brachial index 0.90 considered reflect presence peripheral artery disease the diagnosis peripheral artery disease based ankle brachial index confirmed computed tomographic angiography magnetic resonance angiography catheter angiography based data obtained procedures six patients arterial lumen narrowing 50% excluded analysis results presented mean sd median interquartile range 25 75% continuous variables number proportion patients categorical variables multiple linear regression analysis performed investigate whether possible cardiovascular risk factors evaluated clinical biochemical metabolic tests lifestyle habits associated bapwv patients t2 dm free apparent cvd conventional atherosclerotic risk factors based clinical judgment certain life styles included model all analyses performed using sas software version 9.3 sas institute cary nc among 736 participating patients 12 complete questionnaires and/or bapwv measurement and/or ankle brachial index 0.90 accordingly excluded analysis table 1 summarizes characteristics remaining 724 japanese patients t2 dm mean age 57.8 8.6 years 62.8% subjects males the mean hba1c 7.01.0% estimated duration t2 dm 9.9 7.2 years most subjects attended educational programs past diet exercise therapy received appropriate medical treatments alt alanine aminotransferase ast aspartate aminotransferase bapwv brachial ankle pulse wave velocity egfr estimated glomerular filtration rate hdl c high density lipoprotein cholesterol uae urinary albumin excretion -gtp -glutamyl transpeptidase the results multivariable linear regression analysis shown table 2 regression analysis included age gender major confounding factors atherosclerosis demonstrated age male sex positively associated bapwv model 1 model 2 adjusted conventional cardiovascular risk factors ( model 1 age duration t2 dm hba1c sbp uric acid uae positively associated bapwv bmi negatively associated bapwv almost similar findings observed model 3 adjusted alcohol consumption smoking background therapies cvd risk factors model 2 model 4 adjusted lifestyle habits model 3 age duration t2 dm sbp uric acid uae poor sleep quality positively associated bapwv bmi negatively associated bapwv multiple linear regression analysis included age gender model 1 model 1 plus body mass index estimated duration diabetes hba1c systolic blood pressure diastolic blood pressure total cholesterol high density lipoprotein cholesterol triglyceride ast alt -gtp serum uric acid egfr uae model 2 model 2 plus current smoker alcohol diabetic retinopathy insulin therapy hypertension medication hyperlipidemia medication anti platelet agents model 3 model 3 plus morningness eveningness questionnaire pittsburgh sleep quality index beck depression inventory ii energy intake kcal day physical activity kcal day sleep time model 4 est standardized estimate regression parameter alt alanine aminotransferase ast aspartate aminotransferase bapwv brachial ankle pulse wave velocity egfr estimated glomerular filtration rate hdl c high density lipoprotein cholesterol uae urinary albumin excretion -gtp -glutamyl transpeptidase in agreement previous studies 1 4 6 20 data demonstrated conventional cardiovascular risk factors age duration t2 dm sbp uric acid uae associated increased arterial stiffness patients t2 dm even adjustment lifestyle habits furthermore found poor sleep quality associated arterial stiffness even adjustment several conventional cardiovascular risk factors lifestyle habits regard previous report demonstrated high levels catecholamines caused poor sleep quality may associated progression arterial stiffness healthy middle aged adults high catecholamine levels could promote smooth muscle cell proliferation fibrosis leading structural changes arterial wall although study design allow evaluation causal relations data suggest poor sleep quality may important target interventions prevent arterial stiffness patients t2 dm contrast data failed show close association lifestyle problems energy intake physical activity morningness eveningness sleep duration depressive state arterial stiffness patients t2 dm a recent clinical trial demonstrated life style interventions special focus reduced calorie intake increased physical activity affect rate cvd obese t2 dm patients it possible caloric intake rather dietary composition may need modified prevent delay development cvd patients t2 dm while previous reports demonstrated bmi positively associated arterial stiffness 5 20 24 25 bmi inversely associated arterial stiffness study consistent previous report non t2 dm patients the exact explanation conflicting results unknown however previous study showed higher bapwv levels t2 dm patients normal bmi increased visceral adiposity compared higher bmi normal visceral adiposity generally increased visceral adiposity associated insulin resistance increases production inflammatory cytokines leading arterial stiffness since asians tend insulin resistance even normal bmi evaluation visceral adiposity may helpful assessment effect obesity arterial stiffness subjects a close association serum uric acid arterial stiffness male patients newly diagnosed t2 dm reported previously even adjustment several risk factors atherosclerosis present study demonstrated significant association serum uric acid arterial stiffness wider range t2 dm population study even adjustment lifestyle habits addition numerous cardiovascular risk factors however whether serum uric acid plays important role potentially useful sensitive marker identifying patients high risk cvd clear cross sectional study indeed deleterious effect hyperuricemia arterial stiffness may mediated interactive cardiovascular risk factors and/or effect hyperuricemia may depend stage atherosclerosis 6 29 first cross sectional design allow evaluation causal relationship risk factors atherosclerosis arterial stiffness third rule possible effects lifestyle habits assessed study arterial stiffness conclusion found several conventional cardiovascular risk factors age duration t2 dm sbp serum uric acid uae lower bmi poor sleep quality associated bapwv even adjustment multiple traditional cardiovascular risk factors lifestyle habits in conclusion found several conventional cardiovascular risk factors age duration t2 dm sbp serum uric acid uae lower bmi poor sleep quality associated bapwv even adjustment multiple traditional cardiovascular risk factors lifestyle habits the study approved institutional review board hospital conducted accordance principles described declaration helsinki all patients provided written informed consent participation registered university hospital medical information network clinical trials registry umin000010932 hm employee takeda pharmaceutical co. hw received lecture fees novo nordisk inc eli lilly company sanofi dainippon sumitomo pharma co. fujifilm bayer health care kissei pharmaceutical company mochida pharmaceutical company msd takeda pharmaceutical company boehringer ingelheim pharmaceuticals inc daiichi sankyo ono pharmaceutical co. ltd novartis pharmaceuticals corporation mitsubishi tanabe pharma corporation astrazeneca lp kyowa hakko kirin company ltd sanwa kagaku kenkyusyo company ltd kowa company ltd astellas pharma inc advisory fees novo nordisk inc mochida pharma company astrazeneca lp kowa company astellas pharma inc msd mitsubishi tanabe pharma corporation novartis pharmaceuticals corporation dainippon sumitomo pharma co. takeda pharmaceutical company ono pharmaceutical co. pfizer inc kowa company research funds boehringer ingelheim pfizer mochida pharmaceutical co. sanofi aventis novo nordisk pharma novartis pharmaceuticals sanwakagaku kenkyusho terumo corp takeda pharmaceutical co. msd shionogi pharma dainippon sumitomo pharma kissei pharma astrazeneca all authors contributed study design involved stages manuscript development all authors also involved collection analysis interpretation data reviewed edited manuscript approved final manuscript hw principal guarantors work full access study data takes responsibility integrity data accuracy data analysis this study conducted research grant manpei suzuki diabetes foundation tm the funding agency role study design data collection analysis decision publish preparation manuscript alanine aminotransferase brachial ankle pulse wave velocity brief self administered diet history questionnaire beck depression inventory systolic blood pressure cardiovascular disease estimated glomerular filtration rate -glutamyl transpeptidase high density lipoprotein cholesterol international physical activity questionnaire morningness eveningness questionnaire pittsburgh sleep quality index type 2 diabetes mellitus urinary albumin excretion

backgroundwhile conventional cardiovascular risk factors and certain lifestyle habits are associated with arterial stiffness in patients with type 2 diabetes mellitus ( t2 dm ) , it is still unknown whether they are actually associated with arterial stiffness even after adjustment for conventional cardiovascular risk factors and lifestyle habits . the aim of this study was to identify variables that are associated with brachial - ankle pulse wave velocity ( bapwv).methodsthe study participants comprised 724 japanese t2 dm outpatients free of history of cardiovascular diseases . lifestyle habits were analyzed using self - reported questionnaires . the associations among conventional cardiovascular risk factors and lifestyle habits with bapwv were investigated by multivariable linear regression analysis.resultsthe mean age of the study subjects was 57.8 8.6 years , and 62.8% of those were males . the mean hba1c was 7.01.0% , and the estimated duration of t2 dm was 9.9 7.2 years . multiple linear regression analysis that included age and gender demonstrated that age and male sex were positively associated with bapwv . in a model adjusted for numerous conventional cardiovascular risk factors and lifestyle habits , age , duration of t2 dm , systolic blood pressure , serum uric acid , urinary albumin excretion and poor sleep quality were positively associated with bapwv , while body mass index was negatively associated with bapwv.conclusionsin japanese t2 dm , in addition to several conventional cardiovascular risk factors , poor sleep quality was associated with bapwv even after adjustment for numerous conventional cardiovascular risk factors and lifestyle habits .

began investigation high reliability might mean health care analyzing known highly reliable organizations function weick sutcliffe provide compelling depiction high reliability organizations hros stay safe they describe environment collective mindfulness workers look report small problems unsafe conditions pose substantial risk organization easy fix weick sutcliffe 2007 they prize identification errors close calls lessons extract careful analysis occurred events these lessons point specific weaknesses safety protocols procedures remedied reduce risk future failures the five high reliability principles weick sutcliffe spell elucidate capability high reliability organizations achieve maintain exemplary levels safety hros preoccupied failure never satisfied accident many months many years always alert smallest signal new threat safety may developing people work hros also resist temptation simplify observations experiences environment threats safety complex present many different forms accordingly able identify often subtle differences among threats may make difference early late recognition finding unsafe condition easy correct failing recognize problem getting control hros recognize earliest indicators threats organizational performance typically appear small changes organization operations they thus take great pains ensure workers intimately involved operations always report deviations expected performance addition hros make sure everyone feels free speak concerns also recognizes obligation highly organization values information vital component ability achieve highest priority near perfect safety hros recognize despite best efforts past safety successes errors occur safety threatened the hallmark hro error free errors n't disable resilience refers organization capability recognize errors quickly contain thereby preventing harm results small errors propagate compounded mushroom major problems when confronted new threat hros mechanisms place identify individuals greatest expertise relevant managing new situation place decision making authority hands person group they invoke organizational hierarchy expect person seniority highest rank effective dealing problem how close far away typical hospital today state high reliability ? rarely observe five principles high reliability guiding actions organizations leaders caregivers as opposed preoccupation avoiding failure hospitals health care organizations behave accept failure inevitable feature daily work how else could explain estimates 99,000 americans die hospitals year health care associated infections hand hygiene compliance routinely registers 40 percent range among many examples ? fortunately events happen rarely perspective individual surgeon hospital close eliminating entirely american health care health care rarity adverse events like tends reinforce organizations beliefs never experience leads misplaced confidence safety systems adequate this complacency blunts alertness surgical teams small signs risk surgical fire wrong site surgery hros recognize complacency threat safety take great pains let take root failing resist for example often approach quality problem simple one size fits best practice solution the joint commission universal protocol developed eliminate wrong site surgery one example it consists three simple steps 1 verify identity patient intended procedure 2 mark surgical site 3 conduct time operating room surgery begins order verify patient procedure operative site correctly identified but this overly simple approach eliminated problem large part fails account complexities surgical process different ways risks wrong site procedure may introduced example risks may arise scheduling surgical procedure set problems universal protocol address one pervasive safety problems hospitals relates failure sensitive operations health care workers levels routinely observe unsafe conditions behaviors practices often fail bring problems attention managers placed appropriately daily work flow address problems quickly transitions one care setting another called handoffs fraught risk error due incomplete inaccurate communication crucial patient information when caregivers come expect poor communication become desensitized hazards one analysis culture low expectations explained substantial number errors led patient undergoing invasive procedure intended someone else chassin becher 2002 thus lack recognition unsafe conditions practices one important reason reported in addition health care workers kinds exposed inordinate amount intimidating behavior suppresses reporting safety problems physicians often seen initiators intimidating disrespectful behavior nurses commonly seen targets leape et al 2012 saxton hines enriquez 2009 caregivers kinds involved unsafe situations 2004 institute safe medication practices published results workplace intimidation survey focused process receiving interpreting acting medication orders institute safe medication practices 2004 more two thousand respondents mainly nurses pharmacists reported variety behaviors personally experienced preceding twelve months the common behaviors perceived intimidating flagrantly abusive practices throwing objects using loud profane language rather failure return phone calls pages use condescending language impatience questions topped list between 60 67 percent respondents said personally experienced behaviors initiated physicians three times preceding year 20 28 percent said experienced behaviors ten times the caregivers experienced behaviors employed variety strategies suboptimal risky deal including asking someone else talk intimidating prescriber safety concern regarding medication order 39% refraining contacting prescriber attempting clarify safety drug order 67% asking colleagues help interpret order avoid interact particular prescriber 75% hros tolerate existence intimidating behaviors suppress reporting safety concerns perpetuate existence unsafe conditions a specific example helps illuminate complexities barriers hospitals face trying sensitive safety signals many medical devices employed routine hospital care come equipped alarms make various sounds preset parameters exceeded intravenous infusion pumps cardiac rate rhythm monitors mechanical ventilators blood oxygen monitors common ones caregivers bombarded hourly alarms especially working intensive care areas housing sickest patients greatest number devices per patient the number alarms sound per patient per day total several hundred for a variety reasons vast majority perhaps many 85% 99% alarm sounds signify clinical situations danger these reasons include poor integration devices one another equipment malfunction inappropriate alarm settings gaps staff training the result caregivers experience alarm fatigue may take variety unsafe actions turning alarms entirely turning sound volume point inaudibility resetting alarm unsafe levels ignoring alarm sounds altogether joint commission 2013 the joint commission voluntary adverse event reporting program recorded ninety eight alarm related events 2009 june 2012 eighty resulting death the ecri institute cited problem one top ten health technology hazards year since 2007 ecri institute 2012 comprehensive solution problem would require many stakeholders work together including device manufacturers information technology experts physicians medical informatics professionals nurses clinical engineers hospital administrators imagine risks safety nuclear power plant alarm systems functioned fashion hospitals health care organizations exhibit features resilience characterize hros high reliability environment errors unsafe conditions recognized early prevented rapid remediation causing harm but health care uncoordinated poorly designed maintained mechanical systems like medical device alarms tolerated even though safe intimidating behaviors suppress reporting lead additional unsafe behaviors caregivers create workarounds avoid repetitive exposure intimidators errors seen valuable information essential hospital ability improve patient safety 2012 report results annual patient safety culture survey the federal agency healthcare research quality stated average 65 percent respondents 1,128 hospitals worried mistakes made kept personnel files 50 percent agreed staff felt mistakes held agency healthcare research quality 2012 finally attempting solve safety quality problems hospitals regularly permit expert individual implement solutions pharmacists often difficult time bringing considerable expertise bear avoid medication errors often health care teams multidisciplinary name physicians senior administrators dominating scene westrum coined term sociological analysis pediatricians failed identify child abuse 1960s he suggested one important underlying phenomena pediatricians ingrained belief if something crucial child health physical abuse parent going surely pediatricians would know bring attention pediatricians but n't know therefore n't happening westrum 1982 ) health care risk individual falling prey fallacy centrality would seem increase seniority this mind set particularly risky organizational leaders encourages risky belief news good news hospitals news often means intimidated caregivers recognizing reporting unsafe conditions soon enough harm patients thus available data considerable experience suggest strongly five principles high reliability would unrecognizable average hospital daily work contrary several instances particularly involving rapid identification management errors unsafe conditions appears today hospitals often exhibit opposite high reliability there important corollary observation hospitals currently characterized low reliability this fact implies strongly hospitals solve problems simply directly adopting high reliability principles practices imagine might happen workers hospital suddenly acquired keen sense collective mindfulness began recognize report unsafe conditions errors encountered moment arrived hospital the organization would soon deluged reports capacity fix problems uncovered reports would overwhelmed many unsafe conditions would necessarily remain unaddressed of course transformation organization culture take place night we must take careful note hospitals function today key arenas must change high reliability become possible this possibility become real accurately describe hospitals current state chart plausible feasible pathway toward high reliability one defines specific milestones representing incremental progress is guidance high reliability literature chart pathway much weick sutcliffe offer series audits rating scales assess extent organization behaving like hro give general advice improve weick sutcliffe 2007 chaps 5 6 but reason offers similar assessment tool checklist assessing institutional resilience cair adapted health care carthey de leval reason 2001 these thoughtful contributions help focus us hospitals become highly reliable but give us much insight precisely goals accomplished in brief know well documented blueprints elevating low reliability organization industry highly reliable one sustaining achievement time as noted earlier described elsewhere broad conceptual framework adapting high reliability science health care organizations chassin loeb 2011 this framework derived integration high reliability science considerable experience working thousands health care organizations joint commission accredits certifies studies explaining hospitals started adapt high reliability principles operations dixon shofer 2006 fei vlasses 2008 frankel leonard denham 2006 may 2013 wolterman shabot 2012 we explored three major changes health care organizations would undertake order make substantial progress toward high reliability 1 leadership commitment ultimate goal zero patient harm 2 incorporation principles practices safety culture throughout organization 3 widespread adoption deployment effective process improvement tools methods we elaborate three changes specific respect hospitals health systems leadership commitment we mean aligned agreement governing body typically board trustees directors senior management physician nurse leaders all constituencies leadership formal informal must share singular vision eventually eliminating harms patients this essential initial requirement success changes depends the goal zero also important one salient characteristics high reliability organizations satisfied whatever current level safety might commercial aviation averaged 129 deaths per year accidents logged average 9.3 million flights per year translating death rate 13.9 deaths per million flights next decade however 2002 2011 death rate plummeted remarkable 88 percent 1.6 deaths per million flights even though average annual number flights increased 10.4 million per year number deaths dropped average 16.6 per year u.s department transportation 2012 for past thirty years commercial aviation invested heavily radically changing flight crews culture order advance airline safety this work began following research conducted national aeronautics space administration 1970s demonstrating majority airplane crashes caused catastrophic mechanical failures failures communication among pilots crew the development worldwide deployment focused highly effective training programs establish safety culture aircraft flight decks followed these programs known crew resource management widely credited playing important role dramatic safety improvements industry witnessed time period helmreich merritt wilhelm 1999 one original developers crew resource management airline industry since turned attention health care he colleagues found professional culture operating rooms communication errors related quite similar found among aircraft crews helmreich 2000 this work led series efforts apply principles methods crew resource management health care gordon mendenhall o'connor 2013 since 2009 joint commission required leadership health care organizations accredits create maintain culture safety consequently many hospitals conduct staff surveys assess safety culture agency healthcare research quality 2012 sexton et al few however moved beyond tabulating survey results taking effective actions resulted creating kind safety culture supports high reliability we proven tools methods guide hospital leaders achieve fully functional safety culture the model describe practical framework derived work reason hobbs reason hobbs 2003 organizational culture depict is based reason years studying complex organizations prevent fail prevent accidents cause harm we believe model one adaptable appropriate health care the third major changes relates hospitals carry efforts improve performance care processes it domain high reliability science provides least guidance health care hros safety processes fail 40 60 percent time case health care e.g. hand hygiene handoff communication bodenheimer 2008 erasmus et al the specific tools methods hros use maintain nearly perfect safety procedures directly relevant setting reliability low health care we believe three sets process improvement tools lean six sigma change management constitute effective way health care dramatically enhance capacity create nearly perfect safety processes dellifraine langabeer nembhard 2010 dupree et al we call three robust process improvement rpi joint commission center transforming healthcare 2013 they represent next generation process improvement methods developed industry imported health care they proving far effective addressing complex clinical quality safety problems pdca plan check act immediate predecessors continuous quality improvement total quality management goldberg 2000 one important distinguishing features newer improvement methods systematic attention uncovering specific causes failures safety processes e.g. hand hygiene pinpointing specific causes e.g. improper use gloves faulty maintenance procedures keep hand gel dispensers full measuring ones prevalent particular area hospital tools direct improvement efforts eliminate causes majority failures careful attention unraveling complexities health care quality safety problems tools robust process improvement offer health care means implement reluctance simplify principle high reliability the joint commission adopted rpi internal process improvement methodology first five years program began 2008 trained 35 percent workforce using tools adrian 2009 since 2009 the joint commission center transforming healthcare applying rpi tools together teams hospitals around country also mastered use address number health care persistent quality safety problems table 1 shows rates improvement demonstrated center first four projects the joint commission experience consistent companies ge employed tools many years great benefit bartlett wozny 2005 rao 2011 improvements seen four projects using robust process improvement notes robust process improvement combination three complementary process improvement methods lean six sigma change management percentage times caregivers cleaned hands walking patient room having established three major domains change considered hospitals health systems operate today might evolve slowly rapidly toward high reliability three areas clearly industry contains much heterogeneity observing differences helps better characterize current state directions evolution devising framework identified several specific components three domains change fourteen components four stages maturity would define progress toward high reliability we observed hospitals health systems several components currently reside four stages high reliability but intentionally attempt add fifth stage perhaps labeled arriving future would describe high reliability hospital know hospitals achieved high reliability across activities therefore firsthand observations use description we created framework two year period employing variety methods sources a team joint commission engaged high reliability experts academia industry since 2010 assimilate known hros institutional knowledge gained work health care quality safety these experts include widely published authors officials executives hros commercial aviation chemical petroleum industries nuclear power military among activities joint commission hosted fifth international high reliability conference may 2012 health care executives interacted representatives academia hros ten different industries joint commission 2012 to produce first draft framework combined information gleaned experiences empirical literature characteristics hospitals associated improved safety quality we conducted two rounds pilot testing health care leaders first round a small group five individuals hospital leadership roles chief quality officers chief medical officers chief executive officers examined framework provided qualitative assessments face validity including whether appropriate elements included framework whether eliminated defined differently separately produced review self assessment questionnaire designed elicit information hospital leaders would permit us assign fourteen components high reliability one four stages maturity based first round reviews made appropriate changes framework questionnaire second round testing each team engaged process four six leaders variety different leadership perspectives included chief executive officers chief nursing officers chief medical officers chief quality officers chief information officers others similar responsibilities we compiled data round testing convened face face meeting teams leaders typically chief executive officer seven hospitals discuss experiences teams the results round testing incorporated framework questionnaire finalized field testing table 2 depicts six components leadership one characteristics four stages maturity the six components board trustees chief executive officer senior management including nursing leaders engagement physicians hospital quality strategy use dissemination data measures quality use information technology support quality safety improvement the identification specific components supported published literature linking better quality performance goeschel wachter pronovost 2010 jha epstein 2010 weiner shortell alexander 1997 the hospital leaders commitment high reliability must include prominent role board trustees directors the board must part leadership commitment eventually achieve zero patient harm elevate quality patient safety organization highest strategic goal if board left management find efforts unsupported misunderstood today hospital boards vary wide spectrum involvement quality programs hospitals oversee jha epstein 2010 leadership high reliability stages organizational maturity addition physicians essential success quality initiative hospitals table 2 identifies two vital components physicians role leadership participation order move effectively toward high reliability physicians must routinely champion quality improvement initiatives throughout hospital formally appointed leaders chief medical officer vice president medical affairs informal leaders medical staff president voluntary medical staff leaders need visible active enthusiasts quality including physician leaders employees hospital it difficult imagine hospital getting close high reliability quality merely one many competing priorities memorial hermann health system twelve hospital health care system based houston explicitly committed becoming highly reliable specified importance major ingredients framework efforts shabot et al system ceo pointed ensuring patient safety core value core value ( wolterman shabot 2012 accelerate progress toward zero harm quality must measured data measures must widely available within hospital public not transparency valuable right public reporting also powerful added force drives improvement quality program measures focus meeting needs addressing specific quality problems hospital patient population other incentives judicious use financial rewards staff advancement opportunities based performance quality measures important accelerants well finally leaders obligated employ health information technology effectively service quality improvement it particularly important hro frequently vehicle nearly perfect processes sustain performance if process effectively redesigned highly reliable automating effective way maintain state unfortunately health care automation often deployed unsafely phenomenon increases rather decreases risk harm ash et al in addition various types health often coordinated thereby increasing risk for example programmable infusion devices supported decision support rules govern pharmacy systems physician order entry systems resulting confusion life threatening patients a hospital approaching high reliability adopts health solutions coordinated integrated manner following principles safe adoption joint commission 2008b karsh 2004 table 3 shows five components safety culture manifestations four stages maturity toward high reliability a culture safety fully supports high reliability three central attributes trust report improve reason hobbs 2003 workers exhibit enough trust peers organization management routinely recognize report errors unsafe conditions this trust established organization eliminates intimidating behavior suppresses reporting acts timely way fix problems reported workers communicates improvements consistently individuals reported problems first place communication turn strengthens the trust led reports fosters identification reporting problems even upstream harm when three components safety culture trust report improve working well reinforce one another produce stable organizational culture sustains high reliability safety culture high reliability stages organizational maturity maintaining trust also requires organization hold employees accountable adhering safety protocols procedures hros establish clear equitable transparent processes recognizing separating small blameless errors people make every day unsafe reckless actions blameworthy understanding blameless errors occur part learning process hros employ maintain exemplary safety records recognizing dealing appropriately blameworthy acts equally important dimension hro safety culture vital role maintaining trust unfortunately health care organizations often punish staff blameless acts failing implement equitable disciplinary procedures commit blameworthy acts hospital leaders succeeded eradicating intimidating behaviors institute safe medication practices 2004 these failings explain lack trust among hospital staff noted earlier agency healthcare research quality 2012 hospitals move toward high reliability establish codes behavior modeled leaders including nurses physicians champion efforts eliminate intimidation encourage reward reporting blameless errors unsafe conditions accountability adhering safe practices ingrained employees spurred implementing standards invoking disciplinary procedures apply staff regardless seniority professional credentials for example maimonides medical center new york city established program code mutual respect commits stakeholders including physicians nurses staff students vendors consultants volunteers working harmoniously together eliminate intimidating behaviors the program includes progressive interventions including disciplinary actions individuals repeatedly violate code maimonides medical center 2009 hros also proactively assess strength resilience safety systems organizational defenses prevent errors propagating leading harm today hospitals function primarily reactive mode investigating incidents patients already harmed conducting root cause analyses instituting corrective action plans prevent future occurrences becoming much safer requires caregivers willingness ability recognize report close calls unsafe conditions combined organizational capacity act effectively reports eliminate risks embody furthermore opposed today norm focusing single events hospitals compile results investigations across many harm events close calls identify safety systems defenses need improvement these evaluations lead development proactive assessments key safety systems e.g. relate medication administration infection prevention control weaknesses identified remedied pose significant risk patients finally progress toward establishing elements culture safety measured today many hospitals regularly use one several available staff surveys assess safety culture few however analyze meaning survey data evaluate area hospital falling short undertake specific focused interventions remedy shortcomings as hospitals make progress toward high reliability include safety culture metrics part strategic planning programs set goals improving metrics report metrics boards report metrics related financial performance patient satisfaction hospitals need new process improvement tools methods break current state low reliability we argued robust process improvement rpi)a combination lean six sigma change management much potent set tools health care currently uses address safety quality problems briefly oversimplifying somewhat lean set tools philosophy employee empowered improvement identifies removes wasted effort processes without compromising quality outcome six sigma tools focus improving outcomes process radically reducing frequency defective products outcomes occur change management systematic approach used alongside lean six sigma prepares organization accept implement sustain improved processes result application lean six sigma tools these three sets tools complementary together provide best available methods hospitals achieve major improvements faulty processes table 4 shows three components rpi changes hospital comes closer high reliability like ge best buy companies benefited rpi believe getting benefit requires employed common language throughout entire organization bartlett wozny 2005 rao 2011 nearly employees trained levels appropriate one job the tools used throughout organization improvement work finally proficiency use rpi part every employee performance appraisal required career advancement within organization these elements provide vital support spreading use tools hros quality safety are personal responsibility every employee armed highly effective ways solve complex problems gives employees need exercise responsibility robust process improvement high reliability stages organizational maturity today hospitals generally lag far behind ideal state used elements rpi often starting lean relatively hospitals adopted full suite rpi tools rpi provides highly effective tools obtaining voice customer perspective patients constitutes high quality outcome particular care process vital improvement making substantial progress toward high reliability safety quality requires application tools like rpi generate extremely high rates sustainable improvement applied poorly performing safety processes exist hospitals today see table 1 we know approach process improvement available present capable generating sustaining rates improvement magnitude consistently widest array areas clinical quality business processes chassin 1998 r. chassin 2008 enabling hospitals use high reliability health care framework requires additional work advance toward high reliability hospitals must able assess current state maturity respect framework fourteen components access proven strategies tools methods advance mature levels the joint commission developing testing instrument permit hospital leaders perform assessment the utility assessment hospitals identifying pressing opportunities making progress toward high reliability availability specific tools facilitate progress currently field tested this initiative south carolina safe care commitment led joint commission center transforming healthcare south carolina hospital association may 2013 stakeholders vested interest moving health care further faster toward high reliability include state federal government health agencies consumer patient advocacy groups employers public private payers health care professional organizations hospitals health systems although comprehensive assessment roles beyond scope article several observations nonetheless pertinent regulation modest supportive role dramatic quality safety improvements industries e.g. commercial aviation car manufacturing consumer electronics health care regulators pay attention first foremost identifying eliminating requirements obstruct progress toward high reliability instances they impose unproductive work regulated organizations distracts dealing effectively quality challenges regulators support transformation high reliability example well crafted programs publicly reporting reliable valid measures quality u.s industries undergone transformations quality stimulated primarily competitive pressures e.g. japanese automakers similar occurrence health care is difficult imagine intensely local environment large majority hospitals health systems operate becher chassin 2001 changes health care must undergo become highly reliable thoroughgoing profound primary drive change must ultimately come health care organizations as saying goes takes one psychiatrist change lightbulb lightbulb want change many health care leaders reluctant commit goal high reliability regard unrealistic unachievable distraction current serious fiscal regulatory pressures one important roles policymakers stakeholders encourage persuade demand health care organizations embark journey even committed long take health care organizations reach high reliability unknown none arrived destination yet cincinnati children hospital medical center working toward goal decade current strategic plan calls eliminating serious patient harm 2015 cincinnati children hospital medical center 2013 finally hospitals systems like memorial hermann cincinnati children trailblazers striving high reliability health care developed strategies tools largely trial error for movement broaden deepen next wave hospitals health systems need proven tools methods speed journey higher levels maturity many stakeholders could contribute development evaluation tools policymakers several levels government could facilitate process focused funding efforts table 2 depicts six components leadership one characteristics four stages maturity the six components board trustees chief executive officer senior management including nursing leaders engagement physicians hospital quality strategy use dissemination data measures quality use information technology support quality safety improvement the identification specific components supported published literature linking better quality performance goeschel wachter pronovost 2010 jha epstein 2010 weiner shortell alexander 1997 the hospital leaders commitment high reliability must include prominent role board trustees directors the board must part leadership commitment eventually achieve zero patient harm elevate quality patient safety organization highest strategic goal if board left management find efforts unsupported misunderstood today hospital boards vary wide spectrum involvement quality programs hospitals oversee jha epstein 2010 leadership high reliability stages organizational maturity addition physicians essential success quality initiative hospitals table 2 identifies two vital components physicians role leadership participation order move effectively toward high reliability both formally appointed leaders chief medical officer vice president medical affairs informal leaders medical staff president voluntary medical staff leaders need visible active enthusiasts quality including physician leaders employees hospital it difficult imagine hospital getting close high reliability quality merely one many competing priorities memorial hermann health system twelve hospital health care system based houston explicitly committed becoming highly reliable specified importance major ingredients framework efforts shabot et al system ceo pointed ensuring patient safety core value core value ( wolterman shabot 2012 accelerate progress toward zero harm quality must measured data measures must widely available within hospital public not transparency valuable right public reporting also powerful added force drives improvement the quality program measures focus meeting needs addressing specific quality problems hospital patient population other incentives judicious use financial rewards staff advancement opportunities based performance quality measures important accelerants well finally leaders obligated employ health information technology effectively service quality improvement it particularly important hro frequently vehicle nearly perfect processes sustain performance if process effectively redesigned highly reliable automating effective way maintain state unfortunately health care automation often deployed unsafely phenomenon increases rather decreases risk harm ash et al 2007 2009 joint commission 2008b koppel et al 2005 sparnon marella 2012 in addition various types health often coordinated thereby increasing risk example programmable infusion devices supported decision support rules govern pharmacy systems physician order entry systems resulting confusion life threatening patients a hospital approaching high reliability adopts health solutions coordinated integrated manner following principles safe adoption joint commission 2008b karsh 2004 table 3 shows five components safety culture manifestations four stages maturity toward high reliability a culture safety fully supports high reliability three central attributes trust report improve reason hobbs 2003 workers exhibit enough trust peers organization management routinely recognize report errors unsafe conditions this trust established organization eliminates intimidating behavior suppresses reporting acts timely way fix problems reported workers communicates improvements consistently individuals reported problems first place communication turn strengthens the trust led reports fosters identification reporting problems even upstream harm when three components safety culture trust report improve working well reinforce one another produce stable organizational culture sustains high reliability safety culture high reliability stages organizational maturity maintaining trust also requires organization hold employees accountable adhering safety protocols procedures hros establish clear equitable transparent processes recognizing separating small blameless errors people make every day unsafe reckless actions blameworthy understanding blameless errors occur part learning process hros employ maintain exemplary safety records recognizing dealing appropriately blameworthy acts equally important dimension hro safety culture vital role maintaining trust unfortunately health care organizations often punish staff blameless acts failing implement equitable disciplinary procedures commit blameworthy acts hospital leaders succeeded eradicating intimidating behaviors institute safe medication practices 2004 these failings explain lack trust among hospital staff noted earlier agency healthcare research quality 2012 hospitals move toward high reliability establish codes behavior modeled leaders including nurses physicians champion efforts eliminate intimidation encourage reward reporting blameless errors unsafe conditions accountability adhering safe practices ingrained employees spurred implementing standards invoking disciplinary procedures apply staff regardless seniority professional credentials for example maimonides medical center new york city established program code mutual respect commits stakeholders including physicians nurses staff students vendors consultants volunteers working harmoniously together eliminate intimidating behaviors the program includes progressive interventions including disciplinary actions individuals repeatedly violate code maimonides medical center 2009 hros also proactively assess strength resilience safety systems organizational defenses prevent errors propagating leading harm today hospitals function primarily reactive mode investigating incidents patients already harmed conducting root cause analyses instituting corrective action plans prevent future occurrences becoming much safer requires caregivers willingness ability recognize report close calls unsafe conditions combined organizational capacity act effectively reports eliminate risks embody furthermore opposed today norm focusing single events hospitals compile results investigations across many harm events close calls identify safety systems defenses need improvement these evaluations lead development proactive assessments key safety systems e.g. relate medication administration infection prevention control weaknesses identified remedied pose significant risk patients finally progress toward establishing elements culture safety measured today many hospitals regularly use one several available staff surveys assess safety culture few however analyze meaning survey data evaluate area hospital falling short undertake specific focused interventions remedy shortcomings as hospitals make progress toward high reliability include safety culture metrics part strategic planning programs set goals improving metrics report metrics boards report metrics related financial performance patient satisfaction hospitals need new process improvement tools methods break current state low reliability we argued robust process improvement rpi)a combination lean six sigma change management much potent set tools health care currently uses address safety quality problems briefly oversimplifying somewhat lean set tools philosophy employee empowered improvement identifies removes wasted effort processes without compromising quality outcome six sigma tools focus improving outcomes process radically reducing frequency defective products outcomes occur change management systematic approach used alongside lean six sigma prepares organization accept implement sustain improved processes result application lean six sigma tools these three sets tools complementary together provide best available methods hospitals achieve major improvements faulty processes table 4 shows three components rpi changes hospital comes closer high reliability like ge best buy companies benefited rpi believe getting benefit requires employed common language throughout entire organization bartlett wozny 2005 rao 2011 nearly employees trained levels appropriate one job proficiency use rpi part every employee performance appraisal required career advancement within organization these elements provide vital support spreading use tools hros quality safety are personal responsibility every employee armed highly effective ways solve complex problems gives employees need exercise responsibility robust process improvement high reliability stages organizational maturity today hospitals generally lag far behind ideal state used elements rpi often starting lean relatively hospitals adopted full suite rpi tools fewer still engaged patients using powerful tools redesign care processes rpi provides highly effective tools obtaining voice customer perspective patients constitutes high quality outcome particular care process vital improvement making substantial progress toward high reliability safety quality requires application tools like rpi generate extremely high rates sustainable improvement applied poorly performing safety processes exist hospitals today see table 1 we know approach process improvement available present capable generating sustaining rates improvement magnitude consistently widest array areas clinical quality business processes chassin 1998 r. chassin 2008 enabling hospitals use high reliability health care framework requires additional work advance toward high reliability hospitals must able assess current state maturity respect framework fourteen components access proven strategies tools methods advance mature levels the joint commission developing testing instrument permit hospital leaders perform assessment the utility assessment hospitals identifying pressing opportunities making progress toward high reliability availability specific tools facilitate progress currently field tested this initiative south carolina safe care commitment led joint commission center transforming healthcare south carolina hospital association may 2013 those stakeholders vested interest moving health care faster toward high reliability include state federal government health agencies consumer patient advocacy groups employers public private payers health care professional organizations hospitals health systems although comprehensive assessment roles beyond scope article several observations nonetheless pertinent regulation modest supportive role dramatic quality safety improvements industries e.g. commercial aviation car manufacturing consumer electronics health care regulators pay attention first foremost identifying eliminating requirements obstruct progress toward high reliability instances they impose unproductive work regulated organizations distracts dealing effectively quality challenges regulators support transformation high reliability example well crafted programs publicly reporting reliable valid measures quality u.s industries undergone transformations quality stimulated primarily competitive pressures e.g. japanese automakers a similar occurrence health care difficult imagine intensely local environment large majority hospitals health systems operate becher chassin 2001 changes health care must undergo become highly reliable thoroughgoing profound primary drive change must ultimately come health care organizations saying goes takes one psychiatrist change lightbulb lightbulb want change many health care leaders reluctant commit goal high reliability regard unrealistic unachievable distraction current serious fiscal regulatory pressures one important roles policymakers stakeholders encourage persuade demand health care organizations embark journey even committed long take health care organizations reach high reliability unknown none arrived destination yet cincinnati children hospital medical center working toward goal decade current strategic plan calls eliminating serious patient harm 2015 cincinnati children hospital medical center 2013 finally hospitals systems like memorial hermann cincinnati children trailblazers striving high reliability health care developed strategies tools largely trial error for movement broaden deepen next wave hospitals health systems need proven tools methods speed journey higher levels maturity many stakeholders could contribute development evaluation tools policymakers several levels government could facilitate process focused funding efforts achieving high reliability health care require hospitals undergo substantial changes take place rapidly we outlined framework fourteen components practical application changes hospitals the components distributed three major domains leadership safety culture robust process improvement we described component four evolutionary stages maturity road high reliability each stage provides hospitals specific guidance actions need take order advance toward high reliability further research experience derived application practical framework required assess effectiveness facilitating hospitals advancement toward high reliability finally policymakers stakeholders various positions evaluate support accelerate transformation

contextdespite serious and widespread efforts to improve the quality of health care , many patients still suffer preventable harm every day . hospitals find improvement difficult to sustain , and they suffer project fatigue because so many problems need attention . no hospitals or health systems have achieved consistent excellence throughout their institutions . high - reliability science is the study of organizations in industries like commercial aviation and nuclear power that operate under hazardous conditions while maintaining safety levels that are far better than those of health care . adapting and applying the lessons of this science to health care offer the promise of enabling hospitals to reach levels of quality and safety that are comparable to those of the best high - reliability organizations.methodswe combined the joint commission 's knowledge of health care organizations with knowledge from the published literature and from experts in high - reliability industries and leading safety scholars outside health care . we developed a conceptual and practical framework for assessing hospitals readiness for and progress toward high reliability . by iterative testing with hospital leaders , we refined the framework and , for each of its fourteen components , defined stages of maturity through which we believe hospitals must pass to reach high reliability.findingswe discovered that the ways that high - reliability organizations generate and maintain high levels of safety can not be directly applied to today 's hospitals . we defined a series of incremental changes that hospitals should undertake to progress toward high reliability . these changes involve the leadership 's commitment to achieving zero patient harm , a fully functional culture of safety throughout the organization , and the widespread deployment of highly effective process improvement tools.conclusionshospitals can make substantial progress toward high reliability by undertaking several specific organizational change initiatives . further research and practical experience will be necessary to determine the validity and effectiveness of this framework for high - reliability health care .

acute generalized exanthematous pustulosis agep rare acute reaction drug induced 90% cases characterized widespread sterile pustular rash cefepime fourth generation cephalosporin antibiotic used treat febrile neutropenia severe infections related urinary tract skin nosocomial pneumonia brain abscess intra abdominal septic lateral cavernous sinus thrombosis a 67-year old man renal failure dialysis last 2 years 8-year history cardiac insufficiency admitted hospital complaining 6 days diarrhea the patient taken semi intensive care unit treated ciprofloxacin consequence his long term medications changed consisted acetylsalicylic acid furosemide captopril carvedilol clonazepam seventh day patient became dyspneic chest radiograph showed left lower lobe opacity treatment nosocomial pneumonia promptly initiated cefepime 1 g day five days later presented pruritic erythematous maculopapular eruption affecting abdomen neck skin folds one day later developed disseminated pustular lesions fig 1 temperature 37c laboratory exams evidenced c reactive protein 136 mg l white blood cells 14,700 cells/l normal 3,50010,500 cells/l 11,995 cells/l neutrophils normal 1,7008,000 cells/l histology showed toxic pustuloderma spongiform subcorneal pustules edema papillary dermis perivascular inflammatory infiltrate consisting neutrophils fig after withdrawal cefepime introduction imipenem disseminated skin nonfollicular pustules cleared within 4 days following desquamation the patient denied previous adverse reaction drugs personal family history psoriasis evident agep disease characterized rapid onset many sterile nonfollicular pustules usually arising edematous erythema frequently accompanied leukocytosis fever skin symptoms usually arise rapidly insult resolve spontaneously within days agep often starts predominantly intertriginous areas face spreading rapidly trunk lower limbs the mean duration pustules 9.7 days annular desquamation typically follows days complications rare 1 3 agep validation score euroscar study group used establish diagnosis score 8 12 agep definitive diagnosis table 1 the case score 11 according validation score euroscar study group table 2 the main differential diagnosis agep pustular psoriasis pustules clinically histologically resemble lesions pustular psoriasis number reports patients history plaque psoriasis authors assume agep nothing acute exacerbation psoriasis caused variety exogenous triggers however many studies strongly suggest agep associated psoriasis 1 5 now agep attributed variety causes viral infections chlamydia pneumoniae infection hypersensitivity mercury skin reaction primarily adverse response drugs antibiotics cefepime implicated causative agents 80% individuals group the present case agep well defined criteria correct diagnosis generally leads spontaneous resolution causative drug withdrawn clinicians keep possibility cutaneous drug reaction mind

acute generalized exanthematous pustulosis ( agep ) is a rare cutaneous rash characterized by widespread sterile nonfollicular pustules . cefepime is a fourth generation cephalosporin , used to treat severe infections . a 67-year - old man was admitted with acute gastroenterocolitis . on the seventh day , the patient developed a nosocomial pneumonia and cefepime was initiated . on the fourth day of cephalosporin treatment , he presented with a maculopapular , pruritic eruption affecting the face , neck , abdomen and limbs . one day later he developed disseminated pustular lesions and his temperature was 37c . laboratory analysis evidenced leukocytosis and skin biopsy showed subcorneal pustule , edema in the papillary dermis , perivascular inflammatory infiltrate consisting of neutrophils , leukocytoclasia and red cell extravasation in the epidermis . cefepime was suspended and within 4 days the non - follicular pustules cleared following a desquamation . agep is a disease attributed to a variety of causes , but in 90% of the cases it is due to an adverse drug reaction . antibiotics are implicated in 80% of these cases , mostly penicillins and macrolides . there are few cases associated with cephalosporins . it is very important to consider agep in cases of acute pustular rashes and drugs should be investigated as causative agents .

paediatric age time diagnosis represents negative prognostic factor keratoconus progression increased probability corneal transplant particularly younger patients represent population high risk rapid progression disease 1 2 the long term results reported literature 35 demonstrated ability cross linking slow progression keratoconus photo polymerization reaction stromal collagen fibres cross linking photodynamic reaction induced combined action photosensitizing substance riboflavina vitamin b2 ultraviolet uv light allowing corneal stiffening increasing number intrafibrillar interfibrillar covalent bonds corneal collagen resistance enzymatic degradation 68 the long term effects technique related also process collagen neosynthesis different structure higher molecular weight 912 confers corneal stroma increased resistance lamellar compaction responsible variable functional modifications recorded treatment 1114 according international results 35 cross linking primary choice young patient progressive keratoconus to report comparative prospective long term functional analysis cross linking three different age groups 18 years 1926 years 27 years patients affected progressive keratoconus since 2004 date 610 patients treated combined riboflavin uv corneal collagen cross linking present prospective nonrandomized open study comprised 516 eyes 413 patients aged 10 40 years affected progressive keratoconus the comparative functional analysis comprised following paediatric group 18 years included 152 eyes 105 patients 29.5% intermediate group 1926 years included 286 eyes 243 patients 55.4% adult group 27 years included 78 eyes 65 patients 15.1% all patients included treatment protocol affected progressive keratoconus documented clinical instrumental worsening least last three months observation the parameters considered establish keratoconus progression worsening ucva bscva 0.50 snellen lines increase sph cyl 0.50 increase topographic symmetry index sai si 0.50 increase maximum k reading 0.50 reduction thinnest point optical pachometry 10 clear cornea biomicroscopic examination absence reticular dark striae confocal laser microscopy vivo we considered significant inclusion study variation least 3 parameters listed one clinical plus two instrumental statistical analysis conducted mann whitney u test nonparametric data ucva bscva paired test parametric data maximum curvature power symmetry indices coma values the surgical procedure corneal cross linking riboflavina uva performed patients according siena protocol using vega cbm caporossi baiocchi mazzotta x linker cso florence italy developed italy department ophthalmology siena university authors intellectual property siena university italy the treatment conducted topical anaesthesia 4% lidocaine drops applying eyelid speculum a 9 mm diameter marker used mark corneal epithelium central circle epithelium removed blunt metal spatula after epithelial scraping disposable solution riboflavin 0.1% dextrane 20% ricrolin sooft montegiorgio italy instilled 10 minutes corneal soaking starting uv irradiation the riboflavin dextrane solution administered every 2.5 minutes total 30 minutes uva exposure 3 mw cm treated eyes dressed therapeutic soft contact lens bandage 4 days medicated antibiotics ofloxacin drops 4 times day nonsteroidal anti inflammatory drugs diclofenac drops 4 times day lachrymal substitutes contact lens removal after therapeutic corneal lens removal fluorometholone 0.2% drops 3 times day lacrimal substitutes administered 6 8 weeks ( 1 152 eyes 105 patients 18 years 91 eyes followup 12 months 74 eyes 24 months 25 eyes 36 months 7 48 months 2 ) 286 eyes 243 patients 19 26 years 108 eyes followup 12 months 83 eyes 24 months 56 eyes 36 months 11 48 months ( 3 78 eyes 65 patients 27 35 eyes followup 12 months 25 eyes 24 months 12 eyes 36 months 8 48 months pre- postoperative examination included uncorrected visual acuity ucva best spectacle corrected visual acuity bscva corneal topography surface aberrometry cso eye top florence italy optical pachometry visante oct zeiss meditech jena germany vivo confocal microscopy hrt ii heidelberg rostock cornea module germany according epidemiology findings found male female ratio whole sample 4 1 male female ratio paediatric group 6 1 male female ratio 3 1 patients 19 40 years old these results even small group differ epidemiological data reported literature male female rate 2 1 reported there statistical difference incidence keratoconus right left eye whole population 516 eyes age related groups comparative ucva patients 18 years showed mean gain 0.14 p 0.0037 0.17 p 0.0043 0.16 p 0.0051 0.2 p 0.006 snellen lines 12 24 36 48 months followup respectively patients 19 26 years showed mean gain 0.13 p 0.0034 0.16 p 0.0041 0.12 p 0.0032 0.14 p 0.0073 snellen lines 12 24 36 48 months followup respectively patients 27 years showed mean gain 0.08 p 0.0036 0.09 p 0.005 0.12 p 0.0047 0.12 p 0.0071 snellen lines 12 24 36 48 months followup respectively figure 1 comparative bscva patients 18 years showed mean gain 0.15 p 0.0056 0.19 p 0.0031 0.18 p 0.0059 0.21 p 0.0079 snellen lines 12 24 36 48 months followup respectively patients 19 26 years showed mean gain 0.10 p 0.0052 0.12 p 0.0045 0.13 p 0.0056 0.2 p 0.0075 snellen lines 12 24 36 48 months followup respectively patients 27 years showed mean gain 0.07 p 0.0054 0.06 p 0.0067 0.08 p 0.0069 0.10 p 0.0075 snellen lines 12 24 36 48 months followup respectively figure 2 k max paediatric group varied mean 0.7 p 0.006 0.8 p 0.0045 1.1 p 0.051 0.9 p 0.071 intermediate group patients 19 26 years varied mean 0.6 p 0.0053 0.5 p 0.0051 0.3 p 0.0045 0.6 p 0.0091 adult group patients 27 years varied mean 0.4 p 0.0065 0.6 p 0.0074 0.5 p 0.0095 0.5 p 0.0091 12 24 36 48 months follow respectively group figure 3 surface asymmetry index sai paediatric patients improved mean value 0.42 p 0.0054 0.18 p 0.0066 0.24 p 0.091 0.10 p 0.096 intermediate group improved mean 1.05 p 0.0032 1.14 p 0.0021 0.84 p 0.0036 0.65 p 0.076 adult group improved mean 0.52 p 0.0067 1.0 p 0.0077 0.17 p 0.0081 1.11 p 0.0094 figure 4 topographic superior inferior symmetry index si paediatric patients varied mean 0.3 p 0.0098),+ 0.6 p 0.011 0.2 p 0.017 1.5 p 0.021 intermediate group changed mean 0.42 p 0.0086 0.55 p 0.0079 0.90 p 0.091 0.40 p 0.099 adult patients varied mean 0.26 p 0.0059 0.21 p 0.0048 1.17 p 0.012 0.21 p 0.0011 12 24 36 48 months followup respectively figure 5 coma values paediatric patients improved mean 0.47 p 0.0034 0.52 p 0.0025 0.47 p 0.0022 0.45 p 0.0054 intermediate group coma values decreased mean 0.89 p 0.0034 0.96 p 0.0065 0.93 p 0.0074 0.91 p 0.0081 old patients recorded mean postoperative value 0.2 p 0.0056 0.18 p 0.0045 0.21 p 0.0034 0.19 p 0.0067 12 24 36 48 months followup respectively figure 6 according literature previous reports keratoconus progression frequent faster younger patients 18 years old time diagnoses higher probability undergo corneal transplantation 1 3 therefore paediatric patients represent goal photo induced riboflavin uv corneal collagen cross linking italian pilot study siena cxl paediatrics conducted large cohort patients long term follow demonstrated significant fast functional improvement younger patients riboflavin uv corneal cross linking recently published however impossible exactly predict distribution cross links geometric redistribution newly formed collagen 12 14 long term comparative analysis showed functional results riboflavin uv corneal collagen cross linking among paediatric patients slightly better without statistically significant differences results recorded intermediate group patients hand patients 27 years showed positive poorer functional response compared age groups mean kmax variation topographic surface asymmetry index results statistically significant paediatric sample particularly postoperative 24 months after 24th month 48th month mean data results statistically significant reasonably due reduced number patients longitudinal analysis the comparative aberrometric data coma values showed significant improvement analyzed groups justifying rapid improvement visual acuity treated patients paediatric group cohort minority patients 5% despite treatment showed worsening trend least instability keratoconus opinion concept should remarked disease age group aggressive possibility progression higher others age groups the instability certain cases explained different genetic patterns keratoconus 1719 relative biochemical modifications potentially occurring corneal stroma associated negative influences environmental factors allergy atopy 2124 every time decide treat paediatric patient 18 years affected progressive keratoconus parents patient well informed possibility treatment minority cases could warrant total long lasting stabilization disease possibility repeat cross linking undergo alternative treatments pilot study demonstrated effective ability corneal cross linking retard keratoconus progression age groups better functional response patients 26 years treatment ensured long term keratoconus stabilization 90% treated cases lower functional response observed patients 27 years plasticity adult age well demonstrated literature 25 26 cross linking treatment may result less effectiveness increased failure complication rate particularly adult patients 35 years reported literature according long term comparative age related analysis results standard riboflavin uv cross linking epithelium removal first choice therapy progressive keratoconus paediatric under-26-year old patients corneal thickness least 400 thinnest point

purpose . to report a comparative prospective long - term functional analysis after riboflavin uv a corneal cross - linking ( cxl ) in three different age groups of patients affected by progressive keratoconus ( kc ) . methods . functional analysis comprised paediatric patients ( 18 years ) included 152 eyes ( 29.5% ) ; intermediate group ( 1926 years ) 286 eyes ( 55.4% ) , and adults ( 27 years ) 78 eyes ( 15.1% ) . cxl was performed according to the siena protocol by using the vega cbm ( caporossi - baiocchi - mazzotta ) x linker ( cso , florence , italy ) at siena university by the same authors . pre- and post - op examinations included ucva , bscva , corneal topography , and surface aberrometry ( cso eye top , florence , italy ) , at 48 months followup . results . at 48 months followup paediatrics , intermediate , and adult patients showed a mean gain in ucva of + 0.2 , + 0.14 and + 0.12 snellen lines . bscva gained by a mean of + 0.21 , + 0.2 , and + 0.1 snellen lines . kmax was reduced by a mean value of 0.9 d , 0.6 d , and 0.5 d , respectively . coma values improved by a mean of 0.45 m , 0.91 m , and 0.19 m , respectively . treatment ensured a long - term keratoconus stabilization in over 90% of treated patients . conclusion . according to our long - term comparative results , epithelium - off riboflavin uv a cross - linking should be the first choice therapy of progressive kc , particularly in paediatric age and patients under 26 years .

rufinamide ruf triazole derivative structurally unrelated antiepileptic drugs aed).1 approved united states food drug administration fda 2004 treatment lennox gastaut syndrome lgs patients aged 4 years older.2 ruf authorized indication europe january 2007.2,3 mechanism action involves limiting firing excessive sodium dependent action potentials.1 ruf reported reduce number drop attacks major motor seizure 60% patients lgs subsequently regarded effective adjunctive therapeutic agent.2 recent years studies reported ruf also efficacious well tolerated treatment various epilepsy syndromes lgs including cases refractory epilepsy pediatric patients.3 however still limited data regarding long term treatment results ruf pediatric refractory epilepsy the aim study delineate long term efficacy tolerability ruf children infants intractable epilepsies we performed retrospective study patients met following inclusion criteria 1 followed one year samsung medical center since beginning ruf use time 31 aug 2011 2 less 19 years age time start ruf treatment 3 classified intractable epilepsy meaning presence persistent seizures despite use three antiepileptic drugs 4 whose data clinical characteristics seizure outcome available the data concerning demography clinical characteristics seizure related characteristics laboratory works including electroencephalography eeg brain magnetic resonance imaging mri treatment outcome collected we conformed classification international league epilepsy ilae 1981 1989 classifying seizure epilepsy patients.4,5 efficacy ruf evaluated response rate comparing seizure frequencies baseline last three months patients showed seizure reduction 50% frequency defined responders the retention rate another index efficacy defined portion patients continued ruf treatment one year tolerability evaluated presence side effects additionally investigated influencing factors response rate retention rate chicago il usa used analysis nonparametric measures statistical evaluation performed means chi square test thirty seven patients 27 male 10 female included study the etiology epilepsy epilepsy syndrome classification type seizures number prior concurrent aeds treatment presented table 1 2 the patients taking many concurrent aeds mean=3.9 range 18 valproic acid n=26 clobazam n=19 topiramate n=15 levetiracetam n=13 lamotrigine n=13 etc initial starting dose ruf mean 7.8 2.631.5 mg kg day final maintenance dose mean 31.4 6.165.6 mg kg day the mean duration ruf therapy 10.52.73 months ruf discontinued 17 37 patients mean period 5.6 0.615.7 months thirty seven patients 27 male 10 female included study the etiology epilepsy epilepsy syndrome classification type seizures number prior concurrent aeds treatment presented table 1 2 the patients taking many concurrent aeds mean=3.9 range 18 valproic acid n=26 clobazam n=19 topiramate n=15 levetiracetam n=13 lamotrigine n=13 etc initial starting dose ruf mean 7.8 2.631.5 mg kg day final maintenance dose mean 31.4 6.165.6 mg kg day the mean duration ruf therapy 10.52.73 months ruf discontinued 17 37 patients mean period 5.6 0.615.7 months the overall response rate ruf 21.6% change seizure frequency according etiology response rates patients atonic seizure type higher seizure types statistical difference table 3 ) overall retention rate ruf one year 54% 20/37 patients the reasons discontinuation ineffectiveness n=11 64.7% adverse effects n=3 17.6% ineffectiveness adverse effects n=1 9.8% increased seizure frequency n=2 11.7% the reasons retention follows responders n=8 including two seizure free patients including additive effect aeds n=2 good initial response n=7 improved cognitive function parent record n=2 ruf effective previous aeds n=3 non responders n=12 60% seven patients showed good initial response ruf two patients improved cognitive abilities increased appropriateness parent record the rest three non responders able decrease aeds due adjunctive treatment ruf two patients one seizure free outcome 50% seizure reduction presumed additive effect aeds furthermore five patients showed great improvement specific types seizures atonic seizure type n=2 generalized tonic seizure type n=1 n=2 adverse effects reported ten patients 27% insomnia n=3 loss appetite n=3 somnolence n=2 irritability n=2 vomiting n=1 dizziness n=1 four patients discontinued ruf due adverse effects vomiting insomnia dizziness loss appetite respectively six patients continued ruf the overall response rate ruf 21.6% change seizure frequency according etiology response rates patients atonic seizure type higher seizure types statistical difference table 3 the overall retention rate ruf one year 54% 20/37 patients the reasons discontinuation ineffectiveness n=11 64.7% adverse effects n=3 17.6% ineffectiveness adverse effects n=1 9.8% increased seizure frequency n=2 11.7% the reasons retention follows responders n=8 including two seizure free patients including additive effect aeds n=2 good initial response n=7 improved cognitive function parent record n=2 ruf effective previous aeds n=3 non responders n=12 60% seven patients showed good initial response ruf two patients improved cognitive abilities increased appropriateness parent record the rest three non responders able decrease aeds due adjunctive treatment ruf two patients one seizure free outcome 50% seizure reduction presumed additive effect aeds furthermore five patients showed great improvement specific types seizures atonic seizure type < n=2 generalized tonic seizure type n=1 n=2 adverse effects reported ten patients 27% insomnia n=3 loss appetite n=3 somnolence n=2 irritability n=2 vomiting n=1 dizziness n=1 four patients discontinued ruf due adverse effects vomiting insomnia dizziness loss appetite respectively six patients continued ruf ruf established efficacy lgs approved adjunctive treatment patients older four years age lgs.6 previous studies ruf also efficacious well tolerated treatment various epilepsy syndromes lgs including cases refractory epilepsy pediatric patients.3 however data efficacy ruf epilepsies lgs limited.3,7 study ruf showed response rate 21.6% patients lgs intractable ge lre the retention rate one year 54% fourth patients showed various adverse effects the response rate ruf patients refractory epilepsies including lgs reported 26.746.7%.1,3,7,8 studies included child adult patients mean age ranged 1 50 years according study kluger et al,7 conducted 60 patients 45 children 15 adults treated ruf mean period 14.5 months inadequately controlled epilepsy syndromes range 318 months overall response rate 26.7% another study the patients refractory epilepsies showed response rate 46% 21 weeks ruf therapy.8 study included pediatric patients lgs intractable epilepsy mean age patients 9.5 years range 120 years the response rate present study 21.6% slightly lower previous studies mentioned this difference may result intractability study population including patients lgs highly intractable generalized epilepsy localization related epilepsy time introduction ruf patients taken mean 7.2 range 413 aeds much higher studies kluger et al and joseph et al.7,8 study patients lgs showed slightly higher response rate 30% patients intractable ge lre in one prospective study 43 patients lgs overall response rate ruf 12 months 60.5% without worsening seizures.2 another study involving children young adults lgs response rate 54.8%.3 studies patients lgs took large part total number patients compared study group due reason response rate studies higher present study however ruf found efficacious patients lgs intractable ge lre seizure types the highest response rate shown atonic seizure 27.8% according previous data atonic seizure tonic seizure were improved types seizure ruf adjunctive therapy 63.6% 48.6% respectively).1,9 also long term open label extension study 124 patients lgs aged 437 years treated ruf median 432 days treatment 47.9% patients 50% reduction frequency tonic atonic seizures.6 based previous studies ruf appeared effective adjunctive therapy intractable atonic seizures study partial seizure responded equally ruf similar result reported studies.3,7 retention rate known indicator evaluating efficacy adverse effects.10 according one previous study retention rate ruf 14.5 months 41.7%,7 consistent present study study reasons retention varied 60% non responders less 50% seizure reduction the reasons retention non responders follows excellent initial response definite cognitive improvement parent record ruf effective previous aeds coppola et al1 reported early effectiveness ruf refractory childhood epileptic encephalopathy patients without vpa similarly study seven patients showed good initial response ruf titration study two patients reported noticeable improvement cognitive function even though seizure reduction less 50% one study suggested ruf favorable cognitive profile increasing appropriateness many patients especially cognitive impairment their cognitive ability rather improved seizure free state worsening reported.11 study 27% patients reported mild moderate degree adverse effects the commonly observed adverse events insomnia n=3 loss appetite n=3 somnolence n=2 irritability n=2 vomiting dizziness none previous studies reported life threatening adverse effects.3,7,8 previous studies reported vomiting drowsiness major symptoms adverse effects.3,12 study adverse events insomnia loss appetite mild moderate severity children lgs intractable epilepsy syndromes showed 21.6% response rate 54% retention rate ruf treatment also 27% children showed adverse effects ruf 10.8% children stopped ruf due adverse effects the present data suggest ruf provides good efficacy tolerability pediatric patients refractory generalized localization related epilepsy syndromes well lgs

background and purpose : rufinamide ( ruf ) is a novel antiepileptic drug ( aed ) and its efficacy has been proven in lennox - gastaut syndrome ( lgs ) . however , there is a lack of data regarding the efficacy in pediatric intractable epilepsies other than lgs . the purpose of the study was to explore the efficacy and tolerability of ruf in pediatric patients with intractable epilepsies as well as lgs.methods:this retrospective observation study was conducted in samsung medical center from august 2010 to september 2011 . thirty seven patients ( 27 males , 10 females , aged between 1.8 and 18.4 years ) , with refractory epilepsies or lgs were treated with ruf as an adjunctive drug . efficacy was represented by the response rate and retention rate over the study period . tolerability was measured as the number of patients who showed adverse effects.results:the overall response rate was 21.6% during the 12 months of the study period with 5.4% of seizure - free patients . the retention rate was 54% and ineffectiveness was the most common reason for discontinuation of ruf . the most common adverse effects were insomnia and somnolence.conclusions:ruf may be considered to be an efficacious and safe aed for pediatric patients with intractable epilepsies as well as lgs .

cluster headache form primary neurovascular headache consists unilateral head pain occurs association cranial autonomic features patients striking circannual circadian periodicity excruciating painful attacks accompanied restlessness agitation usually last less 3 h occur bouts months patient one crisis per day the international headache society defines cluster headache attacks severe strictly unilateral pain orbital supraorbital temporal combination sites lasting 15180 min occurring every day 8 times day the attacks associated one following ipsilateral conjunctival injection lacrimation nasal congestion rhinorrhoea forehead facial sweating miosis ptosis eyelid oedema epidemiological data general population scarce five studies carried cluster headache prevalence general population conflicting results prevalence rates vary 56 326 cases every 100,000 inhabitants recent study sample representative italian general population aged 14 years reported estimated prevalence rate 279/100,000 95% ci 173427 227/100,000 95% ci 104431 women 338/100,000 95% ci 175592 men cataract clouding opacity lens eye causes progressive painless loss vision first symptom cataract usually blurred vision other symptoms may progressively appear glare halos double vision perception colours yellow less vibrant three different types cataract identified histological basis nuclear cortical posterior subcapsular investigations 58 possible risk factors development cataract showed positive correlation myopia diabetes smoking 911 use systemic corticosteroids exposition uv b environmental factors added genetic predisposition however underlined different degrees correlation distinct risk factors three types cataract reported 6 15 prevalence studies age related cataracts hampered absence uniform grading system cataract opacities differing definitions visual impairment additional coexisting ocular pathologies causing loss vision nonetheless framingham eye study 1977 reported proportion people age related cataracts causing loss vision 20/30 6/9 worse 15.5% ages 45.9% older 75 years beaver dam eye study 1992 using similar definition loss vision reported proportions 38.8% men 45.9% women older 74 years however clear variations frequency reflect methodological diversity true differences populations interestingly cited studies analyzed cataract prevalence age groups substantially different two cases one single study reported prevalence cataract younger age 40 years older identified risk factors three different histological types cataract this study showed age specific rates different histological cataract type stratifying population sample age prevalence 1% cortical type 0.2% nuclear type 2.0% posterior subcapsular reported individuals 40 49 years prevalence 3.9% cortical type 0.2% nuclear type 2.6% posterior subcapsular reported individuals 50 59 years here report two cases developed cataract age 50 years side affected cluster headache reviewed published similar cases potential mechanisms comorbidity we present 44-year old man history cluster headache diagnosed accordance ihs guidelines since 21 years old daily pain attacks lasting 90 min localized left periorbital area ipsilateral lacrimation rinorrhea the cluster headache initially occurred sporadically age 43 years became chronic at age 39 patient diagnosed cataract left eye year treated surgery the sole risk factor developing cataract anamnesis smoking habit 30 cigarettes day detailed pharmacological anamnesis excluded kind exposure prolonged treatment steroid drugs the second case 53-year old man experienced first cluster headache episode 44 years old cluster periods characterized 34 attack day severe right sided pain localized periorbital region lasting 45 min associated ipsilateral lacrimation the reported risk factor developing cataract smoking habit 10 cigarettes day also case report chronic treatment steroid drugs references identified searches pubmed 1966 october 2007 terms cluster headache cataract our search pubmed shows one report patient cluster like headache began surgical removal crystalline cataract intraocular lens implant therefore knowledge first report cataract occurring cluster headache patients interestingly eye affected pain attack it interest patients developed cataract young age suffering years chronic cluster headache attacks absence main risk factors development early onset cataract including prolonged steroid drugs exposure it noteworthy two patients although relative rather uncommon last name found specifically represented defined area north eastern italy however could investigate pedigree one patient hypothetical genetic link uncommon occurrence early onset cataract cluster headache proposed in addition possible perform either two patients investigation ocular inflammation thus speculation made possible causal relationship repeated episodes pain inflammation autonomic abnormalities considered contribute cluster headache attack precocious occurrence cataract however first evidence occurrence cataract relatively young adult men history chronic cluster headache importance underlining uncommon type comorbidity may favour report additional similar cases

cluster headache ( ch ) consists of attacks of severe , unilateral orbital / supraorbital / temporal pain , lasting for 15180 min , occurring once or more times a day , and associated with ipsilateral conjunctival injection , lacrimation and other symptoms . cataract is clouding of the lens of the eye causing a progressive and painless loss of vision . we describe the cases of two men ( not relative , but with the same last name , which originates from north - eastern italy ) that in young adult age , after years of suffering from chronic ch , developed cataract on the same side of the pain attacks . patient 1 was diagnosed as having cataract 18 years after the onset of episodic ( and subsequently chronic ) ch . patient 2 began suffering from chronic ch at the age of 44 years and after 8 years he developed cataract . this is the first report of cataract in patient suffering of ch and occurring in the eye affected by the pain attack .

obesity risk factor cancer development also factor affecting treatment outcomes endometrial cancer ec first malignancy recognized related obesity it associated lower quality life values among ec survivors higher body mass index bmi measured ec diagnosis associated higher cause endometrial cancer specific mortality early stage ec treatment involves surgery total abdominal hysterectomy bilateral salpingo oopherectomy without pelvic lymph node dissection followed adjuvant radiotherapy selected cases randomized studies shown radiotherapy rt reduces risk pelvic relapse vaginal cuff common site relapse postoperative radiation therapy endometrial carcinoma 2 portec-2 trial demonstrated patients intermediate risk ec treated safely postoperative vaginal cuff brachytherapy vcb absence whole pelvic external beam radiotherapy thus decreasing toxicity although vcb one commonly used adjuvant gynaecological treatments lack studies analysing whether dosimetric factors influenced overweight our aim therefore evaluate effect bmi perivaginal fat dose organs risk fractionated vcb retrospective analysis 220 consecutive brachytherapy fractions derived 59 patients underwent postoperative vcb gynecological cancer conducted fiftysix patients underwent vcb due endometrial cancer three patients due cervical cancer twenty six endometrial cancer patients underwent postoperative vcb alone using six fractions remaining patients treated whole pelvis external beam radiotherapy wprt followed three four vcb fractions 15 patients underwent fractionation patients assessed weekly treatment every 4 6 months afterwards according clinical stage brachytherapy performed largest diameter cylinder could fit comfortably vaginal vault cylinders positioned remain parallel cranio caudal axis patient no instructions given prior vcb procedure apart need evacuate prior coming hospital all patients underwent ct planning scan every application 2-mm thick slice 2-mm overlapping intervals supine position using foley bladder catheter instilling dilute contrast medium 5 ml omnipaque350 ge healthcare bio sciences madrid spain 45 ml saline solution order increase bladder visibility segmentation volume reproducibility treatment cts transferred 3d treatment planning system oncentra v.4.1 nucletron elekta company elekta ab stockholm sweden improve comparisons physician contoured planned every image set conditions iridium-192 remote afterloading unit microhdr nucletron nucletron elekta company regardless treatment administered the entire bladder volume delineated rectum defined 1 cm cylinder tip 1.5 cm last activated source dwell position an active length 2.5 cm used deliver fraction dose 5 gy 5 mm depth vaginal surface d0.1cc d1cc d2cc bladder rectum assessed dvhs fraction the angle vaginal cylinder applicator related horizontal plane parallel craneo caudal patient axis calculated positive values indicating tip directed bladder negative values indicating posterior displacement cylinder tip toward rectum cylinder size rectum bladder volumes also noted the bmi formula weight kilograms divided square height metres categories underweight 18.5 kg normal 18.5 24.9 kg mm overweight 25.0 29.9 kg mm obese 30.0 34.9 kg mm obese ii 35.0 39.9 kg mm obese iii 40.0 kg mm we considered fat confined within 100% isodose first ct planning scan perivaginal fat it segmented thresholding hounsfield units hu 190 30 hu volume recorded perivaginal fat analysis performed first ct planning scan common practice vcb perform one ct translate plan next fractions patient mean dose volume metrics volumes cylinder angle calculated different fractions chi square tests kruskal wallis analysis univariate regression analysis performed a stepwise multiple regression analysis used model organs risk oars dvh parameters function variables significance levels addition removal model set 0.05 0.10 respectively all patients underwent ct planning scan every application 2-mm thick slice 2-mm overlapping intervals supine position using foley bladder catheter instilling dilute contrast medium 5 ml omnipaque350 ge healthcare bio sciences madrid spain 45 ml saline solution order increase bladder visibility segmentation volume reproducibility treatment cts transferred 3d treatment planning system oncentra v.4.1 nucletron elekta company elekta ab stockholm sweden improve comparisons physician contoured planned every image set conditions iridium-192 remote afterloading unit microhdr nucletron nucletron elekta company regardless treatment administered the entire bladder volume delineated rectum defined 1 cm cylinder tip 1.5 cm last activated source dwell position an active length 2.5 cm used deliver fraction dose 5 gy 5 mm depth vaginal surface d0.1cc d1cc d2cc bladder rectum assessed dvhs fraction the angle vaginal cylinder applicator related horizontal plane parallel craneo caudal patient axis calculated positive values indicating tip directed bladder negative values indicating posterior displacement cylinder tip toward rectum cylinder size rectum bladder volumes also noted the bmi formula weight kilograms divided square height metres categories underweight 18.5 kg normal 18.5 24.9 kg mm overweight 25.0 29.9 kg mm obese 30.0 34.9 kg mm obese ii 35.0 39.9 kg mm obese iii 40.0 kg mm we considered fat confined within 100% isodose first ct planning scan perivaginal fat it segmented thresholding hounsfield units hu 190 30 hu volume recorded perivaginal fat analysis performed first ct planning scan common practice vcb perform one ct translate plan next fractions patient mean dose volume metrics volumes cylinder angle calculated different fractions chi square tests kruskal wallis analysis univariate regression analysis performed a stepwise multiple regression analysis used model organs risk oars dvh parameters function variables significance levels addition removal model set 0.05 0.10 respectively a mean 3.7 vcb fractions per patient retrieved according bmi classification 6.8% patients classified normal 35.6% overweight 57.6% obese i. severely obese patients class ii iii the median age iqr bmi overall group 64.3 years 18.4 32 years 9.5 respectively median bmi values every classification 22 27.8 36.6 normal weight overweight obese patients respectively patients significantly older bmi class increased kruskal wallis test p=0.043 the fact obese patients treated larger cylinder applicators 82.4% patients normal range 25% chi square test p=0.037 significant positive significant correlation observed bmi perivaginal fat volume r=0.556 p=0.001 fig bmi also showed positive correlation age r=0.272 p=0.037 negative correlation bladder dvh metrics d0.1cc r=0.366 p=0.004 d1cc r=0.454 p=0.001 d2cc r= 0.451 p 0.001 ) a low positive correlation observed perivaginal fat volume cylinder angle r=0.288 p=0.027 vaginal cylinders tilted towards bladder perivaginal fat volume increased fig 2 univariate regression showed significant association bladder dose metrics cylinder angle multivariate regression performed determine effect variables analysed bladder dvh metrics table 3 age patients use 3.5 cm cylinder diameter showed significance d2cc when reanalysis performed replacing bmi perivaginal fat volume models unable retain perivaginal fat volume among predictors patients developed grade 1 2 acute toxicity higher acute toxicity levels observed late toxicity uncommon one patient developed g3 bladder toxicity two patients developed late rectal toxicity one g1 g2 the main finding study higher bmi values showed association lower bladder dose parameters rectal dose adipose tissues commonly classified subcutaneous visceral fat latter associated metabolic disease visceral fat located periprostatic area associated prostate cancer aggressiveness volume showed direct correlation bmi reduction rectal doses among patients higher bmi prostate brachytherapy this dose reduction considered result inverse square law due increase fatty tissue prostate rectum interface our results analysis large sample high number brachytherapy insertions confirm recent study reported higher bladder doses women lower bmi vcb a positive significant correlation observed bmi perivaginal fat volume r=0.5561 p=0.001 fig unfortunately due limited field view fov used ct scan acquisition unable determine whether correlation existed visceral fat the small fov left part visceral fat outside scanning area precluding analysis our results support increase perivaginal fat application inverse square law main cause lower doses oars due inability perivaginal fat remain multivariate regression models main explanation rectal doses would also decrease higher perivaginal fat volumes obesity class patients strongly associated hazard ratio 3.93 ec association stage grade disease found large women health initiative study the relative risk death cancer among women bmi 32 higher 2.1 compared risk women bmi 19 although obesity described risk factor adverse outcomes treatment many malignancies effect bmi ec treatment outcomes subject debate while analysis national institutes health aarp diet health study demonstrated increased risk overall disease specific mortality among women higher pre diagnosed bmi analysis gog 99 study data associated obesity higher mortality causes ec disease recurrence the analysis astec trial failed find association bmi reduced post treatment survival a recent systematic review suggest association bmi recurrence cancer vaginal brachytherapy vbt one main adjuvant treatments ec since results randomized trials showed vbt inferior pelvic irradiation high intermediate risk patients use extended external beam radiotherapy high risk patients there concerns regarding methods delivery vbt whether use single plan approach customized plan every fraction the first approach requires level homogeneity insertions several variables affect dose deposition oars 17 20 our data influenced bladder filling due routinely placed bladder catheter perivaginal fat volume associated anterior cylinder tilt paradoxically go higher bladder dose therefore believe anatomical visceral fat distribution could cause differences bladder dose according bmi fig a limitation study lack complete pelvic visceral fat segmentation possible due limited pelvic volume scanned insertions strong correlation observed perivaginal fat volume used surrogate visceral fat volume bmi this measure validated abdominal adiposity umbilicus level usual measure visceral subcutaneous fat study relationships the fact severe extremely obese patients bmi obese class ii iii report noteworthy reflects bmi distribution among general population mediterranean countries compared population united states in addition population attributable fraction greater us population 56.8% european population 45.2% population differences bmi obese classes ii iii could modify results although study focus clinical results believe important know much possible variables influence absorbed doses organs risk knowledge important clinical setting first vbt application planned remaining assumed similar due fixed geometry according american brachytherapy society consensus in conclusion results reported study demonstrate significant inverse correlation bmi bladder dose deposition vcb the impact findings late toxicity needs evaluated clinical practice

purposeassociation between body mass index ( bmi ) and doses in organs at risk during postoperative vaginal cuff brachytherapy ( vcb ) treatment has not been evaluated . the aim of this study was to analyse the impact of bmi on the dose delivered to bladder and rectum during high - dose - rate vcb using computed tomography ( ct ) scans at every fraction.materials and methodsa retrospective analysis of 220 planning ct sets derived from 59 patients was conducted . every planning ct was re - segmented and re - planned under the same parameters . rectum and bladder dose - volume histogram values ( d0.1cc , d1cc , and d2cc ) were extracted and evaluated . the mean values for all applications per patient were calculated and correlated with bmi , as well as other factors influencing rectal and bladder doses . multiple regression analysis performed to model organ at risk dose - volume parameters.resultsaccording to world health organization ( who ) , 6.8% of patients were normal , 35.6% were overweight , and 57.6% were class i obese . median rectal doses were 133.5% , 110.9% , and 99.3% for d0.1cc , d1cc , and d2cc , respectively . the corresponding median bladder doses were 96.2% , 80.6% , and 73.3% , respectively . bmi did not show significant association with rectal doses . however , bmi did show a significant association with evaluated bladder dose metrics ( d0.1cc , r=0.366 , p=0.004 ; d1cc , r=0.454 , p < 0.001 ; d2cc , r=0.451 , p < 0.001 ) . bmi was retained in the multivariate regression models ( d0.1cc , p=0.004 ; d1cc , p < 0.001 ; d2cc , p=0.001).conclusionin this group of mediterranean , overweight , and moderately obese patients , bmi showed association with lower bladder dose values , but not with rectal doses .

hepatitis b virus hbv important risk factor developing hepatocellular carcinoma hcc 1 hcc fifth common cancer worldwide causes significant public health problems especially association chronic hepatitis b 2 morphologic lesions hepatocarcinogenesis include dysplastic lesions dysplastic foci df dysplastic nodules dns low- high grade dysplasia small cancerous lesions 2 cm diameter early hcc 4 7 the common cause cirrhosis shiraz transplant center largest liver transplant center country hbv related majority patients hcc center also hbv related 1 therefore attempted determine incidence hcc precursors group cirrhotic patients thorough examination explanted hbv related cirrhotic livers a two year cross sectional study performed 103 explanted hbv related cirrhotic livers pathology department shiraz university medical sciences study period 2014 2015 explanted cirrhotic hbv related livers recipient cirrhotic liver liver transplantation received department pathology fixed formalin 24 48 hours histologic sections hepatic nodule larger 1 cm diameter kind difference background parenchyma color consistency studied figures 1 2 sections stained hematoxylin eosin method slides examined double headed microscope two pathologists df classified either small cell changes scc formerly called small cell dysplasia large cell changes lcc formerly called large cell dysplasia nodules studied classified low grade dysplastic nodules lgdn high grade dysplastic nodules hgdn hepatocellular carcinoma hcc 5 the following criteria used classify different nodules cirrhotic livers cellularity thickness hepatocellular plates nuclear atypia either pleomorphism irregular contour nucleocytoplasmatic ratio cytoplasmic staining eosinophilic basophilic amphophilic clear pseudoacinar pattern stromal invasion portal tracts biliary pigment lipid vacuoles iron status nodules 8) lgdn hepatocytes completely abnormal that show normal slightly increased nucleocytoplasmic n c ratio minimal nuclear atypia mitotic activity portal tracts still present compared lgdn hgdn features except n c ratio higher nuclear atypia obvious cytoplasmic basophilia significant liver plates two cells thick thickened mitotic figures hcc cell size is usually decreased nuclear density least twice normal nuclear atypia definite mitotic figures present pseudoacinar formation may present 9 the term early hcc mean hcc early stage small size usually 2 cm histologically hcc well differentiated lack prominent cellular structural atypia 10 13 convenience used term advanced hcc ahcc hccs greater 2 cm size article the chi square test used evaluate significance relation nodules categorical variables a total 103 patients included study among patients 14 13.16% women 89 86.4% men age ranged 20 73 mean age 51.4 10.5 among explanted cirrhotic livers 92 89.3% df lcc 57 55.3% showed scc well 103 explanted livers 39 37.9% lgdn 38 36.9% hgdn 19 18.4% early hepatocellular carcinoma ehcc 21 20.4% ahcc figures 3 6 note cases ehcc ahcc also scc lcc hgdn lgdn thirteen cases ehcc accompanied ahcc 6 cases ehcc show ahcc size larger 2 cm all 21 ahcc cases transplanted preoperative diagnosis malignancy hepatitis b related cirrhosis however among livers ehcc 6 accompanied ahcc 2 unsuspected surgery diagnosed explanted liver the two ahcc cases smaller 2 cm diameter table 1 our results showed presence scc lgdn hgdn statistically significant association ehcc p 0.05 hcc p 0.05 moreover presence ehcc associated ahcc p 0.05 hcc poor prognostic cancer one common causes cancer death around globe hcc also common countries high incidence hbv related cirrhosis 1 despite improvements treatment modalities hcc survival rate remains low even liver transplantation hcc easily diagnosed advanced stage 14 therefore studies incidence diagnosis precancerous lesions hcc important especially patients develop hcc secondary hbv infection cirrhosis therefore pathologists clinicians able diagnose ahccs ehccs precursor lesions using various techniques preoperatively 10 hepatic precancerous lesions currently divided two categories depending cytological histological changes microscopic df macroscopic dns 11 df recognized microscopic examination smaller 1 mm dns recognizable gross examination hepatic specimens microscopic examination well defined nodule lesions different background cirrhotic liver tissue size color texture degree bulging cut surface hccs either ehcc less 2 cm ahcc larger 2 cm 8 9 globally hbv common cause cirrhosis hcc iran 12 study attempted identify true prevalence hcc precursors 103 explanted livers thorough sectioning least 15 sections liver precise inspection gross specimens among 103 explanted cirrhotic livers study 92 89.3% df lcc according result lcc common finding cirrhotic livers patients chronic hepatitis b seem associated malignant lesion thus confirming lack premalignant potential theses lesions that studies similar found lcc formerly called lcd histogenetic association hcc 13 others concluded lcc relation hbv might innocent bystander might closely related hepatocarcinogenesis 15 it diagnosed presence clusters hepatocytes small size minimal nuclear atypia high n c ratio high nuclear crowding the report plentz et al indicated severely decreased expression p16 p21 telomere shortening accumulation dna damage scc hcc compared lcc cirrhotic nodules suggestive malignant potential scc compared lcc 17 study 103 patients hepatitis b associated liver cirrhosis 57 55.3% scc 57 livers scc 19 70.4% ehcc 21 36.8% hcc our results showed presence scc associated ehcc p 0.05 hcc p 0.05).therefore scc may associated presence ehcc hcc according results thorough sampling advised every patient scc detect malignant lesion for example one study scc proved associated aneuploidy elevated dna index found moderate high risk progression hcc 18 among 103 explanted livers 39 37.9% lgn 38 36.9% hgdn 19 18.4% ehcc 21 20.4% ahcc thus 70% 53% cases lgdn 50% 55% cases hgdn showed ehcc hcc respectively this association statistically significant p value 0.05 according previous studies lgdn hgdn indicators increased risk hcc cirrhotic livers hcc nodules commonly seen within dns i.e. nodule within nodule pattern 14 conclusion scc lgdn hgdn seem common associated findings precursors hcc livers infected hepatitis b. strict follow precise thorough sampling sectioning livers scc abnormal dns especially larger 1 cm highly recommended association malignancy

backgroundthe most common cause of liver transplantation in iran is hepatitis b positive cirrhosis , and it also one of the major and important causes of hepatocellular carcinoma ( hcc ) . most cases with hcc follow a multistep sequence . morphologic lesions during hepatocarcinogenesis include dysplastic lesions and small cancerous lesions ( 2 cm in diameter ; early hcc ) . however , insufficient information is available on the incidence of hcc and its precursors in hepatitis b - related cirrhosis.objectivesin this study , we determined the incidence of hcc and its precursors in hepatitis b - related cirrhosis in the largest liver transplant center in iran.methodsin a two - year study , all explanted livers of patients with hepatitis b virus ( hbv)-positive cirrhosis were completely sectioned and examined . each specimen was investigated grossly and microscopically to determine any abnormal nodule or cellular changes ( at least 15 sections from each liver).resultsamong all explanted cirrhotic livers ( 103 livers ) during the study period ( 2014 - 2015 ) , 92 ( 89.3% ) had dysplastic foci with large cell changes ( lcc ) , 57 ( 55.3% ) of which showed small cell changes ( scc ) as well . thirty - nine cases ( 37.9% ) had low - grade dysplastic nodules ( lgdn ) , 38 ( 36.9% ) high - grade dysplastic nodules ( hgdn ) , 19 ( 18.4% ) were early hepatocellular carcinoma ( ehcc ) , and 21 ( 20.4% ) were hepatocellular carcinoma more than 2 cm . all the cases with ehcc and hcc of more than 2 cm also had scc , lcc , hgdn , and lgdn . thirteen cases of ehcc were accompanied with hccs more than 2 cm , and 6 cases of ehcc did not show any hcc ( larger than 2 cm).conclusionsscc , lgdn , and hgdn are common associated findings and precursors of hcc in livers infected with hepatitis b. a strict follow - up and a precise and thorough sampling of livers with scc and any abnormal dysplastic nodules ( dns ) , especially those larger than 1 cm , are highly recommended because these dns are highly associated with malignancy .

dry eye disease ded continues present clinicians diagnostic dilemma primarily due multiple causative factors result disease complexity currently test set tests considered gold standard diagnosis monitoring without diagnostic standard care however recent study showed 43% asymptomatic patients clinical signs dry eyes.1 left untreated ded significantly impact person vision quality life diagnostic tool globally diagnose ded regardless cause may valuable helping physicians establish earlier accurate dry eye diagnosis based definition dry eye workshop dews 2007 ded defined follows dry eye multifactorial disease .. results symptoms discomfort visual disturbance tear film instability potential damage ocular surface it accompanied increased osmolarity tear film inflammation ocular surface.2 dry eye multifactorial disease .. results symptoms discomfort visual disturbance tear film instability potential damage ocular surface it accompanied increased osmolarity tear film inflammation ocular surface.2 seen definition symptoms likely non specific ocular surface damage may actually late complication the dews identified increased tear osmolarity tear film instability core mechanisms ded regardless etiology.2 tests accurately measure tear osmolarity tear film instability therefore theoretically best identifying determining severity ded tear osmolarity attractive offers objective numerical output monitored commonly used diagnostic tests rely heavily subjective grading criteria past the two commonly used instruments measure tear osmolarity clifton vapor pressure osmometers studies demonstrated accuracy high sensitivity specificity.3,4 main downside instruments require significant amount time involve numerous steps increasing potential tear evaporation practical especially dry eye patients low tear volumes.3,4 newer technology produced tearlab uses micro electrode measure number charged particles tear sample electrode designed avoid direct contact ocular surface thereby reducing chance reflex tearing.3 tearlab measurement method appears accurate involved osmometers differing average 2 mosm l normal dry eye patients.3 tear lab device requires small tear sample ~0.2 l able provide results almost instantly reducing level tear evaporation.3,5 compared vapor pressure osmometer tearlab osmolarity system able measure patients lower volume tear samples.4 compared clifton osmometer tearlab device reported similar sensitivity slightly better specificity positive predictive value.3 aim current review establish whether published studies support use tear osmolarity testing primarily using tearlab device tool ded diagnosis treatment monitoring comments literature related role tear osmolarity pathophysiology ded ocular systemic conditions pharmaceuticals impact tear osmolarity also discussed a literature search conducted using pubmed june 2014 using search term tear osmolarity identify potentially relevant articles date a review abstract article used identify related unrelated tear osmolarity identify articles tear osmolarity primary focus paper doubt the paper classified related tear osmolarity detailed review remaining papers related tear osmolarity tear osmolarity research year 2000 limited scope methodology applicability more recent research generally reflects relevant earlier results decision made articles published year 1999 earlier would included review discussions tear osmolarity include diagnostic component also eliminated provide research basis use osmolarity evaluating tear film diagnostic papers indicative clinical application basic research a rating relevant articles performed using grading recommendations assessment development evaluation grade based likelihood research required confirm significance reported results,6 well university michigan practice guideline based study design.7 grade scoring included consideration study sample size financial interest disclosures evaluating quality evidence the financial interest categorization suggest sponsored studies lower quality independent research reflect potential bias reporting results relevant data entered access database microsoft corporation redmond wa usa analysis an article rated high category fulfilled criteria university michigan practice guideline fulfilled definition high requirement grade system similarly b moderate c low well low were used university michigan practice guideline grade rating systems respectively identify rating relevant articles the articles also subjectively reviewed impression left reader whether positive neutral table 1 contains summary preliminary search data total 407 articles listed potentially relating tear osmolarity the results preliminary analysis identified 164 papers peer reviewed literature reference tear osmolarity use diagnostic tool of 164 articles identified relevant peer reviewed articles impression results positive neutral negative possible 163 one foreign language study excluded all four remaining studies could graded well using combination two grading systems outlined previously table 2 shows breakdown articles grading impression seen 32% 52/163 studies available qualified moderate high quality sample size lack randomization lack control populations primary limiting design factors led lower quality ratings studies subgroup moderate high quality articles 73% 38/52 positive impression 17% neutral impression 10% negative impression this breakdown impression similar studies graded low low 71% positive 24% neutral 5% negative respectively table 3 summarizes financial interest breakdown percentage breakdown financial interest column approximately two thirds articles independent remaining third sponsored tearlab others the impressions 93% much larger group independent articles positive neutral while shown table 3 results independent study quality 104 independent studies rated 31% 32/104 high moderate rating 32 articles 72% 23/32 positive indicating tear osmolarity useful diagnostic tool evaluation dry eye severity a 19% 6/32 neutral 9% 3/32 articles suggested little value use tear osmolarity the following discussion outlines results literature review based primarily studies rated moderate high we discuss tear osmolarity testing performed cut value dry eye diagnosis accuracy repeatability tear osmolarity testing tear osmolarity compares commonly used diagnostic tests finally articles discussing effect external factors ocular and/or systemic disease pharmaceuticals tear osmolarity reviewed evaluating diagnostic ability tear osmolarity requires specifying value discriminates healthy eye eye ded value known threshold value literature tear osmolarity threshold values varied 305 mosm l8 316 mosm l.5 one reported reason variability tear osmolarity threshold values tear film instability hallmark characteristic disease.9,10 normal mild moderate severe dry eyes average tear osmolarity values approximately 302+/8 mosm l 315+/10 mosm l 336+/022 mosm l respectively.10,11 another study lower reported mean values study noted included patients mild moderate ded severe ded.8 seen distribution ded severity different populations may explain variability threshold values studies demonstrated tear osmolarity influenced correlated disease severity.10,11 one study found using tear osmolarity threshold 305 mosm l gave 98.4% positive predictive value.8 using tear osmolarity threshold 316 mosm l 317 mosm l sensitivity varied 59%12 81%,13 specificity varied 78%14 94%,12 positive predictive value 85%,3,13 negative predictive value 74%,13 overall predictive accuracy 89%.12 meta analysis used 316 mosm l threshold noted tear osmolarity may accurate lactoplate schirmer test rose bengal testing.12 currently 316 mosm l threshold believed better discriminate mild moderate severe dry eye 308 mosm l considered widely accepted threshold.5 tear osmolarity threshold 308 mosm l correctly diagnosed severe dry eye normal patients 90.7% 81.3% time respectively value appeared sensitive discriminating normal eyes presenting early stages ded.10 variability tear osmolarity also diagnostic indicator one study variability two eyes normal mild moderate dry eye patients severe dry eye patients 6.95.9 mosm l 11.710.9 mosm l 26.522.7 mosm l respectively.10 variability inter eye measurements repeat measurements eye appears increase severity dry eye mild ded may also manifest variability repeat measurement eye two eyes measured osmolarity low stable without ded.9 tear osmolarity shown good repeatability8 normal subjects significant difference osmolarity values using four readings taken 1 to15 minutes apart.9 concern using osmolarity diagnostic test observed overlap measured values normal dry eyes documented especially high two studies.15,16 however one studies,15 dry eye patients treated eye drops systemic medications would expected lower osmolarity second study16 may suffered selection bias diagnostic tests tear osmolarity used categorize patients dry eye control group this categorization may skewed results favor tests used categorization process.16 different studies suggested tear osmolarity provide sharp diagnostic cut rather serve guide indicate disease severity progression.8,10 tear osmolarity testing beneficial providing numerical value current state dry eyes complementary diagnostic tests valuable discriminating etiology disease.10 compared schirmer test results meibomian gland grading ocular surface disease index osdi tear break time tbut corneal conjunctival staining tear osmolarity better predicting dry eye severity tear osmolarity increased dry eye severity also increased largely linear pattern.8,11 another study also demonstrated tear osmolarity higher sensitivity compared corneal staining conjunctival staining meibomian grading better specificity compared tbut schirmer test results.10 higher severity ded greater correlation tear osmolarity diagnostic tests;8 result may indicate diagnostic tests less sensitive mild ded diagnosis given intermittency disease severity mild dry eye patient performance diagnostic tests variable population segment for instance meibomian gland dysfunction may increase tear osmolarity demonstrated one study.17 one explanation meibomian gland dysfunction alone may sufficient overwhelming homeostatic control patients examining studies present review there indication osmolarity correlated diagnostic tests correlation less apparent looking studies rated moderate quality higher one factor general correlation objective tests subjective tests dry eye poor.1 issue likely due multiple etiologies presentations ded particular tests specific one etiology may lack overall correlation.1 like diagnostic signs tear osmolarity may correlate symptoms general population,18 although shown correlate time effective therapy described pharmacological effects treatment section present review dry eye symptoms non specific result psychiatric disorder depression post traumatic stress disorder conditions increased dry eye symptoms associated dry eye signs increased c reactive protein tear osmolarity.19 one study9 found majority 92% 23/25 patients patient symptomatic maximum tear osmolarity 308 mosm l patient asymptomatic maximum tear osmolarity 308 mosm l study,9 minority patients 8% 2/25 increased tear osmolarity symptoms low tear osmolarity symptoms result may due mild ded former case low osmolarity due condition ded latter case this study9 concluded increased tear osmolarity likely precursor dry eye complaints despite lack correlation tear osmolarity dry eye symptoms certain factors diseases drugs noted associated ded increased tear osmolarity it likely patients exposed external factors diseases drugs dry eyes increased tear osmolarity such reactions function individual response stresses whether eyes able adjust the effect external factors humidity tear osmolarity clear one study subjects exposed low relative humidity 20% 10 minutes.20 another study found exposure humidity 5% 2 hours increase result variability tear osmolarity.21 studies included subjects using artificial tears study although discontinued use 1 hour advance testing one study specify type artificial tear used state airflow velocity such differences may account conflicting results.20,21 addition use less sensitive diagnostic tests may resulted inclusion mild dry eye patients normal group would confounded results the literature divided whether contact lens wear increases2,22,23 effect tear osmolarity.10,24,25 osmolarity measured studies,10,24 appropriate delay contact lens removal osmolarity measurement the differences reported results likely function wearer ability maintain homeostasis tear film overcoming drying effect contact lenses.10 appears certain procedures may influence tear osmolarity others in recent 2013 article change tear osmolarity dry eye diagnostic test observed refractive surgery despite decrease corneal sensitivity.26 cataract surgery tear osmolarity noted increase.27 difference may explained subjects ages average age 26 years refractive surgery study26 71 years cataract surgery study.27 older patients may take longer recover surgically induced ocular trauma.27 trabeculectomy surgery performed patient population average age 71 years also higher tear osmolarity control group result attributed presence bleb use mitomycin c may cause epithelial damage residual effects long term use glaucoma medications surgery.28 difference tear osmolarity noted eye underwent eyelid reconstruction surgery un operated eye patient result may related corrective tearing evidenced higher schirmer test results operated eye.29 table 4 shows studies investigated effect various ocular systemic conditions tear osmolarity of systemic conditions diabetes found increase tear osmolarity.30,31 increase tear osmolarity correlated increase diabetes duration the relation diabetes increased tear osmolarity likely due decrease amount aqueous secretion result injury lacrimal gland small blood vessels lacrimal gland and/or corneal nerve.31 thyroid disease results increase palpebral aperture proptosis also reported significantly increases tear osmolarity compared control.32 increased pain sensation evident patients fibromyalgia related increased tear osmolarity.33 traumatic brain injury patients higher tear osmolarity values healthy control group.34 patients increased tear osmolarity may also dehydrated water intake may important consideration dry eye diagnosis treatment.35 increase estrogen and/or progesterone form birth control pills)25 excess androgens noted patients polycystic ovary syndrome)36 seemed effect tear osmolarity another study found low levels 17-beta estradiol estrone testosterone associated increased tear osmolarity treatment phytoestrogen decreased tear osmolarity compared control group.37 studies area appear warranted understand correlation sex hormones tear osmolarity adequate control hydration measurement hydration status may important studies different ocular conditions also seem different effects tear osmolarity table 4 for instance eyes pseudoexfoliation syndrome condition thought alter goblet cell activity mucin production reported increased tear osmolarity compared control group.38 patients pterygia predominantly inflammatory condition also show increased tear osmolarity compared control group.39 keratoconus demonstrated reduce corneal sensitivity tear osmolarity similar control group the reason similarity clear though may keratoconus generally clinically considered inflammatory disease.40 herpetic keratitis associated higher tear osmolarity affected unaffected eyes compared control group reason unaffected eye higher tear osmolarity may due type keratitis.41 patients nasolacrimal duct obstruction likely compensate decreasing tear production resulting tear osmolarity value similar control group.42 table 5 summarizes findings different studies effect certain drugs tear osmolarity use oral mucolytics often treat respiratory conditions increases tear osmolarity this increase likely due alterations mucin layer tear film.43 glaucoma medications increased tear osmolarity perhaps due preservatives medications significant effect tbut schirmer test results.28 artificial tears noted several studies decrease tear osmolarity,20,44,45 one study found correlation tear osmolarity use artificial tears.16 latter study disclose type artificial tear used this may important factor type eye drop impact tear osmolarity.45 instance lower osmolarity eye drops associated lower tear osmolarity.46,47 noted decrease osmolarity observed use artificial tears may comparable effects heat massage application eyelids using thermal massager.44 effect anti inflammatory medication tear osmolarity less clear especially studies evaluating effect suffer limitations small sample size lack control group randomization.4850 one recent randomized double blind parallel study finland osmolarity observed increase dry winter months.51 participants taking oral sea buckthorn oil containing n-3 n-6 fatty acids antioxidants less increase tear osmolarity compared control group presumed function oil anti inflammatory mechanism.51 evaluating diagnostic ability tear osmolarity requires specifying value discriminates healthy eye eye ded value known threshold value literature tear osmolarity threshold values varied 305 mosm l8 316 mosm l.5 one reported reason variability tear osmolarity threshold values tear film instability hallmark characteristic disease.9,10 normal mild moderate severe dry eyes average tear osmolarity values approximately 302+/8 mosm l 315+/10 mosm l 336+/022 mosm l respectively.10,11 another study lower reported mean values study noted included patients mild moderate ded severe ded.8 seen distribution ded severity different populations may explain variability threshold values studies demonstrated tear osmolarity influenced correlated disease severity.10,11 one study found using tear osmolarity threshold 305 mosm l gave 98.4% positive predictive value.8 using tear osmolarity threshold 316 mosm l 317 mosm l sensitivity varied 59%12 81%,13 specificity varied 78%14 94%,12 positive predictive value 85%,3,13 negative predictive value 74%,13 overall predictive accuracy 89%.12 meta analysis used 316 mosm l threshold noted tear osmolarity may accurate lactoplate schirmer test rose bengal testing.12 currently 316 mosm l threshold believed better discriminate mild moderate severe dry eye 308 mosm l considered widely accepted threshold.5 tear osmolarity threshold 308 mosm l correctly diagnosed severe dry eye normal patients 90.7% 81.3% time respectively value appeared sensitive discriminating normal eyes presenting early stages ded.10 variability tear osmolarity also diagnostic indicator one study variability two eyes normal mild moderate dry eye patients severe dry eye patients 6.95.9 mosm l 11.710.9 mosm l 26.522.7 mosm l respectively.10 variability inter eye measurements repeat measurements eye appears increase severity dry eye mild ded may also manifest variability repeat measurement eye two eyes measured osmolarity low stable without ded.9 tear osmolarity shown good repeatability8 normal subjects significant difference osmolarity values using four readings taken 1 to15 minutes apart.9 concern using osmolarity diagnostic test observed overlap measured values normal dry eyes documented especially high two studies.15,16 however one studies,15 dry eye patients treated eye drops systemic medications would expected lower osmolarity the second study16 may suffered selection bias diagnostic tests tear osmolarity used categorize patients dry eye control group this categorization may skewed results favor tests used categorization process.16 different studies suggested tear osmolarity provide sharp diagnostic cut rather serve guide indicate disease severity progression.8,10 tear osmolarity testing beneficial providing numerical value current state dry eyes complementary diagnostic tests valuable discriminating etiology disease.10 compared schirmer test results meibomian gland grading ocular surface disease index osdi tear break time tbut corneal conjunctival staining tear osmolarity better predicting dry eye severity tear osmolarity increased dry eye severity also increased largely linear pattern.8,11 another study also demonstrated tear osmolarity higher sensitivity compared corneal staining conjunctival staining meibomian grading better specificity compared tbut schirmer test results.10 higher severity ded greater correlation tear osmolarity diagnostic tests;8 result may indicate diagnostic tests less sensitive mild ded diagnosis given intermittency disease severity mild dry eye patient performance diagnostic tests variable population segment for instance meibomian gland dysfunction may increase tear osmolarity demonstrated one study.17 one explanation meibomian gland dysfunction alone may sufficient overwhelming homeostatic control patients examining studies present review indication osmolarity correlated diagnostic tests correlation less apparent looking studies rated moderate quality higher one factor general correlation objective tests subjective tests dry eye poor.1 issue likely due multiple etiologies presentations ded particular tests specific one etiology may lack overall correlation.1 like diagnostic signs tear osmolarity may correlate symptoms general population,18 although shown correlate time effective therapy described pharmacological effects treatment section present review dry eye symptoms non specific result psychiatric disorder depression post traumatic stress disorder conditions increased dry eye symptoms associated dry eye signs increased c reactive protein tear osmolarity.19 one study9 found majority 92% 23/25 patients patient symptomatic maximum tear osmolarity 308 mosm l patient asymptomatic maximum tear osmolarity 308 mosm l study,9 a minority patients 8% 2/25 increased tear osmolarity symptoms low tear osmolarity symptoms result may due mild ded former case low osmolarity due condition ded latter case this study9 concluded increased tear osmolarity likely precursor dry eye complaints despite lack correlation tear osmolarity dry eye symptoms when compared schirmer test results meibomian gland grading ocular surface disease index osdi tear break time tbut corneal conjunctival staining tear osmolarity better predicting dry eye severity tear osmolarity increased dry eye severity also increased largely linear pattern.8,11 another study also demonstrated tear osmolarity higher sensitivity compared corneal staining conjunctival staining meibomian grading better specificity compared tbut schirmer test results.10 higher severity ded greater correlation tear osmolarity diagnostic tests;8 result may indicate diagnostic tests less sensitive mild ded diagnosis given intermittency disease severity mild dry eye patient performance diagnostic tests variable population segment for instance meibomian gland dysfunction may increase tear osmolarity demonstrated one study.17 one explanation meibomian gland dysfunction alone may sufficient overwhelming homeostatic control patients examining studies present review there indication osmolarity correlated diagnostic tests correlation less apparent looking studies rated moderate quality higher one factor general correlation objective tests subjective tests dry eye poor.1 issue likely due multiple etiologies presentations ded particular tests specific one etiology may lack overall correlation.1 like diagnostic signs tear osmolarity may correlate symptoms general population,18 although shown correlate time effective therapy described pharmacological effects treatment section present review dry eye symptoms non specific result psychiatric disorder depression post traumatic stress disorder conditions increased dry eye symptoms associated dry eye signs increased c reactive protein tear osmolarity.19 one study9 found majority 92% 23/25 patients patient symptomatic maximum tear osmolarity 308 mosm l patient asymptomatic maximum tear osmolarity 308 mosm l study,9 minority patients 8% 2/25 increased tear osmolarity symptoms low tear osmolarity symptoms result may due mild ded former case low osmolarity due condition ded latter case this study9 concluded increased tear osmolarity likely precursor dry eye complaints despite lack correlation tear osmolarity dry eye symptoms certain factors diseases drugs noted associated ded increased tear osmolarity it likely patients exposed external factors diseases drugs dry eyes increased tear osmolarity such reactions function individual response stresses whether eyes able adjust the effect external factors humidity tear osmolarity clear one study subjects exposed low relative humidity 20% 10 minutes.20 another study found exposure humidity 5% 2 hours increase result variability tear osmolarity.21 studies included subjects using artificial tears study although discontinued use 1 hour advance testing one study specify type artificial tear used state airflow velocity such differences may account conflicting results.20,21 addition use less sensitive diagnostic tests may resulted inclusion mild dry eye patients normal group would confounded results the literature divided whether contact lens wear increases2,22,23 effect tear osmolarity.10,24,25 osmolarity measured studies,10,24 appropriate delay contact lens removal osmolarity measurement the differences reported results likely function wearer ability maintain homeostasis tear film overcoming drying effect contact lenses.10 appears certain procedures may influence tear osmolarity others recent 2013 article no change tear osmolarity dry eye diagnostic test observed refractive surgery despite decrease corneal sensitivity.26 cataract surgery tear osmolarity noted increase.27 difference may explained subjects ages average age 26 years refractive surgery study26 71 years cataract surgery study.27 older patients may take longer recover surgically induced ocular trauma.27 trabeculectomy surgery performed patient population average age 71 years also higher tear osmolarity control group result attributed presence bleb use mitomycin c may cause epithelial damage residual effects long term use glaucoma medications surgery.28 difference tear osmolarity noted eye underwent eyelid reconstruction surgery un operated eye patient result may related corrective tearing evidenced higher schirmer test results operated eye.29 table 4 shows studies investigated effect various ocular systemic conditions tear osmolarity of systemic conditions diabetes found increase tear osmolarity.30,31 increase tear osmolarity correlated increase diabetes duration the relation diabetes increased tear osmolarity likely due decrease amount aqueous secretion result injury lacrimal gland small blood vessels lacrimal gland and/or corneal nerve.31 thyroid disease results increase palpebral aperture proptosis also reported significantly increases tear osmolarity compared control.32 increased pain sensation evident patients fibromyalgia related increased tear osmolarity.33 traumatic brain injury patients higher tear osmolarity values healthy control group.34 patients increased tear osmolarity may also dehydrated water intake may important consideration dry eye diagnosis treatment.35 increase estrogen and/or progesterone form birth control pills)25 excess androgens noted patients polycystic ovary syndrome)36 seemed effect tear osmolarity another study found low levels 17-beta estradiol estrone testosterone associated increased tear osmolarity treatment phytoestrogen decreased tear osmolarity compared control group.37 studies area appear warranted understand correlation sex hormones tear osmolarity adequate control hydration measurement hydration status may important studies different ocular conditions also seem different effects tear osmolarity table 4 for instance eyes pseudoexfoliation syndrome condition thought alter goblet cell activity mucin production reported increased tear osmolarity compared control group.38 patients pterygia predominantly inflammatory condition also show increased tear osmolarity compared control group.39 keratoconus demonstrated reduce corneal sensitivity tear osmolarity similar control group the reason similarity clear though may keratoconus generally clinically considered inflammatory disease.40 herpetic keratitis associated higher tear osmolarity affected unaffected eyes compared control group reason unaffected eye higher tear osmolarity may due type keratitis.41 patients nasolacrimal duct obstruction likely compensate decreasing tear production resulting tear osmolarity value similar control group.42 table 5 summarizes findings different studies effect certain drugs tear osmolarity the use oral mucolytics often treat respiratory conditions increases tear osmolarity this increase likely due alterations mucin layer tear film.43 glaucoma medications increased tear osmolarity perhaps due preservatives medications significant effect tbut schirmer test results.28 artificial tears noted several studies decrease tear osmolarity,20,44,45 one study found correlation tear osmolarity use artificial tears.16 latter study disclose type artificial tear used this may important factor type eye drop impact tear osmolarity.45 instance lower osmolarity eye drops associated lower tear osmolarity.46,47 noted decrease osmolarity observed use artificial tears may comparable effects heat massage application eyelids using thermal massager.44 effect anti inflammatory medication tear osmolarity less clear especially studies evaluating effect suffer limitations small sample size lack control group randomization.4850 one recent randomized double blind parallel study finland osmolarity observed increase dry winter months.51 participants taking oral sea buckthorn oil containing n-3 n-6 fatty acids antioxidants less increase tear osmolarity compared control group presumed function oil anti inflammatory mechanism.51 the effect external factors humidity tear osmolarity clear one study subjects exposed low relative humidity 20% 10 minutes.20 another study found exposure humidity 5% 2 hours increase result variability tear osmolarity.21 studies included subjects using artificial tears study although discontinued use 1 hour advance testing one study specify type artificial tear used state airflow velocity such differences may account conflicting results.20,21 addition use less sensitive diagnostic tests may resulted inclusion mild dry eye patients normal group would confounded results the literature divided whether contact lens wear increases2,22,23 effect tear osmolarity.10,24,25 osmolarity measured studies,10,24 appropriate delay contact lens removal osmolarity measurement the differences reported results likely function wearer ability maintain homeostasis tear film overcoming drying effect contact lenses.10 appears certain procedures may influence tear osmolarity others recent 2013 article no change tear osmolarity dry eye diagnostic test observed refractive surgery despite decrease corneal sensitivity.26 cataract surgery tear osmolarity noted increase.27 difference may explained subjects ages average age 26 years refractive surgery study26 71 years cataract surgery study.27 older patients may take longer recover surgically induced ocular trauma.27 trabeculectomy surgery performed patient population average age 71 years also higher tear osmolarity control group result attributed presence bleb use mitomycin c may cause epithelial damage residual effects long term use glaucoma medications surgery.28 difference tear osmolarity noted eye underwent eyelid reconstruction surgery un operated eye patient result may related corrective tearing evidenced higher schirmer test results operated eye.29 the effect external factors humidity tear osmolarity clear one study subjects exposed low relative humidity 20% 10 minutes.20 another study found exposure humidity 5% 2 hours increase result variability tear osmolarity.21 studies included subjects using artificial tears study although discontinued use 1 hour advance testing one study specify type artificial tear used state airflow velocity such differences may account conflicting results.20,21 addition use less sensitive diagnostic tests may resulted inclusion mild dry eye patients normal group would confounded results the literature divided whether contact lens wear increases2,22,23 effect tear osmolarity.10,24,25 osmolarity measured studies,10,24 appropriate delay contact lens removal osmolarity measurement the differences reported results likely function wearer ability maintain homeostasis tear film overcoming drying effect contact lenses.10 it appears certain procedures may influence tear osmolarity others recent 2013 article no change tear osmolarity dry eye diagnostic test observed refractive surgery despite decrease corneal sensitivity.26 cataract surgery tear osmolarity noted increase.27 difference may explained subjects ages average age 26 years refractive surgery study26 71 years cataract surgery study.27 older patients may take longer recover surgically induced ocular trauma.27 trabeculectomy surgery performed patient population average age 71 years also higher tear osmolarity control group result attributed presence bleb use mitomycin c may cause epithelial damage residual effects long term use glaucoma medications surgery.28 difference tear osmolarity noted eye underwent eyelid reconstruction surgery un operated eye patient result may related corrective tearing evidenced higher schirmer test results operated eye.29 table 4 shows studies investigated effect various ocular systemic conditions tear osmolarity systemic conditions diabetes found increase tear osmolarity.30,31 increase tear osmolarity correlated increase diabetes duration the relation diabetes increased tear osmolarity likely due decrease amount aqueous secretion result injury lacrimal gland small blood vessels lacrimal gland and/or corneal nerve.31 thyroid disease results increase palpebral aperture proptosis also reported significantly increases tear osmolarity compared control.32 increased pain sensation evident patients fibromyalgia related increased tear osmolarity.33 traumatic brain injury patients higher tear osmolarity values healthy control group.34 patients increased tear osmolarity may also dehydrated water intake may important consideration dry eye diagnosis treatment.35 increase estrogen and/or progesterone form birth control pills)25 excess androgens noted patients polycystic ovary syndrome)36 seemed effect tear osmolarity another study found low levels 17-beta estradiol estrone testosterone associated increased tear osmolarity treatment phytoestrogen decreased tear osmolarity compared control group.37 studies area appear warranted understand correlation sex hormones tear osmolarity adequate control hydration measurement hydration status may important studies different ocular conditions also seem different effects tear osmolarity table 4 for instance eyes pseudoexfoliation syndrome condition thought alter goblet cell activity mucin production reported increased tear osmolarity compared control group.38 patients pterygia predominantly inflammatory condition also show increased tear osmolarity compared control group.39 keratoconus demonstrated reduce corneal sensitivity tear osmolarity similar control group the reason similarity clear though may keratoconus generally clinically considered inflammatory disease.40 herpetic keratitis associated higher tear osmolarity affected unaffected eyes compared control group reason unaffected eye higher tear osmolarity may due type keratitis.41 patients nasolacrimal duct obstruction likely compensate decreasing tear production resulting tear osmolarity value similar control group.42 table 5 summarizes findings different studies effect certain drugs tear osmolarity the use oral mucolytics often treat respiratory conditions increases tear osmolarity this increase likely due alterations mucin layer tear film.43 glaucoma medications increased tear osmolarity perhaps due preservatives medications significant effect tbut schirmer test results.28 artificial tears noted several studies decrease tear osmolarity,20,44,45 one study found correlation tear osmolarity use artificial tears.16 latter study disclose type artificial tear used this may important factor type eye drop impact tear osmolarity.45 instance lower osmolarity eye drops associated lower tear osmolarity.46,47 noted decrease osmolarity observed use artificial tears may comparable effects heat massage application eyelids using thermal massager.44 effect anti inflammatory medication tear osmolarity less clear especially studies evaluating effect suffer limitations small sample size lack control group randomization.4850 one recent randomized double blind parallel study finland osmolarity observed increase dry winter months.51 participants taking oral sea buckthorn oil containing n-3 n-6 fatty acids antioxidants less increase tear osmolarity compared control group presumed function oil anti inflammatory mechanism.51 a systematic review 164 peer reviewed articles related diagnostic value tear osmolarity found 72% positive impression value tear osmolarity diagnostic tool ded these results largely independent financial interest article quality rating many studies graded high moderate might expected review literature type it worth noting grading systems used current review typically applied therapeutic trials gold standard control often exists dry eye multifactorial disease clear gold standard diagnostic test randomized masked trials comparisons relevant control difficult design performing tear osmolarity testing clinical setting appears feasible availability new instruments tearlab osmolarity system reflex tearing concern tear osmolarity collected per label instructions.3,5,9 test results shown repeatable accurate good sensitivity specificity they appear better commonly used diagnostic tests especially early stages disease.8,10,12,13 correlations different diagnostic tests remain issue lack correlation surprising given multi factorial nature ded fact different diagnostic tests reveal different aspects disease.1 seemingly inconsistent threshold values reported literature may reflection disease characteristic tear film instability may also due difference disease severity tested groups given strong correlation tear osmolarity disease severity.911 severe ded higher threshold greater variability tear osmolarity value.9 currently agreed upon value discriminates healthy dry eyes higher tear film osmolarity value two eyes 308 mosm l.5,10 diagnostic tool interpretation must based complete clinical picture including diagnostic tests might helpful determining dry eye etiology.10 certain commonly encountered external factors contact lens wear may may increase tear osmolarity depending individual response.2,24 impact environmental factors temperature humidity airflow tear osmolarity appears unclear effect sex hormones factors would benefit investigation.20,21,25,36,37 effect ocular surgery tear osmolarity may partially dependent patient age type surgery performed ability eye recover.2629 systemic conditions commonly believed result dry eye dehydration diabetes thyroid ophthalmopathy shown increase tear osmolarity expected.31,32,35 ocular conditions herpetic keratitis pterygia pseudoexfoliation syndrome also demonstrated increase tear osmolarity.38,39,41 keratoconus largely viewed clinically non inflammatory condition associated increased tear osmolarity.40 pharmaceuticals mucin altering drugs glaucoma medications appear increase tear osmolarity others artificial tears likely lower tear osmolarity.20,28,43 research effect anti inflammatory medications tear osmolarity produced varied results topic deserves investigation.48,49,51 limitations review nature consequently comments related factors associated osmolarity confining review tear osmolarity related discussions dry eye issues effect hormones papers missed additionally limiting review recent papers foundational work field omitted in summary findings articles evaluated present review appear confirm central role increased tear osmolarity pathophysiology ded described dews.2 majority studies reviewed specifically rated moderate high quality support use tear osmolarity tool diagnose grade severity track therapeutic response ded

objectiveto evaluate the evidence in the peer - reviewed literature regarding the use of tear osmolarity as a physiological marker to diagnose , grade severity , and track therapeutic response in dry eye disease ( ded ) . in addition , to review the evidence for the role of tear osmolarity in the pathophysiology of ded and ocular surface disease.methodsa literature review of all publications after the year 2000 , which included the keywords tear osmolarity , was conducted . relevant articles were graded according to quality of evidence and research , using the university of michigan practice guideline and the grading of recommendations assessment , development , and evaluation ( grade ) rating systems . articles were further categorized by the nature of any reported financial support and by the overall impression they provided related to tear osmolarity.resultsa total of 164 articles were identified as relevant to the search criteria , although some were editorials , and some were written in a foreign language . of the total , it was possible to grade 159 , and an overall impression was generated for 163 . a positive impression of tear osmolarity in ded diagnosis was evident in 72% ( 117/163 ) of all articles , with a neutral impression in a further 21% ( 35/163 ) ; 7% had a negative impression . the percentage of positive impressions appeared independent of the quality of research ; 73% ( 38/52 ) of articles graded high / moderate quality supported the use of tear film osmolarity measurement in ded diagnosis . impressions were also independent of the source of financial support , with 72% ( 75/104 ) of independent studies positive.conclusionthe literature broadly supports the use of tear film osmolarity as an objective numerical measure for diagnosing , grading severity , and managing treatment of ded .

anemia defined hemoglobin less 13 g dl hematocrit less 36% relatively common preoperative finding approximately one third patients anemic undergo joint arthroplasty baseline hemoglobin levels 10 13 g dl veterans undergoing noncardiac surgery the incidence preoperative anemia hematocrit 36% 34% surgical patients refused transfusions religious reasons 28% preoperative hemoglobin levels less 12 g dl the incidence anemia also increases age recent study 296 elderly patients hip fractures mean hemoglobin hospital admission 12.1 1.7 g dl anemia especially prevalent patients cancer treatment plans often include surgery a low initial hemoglobin level measured disease presentation risk factor development anemia throughout course treatment patients colorectal cancer following radical mastectomy breast cancer 38% women anemic hemoglobin 12 g dl first chemotherapy cycle 59% became anemic surgery chemotherapy ensuring adequate tissue oxygenation surgical setting frequently involves administration blood transfusions patients preoperative hemoglobin levels 10 13 g dl may three times likely receive allogeneic blood baseline hemoglobin greater 14 g dl although documented patients prefer receive autologous blood anemic surgery candidates preoperative autologous donation pad patients coronary artery disease impaired hemodynamic nonhemodynamic responses hypoxia autologous blood donation feasible hemodilution ensues may tolerated endogenous erythropoietin normally stimulates bone marrow increase red blood cell rbc mass hypoxic conditions nonhemodynamic response hypoxia impaired hemoglobin levels rebound sufficiently if hemoglobin levels fall even surgery due blood losses commonly occurs likelihood receiving allogeneic blood transfusion increases rbc transfusions associated increased risk postoperative infections tumor recurrence well variety immunologic complications may adversely affect surgical patients longer hospital stays higher health care costs also linked blood transfusions although allogeneic blood reportedly safer ever safety issues blood supply still exist furthermore blood scarce resource becoming even baby boomer population ages two factors converging limit blood availability drive cost first eligible donor pool shrinking second surgery patients whether elective otherwise increase quality care economic factors decision transfuse carefully weighed members surgical team transfusion alternatives considered the importance correcting preoperative anemia overemphasized condition known associated poor outcomes surgery coronary artery disease patients anemia exhibit higher mortality rates experience cardiac abdominal renal complications cardiac noncardiac surgery patients normal hemoglobin levels myocardial ischemia one major complications occur anemic patients without overt cardiac disease elderly men undergoing radical prostatectomy st segment changes postoperative ischemic episodes were seen frequently lower hematocrit another study noncardiac surgical patients various types comprising large veterans administration database perioperative anemia blood transfusions associated increased risk infection higher mortality rates substantial consumption health care resources dunne coworkers recently documented relationship preoperative anemia stage colorectal cancer diagnosis study 198 patients rectal cancer showed low preoperative hemoglobin levels independent risk factor mortality p 0.0001 taken together reports indicate preoperative anemia prevalent anemia blood transfusions associated risks poor outcomes anemia treated preferably strategies exclude allogeneic blood the human recombinant form endogenous erythropoietin rhuepo epoetin alfa indicated treatment anemic patients scheduled undergo elective surgery noncardiac nonvascular reduce need allogeneic blood transfusions multiple clinical studies involving many different surgical types shown preoperative rhuepo safe effective patients initial hematocrits 33% 39% stand lose 3000 ml blood surgery among benefit 21 23 the positive impact rhuepo readily apparent patients undergoing orthopedic surgery often elective associated substantial blood losses oral iron supplementation treatment rhuepo considered anemic patient hemoglobin 10 13 g dl whose risk transfusion estimated exceed 10% patients undergoing major orthopedic surgery received rhuepo 300 units kg per day 10 days preoperatively day surgery 4 days postoperatively nearly six fold reduction allogeneic transfusion risk compared patients received placebo in another study compared rhuepo administered daily 300 units kg per day rhuepo administered weekly 600 units kg per week four doses beginning 21 days surgery transfusion requirements comparable indicating weekly rhuepo regimen also reduces transfusion requirements improving patient convenience rhuepo also demonstrated reduce transfusion requirements elderly patients undergoing major hip knee reconstruction heterogeneous populations patients needing total joint arthroplasty major hip knee surgery even children requiring craniosynostosis repair cardiac surgery all seven randomized studies included meta analysis provided evidence rhuepo without pad produced significant decreases proportion patients transfused allogeneic blood another single blind randomized study patients undergoing coronary artery bypass graft surgery patients treated low dose 100 units kg rhuepo 4 days surgery received less half amount autologous blood p 0.01 compared control group surgery cancer patients represents particular challenge patients may suffer severe anemia caused cancer treatment patients hemoglobin 8.5 g dl colorectal cancer 300 iu kg rhuepo day 4 surgery followed 150 iu kg next 7 days thereafter stimulated erythropoiesis reduced transfusion requirements compared placebo transfusion requirements reduced hemoglobin levels improved preoperative rhuepo treatment patients gastrointestinal tract cancer rectal cancer undergoing surgery other patient groups surgery indicated preoperative rhuepo exerts beneficial effects include prostate cancer head neck cancer women undergoing gynecologic surgery benign conditions surgical blood losses anticipated exceed 3000 ml rhuepo effectively used conjunction pad patients undergo total hip replacement rhuepo facilitated dose dependent increases pad amounting 4.3 units 300 iu kg 3.4 units 150 iu kg 3.0 units 75 iu kg compared 2.1 units placebo minimized reductions hematocrit associated repeated phlebotomy treatment rhuepo resulted rapid increases rbc production 3.5 days nonanemic hematocrit 39% patients participating aggressive program autologous blood donation this treatment allowed higher rbc volumes donated compared placebo treated patients even anemic gastrointestinal cancer patients rhuepo facilitated pad compared patients supplemented iron alone other examples utility rhuepo pad gynecologic surgery orthopedic surgery found although field medicine unto practice bloodless surgery gaining attention therefore deserves brief mention jehovah witnesses refuse transfusions basis religious convictions known survive trauma surgery remarkably low hemoglobin levels many cases patients pharmacological support erythropoietic agents 49 51 case study involving 48 jehovah witness patients rhuepo successfully used avoid transfusions completely elective coronary heart valve surgery used combination products substitute blood ( e.g. hemoglobin based oxygen carriers also known hbocs may complementary effects thus patients wishing avoid transfusions may even options future conclusion preoperative anemia effectively managed minimal exposure allogeneic transfusions virtually surgical specialties rhuepo reduces transfusion requirements facilitates collection preoperative autologous blood effective alone blood conservation strategies severe anemia surgeons enthusiastically adopt available therapies help avoid transfusions accompanying risks conserve blood treat preoperative anemia goal improving surgical outcomes tgm received research support honoraria lectures ortho biotech products l.p pad preoperative autologous donation rbc red blood cell rhuepo recombinant human erythropoietin

preoperative anemia in a surgical patient predisposes to poor outcomes and allogeneic blood transfusions . as an alternative to transfusions , pharmacologic management of preoperative anemia with recombinant human erythropoietin ( rhuepo ) has been well studied in many different types of surgery . rhuepo , when used alone or in combination with preoperative autologous blood donation before elective surgery , stimulates erythropoiesis and helps to avoid or reduce the need for allogeneic blood transfusions . the clinical evidence on preoperative use of rhuepo in orthopedic , cardiac , and cancer surgery , as well as in bloodless surgery , is reviewed .

curcumin,1,7-bis(4-hydroxy-3-methoxypheny l)- 1- 6- heptadiene- 3 5-dione commonly known diferuloylmethane yellow pigment component curry turmeric curcuma longa 1 turmeric extracts extensively used treatment several diseases ayurvedic medicine india several centuries curcumin first extracted turmeric impure form 1815 1910 crystallized structure elucidated 2 it antimicrobial antioxidant immunomodulatory anti inflammatory anti alzheimer anticancer activity 38 it shown toxicity vitro numerous cell culture systems vivo animal models 13 phase human clinical trials it generally recognized safe united states food drug administration fda 2 4 5 curcumin shown affect several targets somatic cells biological activity 38 it inhibits nf-b activity cox-2 5-lox expression modulates release several cytokines 3 4 it also binds number proteins including thioredoxin reductase protein kinases several receptors 3 4 ) however proteins factors may expressed/ present terminally non transcriptional sperm also sperm unique characteristic motility present cells thus curcumin may different molecules/ mechanism(s action unique sperm recently laboratory reported first time ever curcumin affects sperm function motility capacitation acrosome reaction fertilization vitro fertility vivo intravaginal administration curcumin caused significant reversible reduction fertility 9 the molecular mechanism(s curcumin inhibits blocks sperm motility delineated since modulation intracellular ph phi plasma membrane polarization shown involved sperm motility capacitation acrosome reaction several mammalian species 1021 present study conducted investigate effect curcumin sperm intracellular ph plasma membrane polarization it hypothesized curcumin mediated effect sperm motility caused modulation phi and/or membrane polarization the long term objective study understand molecular mechanism(s curcumin affects sperm motility function develop novel non steroidal contraceptive spermicidal properties semen liquefied analyzed volume sperm concentration percent progressive motility semen samples sperm concentration 50x10sperm ml percent motility 60% progressive motility 3 scale 0 5 contamination immature germ cells immune cells < 1% used collect pure swim sperm population 22 the study approved west virginia university institutional review board irb human studies mouse sperm collected cauda epididymis vas deferens mature balb c cd-1 males motile sperm isolated swim procedure washed centrifugation 500 g 10 min ham f-10 medium supplemented human serum albumin 5 mg ml modified sperm washing medium irvine scientific santa ana ca usa the study approved west virginia university animal care use committee acuc animal studies # c27727 obtained sigma aldrich st louis mo usa curcumin dissolved dimethylsulf oxide dmso 25 50 mm stock diluted medium desired concentrations the effect curcumin sperm forward motility examined incubating 10 100 l sperm suspension 100 250x10 motile sperm ml various concentrations curcumin 50 400 final concentration 1 hr percentage forward moving sperm recorded every 5 20 min incubation each experiment including various treatment groups performed 3 5 times using sperm 3 5 men mice different days take care inter individual variation among various sperm specimens different men mice the intracellular ph phi human mouse sperm measured fluorescent ph indicator 2,7-bicarb oxyethy l-5 6-carboxyfluorescein- acetoxymethylester bcecf molecular probes eugene usa following manufacturer protocol described hamamah et al bcecf neutral lipophilic form bis carboxyfluorescein diffuses freely plasma membrane cell hydrolyzed esterases releasing bcecf retained within cytoplasm the motile sperm isolated swim procedure centrifuged pellet washed resuspended 1 ml phosphate buffered saline pbs ph=7.4 sperm 8 15x10/ml loaded 2 bcecf final concentration incubated 37c 35 min dark following incubation sperm centrifuged washed x2 resuspended pbs intracellular ph calibration curve sperm loaded bcecf various extracellular phs phe 6.8 7.0 7.2 7.4 7.6 7.8 treated 0.1% triton x100 subsequently fluorescence intensity measured described calibration curve constructed plotting fluorescence intensity versus extracellular ph 24 using calibration curve the fluorescence intensity bcecf dependent upon ph maximum response excitation =535 nm f1 =490 nm f2 intensity independent ph the phi determined graphically using ratio f1/f2 calibration curve obtained permeabilization spermatozoa 0.1% triton measuring maximum fluorescence intensity adding naoh minimum adding hcl the f1/f2 ratio represents pseudo linear function ph 23 examine effect curcumin bcecf loaded sperm incubated 37c 5 10 min various concentrations curcumin 50 400 washed the treated control sperm transferred wells 200 l well black 96-well microplate fluorescence intensity measured biotek synergy2 multiplatform automated plate reader using excitation emission wavelengths 490 535 nm respectively each experiment including various treatment groups performed 3 5 times using sperm 3 5 men mice different days take care inter individual variation among various sperm specimens different men mice moreover pertinent examine effect cur cumin per se fluorescence intensity similar experiments carried various concentrations 50 400 curcumin without sperm affect fluorescence intensity indicating curcumin per se without sperm affect fluorescence intensity the changes sperm plasma membrane polarization examined using fluorescence sensitive dye bis 1,3-dibarbituric acid)-trimethine oxanol dibac4(3 molecular probes eugene usa following manufacturer protocol described rossato et al the motile sperm swim fraction centrifuged washed x2 pbs incubated 37 c 1 hr 2 dibac4(3 dibac4(3 enter depolarized cells bind intracellular proteins membrane exhibits enhanced fluorescence increased depolarization results additional influx anionic dye increase fluorescence sperm 8 15x 10/ml centrifuged washed twice pbs the dibac4(3)-incubated sperm aliquoted different tubes equal volumes incubated 5 10 min various concentrations curcumin 100 200 300 400 after final washing pbs sperm transferred wells 200 l well black 96-well microplate fluorescence intensity measured biotek synergy2 multiplatform automated plate reader using excitation emission wavelengths 485 530 nm respectively each experiment including various treatment groups performed 3 5 times using sperm 3 5 men mice different days take care inter individual variation among various sperm specimens different men mice similar experiments carried various concentrations 50 400 curcumin without sperm affect fluorescence intensity indicating curcumin per se without sperm affect fluorescence intensity the significance difference among various groups analyzed using analysis variance anova tukey kramer multiple comparison test semen liquefied analyzed volume sperm concentration percent progressive motility semen samples sperm concentration 50x10sperm ml percent motility 60% progressive motility 3 scale 0 5 contamination immature germ cells immune cells < 1% used collect pure swim sperm population 22 the study approved west virginia university institutional review board irb human studies mouse sperm collected cauda epididymis vas deferens mature balb c cd-1 males motile sperm isolated swim procedure washed centrifugation 500 g 10 min ham f-10 medium supplemented human serum albumin 5 mg ml modified sperm washing medium irvine scientific santa ana ca usa the study approved west virginia university animal care use committee acuc animal studies # c27727 obtained sigma aldrich st louis mo usa curcumin dissolved dimethylsulf oxide dmso 25 50 mm stock diluted medium desired concentrations the effect curcumin sperm forward motility examined incubating 10 100 l sperm suspension 100 250x10 motile sperm ml various concentrations curcumin 50 400 final concentration 1 hr percentage forward moving sperm recorded every 5 20 min incubation each experiment including various treatment groups performed 3 5 times using sperm 3 5 men mice different days take care inter individual variation among various sperm specimens different men mice the intracellular ph phi human mouse sperm measured fluorescent ph indicator 2,7-bicarb oxyethy l-5 6-carboxyfluorescein- acetoxymethylester bcecf molecular probes eugene usa following manufacturer protocol described hamamah et al bcecf neutral lipophilic form bis carboxyfluorescein diffuses freely plasma membrane cell hydrolyzed esterases releasing bcecf retained within cytoplasm the motile sperm isolated swim procedure centrifuged pellet washed resuspended 1 ml phosphate buffered saline pbs ph=7.4 sperm 8 15x10/ml loaded 2 bcecf final concentration incubated 37c 35 min dark following incubation sperm centrifuged washed x2 resuspended pbs intracellular ph calibration curve sperm loaded bcecf various extracellular phs phe 6.8 7.0 7.2 7.4 7.6 7.8 treated 0.1% triton x100 subsequently fluorescence intensity measured described calibration curve constructed plotting fluorescence intensity versus extracellular ph 24 using calibration curve the fluorescence intensity bcecf dependent upon ph maximum response excitation =535 nm f1 =490 nm f2 intensity independent ph the phi determined graphically using ratio f1/f2 calibration curve obtained permeabilization spermatozoa 0.1% triton measuring maximum fluorescence intensity adding naoh minimum adding hcl the f1/f2 ratio represents pseudo linear function ph 23 examine effect curcumin bcecf loaded sperm incubated 37c 5 10 min various concentrations curcumin 50 400 washed the treated control sperm transferred wells 200 l well black 96-well microplate fluorescence intensity measured biotek synergy2 multiplatform automated plate reader using excitation emission wavelengths 490 535 nm respectively each experiment including various treatment groups performed 3 5 times using sperm 3 5 men mice different days take care inter individual variation among various sperm specimens different men mice moreover pertinent examine effect cur cumin per se fluorescence intensity similar experiments carried various concentrations 50 400 curcumin without sperm affect fluorescence intensity indicating curcumin per se without sperm affect fluorescence intensity the changes sperm plasma membrane polarization examined using fluorescence sensitive dye bis 1,3-dibarbituric acid)-trimethine oxanol dibac4(3 molecular probes eugene usa following manufacturer protocol described rossato et al the motile sperm swim fraction centrifuged washed x2 pbs incubated 37 c 1 hr 2 dibac4(3 dibac4(3 enter depolarized cells bind intracellular proteins membrane exhibits enhanced fluorescence increased depolarization results additional influx anionic dye increase fluorescence sperm 8 15x 10/ml centrifuged washed twice pbs the dibac4(3)-incubated sperm aliquoted different tubes equal volumes incubated 5 10 min various concentrations curcumin 100 200 300 400 after final washing pbs sperm transferred wells 200 l well black 96-well microplate fluorescence intensity measured biotek synergy2 multiplatform automated plate reader using excitation emission wavelengths 485 530 nm respectively each experiment including various treatment groups performed 3 5 times using sperm 3 5 men mice different days take care inter individual variation among various sperm specimens different men mice similar experiments carried various concentrations 50 400 curcumin without sperm affect fluorescence intensity indicating curcumin per se without sperm affect fluorescence intensity the significance difference among various groups analyzed using analysis variance anova tukey kramer multiple comparison test curcumin caused concentration dependent decrease human sperm forward motility table 1 50 concentration apparent p>0.05 effect sperm forward motility 5 10 min slight decrease time 1 hr decreased 25% p 0.001 100 concentration significant p<0.001 decrease sperm forward motility 5 10 min decreased 80% 1 hr concentrations 200 complete block sperm forward motility within 5 10 min dmso vehicle affect p>0.05 sperm forward motility compared medium control effect curcumin human sperm forward motility versus control significantly different p<0.001 others non significant curcumin caused similar effect mouse sperm forward motility table 2 50 concentration apparent p>0.05 effect sperm forward motility 5 10 min slight decrease time 1 hr decreased 40% p<0.001 100 concentration significant p<0.001 decrease sperm forward motility 5 10 min decreased 70% 1 hr concentrations 200 complete block forward motility within 5 10 min dmso vehicle affect p>0.05 sperm forward motility compared medium control effect curcumin murine sperm forward motility versus control significantly different p<0.001 others non significant linear relationship extracellular ph fluorescence intensity human r=0.9513 mouse r=0.9835 sperm figure 1 panel b respectively increase extracellular ph corresponding increase fluorescence intensity the ph=7.4 selected subsequent experiments examine effect curcumin intracellular sperm ph phi calibration curve human panel mouse panel b sperm fluorescence intensity various extracellular ph bcecf loaded sperm treated 0.1% triton x 100 various extracellular ph phe fluorescence intensity measured fluorescence intensity values plotted phe values obtain calibration curve human mouse sperm curcumin caused concentration dependent decrease intracellular ph figure 2 human panel mouse panel b sperm in human sperm control sperm phi 7.30.003 significantly different p>0.05 dmso treated sperm phi 7.30.105 figure 3 panel treatment curcumin significantly p<0.001 decreased intracellular ph concentration dependent manner compared control/ dmso treated sperm 50 curcumin concentration phi 7.210.010 100 phi 7.040.008 200 phi 6.950.02 300 phi 6.880.0067 400 phi 6.810.014 comparing change phi within curcumin treated groups the difference significant p<0.05 among 50 100 400 groups human panel mouse panel b sperm intracellular ph phi curcumin treatment bcef loaded sperm treated increasing concentrations curcumin fluorescence intensities measured converted intracellular ph using calibration curve intracellular ph phi values meanse 3 5 independent experiments using sperm 3 5 different men mice shown there effect concentration curcumin tested per se fluorescence intensity without sperm effect curcumin plasma membrane potential human panel mouse panel b sperm fluorescence intensity values meanse 3 5 independent experiments using sperm 3 5 different men mice shown fluorescence intensity values lower control indicate hyperpolarization effect concentration curcumin tested per se fluorescence intensity without sperm similar results obtained mouse sperm figure 2 panel b mouse sperm control sperm phi 7.150.005 significantly different p>0.05 dmso treated sperm phi 7.150.00 figure 2 panel b treatment curcumin significantly p<0.001 decreased intracellular ph concentration dependent manner compared control dmso treated sperm 50 curcumin concentration phi 7.070.017 100 phi 6.970.017 200 phi 6.91 0.012 300 phi 6.880.02 400 phi 6.830.017 comparing changes phi within curcumin treated groups difference significant p<0.05 among 50 100 400 groups the effect various concentrations curcumin plasma membrane polarization evaluated using human mouse sperm human sperm treatment dmso significantly p>0.05 affect fluorescence intensity compared control sperm control 286143.016 dmso 3252 215.860 figure 3 panel treatment curcumin caused significant p<0.001 hyperpolarization sperm plasma membrane compared control dmso treated sperm figure 3 panel 50 curcumin fluorescence intensity 60151.052 100 59720.664 200 52272.421 300 45847.576 400 41429.464 comparing changes fluorescence intensities within curcumin treated groups differences significant similar results obtained mouse sperm figure 3 panel b mouse sperm treatment dmso significantly affect fluorescence intensity compared control control 5592.0852.186 dmso 5130.905130.770 figure 3 panel b treatment curcumin caused significant p<0.001 hyperpolarization sperm plasma membrane compared control dmso treated sperm figure 3 panel b 50 curcumin fluorescence intensity 997.50222.229 100 877.35641.328 200 624.33142.116 300 578.92943.245 400 522.43157.900 comparing changes fluorescence intensity within curcumin treated groups difference significant p<0.05 50 400 groups curcumin caused concentration dependent decrease human sperm forward motility table 1 50 concentration apparent p>0.05 effect sperm forward motility 5 10 min slight decrease time 1 hr decreased 25% p 0.001 100 concentration significant p<0.001 decrease sperm forward motility 5 10 min decreased 80% 1 hr concentrations 200 complete block sperm forward motility within 5 10 min dmso vehicle affect p>0.05 sperm forward motility compared medium control effect curcumin human sperm forward motility versus control significantly different p<0.001 others non significant curcumin caused similar effect mouse sperm forward motility table 2 50 concentration apparent p>0.05 effect sperm forward motility 5 10 min slight decrease time 1 hr decreased 40% p<0.001 100 concentration significant p<0.001 decrease sperm forward motility 5 10 min decreased 70% 1 hr concentrations 200 complete block forward motility within 5 10 min dmso vehicle affect p>0.05 sperm forward motility compared medium control effect curcumin murine sperm forward motility versus control significantly different p<0.001 others non significant there linear relationship extracellular ph fluorescence intensity human r=0.9513 mouse r=0.9835 sperm figure 1 panel b respectively increase extracellular ph the ph=7.4 selected subsequent experiments examine effect curcumin intracellular sperm ph phi calibration curve human panel mouse panel b sperm fluorescence intensity various extracellular ph bcecf loaded sperm treated 0.1% triton x 100 various extracellular ph phe fluorescence intensity measured fluorescence intensity values plotted phe values obtain calibration curve human mouse sperm curcumin caused concentration dependent decrease intracellular ph figure 2 human panel mouse panel b sperm in human sperm control sperm phi 7.30.003 significantly different p>0.05 dmso treated sperm phi 7.30.105 figure 3 panel treatment curcumin significantly p<0.001 decreased intracellular ph concentration dependent manner compared control/ dmso treated sperm 50 curcumin concentration phi 7.210.010 100 phi 7.040.008 200 phi 6.950.02 300 phi 6.880.0067 400 phi 6.810.014 comparing change phi within curcumin treated groups the difference significant p<0.05 among 50 100 400 groups human panel mouse panel b sperm intracellular ph phi curcumin treatment bcef loaded sperm treated increasing concentrations curcumin fluorescence intensities measured converted intracellular ph using calibration curve intracellular ph phi values meanse 3 5 independent experiments using sperm 3 5 different men mice shown there effect concentration curcumin tested per se fluorescence intensity without sperm effect curcumin plasma membrane potential human panel mouse panel b sperm fluorescence intensity values meanse 3 5 independent experiments using sperm 3 5 different men mice shown there effect concentration curcumin tested per se fluorescence intensity without sperm similar results obtained mouse sperm figure 2 panel b mouse sperm control sperm phi 7.150.005 significantly different p>0.05 dmso treated sperm phi 7.150.00 figure 2 panel b treatment curcumin significantly p<0.001 decreased intracellular ph concentration dependent manner compared control dmso treated sperm 50 curcumin concentration phi 7.070.017 100 phi 6.970.017 200 phi 6.91 0.012 300 phi 6.880.02 400 phi 6.830.017 comparing changes phi within curcumin treated groups difference significant p<0.05 among 50 100 400 groups the effect various concentrations curcumin plasma membrane polarization evaluated using human mouse sperm human sperm treatment dmso significantly p>0.05 affect fluorescence intensity compared control sperm control 286143.016 dmso 3252 215.860 figure 3 panel treatment curcumin caused significant p<0.001 hyperpolarization sperm plasma membrane compared control dmso treated sperm figure 3 panel 50 curcumin fluorescence intensity 60151.052 100 59720.664 200 52272.421 300 45847.576 400 41429.464 comparing changes fluorescence intensities within curcumin treated groups similar results obtained mouse sperm figure 3 panel b mouse sperm treatment dmso did significantly affect fluorescence intensity compared control control 5592.0852.186 dmso 5130.905130.770 figure 3 panel b treatment curcumin caused significant p<0.001 hyperpolarization sperm plasma membrane compared control dmso treated sperm figure 3 panel b 50 curcumin fluorescence intensity 997.50222.229 100 877.35641.328 200 624.33142.116 300 578.92943.245 400 522.43157.900 comparing changes fluorescence intensity within curcumin treated groups difference significant p<0.05 50 400 groups the data indicated curcumin affects sperm forward motility starting 100 concentration complete block 200 concentration within 5 10 min human murine sperm these findings correlate well earlier published data 9 general forward motility corresponded well overall motility progressive motility decrease forward motility corresponded decrease overall progressive motility also loss sperm viability decrease forward motility the totally immotile sperm completely non viable tested eosin nigrosin staining data shown the present study conducted examine mechanism curcumin affects sperm forward motility the findings indicated curcumin acidified sperm intracellularly hyperpolarized cell membrane human mouse sperm there concentration dependent decrease phi 7.3 6.81 highest concentration 400 curcumin tested these findings correlate well effect curcumin human murine sperm forward motility starting 100 concentration significant effect within 5 10 min total block 200 concentration the phi plays important role modulating mammalian sperm motility 12 17 20 these cumulative findings indicate acidic phi inhibits sperm motility agreement findings the phi 6.6 6.8 reported one study uncapacitated murine sperm 13 study this may due different methodologies media and/or strain mice used and/or various degrees capacitation sperm preparations the sodium proton membrane exchange mechanism modulate phi affect motility 26 the phi changes also involved sperm motility sperm cell passes epididymis 27 28 also ph epididymal cauda fluid acidic almost species 29 acts directly phi decrease sperm motility 28 our extensive data search pubmed google scholar yield publication examined effect curcumin intracellular ph sperm cell types the degree decrease intracellular florescence intensity indicative degree hyperpolarization the change sperm membrane potential shown involved sperm motility function 18 19 21 24 25 28 3033 for mouse sperm intracellular alkalinization produces hyperpolarization sperm membrane potential may important hyperactivated motility acrosome reaction study curcumin induced hyperpolarization intracellular acidification correlate reduction block sperm motility there drastic effect hyperpolarization change phi 50 concentration significantly decrease phi showed significant effect membrane polarization human mouse sperm it possible curcumin may affect phi membrane potential interacting different molecules signal transduction cascades besides sperm hyperpolarization also connected intracellular phi changes several cell systems 34 although publication examined effect sperm curcumin reported affect membrane potential cell systems curcumin inhibits sarco endoplasmic reticulum caatpase ser ca pump major role maintaining lower levels intracellular ca importing extracellular protons rabbit skeleton muscle 36 study cao et al ( 37 examined effect curcumin changes mitochondrial membrane potential human hepatomag2 hepg2 cells their study showed hepg2 cells incubated curcumin induced oxidative damage mitochondrial dna mitochondrial membrane hyperpolarization several molecules mechanisms involved regulation sperm intracellular ph membrane polarization sodium hydrogen exchange suggested mediator ph regulation various mammalian sperm 27 38 39 at least two sodium hydrogen exchangers nhe1 nhe5 shown expressed spermatozoa the catalytic subunit protein kinase pka involved activation motility evoked hco3 anion 40 capacitation associated hyperpolarization involves decrease intracellular na+ regulated pka activation cystic fibrosis transmembrane conductance regulator cftr 41 alkalinization activates intracellular ph sensitive i(ksper inducing membrane potential approach negative potentials ca entry via i(catsper maximized 33 42 the exact molecular mechanism(s signal transduction pathway involved modulation sperm intracellular ph membrane potential curcumin need investigation there several mechanisms affect sperm motility function leading infertility 43 44 this preliminary data indicates curcumin may inhibit tyrosine phosphorylation subset sperm surface proteins ca channels unpublished data tyrosine phosphorylation shown involved sperm motility capacitation acrosome reaction function 16 the findings indicate curcumin causes intracellular acidification membrane hyperpolarization may involved inhibiting sperm forward motility this first study knowledge examined effect curcumin sperm phi membrane potential these exciting findings application deciphering molecular mechanism(s involved curcumin action delineating signal transduction pathway relevant sperm motility function also data may clinical application development novel non steroid contraceptive spermicidal properties

backgroundcurcumin has shown to affect sperm motility and function in vitro and fertility in vivo . the molecular mechanism(s ) by which curcumin affects sperm motility has not been delineated . since modulation of intracellular ph ( phi ) and plasma membrane polarization is involved in sperm motility , the present study was conducted to investigate the effect of curcumin on these sperm ( human and murine ) parameters.methodsthe effect of curcumin on sperm forward motility was examined by counting percentages of forward moving sperm . the effect of curcumin on intracellular ph ( phi ) was measured by the fluorescent ph indicator 2,7-bicarboxyethyl-5,6-carboxyfluorescein - acetoxymethyl ester ( bcecf - am ) . the effect of curcumin on plasma membrane polarization was examined using the fluorescence sensitive dye bis ( 1,3-dibarbituric acid)-trimethine oxanol [ dibac4(3)].resultscurcumin caused a concentration - dependent ( p<0.05 ) decrease in forward motility of both human and mouse sperm . it also caused a concentration - dependent decrease in intracellular ph ( phi ) in both human and mouse sperm . curcumin induced significant ( p<0.05 ) hyperpolarization of the plasma membrane in both human and mouse sperm.conclusionthese findings indicate that curcumin inhibits sperm forward motility by intracellular acidification and hyperpolarization of sperm plasma membrane . this is the first study to our knowledge which examined the effect of curcumin on sperm phi and membrane polarization that affect sperm forward motility . these exciting findings will have application in deciphering the signal transduction pathway involved in sperm motility and function and in development of a novel non - steroidal contraceptive for infertility .

incidence splenic abscess clinically uncommon disease current literature reporting 0.140.7% occurrence rate 1 2 although splenic abscesses clinical rarity potential fatal the presentation disease often vague insidious including left upper quadrant abdominal pain fever chills additionally patients may present leukocytosis left upper quadrant mass pleural effusion chest x ray splenic abscesses generally occur patients underlying comorbidities commonly include neoplasia immunodeficiency trauma metastatic infection splenic infarct diabetes the best management splenic abscesses still debatable various modalities including antibiotics percutaneous drainage splenectomy the current literature supports 67100% success rate percutaneous drainage however tung et al states optimal treatment splenic abscess splenectomy 600 cases splenic abscess documented current literature most described air confined left upper quadrant chest x ray we report young patient ruptured splenic abscess resulting acute abdomen pneumoperitoneum knowledge four reported cases ruptured splenic abscess causing pneumoperitoneum 3 4 a 48-year old female past medical history diabetes coronary artery disease psoriasis presented acute onset diffuse abdominal pain 2 days prior admission hospital upon presentation tachycardic heart rate 130 hypotensive blood pressure 78/45 she resuscitated emergency department intravenous fluids blood pressure heart rate responded appropriately her initial labs showed normal wbc level 8.3 thou mcl however patient acidotic ph 7.29 lactic acid level 3.5 mmol l an acute abdominal series obtained demonstrating free air right hemidiaphragm fig 1 the patient subsequently boarded exploratory laparotomy repair perforated viscus usual cause pneumoperitoneum especially right hemidiaphragm because patient vital signs stabilized resuscitated decision made obtain ct scan better assess location perforated viscus much surprise patient perforated viscus splenic abscess ruptured causing pneumoperitoneum fig 2 figure 2:ct scan demonstrating gas forming splenic abscess free air peritoneal cavity ct scan demonstrating gas forming splenic abscess free air peritoneal cavity the patient became increasingly confused vital signs deteriorated demonstrating worsening sepsis the patient taken operating room laparoscopic splenectomy attempted quickly converted laparotomy splenectomy due gross contamination abdomen the patient continued antibiotics taken intensive care unit post operative care the splenic abscess grew prevotella intermedia bacterium commonly found oral flora the patient underwent full work looking source splenic abscess a transesophageal echocardiogram performed negative masses thrombus vegetation furthermore panorex performed negative patient reported tooth pain 1 week prior admission she returned clinic post operative day 14 receive splenectomy vaccinations although rare disease splenic abscess included differential diagnosis patient presenting hospital peritonitis pneumoperitoneum this especially true patients immunocompromised underlying comorbidities including neoplasia diabetes trauma history splenic infarct embolization case ct scan provided important information cause patient pneumoperitoneum assumed due perforated ulcer diverticulitis this change operative management attempted perform less invasive surgery patient starting laparoscopic technique however convert open splenectomy due gross contamination adhesions splenic abscess in retrospect open laparotomy best approach patient care allowed better visualization irrigation abdominal cavity it unknown successfully performed splenectomy laparoscopically would decreased recovery time the current literature supports laparoscopic splenectomy safe effective procedure patients splenic abscess demonstrating average length stay 14 days case the patient remained hospital 10 days converting open procedure the current literature reports common organisms found splenic abscesses aerobic microbes particularly streptococci escherichia coli this microbe commonly found oral flora associated periodontal disease although transesophageal echocardiogram panorex negative believe splenic abscess developed result translocation oral flora patient complaining tooth ache 1 week prior presentation summary presented case ruptured splenic abscess cause pneumoperitoneum peritonitis although rare encourage splenic abscess included differential diagnosis patients presenting peritonitis pneumoperitoneum although still controversy current literature managing splenic abscesses recommendation splenectomy especially case ruptured splenic abscess resulting hemodynamic instability whether better perform splenectomy laparoscopically open still debatable important thing obtain source control furthermore full work required patients presenting splenic abscess underlying cause could cause serious illnesses patient future there conflicts interests financial funding disclose contributing authors

we encountered a case of ruptured splenic abscess presenting as peritonitis and pneumoperitoneum . our patient did not have an underlying neoplasm nor was she immunosuppressed . in our case , splenectomy was the treatment of choice in combination with antibiotics , which proved to be a good outcome for the patient . work - up for the cause of the abscess was negative , although bacteria predominately found in the oral flora were isolated from the abscess . we strongly encourage that splenic abscess be considered on the differential diagnosis of patients presenting with pneumoperitoneum and peritonitis , although a clinical rarity .

squamous cell carcinoma head neck scchn fifth common neoplasm estimated annual global incidence 500,000 cases diagnosed worldwide the treatment usually interdisciplinary mainly involves surgeons radiation oncologists medical oncologists clinical nurse specialists speech language specialists dieticians dependent situation goals treatment obtain high locoregional control survival rates patients limited disease ii increased survival patients advanced disease improved locoregional control reduced probability distant metastasis second malignancies iii increased organ function preservation resectable unresectable tumors vi increased therapeutic ratio cure toxicity ratio single modality treatment recommended patients early stage disease stage stage ii approximately 40% patients scchn combined modality treatment patients locally advanced disease the combined modality treatment may include surgery followed adjuvant radiotherapy radiochemotherapy concomitant radiochemotherapy using conventional alternative fractionation regimen induction chemotherapy followed radiotherapy radiochemotherapy 3 4 a precise understanding prognostic factors important select optimal treatment individual patient stratify patients clinical trials statistical analyses retrospective single institutional study the role potential prognostic factors evaluated compared patients squamous cell carcinoma oro- hypopharynx treatment definitive radiotherapy radiochemotherapy drt versus surgery followed postoperative radiotherapy prt between 1992 2000 288 patients squamous cell carcinoma oropharynx hypopharynx received radiation therapy definitive n 162 postoperative n 126 treatment eligibility criteria retrospective single institutional study histologically proven squamous cell carcinoma oropharynx hypopharynx distant metastasis synchronous cancer time diagnosis definitive postoperative radiotherapy minimum total dose 60 gy one hundred thirty eight 162 85% patients treated definitive radiotherapy received concomitant boost fractionation regimen 24 15% treated conventional fractionation single fraction dose 2 gy one fraction day five fractions week two concomitant boost regimen used regimen 1 consisted total dose 66 gy five weeks daily fraction dose 2 gy concomitant boost 1.6 gy last two weeks n 76 regimen 2 total dose 69.9 gy 5.5 weeks daily fraction dose 1.8 gy concomitant boost 1.5 gy last 2.5 weeks n 62 ninety four 138 68% patients treated concomitant boost fractionation regimen received simultaneous chemotherapy well five 4% patients treated conventional fractionation the simultaneous chemotherapy consisted 70 mg carboplatin days 15 2933 n 73 70 mg carboplatin 600 mg 5-fluorouracil days 15 2933 n 26 carboplatin administered daily short term intravenous infusion 5-fluorouracil intravenous continuous infusion 120 hours postoperative radiotherapy conventional fractionation without simultaneous chemotherapy used n 123 the radiotherapy performed opposed lateral fields upper neck one anterior field lower neck using 6 mev photons patients treated thermoplastic mask immobilization individual blocks used spare normal tissue possible dose 30 36 gy reference point the spinal cord spared photon fields uninvolved posterior neck treated electrons selected energy according ct findings daily doses 2.5 gy five times week prescribed total dose target volumes defined ct scans dose calculated midplane selected patients pre treatment ct scans patients digitized automatic laser scanner fips plus the stored images transferred personal computer macroscopic tumor shape primary tumor locoregional lymph node metastases ) was defined every ct slice slice thickness 5 mm 8 mm using drawing tool software photostyler number pixels n enclosed contour determined custom shaped image processing program software interactive data language the area ai ith slice determined ai pixel size length)pixel size width)n pixel size determined using scaling given ct hardcopy the determined tumor area slice multiplied slice thickness di ttv approximated 1)vtumor(cm3)=i1mai(mm2)di(mm)/1000 repeated measurements using irregularly shaped tumor phantoms showed difference reference volumes ct based volume measurements depending slice thickness 5 mm 8 mm 1.4% 4.5% in addition quantitative tumor volumetry based digitised pretreatment ct scans ttv estimated based postoperative histopathological report patients treated adjuvant radiotherapy the ttv approximated using equation v 4/3 abc b c represent orthogonal maximal tumor diameters the overall survival defined time first day treatment death cause the overall survival estimated using kaplan meier method treatment groups compared using two sided log rank test the locoregional failure free survival defined time first day treatment locoregional failure the distant metastasis free survival defined time first day treatment distant failure the locoregional failure free survival distant metastasis free survival estimated cumulative incidence method treatment groups compared using gray test 5 6 the simultaneous relationship multiple prognostic factors overall survival assessed using cox proportional hazard regression analysis the simultaneous relationship multiple prognostic factors locoregional failure free distant metastasis free survival assessed using hazards cumulative incidence function model estimate reliability tumor volumetry ttv derived digitised pretreatment ct scans was compared ttv derived calculations based tumor diameters provided postoperative histo pathological report patients method comparison the median follow time censored patients treated definitive radiotherapy radiochemotherapy 28.5 months patients treated adjuvant postoperative radiotherapy 36.5 months the 5-year overall survival patients treated definitive radiotherapy radiochemotherapy 0.27 95% ci 0.180.35 patients treated postoperative radiotherapy 0.69 95% ci 0.590.79 patients treated definitive radiotherapy radiochemotherapy greater proportion stage 4 tumors 96.9% versus 66.9% much larger median ttv 68.4 cm versus 21.2 cm lower proportion preradiotherapy hemoglobin level 12 g dl 17.6% versus 42.6% the performance status considered study consistently documented patient files on univariate analysis ttv figures 1 2 pre radiotherapy hemoglobin level statistically significant impact overall survival locoregional control patients treated definitive radiotherapy radiochemotherapy simultaneous chemotherapy statistically significant effect overall survival incidence distant metastasis in addition statistical trend association simultaneous chemotherapy locoregional control p 0.08 the n classification statistically significant impact locoregional control significant impact overall survival metastasis free survival on multivariate analysis ttv simultaneous chemotherapy maintained statistical significance table 3 univariate analysis the ttv statistically significant impact locoregional control figure 3 the n classification statistically significant impact incidence distant metastasis statistical trend locoregional control p 0.06 on multivariate analysis ttv p 0.05 maintained statistical significance table 5 the ttv estimated using quantitative tumor volumetry digitized pre treatment ct scans patients in addition ct based volumetry 34 patients ttv also estimated based tumor diameters reported histo pathological report the tumor volumes based two methods compared estimate precision tumor volume measurements the method comparison 34 patients showed 95% limit agreement two total tumor volume measurements approximately 150% average total tumor volume measurement methods figure 4 this retrospective single institution analysis investigated possible prognostic factors patients squamous cell cancer oro- hypopharynx treated definitive radiotherapy radiochemotherapy ii surgery followed postoperative radiotherapy the two patient groups analysed separately differed considerably respect patient- treatment related characteristics prognosis the tumor volume stated one precise relevant predictor radiotherapy outcome patients oro- hypopharyngeal cancer treated definitive radiotherapy radiochemotherapy the quantitative tumor volume identified significant prognostic factor majority studies 1020 studies prognostic factor marginal 21 22 significance multivariate analysis study total tumor volume statistically highly significant impact overall survival locoregional control patients treated definitive radiotherapy radiochemotherapy a new finding study total tumor volume also statistically significant impact locoregional control patients treated surgery followed postoperative radiotherapy patient group our data suggest total tumor volume used select patients intensified definitive postoperative adjuvant treatment patients treated adjuvant simultaneous radiochemotherapy compared adjuvant radiotherapy alone 24 25 a comparative analysis studies revealed extracapsular extension tumor neck nodes and/or microscopically involved surgical margins significant clinical risk factors poor outcome the significant prognostic factor multivariate analysis patients treated definitive radiotherapy study application simultaneous chemotherapy the simultaneous chemotherapy significantly associated improved overall survival distant metastasis free survival showed statistical trend improved locoregional control p 0.15 randomized clinical trials 2735 meta analyses 36 37 shown significantly improved local control survival definitive simultaneous radiochemotherapy compared definitive radiotherapy alone patients advanced squamous cell cancer head neck interestingly study preradiotherapy hemoglobin level significant prognostic factor univariate analysis patients treated definitive radiotherapy radiochemotherapy lost statistical significance multivariate analysis if total tumor volume removed multivariate model pre radiotherapy hemoglobin level retained significance our data suggest total tumor volume stronger prognostic factors a significant association pre radiotherapy hemoglobin concentration treatment outcome definitive radiotherapy radiochemotherapy absence quantitative tumor volume data reported several studies literature 3846 patients treated postoperative radiotherapy pre radiotherapy hemoglobin level showed prognostic significance study literature one study found significant impact pre radiotherapy hemoglobin level survival patients head neck cancer treated adjuvant radiotherapy other studies evaluated prognostic significance hemoglobin level different time points treatment studies no prognostic significance found pre radiotherapy hemoglobin level hemoglobin level surgery surgery 49 50 duration low hemoglobin level interval surgery radiotherapy difference hemoglobin concentration adjuvant radiotherapy several studies using po2 histography shown impact tumor oxygenation survival patients head neck cancer radiotherapy 5255 clear correlation found tumor oxygenation means po2 histography hemoglobin concentration 56 57 multivariate analysis potential prognostic factors gender age pre radiotherapy hemoglobin level tumor site t- n- r status rt interruptions 5 days interval surgery rt 32 days showed statistical significance study prognostic factors multivariate analysis locally advanced head neck cancers reported studies performance status 45 46 high grade acute organ toxicity uicc stage our data suggest total tumor volume predominant prognostic factor patients squamous cell cancer oro- hypopharynx treated definitive radiotherapy radiochemotherapy surgery followed postoperative radiotherapy the total tumor volume used identify high risk patients stratify patients clinical trials statistical analyses

purpose . to compare the impact of prognostic factors of patients treated with definitive radio(chemo)therapy versus patients treated with surgery and postoperative radiotherapy for squamous cell carcinoma of the oro- and hypopharynx . patients and methods . 162 patients treated with definitive radiotherapy and 126 patients treated with postoperative radiotherapy were retrospectively analysed . the impact of the prognostic factors gender , age , total tumor volume ( ttv ) , pre - radiotherapy hemoglobin level ( hb - level ) , tumor site , t- and n - classification , radiotherapy interruptions > 5 days , radiotherapy versus simultaneous radiochemotherapy , r - status and time interval between surgery and radiotherapy were investigated . results . the median follow - up time for the censored patients treated with definitive radio(chemo)therapy was 28.5 months and for postoperative radiotherapy 36.5 months . on univariate analysis , the ttv , hb - level , and simultaneous radiochemotherapy had a significant impact on the survival of patients treated with definitive radio(chemo)therapy . for patients treated with postoperative radiotherapy , only the ttv showed a statistical trend for the survival ( p = 0.13 ) . on multivariate analysis , the ttv and simultaneous radiochemotherapy maintained their statistical significance for patients treated with definitive raditherapy , and the ttv , the statistical trend for patients treated with postoperative radiotherapy ( p = 0.19 ) . conclusions . the ttv was the predominant prognostic factor for both , patients treated with definitive or postoperative radiotherapy .

measures intellectual abilities vocabulary crystallized abilities resistant age effects fluid abilities attention executive functions memory functions seem affected particularly related source memory episodic explicit women perform better verbal memory tasks whereas men excel spatial tasks however sex differences aging effects some evidence suggests women show less age associated cognitive decline men data young adults age 1 8 45 years indicate men show significant decline several neurocognitivc domains women evince decline we initially studied sex difference neurocognitivc measures standardized battery examines 8 neurobehavioral domains sample 241 healthy young adults aged 18 45 124 men 117 premenopausal women women better verbal memory men performed better spatial motor tasks however observe better performance women language domain figure 1 examining components domains show sex differences find verbal memory advantage women accounted primarily performance california verbal learning test cvlt/ figure 2 whereas advantage men spatial abilities accounted performance benton line orientation test some sex differences manifested profile abilities effects aging within range young adulthood sample significant correlations observed age behavioral measures women correlations ranged -0.15 0.09 men however increased age associated decrease performance attention r=0.43 p=0.0001 verbal memory r=-0.20 p=0.029 spatial memory r=-0.34 p=0.0001 spatial abilities r=-0.33 p=0.0002 df=122 p values 2-tailed figure 3 figure 4 the battery lengthy test provides measures difficult link current knowledge brain systems regulating behavior furthermore tests comprising batteries readily applied functional imaging studies alternative forms available repeated testing address limitations our general approach task development validation process detailed gur et al advantages computerized battery include measure designed probe narrow well defined neurobehavioral domain ii uniform presentation test stimuli iii errorless data entry scoring iv availability reaction time data v shorter time administration vi alternative forms readily generated using set algorithms is impersonal ii participants particularly elderly dislike computers require training iii tests yet available well validated indices language functioning particularly involving verbal output eg vocabulary verbal fluency however experience computerized testing indicates first two disadvantages overcome third addressed available technology most older adults respond well computerized testing approached properly developed short module trains participants use mouse level required testing the normative data shown favorable psychometric characteristics high inter item consistency cronbach alpha test retest reliability comparable levels difficulty 70% 80% correct normative sample true score variance our efforts generate kind sensitivity permit differentiation within healthy people also successful seen figure 5 summary scores show sex differences young adults the pattern sex differences duplicates obtained traditional batteries adds finding women better facial memory available traditional battery measures reaction testing time provide efficiency accuracy time index used calculate comparable z scores across tests algorithms generate multiple forms repeated administration error analysis performed items parameters examine strategy persistence perceiving experiencing expressing emotions seem essential capacities recently study emotion benefited converging methodologies animals humans the face main target study humans methods applied quantify expression emotion cross cultural consistency standardized tools developed measuring emotion discrimination mood induction affective valence arousal emotional displays reliably coded face happiness sadness anger fear disgust surprise controversial seems organized perhaps parallel cognitive system along laterality anterior posterior dimensions ittiere controversy whether emotional expression lateralized although meta analysis borod et al seems confirm negative emotions expressed intensely left side face whereas opposite holds positive emotions agreement though fewer data receptive experiential expressive aspects emotion processing mapped frontal temporal parieto occipital involvement respectively this interaction emotion cognitive systems particularly applies memory issue current interest large scale studies standardized measures indicated elderly people better mood young counterparts gross et al examined cross cultural samples age differences concluded older adults reported fewer negative emotional experiences greater emotional control however findings regarding emotional expressivity less consistent older participants reporting less expressivity there also evidence elderly vulnerable young adverse effects negative emotional states memory cognitive abilities indeed suggested depressed mood strongest predictor health decline elderly women perform better speeded emotion recognition tasks tasks requiring facial expression emotions study facial recognition reported sex differences sensitivity happy sad expressions depending poser sex women sensitive opposite sex expressions whereas men differentially poor detecting sadness female faces regarding emotional experience women prone clinical depression mood fluctuations associated phases menstrual cycle documented phase associated hormonal changes may relate cognitive performance magnetic resonance imaging mri studies brain documented aging associated progressive parenchymal volume decrease cerebrospinal fluid csf volume increase some studies suggest volume decrease age gray matter gm tissue others report decrease also white matter wm compartment there consistency regional distribution effects mesolimbic temporal frontal regions showing greater vulnerability sex differences observed compartmental composition intracranial volumes volume density language associated cortex rate age associated changes data indicate less parenchymal loss women men particularly frontal temporal regions samples limited elderly cohorts our data elderly suggest similar rates tissue loss men women perhaps reflecting acceleration following menopause henkel et al concluded age related decline source memory affects processes involved binding features complex memories ... contextual features memories neural substrates source memory system implicate mesial temporal frontal regions henkel ct al hypothesis based behavioral data supported raz et al study volume limbic structures unrelated cognitive functions across age range older participants reduction predicted declines explicit memory neural substrates age related changes affect less clearly defined although recent work affords hypotheses tested studies networks emotion implicated amygdala hypothalamus mesocorticolimbic dopaminergic systems projections orbital dorsolateral frontal temporal parietal cortex studies patients brain lesions support role regions emotion regulation the link age related changes neuroanatomy performance tasks established evaluate effects extend across agegroups the correlation age total intracranial volume nil r=0.02 indicating secular drift head size for young 50 years sample considered separately a. small yet significant correlation age gm volume r=-0.17 df=130 p<0.05 this correlation higher men r=-0.27 df=74 p<0.01 women r=-0.01 df=54 ns age correlate significantly wm csf volumes age group for entire age range gm continues decline volume aging r(184)=-0.49 r(92)=-0.52 r(90)=-0.40 whole sample males females respectively p<0.0001 whereas volume csf continues increase age corresponding correlations 0.31 0.45 0.29 p<0.0001 wm changes less clear although correlation age significant entire sample r=0.09 within sex correlations small positive r=0.28 p=0.01 r=0.24 p=0.02 we conclude gm volume reduced aging csf volume increases concomitantly wm volume change appreciably perhaps increased slightly effects gm csf agree recent study guttmann et al conclusion regarding wm percentage intracranial compartments appears examined percentages raw volumes case guttmann et al study reduced wm could reflect age effects another compartment example increased csf perhaps paucity subjects elderly group matched paucity young group 10 participants 40 years our sample also enabled examination whether volume changes related cognitive performance while young elderly participants received age appropriate neuropsychological batteries groups received several identical tests the total number items recalled memorization trials correlated volumes brain parenchyma gm as seen figure 6 parenchymal volume goes age number words recalled also declines parenchymal volume associated memory even age effects partialled the feasibility studying neural substrates behavior enhanced functional imaging methods measuring regional brain activity activation patterns linked performance cognitive tasks requiring verbal spatial attention memory facial processing the study age effects regional brain physiology quite extensive considerable agreement cerebral blood flow cbf shows age related decline even people screened cerebrovascular disorders similarly measures neurotransmitter function shown reduced availability dopaminergic serotonergic transmitter activity the decline linked performance behavioral tasks related dopaminergic system the effects aging glucose metabolism less clear although shows decline particularly frontal temporal regions relevance study memory emotion systems cherrier et al examined correlations mood state self ratings cerebral metabolism pet 18f fdg 27 persons age associated memory impairment aged 44 81 years specifically regions involved mood memory similar abnormalities reports boredom related mesial temporal parietal asymmetry well decreased parietal metabolism thse findings suggest mood changes may influence metabolism brain regions implicated emotion memory function there fewer data effects aging regional brain activation response task demands using 133xe method found substantial age related decline overall cbf values pattern lateralized changes response verbal spatial tasks identical young elderly people however used tasks quite resistant age effects results may differ tasks showing greater age related decline indeed cabeza ct al found less frontal activation measured h2 0 pet cbf elderly people memory task madden et al cbf study using spect reported several cbf activations greater young older adults however observed although performance demonstrated greater age related slowing visual encoding semantic retrieval changes associated corresponding changes rcbf activation association cbf activation performance effects aging association merit elucidation several studies examined emotion processing functional neuroimaging methods healthy people xe method measuring cortical cbf processing emotional facial expressions noted increased right temporoparietal activity mood induction procedure increased cbf observed sad induction correlated mood change pet study h2 0 reported cbf increase left amygdala cbf decrease right amygdala sad mood subsequent studies pet functional mri fmri confirmed lesion data implicating amygdala anterior brain regions mood posterior regions seem activated visual emotion discrimination unlike neuroanatomic studies consistently examined sex differences age effects paucity neurophysiologic studies examined factors using xe cbf method reported women higher rates cortical cbf finding replicated extended methods measure cbf entire cranium we found equal rates age associated reduction cbf men women also reported studies although samples usually small elderly range no studies linked changes cbf activation sex differences agerelated neuroanatomic changes performance memory emotion tasks the method several potential advantages higher spatial temporal resolution noninvasiveness lack ionizing radiation direct correlation anatomical imaging greater repeatability economy the disadvantages fmri techniques include loud background noise generated gradients difficulties presenting stimuli performing tasks magnet bore claustrophobia low signal noise methods lack quantification physiologic units methods among various fmri methods blood oxygenation level dependent this technique relies magnetic susceptibility effects deoxyhemoglobin cause regional decreases signal imaging sequences sensitive susceptibility eg echoplanar regional brain activation studies net increase signal intensity observed regions known activated task this attributed increase regional blood flow compared regional oxygen consumption a typical response 1 25% increase regional image intensity develops 3 8 seconds following task initiation susceptibility effects field dependent using 4-t magnet available us initial decrease signal intensity detectable first 1 2 seconds following stimulation corresponding focal increase deoxyhemoglobin combined ultrafast echoplanar imaging -100 ms per slice time course signal change response individual stimuli thus observed evaluate task induced regional activation we applied verbal spatial task previously demonstrating regional activation methods this study examined activation spatial task judgment line orientation compared a. verbal reasoning task analogies sample 29 healthy participants 15 men 14 women an image based multisubject analysis performed registering brains different subjects well characterized brain registration algorithm used register tl images different subjects one particular subject once registered statistics summed across subjects divided square root number subjects appropriate independent normally distributed variables the statistical images smoothed convolution gaussian kernel full width half maximum 12 mm thresholded a. p value 0.05 corrected multiple comparisons using theory gaussian random fields the activation map figure 7 indicates hypothesized left lateralized changes seen verbal task posterior temporal inferior parietal regions right latcralizcd increase seen spatial task regions this imagebased analysis revealed a. distributed network cortical regions expanded hard verbal task became circumscribed hard spatial task the task hemisphere interactions hypothesized inferior parietal superior temporal planum temporale regions significant order magnitude comparable obtained methods f<0.001 appears spatial processing requires harder tasks greater reliance visual association cortex minimal activation areas poorer performance women may relate continued reliance supplementary strategies perhaps verbal ineffective success harder spatial items effects aging regional activation relation cognitive strategics yet examined fmri as generally case age related changes hormonal environment pervasive effects require scrutiny early development also perimenopausal phase menopause single event progressive process ovarian aging beginning increased frequency menstrual disturbances anovulation follicular units depleted the process accelerated age 37 ultimately culminating virtual absence follicles capacity generate significant quantities estradiol the median chronological age menopause usa 51.4 years range 48 55 years estrogen decrease associated substantial central nervous system cns alterations including vasomotor instability insomnia depression cognitive decline recent studies suggest estrogen protective effect respect onset alzheimer disease cognitive decline there evidence neurobiologie processes triggered hormonal changes exert influence affecting neurotransmitter availability cerebral perfusion perhaps eliminating neuroprotective effects estrogen recent study matteis et al using transcranial doppler ultrasonography found higher flow estimates women men overall however subgroup 15 postmenopausal women aged 48 53 years lower flow values 15 premenopausal women age group there increasing evidence across behavioral neuroanatomic neurophysiologic domains sex differences play prominent role modulating effects aging brain function the overall finding age related decline begins earlier men women the decline pronounced frontotemporal regions associated attention inhibition memory more specific tasks using computerized approach help better delineate associations agerelated decline aspects cognitive emotion processing the sex differences brain aging may investigated molecular level data physiologic parameters glucose oxygen metabolism receptor function could help elucidate mechanisms explaining differences such studies could ultimately help explain range phenomena related sex differences including cognition emotion processing although focused findings healthy people effects implications brain disorders gender differences observed across life span for example neurodevelopmental disorders attention deficit learning disabilities common boys schizophrenia severe young men depression common women understanding neural basis disorders advanced considering sex differences brain function the clinical implications findings need examined relation disease presentation course view greater vulnerability males prefrontal regions one would expect brain disorders affecting regions severe perhaps requiring multimodal therapeutic interventions for females improved understanding regional brain activity emotion processing maybe position explain neurobiology increased vulnerability depression finally measures employed work seem sensitive variability healthy people may therefore serve endophenotypic markers vulnerability neuropsychiatrie disorders sex differences evident may contribute developing genetic models

gender and aging moderate brain - behavior relationships . advances in neuroscience enable integration of neurobehavioral , neuroanatomic , and neurophysiology measures . here we present neurobehavioral studies thai examine cognitive and emotion processing in healthy men and women and highlight the effects of sex differences and aqinq . neuroanatomic studies with maqnetic resonance imaging ( mri ) indicate that the progressive decrease in brain volume affects froniotemporal brain regions in men more than in vi / omen , functional imaging methods suggest sex differences in rate of blood flow , pattern of glucose metabolism , and receptor activity . the role of ovarian hormones is important in elucidating the observed relationships . a life span perspective on gender differences through the integration of available methodologies will advance understanding healthy people and the effects of brain disorders .

purpose process destroy remove injurious agents injured tissues thereby promoting tissue repair when beneficial response occurs uncontrolled manner result excessive cellular tissue damage results chronic inflammation destruction normal tissue moreover inflammatory airway lung diseases asthma chronic obstructive pulmonary disease copd characterized chronic inflammation many known inflammatory target proteins matrix metalloproteinase-9 mmp-9 intercellular adhesion molecule-1 icam-1 vascular cell adhesion molecule-1 vcam-1 cyclooxygenase-2 cox-2 cytosolic phospholipase a2 cpla2 associated inflammatory signaling pathways induced various stimuli including tumor necrosis factor- tnf- interleukin-1 il-1 adenosine-5-triphosphate atp cigarette smoke extract cse lipoteichoic acid lta lipopolysaccharide lps 26 airway smooth muscle considered end response effector regulating regional differences ventilation contracting response various proinflammatory mediators exogenous substances released homeostatic pathologic conditions asthma lung cells particular alveolar epithelial type ii cells susceptible injurious effects oxidants it shown lung cells release inflammatory mediators cytokines chemokines il-1 il-6 il-8 tnf- response oxidative stress moreover the sfks pkc growth factor tyrosine kinase receptors nadph oxidase ros pi3k akt mapks components signaling cascades respond extracellular stimuli targeting transcription factors nf-b ap-1 resulting modulation inflammatory gene expression thus review focus general aspects inflammatory signaling regulation summarize current knowledge regarding presence functional roles inflammatory signal molecules within respiratory system proposed involvement expression inflammatory target proteins response proinflammatory mediators airway lung inflammation the pharmacological interventions protect inflammation induced airway lung diseases discussed leukocytes continuously circulate throughout body order come contact antigens sequestered within tissues enter tissues circulating leukocytes migrate blood vascular endothelial cells tissue during migration leukocytes initially bind endothelial cells via low affinity adhesion molecules low affinity adhesion combination force blood flow results rolling leukocytes endothelial cells vcam-1 one inducible cell transmembrane glycoproteins immunoglobulin supergene family expressed several cell types plays important role number inflammatory immune responses it first identified adhesion molecule induced endothelial cells proinflammatory cytokines lps 11 12 normal processes vcam-1 important development since vcam-1 knockout embryonic lethal pathogenesis vcam-1 expression induced endothelial cells inflammatory bowel disease atherosclerosis infection asthmatic responses 1315 upregulation vcam-1 expression cytokine triggered vascular endothelial cells enhances targeted transmigration pmns extravascular space inflammation airways reach submucosa airway lumen circulating pmns must first recruited across vascular endothelium migrate interstitial matrix interacting airway epithelium accumulation inflammatory cells within airways influenced expression adhesion molecules airway epithelium thus similar processes govern pmns adhesion lung airway resident cells may occur contribute damage cells seen inflammatory responses asthma this event crucial development allergic inflammation mediated adhesion molecules cytokines icam-1 endothelial- leukocyte associated transmembrane protein long known importance stabilizing cell cell interactions facilitating leukocyte endothelial transmigration more recently icam-1 characterized site cellular entry human rhinovirus because associations immune responses many researchers hypothesized icam-1 could function signal transduction earlier studies showed icam-1 gene highly expressed pulmonary fibroblasts copd patients in addition blocking pulmonary icam-1 expression ameliorates lung injury established diet induced pancreatitis thus adhesion molecules play key role regulating inflammation respiratory disorders figure 1 ) the first class pla2 secretary pla2 spla2 expressed variety cell types preference aa sn-2 position requires millimolar amounts ca activity sensitive sulfhydryl reducing agents dithiothreitol dtt resistant heat acid conditions the second class pla2 calcium independent pla2 ipla2 require ca catalytic activity ipla2 prefers plasmalogen substrates appear preference type fatty acid sn-2 position the third class novel high molecular weight 85 kda cytosolic pla2 cpla2 cpla2 enzymes catalyze hydrolysis sn-2 position membrane glycerophospholipids leading production free fatty acids lysophospholipids this reaction particular importance esterified fatty acid arachidonic acid aa converted downstream metabolic enzymes various bioactive lipophilic compounds called eicosanoids including prostaglandins pgs leukotrienes lts the increase cpla2 activation expression following external stimuli including proinflammatory cytokines growth factors microbial toxin often observed several systems the implication cpla2 inflammatory diseases confirmed airway anaphylactic response cpla2 knockout mice markedly reduced compared wild type mice moreover cpla2-deficient mice provided definitive evidence central role cpla2 eicosanoid well pathogenesis several inflammatory diseases acute respiratory distress syndrome ards due bacterial sepsis 30 31 these studies demonstrated reduction bronchial lumen alveolar thickening control mice remarkably absent cpla2 knockout mice this outcome also appeared 5-lipoxygenase lo)-knockout mice mice pgd2 receptor deficiency thus cpla2 seems function crucial upstream regulator production eicosanoids airway resistance allergic inflammation correlated process asthma figure 1 the inhibition cpla2-mediated pathways may also provide therapeutic approach airway lung injury cox metabolites diverse effects lung known modify airway tone well inflammatory responses cox-1 constitutively expressed tissues considered housekeeping isoform produces pgs required maintenance normal cell organ function in contrast cox-2 primarily inducible isoform whose expression upregulated many cell types cytokines mitogens endotoxin 3 4 it highly expressed inflamed tissues believed produce pgs involved inflammatory processes cox-2 multiple transcriptional regulatory sequences promoter region including tata box nf il6 motif two ap-2 sites three sp1 sites two nf-b sites cre motif e box cox-2 gene expression induced multiple cytokines growth factors via activation transcriptional regulatory proteins act promoter sites thus cox-2 appears primary cox controlling pge2 synthesis response inflammation figure 1 cox effects widespread extremely complex however studies knockout mice cox-1 versus cox-2 reveal sometimes overlapping altogether predictable roles two enzymes the levels prostanoids bronchoalveolar lavage fluid increased asthma several studies found enhanced expression cox-1 cox-2 airways asthmatics 36 37 a recent research renewed interest role prostanoids allergic airway disease moreover expression cox-2 protein induced lactobacillus rhamnosus gg lgg endotoxin lipoteichoic acid lta t84 epithelial cells the presence cox-3 mrna transcript size approximately 5.2 kb subsequently confirmed human cells cox-3 highest concentrations cerebral cortex heart tissue however retention intron 1 cox-3 seems slow enzymatic activity comparison cox-1 cox-2 thus inhibition cox-2-mediated inflammatory pathway may provide therapeutic approach respiratory diseases mmps proteolytic enzymes able degrade extracellular matrix ecm components thus play role cell migration tissue remodeling moreover splice in)activate cytokines chemokines thereby influencing recruitment function inflammatory cells date 24 mmps identified mammals cellular sources include inflammatory stromal epithelial cells some mmps anchored cell surface whereas others secreted extracellular space they released inactive proenzymes activated proteolytic cleavage n terminal domain most mmps constitutively secreted become translated gelatinase subfamily mmps mmp-2 mmp-9 catalytic domain includes zn binding site also contains repeats fibronectin motifs allowing ability bind gelatin major substrate patients asthma increased gelatinolytic activity linked mmp-2 mmp-9 higher levels tissue inhibitor metalloproteinase-1 timp-1 natural inhibitor mmps sputum the activated form mmp-9 85 kda found sputum 60% asthmatics absent control subjects although less frequently detectable pro mmp-9 pro mmps catalytically inactive activated active mmp cleaving prodomain pro mmp-2 72 kda also found frequently asthmatics 50% control subjects 5% in addition patients copd increased gelatinolytic activity sputum linked mmp-2 mmp-9 smokers emphysema mmp-8 mmp-9 levels bronchoalveolar lavage bal fluid were significantly higher smokers without emphysema vitro cultured human airway smooth cells a549 cells tnf- il-1 induce mmp-9 expression cell migration 2 43 44 via various signaling pathways pkc mapks nf-b ap-1 thus mmps inhibitors timps play multiple functions physiological processes interact many mediators regulating inflammatory processes cell behavior angiogenesis these mediators implicated many intricate loops reciprocal interactions rendering understanding role mmps regulatory processes difficult many respiratory diseases mmps overexpressed oversecreted leading deregulation physiological homeostatic processes ecm degradation disorganization figure 1 pkcs important many cellular responses lung including permeability contraction migration hypertrophy proliferation apoptosis secretion pkc family serine threonine kinases characterized least eleven different isotypes pkc isotypes differentially regulated calcium ca diacylglycerol phospholipids differ structure expression intracellular localization substrate utilization mechanisms activation the pkc isotypes subdivided three groups classical novel atypical this subdivision based structural functional differences conserved domains c1c4 the classical pkc pkci ii pkc isotypes ca diacylglycerol dependent the novel pkc pkc pkc pkc pkc isotypes contain c2 domains lack ca binding ability still retain functional c1a c1b domains bind endogenous diacylglycerol exogenous phorbol esters the atypical pkc pkc pkc isotypes lack functional c2 domain contain single c1 domain lacks ability bind diacylglycerol phorbol esters therefore mechanism activation atypical pkc isotypes ca diacylglycerol independent moreover pkcs important signaling intermediates chronic airway diseases like asthma copd resident airway epithelial cells produce proinflammatory mediators regulation pkc increased pkc activity increases nf-b dependent proinflammatory cytokine generation human airway epithelial cells expression dominant negative pkc mutant inhibiting effects human airway smooth muscle cells pkc found cytosol ii membrane basal conditions the proinflammatory neuropeptide bradykinin bk causes activation pkc applied airway smooth muscle cells bk also induces cox-2 protein expression pge2 accumulation human airway smooth muscle cells via pkc-dependent signaling pkc increased lungs patients copd thought important hypertrophy proliferation airway smooth muscle cells pkc activity also increased proliferating human airway smooth muscle cells hand pkc important mediating effects proinflammatory cytokines phosphorylating cpla2 leading release aa phospholipids subsequent production bioactive eicosanoids activated cells at least three pkcs expressed interstitial fibroblasts including pkc activation pkc causes decreased collagen expression via extracellular signal regulated kinase kinase mek)/erk signaling cascade response opposed pkc selected pkcs activated lps leading production proinflammatory cytokines tnf- il- il-6 in addition thrombin causes increase cytosolic ca activation selected pkcs tnf- shown induce mmp-9 expression via pkc-dependent pathway a549 cells taken together studies indicated pkcs play critical role mediating inflammation respiratory diseases figure 2 because multiple signaling pathways contribute key cellular responses important lung biology therapeutic strategies targeting pkcs may effective combined inhibitors pathways additive synergistic effect mechanisms regulate pkc activity including phosphorylation interaction isozyme specific binding proteins also potential therapeutic targets respiratory diseases ros products normal cellular metabolism known act second messengers physiological conditions ros participate maintenance cellular redox homeostasis protect cells oxidative stress in addition regulation redox state important cell activation viability proliferation organ function however overproduction ros frequently due excessive stimulation either reduced nadph proinflammatory cytokines mitochondrial electron transport chain xanthine oxidase results oxidative stress oxidative stress deleterious process leads airway lung damage consequently several respiratory inflammatory diseases injuries ros intracellularly generated several sources including mitochondrial respiration cytochrome p450 nadph oxidase system xanthine xanthine oxidase however major ros generating enzyme nadph oxidase membrane bound multicomponent enzyme complex present phagocytes well nonphagocytic cells first superoxide produced nadph oxidase 2 required respiratory burst occurs phagocytes leading microbial killing ros derived nadph oxidase specifically reversibly react proteins altering activity localization half life activated phagocytic cells generate ros via assembly activation nadph oxidase complex comprises membrane associated flavocytochrome b558 gp91 p22 various cytosolic cofactors p47 p67 p40 gtpase rac1 mediates transmembrane electron transfer major cellular electron donor nadph reduce molecular o2 superoxide anion o2 hydrogen peroxide h2o2 number homologs main business end nadph oxidase gp91 discovered mammalian systems known contain seven nadph oxidase homologs comprising nadph oxidase 15 nadph oxidase 2 new name gp91 two larger dual oxidases duox1 duox2 widely expressed many cell types mediate variety biological functions cell mitosis differentiation migration immune regulation vitro studies using macrophages alveolar bronchial epithelial cells ros shown induce gene expression inflammatory mediators il-1 tnf- 57 58 patients asthma demonstrate increased generation ros superoxide anion hydrogen peroxide hydroxyl radicals increased production ros demonstrated many cell types within lung asthma including macrophages antigen presenting cells apcs neutrophils eosinophils excessive production ros correlates degree airway hyperresponsiveness quantified methacholine challenge in addition oxidative stress also contributes proteinase antiproteinase imbalance inactivating antiproteinases 1-antitrypsin secretory leukocyte proteinase inhibitor activating proteinases mmps hand oxidants also promote inflammation activating nf-b ap-1 orchestrates expression multiple inflammatory genes recognized important copd tnf-. recently phagocytic nadph oxidase ros signaling shown play critical role promoting tnf--induced nf-b dependent acute inflammatory responses tissue injury specifically lungs effected preferential leukocyte infiltration thus oxidative stress plays critical role inflammatory responses airway lung diseases via upregulation redox sensitive transcription factors ap-1 nf-b thereby proinflammatory genes mmp-9 vcam-1 icam-1 cox-2 cpla2 expression taken together nadph oxidase ros play critical role development airway lung diseases figure 2 the pi3k family central signaling elements diverse array cellular functions including growth proliferation migration survival it therefore understandable dysregulation pi3k implicated induction and/or progression variety disease states including respiratory tract ranging asthma cancer pi3ks type pi3ks activated cell surface receptors growth factors insulin g protein coupled receptors gpcrs class ii pi3ks comprised isoforms characterized presence c2 domain c terminus structurally pi3ks ia exist heterodimeric complexes catalytic p110 subunit designated association particular regulatory subunit designated p85 p55 p50 importantly pi3k ia signals downstream receptor tyrosine kinase ras single class pi3k ib consists p110 catalytic subunit complexed p101 regulatory subunit signals downstream gpcrs ras activated subunits gpcrs receptors chemokines despite limitations selectivity the two commercially available pi3k inhibitors wortmannin ly294002 contributed greatly understanding biological role pi3k lung inflammation moreover previous study indicated intratracheal administration ly294002 significantly reduced ovalbumin-(ova- induced increases total cell counts eosinophil counts il-5 il-13 ccl11 eotaxin levels bal fluid dramatically inhibited ova induced tissue eosinophilia airway mucus production this study confirmed ly294002 markedly attenuated ova induced serine phosphorylation akt direct downstream substrate pi3k in addition studies also showed ly294002 wortmannin attenuated eosinophilic airway inflammation airway hyperresponsiveness murine asthma model thus pi3k inhibition indicated therapeutic potential treatment asthmatic airway inflammation ly294002 shown reduce chemokine induced ros generation phagocytes confirmed studies using pi3k knockout mice it also reported serum withdrawal sw killed human u937 blood cells elevating cellular ros levels occurred pi3k activation thus pi3k family plays prominent role various airway lung inflammation figure 2 moreover inhibitors pi3k akt may prove useful novel therapies treatment respiratory diseases sfks signaling enzymes long recognized regulate critical cellular processes proliferation survival migration metastasis src protein tyrosine kinase ptk family categorized nonreceptor tyrosine kinases consists nine members src fyn yes yrk ubiquitously expressed whereas blk fgr hck lck lyn expressed restricted patterns src ptk family members activated response stimulation variety cell surface receptors tyrosine kinase receptors integrin receptors g protein coupledreceptors cellular stress moreover reported tnf- il-1 induces vcam-1 icam-1 expression via c src dependent pathway human airway smooth muscle cells in addition c src shown regulate cox-2/pge2/il-6-dependent airway inflammation via nadph oxidase ros human lung epithelial cells addition activating nf-b inducing kinase nik via traf2 tnf- could activate c src pkc systemic inhibition kinases using specific small molecule inhibitors src ptks either pp2 su-6656 significantly attenuated lps induced lung injury capillary permeability reduced lps dependent cytokine chemokine levels lung serum thus role src family ptks inflammatory responses rising area research figure 2 however application small chemical inhibitors effectively specifically block src ptks could great clinical implication airway lung diseases inflammatory responses underlying mechanisms cell surface tyrosine kinases receptors play pivotal roles development tissue repair normal cellular homeostasis aberrant expression signaling patterns receptors also linked progression diversity diseases including asthma two major families tyrosine kinases receptors epidermal growth factor receptor egfr platelet derived growth factor receptor pdgfr families received great deal attention potentially therapeutic targets respiratory diseases receptors shown play key roles chronic tissue remodeling asthma bronchitis pulmonary fibrosis the egfr system epithelial cells underlying mesenchymal cells fibroblasts myofibroblasts smooth muscle cells drives numerous phenotypic changes progression pulmonary diseases including mesenchymal cell hyperplasia differentiation ecm production pdgfr system located primarily mesenchymal cells transduces signals cell survival growth chemotaxis the variety egfr pdgfr ligands produced airway epithelium adjacent mesenchymal cells allows intimate epithelial mesenchymal cell communication humans airway epithelium expresses egfr ligands constitutively including egf tgf- hb egf amphiregulin heregulin betacellulin expression several egfr ligands also investigated diseases copd asthma showed p. aeruginosa bacterial supernatant induces mucin production human airway epithelial cells nci h292 via egfr activation multiple studies also reported stimulation airway epithelial cells lps induces secretion il-8 via cellular cascade involving tlr4/myeloid differentiation primary response gene myd)88/nf-b dependent pathway in addition ros shown stimulate pdgfr activation via c src family kinases there accumulating evidence pkc dependent phosphorylation p47 essential pdgf stimulated ros generation important pdgf induced mapks activation taken together studies suggest growth factor tyrosine kinase receptors may also play key role mediating expression inflammatory genes figure 2 mapks important components signaling modules activated neurotransmitters cytokines growth factors well chemical mechanical stressors airway these external signals produce acute responses modify smooth muscle contraction may also induce chronic responses modify airway structure acute chronic events airway remodeling result altered expression multiple genes encoding protein mediators cell cell signaling ecm remodeling cell cycle control intracellular signaling pathways mammals three groups mapks identified extracellular signal regulated protein kinases erks c jun nh2-terminal kinases jnks p38 mapk the p38 mapk activated cellular stresses including uv radiation lps growth factors cytokines the jnk activated many stimuli activate p38 mapk cellular stresses numerous cytokines thus inhibition mapks activity via pharmacological genetic approaches blocks allergic inflammation airways moreover asthmatic patients demonstrated increased immunostaining phospho p)-erk p p38 mapk p jnk the phosphorylation p38 mapk primarily observed basal layer columnar epithelium it likely p38 mapk drives basal metabolic processes particular cell type there significant correlation clinical severity asthma intensity immunostaining p erk p p38 mapk p erk number tissue eosinophils neutrophils airways early studies p38 mapk demonstrated il-1 tnf- activate p38 mapk monocytes furthermore inhibition p38 mapk pathway shown exert anti inflammatory effects inhibition il-1 il-6 tnf- expression airway smooth muscle cells solid evidence erk p38 mapk pathways contribute il-1-induced cox-2 expression pge2 synthesis 76 77 mouse model chronic lung inflammation allergic inflammation significant inhibition tnf- il-4 il-13 rantes regulated activation normal cell expressed secreted lung homogenates observed jnk inhibitor sp600125 addition also found lta il-1 could induce cpla2 cox-2 mmp-9 human airway smooth muscle cells a549 cells 3 44 therefore mapks play important role mediating airway lung inflammation figure 2 nf-b viewed master regulator inflammatory responses plays essential role evolution well resolution phase inflammation nf-b controls wide spectrum biological effects ranging immune stress induced responses cell fate decisions proliferation differentiation tumorigenesis apoptosis tissue remodeling nf-b usually exists heterodimeric complex p50 p65/rela subunits unstimulated cells nf-b found cytoplasm inactive non dna binding form associated inhibitor protein called inhibitory b ib masks nuclear translocation signal prevents nf-b entering nucleus upon cell stimulation various nf-b inducers ib rapidly phosphorylated two serine residues targets inhibitor protein ubiquitination e3 ubiquitin ligases e3rsib subsequent degradation 26s proteasome the released nf-b dimer translocated nucleus activate target genes binding high affinity b elements promoters nf-b activated numerous extracellular stimuli including cytokines tnf- il-1 viruses environmental particulates pm10s oxidative stress exogenous h2o2 also activated nf-b murine model ros induced acute lung injury administration otc l-2-oxothiazolidine-4-carboxylate resulted significant reduction nf-b translocation nucleus expression adhesion molecules chemokines cytokines previous study demonstrated nf-b activation occurred rapidly ovalbumin ova model allergic airways disease nf-b activation predominantly occurred epithelial cells conducting airways association enhanced mrna expression nf-b regulated chemokine genes including mip-2 eotaxin a novel cyclin dependent kinase inhibitor bai shown downregulate tnf--induced expression cell adhesion molecules inhibition nf-b activation human pulmonary epithelial cells recently also demonstrated overexpression ho-1 protects tnf--mediated airway inflammation downregulation tnfr1-dependent oxidative stress nf-b activation taken together results show nf-b plays key role mediating expression inflammatory proteins airway lung inflammation injury figure 2 ap-1 transcription factor typically consists combinations jun c jun jun b jun fos proteins c fos fos b fra-1 fra-2 bind promoters target genes it found responsible transcriptional activation various genes activated phorbol esters pma via activation pkc ap-1 may activated via pkc various cytokines including tnf- il-1 via several types ptk mapks activate cascade intracellular kinases certain signals rapidly increase transcription fos gene resulting increased synthesis fos protein other signals lead activation kinases phosphorylate c jun resulting increased activation specific jun fos kinases recognized may play key role regulation cellular responsiveness cytokine signals recent studies showed sirtuin 1 sirt1 directly interacted c jun repressed transcriptional activity ap-1 thus decreasing mmp-9 expression more recently reported sirt1 decreased c fos c jun acetylation induced p300 inhibited transcriptional activity ap-1 subsequent cox-2 expression pge2 generation thus ap-1 may play critical role mediating expression various inflammatory proteins there evidence increased expression c fos epithelial cells asthmatic airways many stimuli relevant asthma activate nf-b also activate ap-1 thus ap-1 also key factor respiratory diseases figure 2 kinase pathways become recognized key cellular signal transducers several protein kinase inhibitors development treatment respiratory diseases the pyridinylimidazole compounds exemplified sb203580 originally prepared inflammatory cytokine synthesis inhibitors subsequently found selective inhibitors p38 mapk sb203580 shown attenuate bal tnf- production ovalbumin challenged rat model asthma sb2439063 reduced neutrophilia mediator expression rat copd models in addition recent study also indicated acute chronic animal models asthma sp600125 jnk inhibitor reduces bal accumulation eosinophils lymphocytes cytokine release serum ige production smooth muscle proliferation repeated allergen exposure intratracheal administration ly294002 reduced ova induced increases total cell counts eosinophil counts il-5 il-13 ccl11 eotaxin levels bal fluid dramatically inhibited ova induced tissue eosinophilia airway mucus production inhibition sfks using specific inhibitors src ptks either pp2 su-6656 attenuated lps induced lung injury capillary permeability reduced lps dependent cytokine chemokine levels lung serum rnai process sequence specific post transcriptional transcriptional gene silencing small interfering rna sirna rnai popular method controlling gene expression potential development drugs several diseases various types cancer viral infections gene therapy asthma already developed demonstrated promising results animal models recent progress delivering sirna respiratory system also improved therapeutic feasibility rnai asthma in context allergic immune responses activation stat6 pivotal th2-mediated ige production development airway inflammation hyperreactivity moreover stat6 sirna shown inhibit allergic airway inflammation hyperreactivity mice inflammatory airway lung diseases characterized chronic inflammation oxidant antioxidant imbalance major cause cell damage injury numerous studies shown effectiveness polyphenols limiting progression chronic diseases this likely occur least part antioxidant capacity molecules extends availability hydroxyl groups presence conjugated double bonds resveratrol reported increase antioxidant capacity reduce various markers oxidative stress recently lee et al indicated resveratrol inhibits activation nf-b p65 tnf- pma reduces atp induced mucin secretion cultured primary rat tracheal surface epithelial rtse cells on hand edaravone 3-methyl-1-phenyl-2-pyrazolin-5-one novel radical scavenger protects neurons reducing endothelial injury ameliorating neuronal damage caused brain ischemia treatment edaravone decreases interstitial edema inflammatory cell infiltration well prevents process pulmonary fibrosis in addition reports clinical benefit airway lung diseases increased vitamins c e dietary antioxidants varied indicated vitamin e treatment prior injury largely prevents increase pulmonary permeability index moderates increase lung lymph flow increases pao2/fio2 ratio ameliorates peak pause airway pressure increases decreases plasma conjugated dienes nitrotyrosine erdosteine thiol antioxidant mucoactive properties ability reduce bacterial adhesiveness this compound introduced mucolytic agent treatment chronic airway pulmonary diseases erdosteine breaks disulfide bonds mucus glycoproteins affecting physical properties mucus thus leading increased cough clearance in addition erdosteine reported antioxidant anti inflammatory antibacterial activity negro et al showed erdosteine dose 600 mg day proved effective significantly reducing ros levels peripheral blood stable copd patients current smokers together reduction levels chemotactic proinflammatory cytokines il-6 il-8 bronchial secretions recently greene gaughan represent new therapeutic targets medicines target specific micrornas mirnas may potential treatment asthma there number studies field mirna implicate specific mirnas pathophysiology asthma for example studies using mouse models identified mirnas altered response allergen challenge certain mirnas involved regulation il-13 th2 response key components asthmatic response shown amenable modulation pre mirs anti mirs other studies identified mirnas implicated bronchial smooth muscle hyperresponsiveness proliferation thus developing mirna based medicines treat pulmonary manifestations asthma could yield therapeutics new properties potential treat inflammation hyperresponsiveness associated disease there increasing evidence inflammatory proteins vcam-1 icam-1 cpla2 cox-2 mmp-9 involved pathogenesis respiratory diseases asthma copd figure 3 moreover various inflammatory signaling pathways including pkcs nadph oxidase ros egfr pdgfr c src pi3k akt mapks ap-1 nf-b involved regulation inflammatory proteins figure 3 further exploration role vcam-1 icam-1 cpla2 cox-2 mmp-9 highly prevalent diseases crucial identify possible therapeutic targets the development new inhibitors highly specific major adverse effects essential targeted delivery inflammatory proteins inhibitors directly airway lung might result fewer side effects option also explored although use inhibitors inflammatory signaling pathways treatment respiratory diseases seems attractive studies needed identify exact role inflammatory signaling molecules diseases develop highly specific inhibitors

in respiratory diseases , there is an increased expression of multiple inflammatory proteins in the respiratory tract , including cytokines , chemokines , and adhesion molecules . chemokines have been shown to regulate inflammation and immune cell differentiation . moreover , many of the known inflammatory target proteins , such as matrix metalloproteinase-9 ( mmp-9 ) , intercellular adhesion molecule-1 ( icam-1 ) , vascular cell adhesion molecule-1 ( vcam-1 ) , cyclooxygenase-2 ( cox-2 ) , and cytosolic phospholipase a2 ( cpla2 ) , are associated with airway and lung inflammation in response to various stimuli . injuriously environmental stimuli can access the lung through either the airways or the pulmonary and systemic circulations . the time course and intensity of responses by resident and circulating cells may be regulated by various inflammatory signalings , including src family kinases ( sfks ) , protein kinase c ( pkc ) , growth factor tyrosine kinase receptors , nicotinamide adenine dinucleotide phosphate ( nadph)/reactive oxygen species ( ros ) , pi3k / akt , mapks , nuclear factor - kappa b ( nf-b ) , activator protein-1 ( ap-1 ) , and other signaling molecules . these signaling molecules regulate both key inflammatory signaling transduction pathways and target proteins involved in airway and lung inflammation . here , we discuss the mechanisms involved in the expression of inflammatory target proteins associated with the respiratory diseases . knowledge of the mechanisms of inflammation regulation could lead to the pharmacological manipulation of anti - inflammatory drugs in the respiratory diseases .

polycystic liver disease condition characterized presence multiple cysts liver may inherited sporadic cases adult polycystic liver disease apld related autosomal dominant polycystic kidney disease adpkd 1964 there reports till date describe association malignant neoplasms potter type iii cystic disease liver kidney 2 3 4 5 6 7 8 however describe pancreatic hepatobiliary neoplasms arising patients polycystic liver kidney diseases including adpkd report described association gastric carcinoma potter type iii cystic disease liver kidney this first case report multiple gastric carcinomas associated potter type iii cystic disease liver mesenterium kidney although unclear whether development multiple gastric carcinomas directly associated pathogenesis multiple cysts liver mesenterium kidney case provides perspective etiology gastric carcinoma polycystic disease he receiving hemodialysis treatment 3 times week 2 years chronic renal failure due polycystic kidneys he stated sister suffered polycystic kidney disease physical examination admission revealed distended abdomen due polycystic disease the patient high serum levels carcinoembryonic antigen 7.9 ng ml carbohydrate antigen 19 9 202 mg dl an abdominal computed tomography scan showed multiple cysts liver cystic lesions sides kidney mesenterium fig gastrointestinal fiberscopy showed presence two ulcerated lesions lesser curvature middle lower portions stomach histopathological analysis gastric tumor biopsy specimens revealed papillary adenocarcinoma one tumor poorly differentiated adenocarcinoma another distal gastrectomy lymph node dissection performed gastric tumors surgically diagnosed t2n1m0 stage ii according tnm classification gastric carcinoma macroscopic examination showed two tumors type ii present middle lower portions stomach fig the distal lesion diagnosed poorly differentiated adenocarcinoma 38 39 6 mm within muscularis propria marked lymphatic invasion ly2 vascular invasion v2 fig 3b lesion proximal side diagnosed papillary adenocarcinoma 58 47 6 mm within submucosal layer marked lymphatic invasion ly2 vascular invasion v1 fig adpkd common inherited renal cystic diseases various external manifestations including cysts pancreas seminal vesicles arachnoid membrane liver the accepted among osathanondh potter system established 1964 according polycystic kidneys classified three types the cysts may present portion nephron tubule commonly found bowman space angle loop henle older classification potter type iii cystic disease considered represent adult type polycystic disease kidney 1972 they also observed cysts liver and/or pancreas 58 patients hence described condition potter type iii polycystic disease present case computed tomography ultrasonography scans showed multiple renal hepatic cysts addition multiple cysts mesenterium therefore according hatfield pfister criteria considered case association apld multiple renal cysts could classified potter type iii cystic disease the patient stated sister suffered polycystic kidney disease however could confirm presence family history polycystic kidney disease sister contact long absence family history adpkd bilateral multiple renal cysts hepatic cysts together absence manifestations suggesting different renal cystic disease provide evidence diagnosis instances a family history however may absent 2040% new patients diagnosis adpkd first suspected imaging studies due de novo mutation pkd-1 pkd-2 genes although could confirm mutation pkd-1 pkd-2 case family history clinical findings imaging studies suggested patient suffered adpkd reports association malignant neoplasm apld adpkd potter type iii cystic disease extremely rare to knowledge 8 cases reported english language literature table 1 2 3 4 5 6 7 8 till date cases neoplasms associated polycystic disease liver kidney reported either pancreatic hepatobiliary neoplasms namely 5 cases pancreatic neoplasms 3 cases cholangiocarcinoma since 5 cases pancreatic neoplasms think adpkd genetic influence development pancreatic neoplasms aziz et al reported 2 patients family brother sister adpkd developed adenocarcinoma gastroesophageal junction however patients cysts liver organs except kidney present case although detailed family history could obtained multiple cysts observed liver kidneys mesenterium these genes encode member polycystin protein family integral membrane protein functions regulator calcium permeable cation channels intracellular calcium homoeostasis some studies described association pkd genes cancer cells polycystin-1 encoded pkd-1 reported induce apoptosis cell cycle arrest g0/g1 phase cancer cells moreover pkd-1 inhibits cancer cell migration invasion may considered new member tumor suppressor family genes therefore adpkd caused defective pkd-1 pkd-2 may carcinogenic condition in addition mutations two genes namely prksch codes hepatocystin sec63 found cause apld however mutations genes found families undiscovered genes responsible causing apld probably still exist although unclear whether development multiple gastric carcinomas directly associated pathogenesis cysts liver mesenterium kidney present case demonstrates gastric carcinoma external complication patients polycystic disease

we report a case of multiple gastric carcinomas associated with potter type iii cystic disease of the liver , mesenterium and kidney . a 65-year - old man with chronic renal failure due to polycystic kidneys and under hemodialysis treatment 3 times a week for 2 years was admitted to our hospital because of anemia . he stated that his sister had suffered from polycystic kidney disease . gastrointestinal fiberscopy showed two lesions in the lesser curvature in the lower portion of the stomach , and histopathological analysis of the gastric tumor biopsies revealed that one of the tumors was a papillary adenocarcinoma and the other a poorly differentiated adenocarcinoma . helicobacter pylori infection was not detected in the stomach mucosa . abdominal computed tomography scan revealed polycystic lesions in the liver , mesenterium and both kidneys . these imaging findings and family history suggested that the patient suffered from multiple gastric carcinomas associated with potter type iii cystic disease of the liver , mesenterium and kidney . reports on the association of malignant neoplasm with potter type iii cystic disease are extremely rare . especially , no case of the association of gastric carcinoma with potter type iii cystic disease of the liver and kidney has been described previously . this is a first report of the association of gastric carcinoma with potter type iii cystic disease . we also review reports of other malignant neoplasms associated with polycystic disease .

canaliculitis common encounter ophthalmic practice supernumerary puncta canaliculi spc rare congenital disorders large series a 59-year old gentleman presented painful swelling left lower lid week associated epiphora the swelling confined nasal aspect left lower lid 0.50.5 mm inflamed overlying skin figure 1a eversion lower eyelid revealed two puncta 0.5 mm apart figure 1b the outer punctum situated normal anatomical position whereas inner punctum caruncle gentle pressure result regurgitation puncta the patient treated oral cloxacillin 500 mg 6 hourly 5 days the outer punctum soft stop regurgitation fluid punctum the outer punctum canaliculus system cul de sac figure 1c c dacryocystography showed pooling dye cul de sac white arrow c dacryocystography showed pooling dye cul de sac white arrow most spcs 78% present epiphora among 23 patients reported satchi et al none presented canaliculitis sequestration tear debris cul de sac served nidus infection resultant canaliculitis surrounding edema caused obstruction lacrimal drainage hence epiphora epiphora however may develop despite patent lacrimal drainage system 2-compartment model lacrimal canalicular drainage kakizaki et al suggested muscle duverney horner may deviate normal flow within accessory canaliculus thence transport tears back lacrimal tear lake leading epiphora a solid epithelial cord forms region medial lower eyelid figure 2a sends projections form canaliculi nasolacrimal duct figure 2b spc due extra budding solid epithelial cord figure 2c canalization begins 4 months gestation disintegration central ectodermal core forming lacrimal drainage outflow system case extra inner canalicular epithelial bud nearer main epithelial cord underwent complete canalization remained connected main epithelial cord the outer canalicular epithelial bud although punctum located normal anatomical position separated main epithelial cord forming cul de sac figure 2d c extra budding solid epithelial cord supernumerary puncta canaliculi d outer canalicular epithelial cord separated main epithelial cord forming cul de sac c extra budding solid epithelial cord supernumerary puncta canaliculi d outer canalicular epithelial cord separated main epithelial cord forming cul de sac

we report the first case of supernumerary puncta and canaliculi presented with canaliculitis . a-59 year - old gentleman presented with painful swelling of the left lower lid for a week , which was associated with epiphora . the swelling was confined to the nasal aspect of the left lower lid ( 0.50.5 mm ) with inflamed overlying skin . two puncta ( 0.5 mm apart ) were noted . the outer punctum at the normal anatomical position was a cul - de - sac while the inner punctum it the caruncle was patent . we described the embryology leading to supernumerary puncta and canaliculi to explain the paradoxical patency of the abnormally located punctum as well as the pathomechanism leading to canaliculitis . the patient was treated with oral cloxacillin 500 mg , 6 hourly for 5 days ; the cellulitis subsided after three days .

mypro is a software pipeline for high - quality prokaryotic genome assembly and annotation . it was validated on 18 oral streptococcal strains to produce submission - ready , annotated draft genomes . mypro installed as a virtual machine and supported by updated databases will enable biologists to perform quality prokaryotic genome assembly and annotation with ease .

diabetes imposes large economic burden individual national healthcare systems countries.[15 healthcare expenditures due diabetes account 11% total healthcare expenditures world 2011 estimated global healthcare expenditures treat diabetes prevent complications totaled 465 billion 2011 2030 this number projected exceed 595 billion average estimated healthcare spending due diabetes was 5,063 per person diabetes high income countries compared 271 low- middle income countries worldwide estimated number adults living diabetes soared 366 million representing 8.3% global adult population this number projected increase 552 million people 2030 9.9% adults equates approximately three people diabetes every 10 almost 10 million per year.[78 kingdom saudi arabia exception global pandemic past four decades the ageing populations together rapid socio economic development progressive urbanization decreasing infant mortality increasing life expectancy tremendous changes lifestyle towards westernized pattern reflected changes nutrition less physical activity tendency increased obesity smoking resulted dramatic increase diabetes prevalence.[916 worldwide desire many healthcare players governments diabetes associations health professionals health economists people diabetes reduce economic burden diabetes related complications costs makes question economic impact diabetes important component determining present economic burden diabetes related complications government healthcare systempredicting likely future economic burden diabetes related complications anddeveloping effective policies strategies treatment diabetes related complications future public health spending related diabetes healthcare costs determining present economic burden diabetes related complications government healthcare system predicting likely future economic burden diabetes related complications developing effective policies strategies treatment diabetes related complications future public health spending related diabetes healthcare costs the purpose research study therefore determine economic impact diabetes mellitus dm saudi arabia healthcare system future this research study however focuses prevalence diabetes aims estimating predicting total per capita healthcare expenditure people diagnosed diabetes to examine predict economic impact diabetes costs saudi arabia healthcare system study relied data provided ministry health moh ministry finance mof years 1992 2010 databases.[1921 1992 2010 time period used study time frame population figures 1992 2010 moh databases patients reported diabetes selected study data government budget total healthcare budget per capita health expenditure obtained directly moh mof databases data population nationality sex age groups undiagnosed diabetes obtained directly central department statistics information databases it noted study based single source diabetic data moh database therefore account considerable number people diagnosed diabetes healthcare providers private semi private healthcare services military universities arabian american oil company aramco healthcare services etc consequently diabetes prevalence probably much higher figures presented study significant number people diagnosed diabetes included analysis it also noted perspective adopted study moh if perspective used payer society health care provider results would different in addition data diabetes obtained moh database based diabetic clinics visits moh hospitals healthcare centers combined in- diabetic patients categorized nationality saudi non saudi sex male female age groups 1 1 4 5 14 15 44 45 60 60 it noted clear whether data provided moh database based first visits related diabetes therefore certain whether diabetic data protected double counting this research study used prevalence based approach combines demographics population without diagnosed diabetes 1992 2010 general principle estimating healthcare costs diabetes straightforward.[2427 healthcare use attributable diabetes determined comparison healthcare use patterns individuals without diabetes dividing healthcare expenditures size population without diabetes provides estimate per capita healthcare expenditures.by multiplying diabetes population figures per capita healthcare expenditures total healthcare budget people diagnosed diabetes derived.assuming diabetes prevalence rates remained constant time exponential projection method i.e. annualized change used reflect growing prevalence diabetes saudi arabia increasing costs healthcare expenditures next 10 years i.e. 2020 healthcare use attributable diabetes determined comparison healthcare use patterns individuals without diabetes dividing healthcare expenditures size population without diabetes provides estimate per capita healthcare expenditures multiplying diabetes population figures per capita healthcare expenditures total healthcare budget people diagnosed diabetes derived assuming diabetes prevalence rates remained constant time exponential projection method i.e. annualized change used reflect growing prevalence diabetes saudi arabia increasing costs healthcare expenditures next 10 years i.e. 2020 it mentioned modern approach conducting cost illness studies based system health account sha approach sha used describe health care system expenditure perspective however sha saudi arabia approaches used knowledge costs diabetes improves understanding importance addressing healthcare prevention issues associated diabetes help inform motivate decisions reduce national burden disease based moh database approximately 0.9 million people 1992 2.5 million people 2010 diagnosed diabetes analysis 1992 2010 moh database suggests number people diagnosed diabetes increased 1.6 million 183% last 18 years this increases proportion total population diabetes 5.3% 1992 9.3% 2010 comparison 2010 diabetes figure 1992 figure suggests net number people diagnosed diabetes growing 0.1 million per year increased prevalence diabetes period study attributable changing pattern saudi lifestyle due rapid socio economic development together increased obesity smoking less physical activities may also attributed increased awareness programs related diabetes health complications community screening campaigns diabetes better diagnostic facilities specially health centers healthcare units better diabetes management systems protocols in fact several community based screening campaigns studies dm hypertension conducted different parts saudi arabia revealed quite large percentage participants diabetic patients detected remain long time without significant symptoms these studies concluded community based screening campaigns extremely efficient identifying undiagnosed diabetic hypertensive individuals society.[2933 table 1 summarizes demographic component population diagnosed diabetes the moh database population diagnosed diabetes nationality sex age group suggests saudi citizens comprised 84% 96% total population diagnosed diabetes 1992 2010 respectively non saudi population diagnosed diabetes comprised 16% 1992 4% 2010 the demographic analysis also revealed half total population diagnosed diabetes 1992 2010 males females represented 47% 48% total population diagnosed diabetes 1992 2010 respectively the analysis revealed 1 11 males diabetic 2010 compared 1 19 males 1992 for females 1 9 diabetic 2010 compared 1 17 1992 population millions percent population without diabetes 1992 2010 population 45 60 years represented highest among population diagnosed diabetes period analysis this age group comprised 52% 45% total population diagnosed diabetes 1992 2010 respectively remaining population age 15 age 15 44 60 years comprised 3% 21% 15% 1992 4% 27% 24% 2010 respectively it important mention population age 60 years represents highest percentage increase among population diagnosed diabetes last 18 years analysis the population age 60 years diagnosed diabetes growing 33.6 thousand people per year the analysis revealed ratio population age 60 years diagnosed diabetes undiagnosed diabetes rose 1:4 1992 almost 1:1 2010 it well documented population highest use healthcare resources attributed diabetes population aged 60 years above.[3438 healthcare expenditure attributed diabetes reflects additional expenditures nation incurs diabetes this equals total healthcare expenditures people diabetes minus projected level expenditures would incurred absence diabetes effect estimates excess healthcare use theoretically due caused diabetes related complications the term attributed also means difference healthcare use people diabetes compared healthcare use would absence diabetes approximately 0.9 billion healthcare expenditures incurred people diabetes reflecting 1 every 11 moh healthcare dollars healthcare expenditures incurred people diabetes increased 500% last 18 years 2010 average people diagnosed diabetes medical healthcare expenditures ten times higher 3,686 vs. 380 expenditures would absence diabetes 1 us dollar 3.75 saudi riyal healthcare expenditures diabetes status nationality sex age groups 1992 2010 usa dollars dividing total attributed healthcare expenditures number people diagnosed diabetes gives estimate average annual excess expenditures population age 15 age 15 44 age 45 60 age 60 9,244 1,255 767 1,442 respectively the population age 45and 60 highest per capita healthcare expenditure last 18 years per capita expenditure population age 45 60 increased 145% 1992 2010 followed age 15 44 137% age 15 45% age 60 years 36% the analysis revealed per capita healthcare expenditure population age 15 almost nine times higher 9,244 vs. 1,048 healthcare expenditures otherwise similar age group without diabetes the analysis also revealed per capita healthcare expenditure population age 15 44 two times higher 1,255 vs. 590 healthcare expenditures otherwise similar age group without diabetes per capita healthcare expenditure male female population diagnosed diabetes equal extent per capita healthcare expenditure male female population without diabetes similarly per capita saudi citizens diagnosed diabetes extent equal per capita saudi citizens undiagnosed diabetes per capita healthcare expenditure non saudi population eight times higher 8,589 vs. 1,018 healthcare expenditures non saudi nationality without diabetes mentioned earlier the rapid increase socio economic development saudi arabia coupled improved living standards technological advancements last four decades resulted dramatic change saudi lifestyle nutrition increasing obesity smoking rate less physical activity many factors reflecting substantial increase size saudi population diagnosed diabetes.[4045 factors likely remain diabetes prevalence rates remained constant time number people diagnosed diabetes expected rise 4.2 million 2015 another seven million 2020 the economic burden diabetes saudi arabia expected approximately 2.4 billion 2015 table 3 this increase 1.5 billion nearly three times level 2010 cost also expected rise another 6.5 billion 2020 given expected increase number people diagnosed diabetes saudi arabia proportion public healthcare spending expected escalate 9.3% 2010 13.1% 2015 18.3% 2020 projection population diagnosed diabetes healthcare expenditure usa dollars actual economic burden diabetes future years expected higher cost health care outpaces overall cost living growing problem obesity increases prevalence rate diabetes even estimates account lost productivity losses attributable pain suffering incurred people diagnosed diabetes well families friends diabetes based moh database approximately 0.9 million people 1992 2.5 million people 2010 diagnosed diabetes analysis 1992 2010 moh database suggests number people diagnosed diabetes increased 1.6 million 183% last 18 years this increases proportion total population diabetes 5.3% 1992 9.3% 2010 comparison 2010 diabetes figure 1992 figure suggests net number people diagnosed diabetes growing 0.1 million per year increased prevalence diabetes period study attributable changing pattern saudi lifestyle due rapid socio economic development together increased obesity smoking less physical activities may also attributed increased awareness programs related diabetes health complications community screening campaigns diabetes better diagnostic facilities specially health centers healthcare units better diabetes management systems protocols in fact several community based screening campaigns studies dm hypertension conducted different parts saudi arabia revealed quite large percentage participants diabetic patients detected remain long time without significant symptoms these studies concluded community based screening campaigns extremely efficient identifying undiagnosed diabetic hypertensive individuals society.[2933 table 1 summarizes demographic component population diagnosed diabetes the moh database population diagnosed diabetes nationality sex age group suggests saudi citizens comprised 84% 96% total population diagnosed diabetes 1992 2010 respectively non saudi population diagnosed diabetes comprised 16% 1992 4% 2010 the demographic analysis also revealed half total population diagnosed diabetes 1992 2010 males females represented 47% 48% total population diagnosed diabetes 1992 2010 respectively the analysis revealed 1 11 males diabetic 2010 compared 1 19 males 1992 for females 1 9 diabetic 2010 compared 1 17 1992 population millions percent population without diabetes 1992 2010 population 45 60 years represented highest among population diagnosed diabetes period analysis this age group comprised 52% 45% total population diagnosed diabetes 1992 2010 respectively remaining population age 15 age 15 44 60 years comprised 3% 21% 15% 1992 4% 27% 24% 2010 respectively it important mention population age 60 years represents highest percentage increase among population diagnosed diabetes last 18 years analysis the population age 60 years diagnosed diabetes growing 33.6 thousand people per year the analysis revealed ratio population age 60 years diagnosed diabetes undiagnosed diabetes rose 1:4 1992 almost 1:1 2010 it well documented population highest use healthcare resources attributed diabetes population aged 60 years above.[3438 healthcare expenditure attributed diabetes reflects additional expenditures nation incurs diabetes this equals total healthcare expenditures people diabetes minus projected level expenditures would incurred absence diabetes effect estimates excess healthcare use theoretically due caused diabetes related complications the term attributed also means difference healthcare use people diabetes compared healthcare use would absence diabetes approximately 0.9 billion healthcare expenditures incurred people diabetes reflecting 1 every 11 moh healthcare dollars healthcare expenditures incurred people diabetes increased 500% last 18 years 2010 average people diagnosed diabetes medical healthcare expenditures ten times higher 3,686 vs. 380 expenditures would absence diabetes 1 us dollar 3.75 saudi riyal healthcare expenditures diabetes status nationality sex age groups 1992 2010 usa dollars dividing total attributed healthcare expenditures number people diagnosed diabetes gives estimate average annual excess expenditures population age 15 age 15 44 age 45 60 age 60 9,244 1,255 767 1,442 respectively the population age 45and 60 highest per capita healthcare expenditure last 18 years per capita expenditure population age 45 60 increased 145% 1992 2010 followed age 15 44 137% age 15 45% age 60 years 36% the analysis revealed per capita healthcare expenditure population age 15 almost nine times higher 9,244 vs. 1,048 healthcare expenditures otherwise similar age group without diabetes the analysis also revealed per capita healthcare expenditure population age 15 44 two times higher 1,255 vs. 590 healthcare expenditures otherwise similar age group without diabetes per capita healthcare expenditure male female population diagnosed diabetes equal extent per capita healthcare expenditure male female population without diabetes similarly per capita saudi citizens diagnosed diabetes extent equal per capita saudi citizens undiagnosed diabetes per capita healthcare expenditure non saudi population eight times higher 8,589 vs. 1,018 healthcare expenditures non saudi nationality without diabetes as mentioned earlier rapid increase socio economic development saudi arabia coupled improved living standards technological advancements last four decades resulted dramatic change saudi lifestyle nutrition increasing obesity smoking rate less physical activity many factors reflecting substantial increase size saudi population diagnosed diabetes.[4045 factors likely remain diabetes prevalence rates remained constant time number people diagnosed diabetes expected rise 4.2 million 2015 another seven million 2020 the economic burden diabetes saudi arabia expected approximately 2.4 billion 2015 table 3 this increase 1.5 billion nearly three times level 2010 cost also expected rise another 6.5 billion 2020 given expected increase number people diagnosed diabetes saudi arabia proportion public healthcare spending expected escalate 9.3% 2010 13.1% 2015 18.3% 2020 projection population diagnosed diabetes healthcare expenditure usa dollars actual economic burden diabetes future years expected higher cost health care outpaces overall cost living growing problem obesity increases prevalence rate diabetes even estimates account lost productivity losses attributable pain suffering incurred people diagnosed diabetes well families friends diabetes diabetes personal crisis people living disease families treatment disease related complications consuming ever larger share healthcare budgets soon force tremendous increase budgets although population diagnosed diabetes comprises 9% saudi population 2010 almost 1 11 people saudi arabia diabetes present prevalence rate remains unchanged time ratio people without diabetes increase almost 1 5 people disease 2020 people diagnosed diabetes medical healthcare expenditures ten times higher expenditures would absence diabetes this cost estimated 6.5 billion 2020 reflecting increase 5.6 billion 7 times higher cost 2010 within next 10 years although diabetes cost estimates presented research study might conservative several reasons omitted analysis due data limitations number people reported diabetes private governmental e.g. universities hospitals armed forces medical services security forces hospital national guard medical services royal commission hospitals youth welfare hospital saudi aramco healthcare service providers time writing 2012 148 hospitals 2,360 health clinics related moh healthcare system this research study relied data obtained directly moh healthcare database people diagnosed diabetes ignored sources data due difficulty obtaining consequently diabetes prevalence probably much higher figures presented study significant number cases included analysis.omitted cost estimates indirect cost associated diabetes lost productivity due disease related absenteeism unemployment due disease related disability lost productivity due early mortality disease well social cost intangibles pain suffering care provided non paid caregivers the prevention programs targeted people diabetes research activities administration costs associated diabetes also omitted diabetes cost estimates study if portion costs attributed diabetes national healthcare cost diabetes would billions dollars higher estimate suggest study.sensitivity analysis related diabetic mellitus related complications carried study omitted analysis due data limitations number people reported diabetes private governmental e.g. universities hospitals armed forces medical services security forces hospital national guard medical services royal commission hospitals youth welfare hospital saudi aramco healthcare service providers time writing 2012 148 hospitals 2,360 health clinics related moh healthcare system this research study relied data obtained directly moh healthcare database people diagnosed diabetes ignored sources data due difficulty obtaining consequently diabetes prevalence probably much higher figures presented study significant number cases included analysis omitted cost estimates indirect cost associated diabetes lost productivity due disease related absenteeism unemployment due disease related disability lost productivity due early mortality disease well social cost intangibles pain suffering care provided non paid caregivers the prevention programs targeted people diabetes research activities administration costs associated diabetes also omitted diabetes cost estimates study if portion costs attributed diabetes national healthcare cost diabetes would billions dollars higher estimate suggest study sensitivity analysis related diabetic mellitus related complications carried study worldwide resources allocated diabetes prevention treatment growing prevalence also growing steadily several international reports preventing dm indicated people diabetes greater risk neurological disease peripheral vascular disease cardiovascular disease renal disease endocrine metabolic complications ophthalmic disease chronic complications compared individuals without diabetes.[18 chronic complications main cause death among diabetic patients account higher costs hospitalization drugs costs drugs complications 2.5 times higher general population these international reports suggested better access preventive healthcare system widespread diagnosis intensive disease management advent new medical technologies could significantly eliminate reduce health problems caused diabetes these factors could also improve quality life people diabetes families time potentially reduce national expenditures healthcare services increasing productivity national economy sum further research studies needed improve understanding economic costs diabetes substantial burden saudi healthcare system special attention also given role diabetic awareness programs community based screening campaigns different health educational programs reducing health problems caused diabetes long run helps reduce national burden disease sha also adopted saudi health providers deal developments financial health flows related consumptions healthcare goods services

background : diabetes imposes a large economic burden on the individual , national healthcare systems , and countries.objective:to determine the economic impact of diabetes mellitus on saudi healthcare system , both now and in the future.materials and methods : this research study uses a prevalence - based approach that combines the demographics of the population ( classified by nationality , sex and age group ) with and without diagnosed diabetes in 1992 and 2010 . the economic impact of diabetes is estimated in this study , using secondary sources of information provided by ministry of health , ministry of finance and central department of statistics and information databases.results:people diagnosed with diabetes , on average , have medical healthcare expenditures that are ten times higher ( $ 3,686 vs. $ 380 ) than what expenditures would be in the absence of diabetes . over 96% of all medical healthcare expenditures attributed to diabetes are incurred by persons of saudi nationality , with the remaining 4% incurred by persons of non - saudi nationality . the population age 45 - 60 incurs 45% of diabetes - attributed costs , with the remaining population under age 15 incurs 3.8% , age 15 - 44 incurs 27.5% , and age 60 and above incurs 23.8%.conclusion : the actual national healthcare burden because of diabetes is likely to exceed the $ 0.87 billion estimated in this study , because it omits the indirect costs associated with diabetes , such as absenteeism , lost productivity from disease - related absenteeism , unemployment from disease - related disability , lost productivity due to early mortality by disease . the social cost of intangibles such as pain and suffering and care provided by non - paid caregivers as well as healthcare system administrative costs , cost of medications , clinician training programs , and research and infrastructure development is also omitted from this research study . further studies are needed to confirm the present findings and to improve our understanding of economic costs of diabetes and its related complications .

prospective study pravastatin elderly risk prosper prospective multicenter randomized placebo controlled trial assess whether treatment pravastatin diminishes risk major cardiovascular events elderly 12,13 december 1997 may 1999 total 5,804 participants aged 7082 years preexisting vascular disease increased risk disease due history smoking hypertension diabetes recruited scotland ireland netherlands the institutional ethics review boards centers approved protocol participants gave written informed consent participants severe cognitive impairment mini mental state examination mmse score 24 excluded inclusion study the rotterdam study large prospective population based cohort study conducted among inhabitants aged 55 years ommoord district rotterdam netherlands 14 the medical ethics committee erasmus university rotterdam approved study written informed consent obtained participants of 10,275 eligible subjects 7,983 individuals 78% participated baseline examinations 1990 1993 mean age 71 25 years range 55106 years all participants interviewed home visited research center examinations prosper fasting glucose levels assessed baseline 5,599 5,804 participants 5,599 participants 580 cognitive function tests available baseline all resulting 5,019 participants full data available cardiovascular risk factors including bmi systolic diastolic blood pressure hdl cholesterol levels baseline this resulted study sample 5,019 participants prosper rotterdam study fasting glucose levels assessed third survey 3,795 participants participants 3,664 free dementia 3,550 data available cardiovascular risk factors including bmi systolic diastolic blood pressure hdl cholesterol levels of 3,550 participants 122 cognitive function tests available start third survey additionally 3,342 3,428 participants fasting insulin levels assessed third survey prosper rotterdam sample there 4,690 5,019 participants prosper sample 2,364 3,428 participants rotterdam sample underwent least one additional measurement fasting glucose level addition initial examination these data used study variability fasting glucose levels time assess appropriateness using single baseline fasting glucose measurement assess relationship fasting glucose cognitive function decline baseline history diabetes defined self reported history diabetes reporting use oral antidiabetes medication use insulin treatment diet registration general practitioner diabetes study samples in addition dedicated neuropsychological test battery used assess executive function memory executive function assessed letter digit substitution task ldst 16 abbreviated stroop test part 3 17 studies well word fluency test wft 18 rotterdam study memory assessed 12-picture learning test 12-plt immediate delayed recall 19 prosper individual test scores transformed standardized z scores z score individual score a compound cognitive test score global cognitive function calculated averaging z scores mmse ldst abbreviated stroop test part 3 a compound cognitive test score 12-plt calculated averaging z scores 12-plt immediate 12-plt delayed recall test prosper cognitive function measured six time points study randomization baseline 9 18 30 months end study the time point last measurement different participants ranged 36 48 months baseline therefore performed analyses individual varying time point report results mean time points 42 months change cognitive function could assessed 4,767 participants least one follow examination cognitive function available initial measurement rotterdam study cognitive function assessed third survey 19971999 additionally fourth survey 20022004 of 3,428 participants rotterdam sample present third survey 2,601 remained study end follow fourth survey available assessment change cognitive function in samples level education bmi systolic diastolic blood pressure hdl cholesterol level apoe 4 carriership assessed baseline prosper third survey rotterdam study level education dichotomized primary education less low primary education high rotterdam study age leaving school 13 years low age leaving school 13 years high prosper the relationship baseline prosper third survey rotterdam study fasting glucose levels cognitive function decline assessed use linear mixed models data prosper sample rotterdam study sample merged one large sample 8,447 participants all analyses adjusted age sex level education study prosper rotterdam study bmi systolic diastolic blood pressure hdl cholesterol level apoe 4 carriership country use pravastatin appropriate test version analyses carried using spss statistical package release 12.0.1 spss chicago il data fasting glucose fasting insulin levels rotterdam study sample used calculate degree insulin resistance according homa 20 the homa index calculated dividing product fasting levels glucose insulin constant shown correlate well r 0.82 p 0.0001 euglycemic hyperinsulinemic clamp method 21 the prospective study pravastatin elderly risk prosper prospective multicenter randomized placebo controlled trial assess whether treatment pravastatin diminishes risk major cardiovascular events elderly 12,13 december 1997 may 1999 total 5,804 participants aged 7082 years preexisting vascular disease increased risk disease due history smoking hypertension diabetes recruited scotland ireland netherlands the institutional ethics review boards centers approved protocol participants gave written informed consent participants severe cognitive impairment mini mental state examination mmse score 24 excluded inclusion study the rotterdam study large prospective population based cohort study conducted among inhabitants aged 55 years ommoord district rotterdam netherlands 14 the medical ethics committee erasmus university rotterdam approved study written informed consent obtained participants of 10,275 eligible subjects 7,983 individuals 78% participated baseline examinations 1990 1993 mean age 71 25 years range 55106 years in prosper fasting glucose levels assessed baseline 5,599 5,804 participants 5,599 participants 580 all resulting 5,019 participants full data available cardiovascular risk factors including bmi systolic diastolic blood pressure hdl cholesterol levels baseline this resulted study sample 5,019 participants prosper rotterdam study fasting glucose levels assessed third survey 3,795 participants participants 3,664 free dementia 3,550 data available cardiovascular risk factors including bmi systolic diastolic blood pressure hdl cholesterol levels of 3,550 participants 122 cognitive function tests available start third survey additionally 3,342 3,428 participants fasting insulin levels assessed third survey prosper rotterdam sample fasting glucose levels additionally measured follow 4,690 5,019 participants prosper sample 2,364 3,428 participants rotterdam sample underwent least one additional measurement fasting glucose level addition initial examination these data used study variability fasting glucose levels time assess appropriateness using single baseline fasting glucose measurement assess relationship fasting glucose cognitive function decline at baseline history diabetes defined self reported history diabetes reporting use oral antidiabetes medication use insulin treatment diet registration general practitioner diabetes study samples global cognitive function measured mmse 15 studies addition dedicated neuropsychological test battery used assess executive function memory executive function assessed letter digit substitution task ldst 16 abbreviated stroop test part 3 17 studies well word fluency test wft 18 rotterdam study memory assessed 12-picture learning test 12-plt immediate delayed recall 19 prosper individual test scores transformed standardized z scores z score individual score a compound cognitive test score global cognitive function calculated averaging z scores mmse ldst abbreviated stroop test part 3 a compound cognitive test score 12-plt calculated averaging z scores 12-plt immediate 12-plt delayed recall test prosper cognitive function measured six time points study randomization baseline 9 18 30 months end study the time point last measurement different participants ranged 36 48 months baseline therefore performed analyses individual varying time point report results mean time points 42 months change cognitive function could assessed 4,767 participants least one follow examination cognitive function available initial measurement rotterdam study cognitive function assessed third survey 19971999 additionally fourth survey 20022004 of 3,428 participants rotterdam sample present third survey 2,601 remained study end follow fourth survey available assessment change cognitive function in samples level education bmi systolic diastolic blood pressure hdl cholesterol level apoe 4 carriership assessed baseline prosper third survey rotterdam study level education dichotomized primary education less low primary education high rotterdam study age leaving school 13 years low age leaving school 13 years high prosper the relationship baseline prosper third survey rotterdam study fasting glucose levels cognitive function decline assessed use linear mixed models data prosper sample rotterdam study sample merged one large sample 8,447 participants all analyses adjusted age sex level education study prosper rotterdam study bmi systolic diastolic blood pressure hdl cholesterol level apoe 4 carriership country use pravastatin appropriate test version analyses carried using spss statistical package release 12.0.1 spss chicago il data fasting glucose fasting insulin levels rotterdam study sample used calculate degree insulin resistance according homa 20 the homa index calculated dividing product fasting levels glucose insulin constant shown correlate well r 0.82 p 0.0001 euglycemic hyperinsulinemic clamp method 21 table 1 shows baseline characteristics total sample participants history diabetes baseline prosper third survey rotterdam study prosper fasting glucose levels sd differed among three countries participants enrolled scotland 5.62 1.27 ireland 5.09 1.34 netherlands 5.76 1.64 mmol l this resulted lower mean fasting glucose level prosper study sample compared rotterdam study sample participants history diabetes higher fasting glucose level bmi systolic blood pressure lower levels hdl cholesterol compared participants without history diabetes * fasting insulin levels available 3,342 participants rotterdam study prosper fasting glucose levels assessed follow 3 6 12 24 36 months addition baseline assessment 3,491 participants without history diabetes fasting glucose levels available baseline 36 months follow the quintiles mean fasting glucose sd baseline 36 months 4.30 0.20 4.77 0.67 mmol l lowest quintile quintile 1 4.70 0.08 4.95 0.47 mmol l quintile 2 5.00 0.08 5.21 0.67 mmol l quintile 4 6.25 0.83 6.30 1.40 mmol l highest quintile quintile 5 rotterdam sample fasting glucose levels assessed third survey well end follow fourth survey 2,209 participants without history diabetes the quintiles fasting glucose levels third survey end follow 4.82 0.24 5.07 0.39 mmol l lowest quintile quintile 1 5.20 0.08 5.36 0.42 mmol l quintile 2 5.50 0.08 5.55 0.50 mmol l highest quintile quintile 5 8,447 participants present baseline 6,641 remained study sample end follow 985 withdrew study 821 died follow the 1,806 participants without final examination comprised 20.8% participants without history diabetes 26.3% participants history diabetes baseline prosper third survey rotterdam study figure 1a shows relationship fasting glucose levels cognitive function baseline 8,447 participants merged study sample study specific quintiles distribution fasting glucose levels participants without history diabetes constructed account differences fasting glucose levels studies cognitive test scores shown quintiles distribution fasting glucose levels participants without history diabetes participants history diabetes participants without history diabetes rise fasting glucose levels nondiabetes range associated impairment cognitive function cognitive tests additionally compared cognitive test scores baseline participants without history diabetes fig 1a showed participants history diabetes worse cognitive function across majority tests baseline compared participants without history diabetes p 0.05 tests except wft rotterdam study sample z scores sem different cognitive test scores plotted study specific quintiles fasting glucose levels nondiabetic participants lowest quintile 1 highest quintile 5 levels fasting glucose participants history diabetes dm p values reflect trend quintiles fasting glucose levels well difference participants without history diabetes n 7,592 participants history diabetes n 855 estimates based maximum number participants available per cognitive test global cognitive function n 8,447 wft n 3,518 12-plt n 5,223 z scores sem represent annual change cognitive test scores study specific quintiles fasting glucose levels nondiabetic participants lowest quintile 1 highest quintile 5 levels fasting glucose participants history diabetes p values reflect trend quintiles fasting glucose levels well difference participants without history diabetes n 6,649 participants history diabetes n 719 estimates based maximum number participants available per cognitive test global cognitive function n 7,368 wft n 2,639 12-plt n 4,960 linear mixed models used adjusted age sex level education study prosper rotterdam study bmi hdl level systolic blood pressure diastolic blood pressure country treatment group test version applicable longitudinal analyses study population clear association baseline fasting glucose levels change cognitive function follow participants without history diabetes fig higher levels fasting glucose associated decreased rate decline 12-plt ptrend 0.039 seen cognitive tests furthermore participants history diabetes show increased rate decline cognitive tests additionally assessed relationship insulin resistance cognitive function decline 3,342 participants rotterdam study sample fasting insulin levels available fig the homa calculated participants correlated fasting glucose levels r 0.54 p 0.001 although overlap quintiles homa index quintiles fasting glucose levels limited 35% participants without history diabetes quintile distribution fasting glucose homa index the relationship insulin resistance cognitive function accordance findings fasting glucose levels cognitive function participants without history diabetes rising insulin resistance associated cognitive function similarly clear relationship levels insulin resistance change cognitive function follow fig when data prosper sample rotterdam study sample analyzed separately findings consistent results merged sample 8,447 participants insulin resistance homa cognitive function rotterdam study z scores sem different cognitive test scores plotted quintiles insulin resistance homa nondiabetic participants lowest quintile 1 highest quintile 5 levels insulin resistance homa participants history diabetes dm p values reflect trend quintiles fasting glucose levels well difference participants without history diabetes n 3,039 participants history diabetes n 303 estimates based maximum number participants available per cognitive test global cognitive function n 3,342 wft n 3,424 z scores sem represent annual change cognitive test scores quintiles insulin resistance homa nondiabetic participants lowest quintile 1 highest quintile 5 levels insulin resistance homa participants history diabetes p values reflect trend quintiles fasting glucose levels well difference participants without history diabetes n 2,331 participants history diabetes n 203 estimates based maximum number participants available per cognitive test global cognitive function n 2,534 wft n 2,567 linear mixed models used adjusted age sex level education bmi hdl level systolic blood pressure diastolic blood pressure our study shows unprecedented large number individuals two independent prospective studies higher levels fasting glucose absence history diabetes associated cognitive function cognitive decline furthermore association insulin resistance homa index cognitive function decline people without history diabetes however participants history diabetes worse cognitive function baseline without diabetes although magnitude observed effects relatively small results analyses fully correspond previous findings rotterdam study diabetes found related increased risk developing dementia 22 although data show history diabetes worse cognitive function baseline without one could argue effect sizes lower expected based previous report almost twofold increased risk dementia people diabetes 22 it possible measurement cognitive function compared assessment participants dementia rotterdam study underlies discrepancy rotterdam study able continuously monitor total cohort incident dementia computerized linkage study database digitalized medical records general practitioners regional institute outpatient mental health care in contrast rotterdam sample cognitive function assessments performed 4.6-year interval could led selective nonparticipation dementia diagnosed cognitive examinations n 85 visit research center follow assessment furthermore previous report nurses health study showed higher insulin levels nondiabetic participants related faster cognitive decline 23 these results agreement results relationship insulin resistance cognitive decline rotterdam study sample the prolonged length follow 10 years compared 4.6 years nurses health study could affected results effect insulin resistance cognitive function might long term on hand results ongoing memory diabetes mind substudy action control cardiovascular risk diabetes accord trial showed higher a1c fasting plasma glucose levels associated worse cognitive function sample almost 3,000 participants 24 line results individual participants diabetes impaired fasting glucose levels often co occur cardiovascular risk factors the metabolic syndrome seen clustering number risk factors abdominal obesity hypertriglyceridemia low hdl cholesterol hypertension hyperglycemia subject ongoing discussion clinical use syndrome individual components establishing risk cardiovascular disease diabetes 25 other studies suggested relationship metabolic syndrome risk cognitive impairment dementia 2628 study clustering factors metabolic syndrome fasting glucose levels could influenced results however adjustment bmi systolic diastolic blood pressure hdl cholesterol differ analyses unadjusted covariates furthermore participants prosper included based increased cardiovascular risk profile either preexisting vascular disease increased risk disease due history smoking hypertension diabetes the known association cardiovascular risk factors cognitive function decline might interfered investigation relationship fasting glucose levels cognitive function decline however when excluded 2,823 participants prosper history vascular disease sample 5,019 participants relationship fasting glucose levels cognitive function baseline markedly differ findings total sample clear relationship fasting glucose levels cognitive function participants without history vascular disease moreover exclusion 2,823 participants significant relationship anymore history diabetes baseline global cognitive function the relationship fasting glucose levels change cognitive function markedly change exclusion participants history vascular disease the seen analyses relationship history diabetes change cognitive function therefore think inclusion participants preexisting vascular disease prosper masked possible association fasting glucose insulin resistance cognitive function we used fasting insulin levels available almost entire sample rotterdam study calculate homa index measure insulin resistance investigate relationship glucose metabolism cognitive function however relationship insulin resistance cognitive function decline showed similarities association fasting glucose levels cognitive function decline participants without history diabetes insulin resistance associated cognitive function decline previous population based studies investigated relationship glucose metabolism cognitive functions suggested number possible biological mechanisms could involved ranging accumulation advanced glycation end products 2 accelerated cerebrovascular disease 1 role insulin degrading enzyme amyloid metabolism 3 although difficult address role suggested mechanisms study 8,000 participants shows effect increased fasting glucose levels cognitive function seems long term independent cardiovascular risk factors like bmi blood pressure hdl cholesterol levels the observed differences cognitive test scores people without history diabetes relatively moderate may therefore lack clinical significance individuals however small effect sizes automatically imply irrelevance observed effect small effects group level indeed represent large effects number participants analyses annual decline cognitive function the prosper sample failed show clear decline mmse score time although seen rotterdam sample participants comparable age it possible potential learning effect mmse higher impact prosper sample compared rotterdam study sample shorter time span cognitive measurements 19 additionally selection criteria participants prosper baseline mmse score 24 may resulted sample participants slightly better cognitive function also represented difference mmse scores samples baseline might effect annual decline mmse score measured prosper the strengths study consist prospective design large number participants studies dedicated neuropsychological test battery used samples furthermore possibility studying variability fasting glucose levels follow examining appropriateness using single measurement fasting glucose level assess association fasting glucose levels cognitive function decline a large variation fasting glucose levels time could disturbed analyses phenomenon regression mean however levels fasting glucose follow materially differ baseline third survey study samples therefore decided use baseline third survey fasting glucose measurement analyses participants present baseline undergo follow examinations predominantly present group history diabetes they worse cognitive function baseline compared participants stayed study end follow this selective attrition participants relatively high levels fasting glucose concurrent low levels cognitive function could resulted underestimation estimates cognitive decline participants history diabetes we also recognize individuals diabetes would missed lack oral glucose tolerance testing more importantly undiagnosed diabetes would prevalent higher quintiles fasting glucose would biased study toward association higher quintiles cognitive decline way around thus lack oral glucose tolerance testing negate findings rather gives us added confidence observations valid conclusion elevations fasting glucose levels clearly associated impaired cognitive function accelerated rate cognitive decline participants without history diabetes furthermore clear relationship insulin resistance homa index cognitive function decline participants without history diabetes these data suggest cognitive decline accelerates strongly person diabetic lesser degrees dysglycemia result preventing individuals risk developing diabetes lifestyle changes may also lead large societal gains preventing individuals undergoing accelerated cognitive decline

objectiveto investigate the relationship between fasting glucose levels , insulin resistance , and cognitive impairment in old age . diabetes is associated with cognitive impairment in older people . however , the link between elevated fasting glucose levels and insulin resistance in nondiabetic individuals , and the risk of cognitive impairment is unclear.research design and methodswe analyzed data from , in total , 8,447 participants in two independent prospective studies : the prospective study of pravastatin in the elderly at risk ( prosper ) , 5,019 participants , aged 6984 years , and the rotterdam study , 3,428 participants , aged 6197 years . fasting glucose levels were assessed at baseline in both studies ; fasting insulin levels were assessed in the rotterdam study only . cognitive function was assessed in both studies at baseline and during follow-up.resultssubjects with diabetes had impaired cognitive function at baseline . in contrast , in people without a history of diabetes , there was no clear association between baseline fasting glucose levels and executive function and memory , nor was there a consistent relationship between elevated baseline fasting glucose levels and the rate of cognitive decline in either cohort . insulin resistance ( homeostasis model assessment index ) was also unrelated to cognitive function and decline.conclusionselevated fasting glucose levels and insulin resistance are not associated with worse cognitive function in older people without a history of diabetes . these data suggest either that there is a threshold for effects of dysglycemia on cognitive function or that factors other than hyperglycemia contribute to cognitive impairment in individuals with frank diabetes .

mentioned report national eye institute dry eye disorder tear film due tear deficiency excessive tear evaporation causes damage interpalpebral ocular surface associated symptoms ocular discomfort since contact lenses disrupt integrity tear film thin lipid layer thus increasing tear evaporation potentially cause symptoms eye dryness wearers . found ocular dryness affects 20% wearers new generation silicone hydrogel contact lenses well 24% wearers older generation low oxygen permeable phema lenses brennan efron found 75% low oxygen permeable lens wearers report feeling discomfort end day related dry eye in general believed 50% contact lens wearers symptomatic dry eye they may also present clinical signs dryness damage surface eye well instability and/or hyperosmolarity tear film however symptoms always correlate objective signs cases subject develops discomfort absence ocular pathology could trigger dryness all factors discarded contact lens wear must therefore considered likely etiology subject symptoms subjects induced ocular dryness contact lens wear usually tend drop modality benefits lenses outweighed discomfort feel especially last hours wear day day this main reason 20% low oxygen permeable lens wearers 13% silicone hydrogel lens wearers stop wearing contact lenses every year this discontinuation rate represents continuous loss industry eyecare professionals quest find new methods prevent induced dryness ocular surface ongoing concern manufacturers first tried deal materials used contact lenses among things comfort depends given material rate eye dehydration 711 wettability 12 13 presence surface deposits 810 silicone hydrogel lenses currently considered material choice compared hydrogels clinically many benefits 14 15 recognized studies shown silicone hydrogel lenses altered end day discomfort described least expected exception senofilcon found improve comfort adverse challenging environments materials proved solution problem dry eye researchers turned attention lens design spherical versus aspherical edge profile sharp thin versus rounded thicker fit lens eye flat steep replacement rate 2 weeks versus 1 month many aspects care products also considered triggering ocular dryness symptoms chemical products known cause short- medium term toxic allergic reactions leading patient discomfort preservatives identified main components products trigger reactions among contact lens wearers 1822 it also known specific biocides found contact lens solutions related toxic allergic reactions produce severe symptoms wearers accordingly solutions containing polyhexanides specifically polyaminopropyl biguanide papb polyhexamethylene biguanide phmb known increase potential adverse reactions polyhexanides polycationic agents relatively high molecular weight theory limit absorption contact lens however studies shown certain materials easily absorb large quantity polycationic agents lens soaking phase night other factors explain absorption number nature buffers solution presence sodium chloride determines ionic strength lens solution interaction on insertion lens balances surrounding ocular environment biocides absorbed lens matrix released eye possibly contributing temporary alteration ocular surface especially first four hours wear absorption release rates vary depending agents materials used surface treatment determines compatibility certain solutions certain materials for example aldox penetrates phmb released rapidly 0 60 min eye insertion contact lens this prompted certain authors suggest wearers neutralize polycationic agents phmb absorbed soaking adding drop anionic solution lenses insertion the anionic cationic reaction creates chemically neutral environment sparing cornea temporary structural changes wear this proven contribute increased patient comfort even end day the simple use lubricant morning would therefore significantly improve end day symptoms creating chemically neutral environment cornea outset thus eliminating potential toxic allergic reactions the agent tested regard confirmed hypothesis carboxymethyl cellulose cmc anionic polymer found certain lubricants refresh allergan irivne ca cmc water soluble synthetic cellulose derivative marked hydrophilic bioadhesive properties promote faster repair eye surface this study aims evaluate performance sodium hyaluronate sh way in fact believed use product instilled lens insertion would chemically neutralize biocide effects ocular surface helping alleviate initial long term discomfort associated contact lens wear sh viscoelastic agent used several eye care products topically surgery protect maintain integrity ocular tissues sixty four patients recruited among 4 optometric offices greater montreal area selected representative average optometric practice north america based number type patients seen one optometrist per office designated appropriately trained conduct study order maximize interobserver agreement rate training coordination study responsibility l'cole d'optomtrie de l'universit de montral regular contacts email phone maintained study university coordinator lm associate researchers this study received approval ethics committee health sciences universit de montral inclusion exclusion criteria subjects listed table 1 enrolled subjects asked observe wash period 72 hours without contact lenses beginning study subjects established wearers least 3 months silicone hydrogel lenses comfilcon coopervision fairport ny subjects randomly assigned four groups based care regimen used length study complete amo abbott irvine ca b opti free replenish alcon forth worth tx c renu fresh b&lomb rochester ny clear care ciba vision duluth ga once recruited subject provided new pair contact lens care regimen steps reviewed according manufacturer recommendations there one base curve available lens limiting customization fit however every lens fitted centered offer movement 0.2 mm maximum upon blinking expose limbus manner gaze half subjects told wear lens month daily wear basis use drops prior insertion lenses for half told instill sodium hyaluronate based comfort drop blink contacts abbott amo irvine ca lens eye insertion nothing said subjects potential clinical effects product justify use insertion subjects seen back control exam assessed 1-month visit case history performed identify subjects level compliance protocol answer questions might raised visual acuity measured using etdrs high contrast chart dim standard illumination a slit lamp exam performed evaluate conjunctival corneal staining conjunctival hyperemia tear break time tbut efron grading scale used evaluate presence severity clinical signs grades varying 0 absence 4 severe fluorescein observed using cobalt blue filter yellow filter kodak wratten filter number 12 reveal contrasts the filters also used evaluate tbut measured 2 times averaged the first questionnaire used study contact lens dry eye questionnaire cldeq it consists 36 questions specific symptoms ocular dryness related contact lens wear there nine symptom subscales discomfort dryness visual changes soreness irritation grittiness scratchiness foreign body sensation burning photophobia finally itching subscales occurrence symptom was also evaluated term intensity different times 2 hours insertion middle end wearing period questionnaire already validated sensitive specific predicting diagnosis dry eye associated wearing contact lenses in addition cldeq questionnaire subjects asked complete brief survey contact lens experience last month this made forced choice questionnaire subject click box select best answer questions questionnaire related subjective evaluation comfort lenses different times day morning afternoon evening subjects select 5 options comfortable score 5 uncomfortable score 1 the 1-month visit ended review care regimen procedures instructions subject wear lenses least 8 hours day 5 days week sleep take nap wearing contact lenses new pair lenses was provided subjects asked observe 48 h00 wash period time without lenses worn next month subjects use sh asked put drop sodium hyaluronate- sh- based product blink contacts amo abbott irvine ca lens insertion the ones cease use drop subjects seen month later final visit clinical procedures repeated the results provided without use sh drops considered comparison end second visit the second questionnaire slightly different first one additional question care regimen would prefer use future without sh drops one file rejected due missing clinical data one visit another one discarded subject admitted worn lenses extended wear basis month a total 61 patients completed study successfully 18 males 30% 43 females 70% the mean subject age 25.0 7,3 years old range 1835 beginning study subjects reported spending 5 hours day average 3 hours work 2 hours outside computer work only 5/61 8.1% received positive diagnosis dry eye past 19 31.1% self reported suffering ocular dryness periodically others 37 subjects60.6% uncertain issue symptoms past fifteen subjects assigned group thirteen group b seventeen group c sixteen group d. based population sample characteristics group found similar following visit 1 v1 ) is related clinical results obtained subject free sh drops visit 2 v2 represents results obtained usage average total 76 22.00 hours wear week estimated v1 slightly less 81 29.50 hours week v2 however difference considered statistically significant test p 0.12 table 2 shows results corneal conjunctival staining conjunctival hyperemia tear break time tbut more subjects 41 versus 38 showed staining use drops also 50% fewer subjects showed clinically significant staining grades 2 3 usage the differences presence corneal staining show tendency statistical significance f(2,122 0.6323 p 0.05 care regimen associated significant increase corneal staining conjunctival staining grade 1 noticed temporal nasal quadrants 24% subjects visit 1 17% visit 2 this ocular sign markedly reduced use sh drop insertion lens there increase 50% subjects 23 versus 14 showing hyperemia v2 compared v1 clinically significant hyperemia grade 2 also markedly reduced 10 subjects v1 versus 3 v2 moreover 2/3 reduction severity hyperemia observed this difference statistically significant pearson f(2,122 3.1217 p 0.04 r 0.0281 overall average tear break time increased slightly second visit 5.8 3.1 sec 6.3 3.3 sec od 5.8 3.2 sec 6.5 3.5 sec os this difference considered statistically significant od p 0,2372 os p 0,1598 hand paired results individual subject compared v1 v2 increase tbut value considered statistically significant tukey kramer q 1,9801 p 0,05 od q 1,9765 p 0,05 os finally visual acuity remained stable study v1/v2 od 1.1 0.2)/1.0 0.2 os 1.1 0.1)/1.0 0.2 questionnaire kept analysis symptom reported least 10% subjects one visit intensity symptoms analyzed subquestions accordingly discomfort q4 dryness q5 visual changes q6 soreness irritation q7 grittiness scratchiness q8 foreign body sensation q9 burning q10 photophobia q11 itching q12 analyzed grittiness scratchiness q8 foreign body sensation q9 burning q10 photophobia q11 itching q12 reported sufficiently considered analysis table 3 shows results average 10% increase subjects reporting comfortable comfortable wear second visit this difference considered significant chi square f(4,61 5.16 p 0.0353 r 0.118 this result applies regardless care regimen used f(6,61 4.675 p 0.15 looking intensity symptom table 4 greater difference found beginning day 2 hours wear p 0.00 this difference decreases midday p 0.01 end day p 0.03 remains statistically significant every time point the difference highly significant chi square f(4,61 20.2312 p 0.00 strong correlation r 0.3191 showing positive effect use sh drop reducing presence intensity symptom in fact decrease 20% subjects reporting symptom constant visit 2 compared visit 1 the severity symptoms also analyzed cldeq questions 5b c overall marked difference reduction severity eye dryness felt patient q5b chi square f4,61 9.538 p 0.0008 r 0.1847 2 hours wear q5c f2,61 2.8366 p 0,05 r 0.1439 end wearing time q5d f4,61 11.6696 p 0.00 r 0.1999 there respective increase 13% 2 hours 8% mid day 14% end day subjects reporting reduced symptoms v2 compared v1 use sh drop 8 10 patients report symptom intense considering astigmatism play among study subjects exclusion criteria blurry vision considered related tear film instability contact lens dehydration the use sh drop helps reduce almost half number subjects experiencing blurry vision contact lens wear sixty percent subjects study reported feeling symptom using sh drop insertion chi square f4,61 13.964 p 0.00 this result shows strong correlation r 0.2154 verified way care system groups questions 6 b c provide details issue asking noticeable blurry vision different times 2 hours q6b middle day q6c end wearing period q6d there statistical difference subjects first two time points 6d end day 15% patients report symptom use sh drop v2 f4,61 5.4858 p 0.03 even many hours wear result care system used significant differences among groups visit 1 84.7% wearers report symptom never infrequently present 15.3% report frequent constant visit 2 5.1% wearers still experienced symptom this considered significant difference favor use sh drop chi square f1,61 2.1899 p 0.04 r 0.0869 in words 10% subjects experiencing eye soreness irritation became asymptomatic use comfort drop lens insertion this difference present 2 hours wear q7b p 0.90 significant middle day q7c p 0.00 highly significant end wearing time q7d p 0.00 this validated across groups b c a highly significant difference exists visit 1 2 chi square f4,61 14.0198 p 0.00 strong correlation r 0,2463 v1 21% subjects admit removing lenses many times week discomfort 11% report v2 this 50% reduction symptomatic patients use sh based drop lens insertion ocular discomfort ocular dryness respectively number one two reasons identified subjects explain need withdraw lenses looking intensity two symptoms positive effect comfort drop applied insertion found in fact highly significant difference grading severity ocular discomfort ocular dryness q13b item b v1 v2 p 0.00 symptom table 9 shows results significant difference every time point obvious difference evening f4,61 10.943 p 0,00 r 0.1817 we find 10% positive difference favor use sh drops confirming cldeq results over 60% subjects reported comfortable comfortable lens evening 10 hours wear compared 52% without use drops this positive effect already measured afternoon 7% increase number wearers reporting comfortable instead average wear these results indicate subjective positive effect use sh drops starts early wear increases time last visit subjects rated ease use 2 care regimens used past 2 months without sh drops ninety percent 90% subjects rated easy easy care regimen without statistically significant differences the care regimen used v1 rated easy 20% easy 70% second one including drops insertion rated easy 25% easy 65% when asked near 9 10 subjects would recommend care regimen sh drops 58.3% strongly 30% certainly none subjects would recommend system item 4 5 finally 75% subjects would definitely opt system sh drops future see figures 2 3 study aimed evaluate clinical outcome instillation sh drop additional step wearing regimen contact lens wearers contact lens related comfort visual performance day the cross design study established alleviate hawthorne like effects well sequencing effects one could argue lack control group using another type lubricating drop would beneficial even true believe cross design eliminates bias raised helps determine real effect usage sh drop instilled lens insertion the results present study prove sodium hyaluronate based drops added list products help symptomatic contact lens wearers reduce overall discomfort cldeq q4 especially end day q4 subquestions matters even symptoms remain symptom severity greatly reduced use sh drops cldeq q5 sh drops also positive impact subjective visual acuity cldeq q6 there significant reduction blurry vision reported subjects visit 2 means use sh drops probably helps maintain tear film stability also contributes reduce lens dehydration we consider blurry vision represents number 2 reason first one astigmatic patients drop contact lens wear study the use sh drop initiated switch significant percentage wearers symptomatic asymptomatic eye soreness irritation cldeq q7 for subjects discomfort longer reason remove contact lenses prematurely cldeq q13 more 60% wearers reported remaining comfortable lenses evening compared 50% use sh drop insertion comfort questionnaire clinical findings observed slit lamp also improved use sh drops lens insertion observed reduction level corneal staining whereas clinically significant hyperemia levels reduced 50% pairing results indicates improvement tbut subjects compared use sh drop these objective findings correlate subjective reports increased comfort use sh drops part regular care regimen they surprising considering nature sodium hyaluronate known beneficial effects ocular surface restoration namely dry eye patients consequently say use sh drops insertion helps address two major reasons patients cease wear contact lens wear discomfort vision contributes reduction physiological impact contact lens wear this anecdotal represent significant step forward keep patients contact lens market it easy realize impact wearers practitioners contact lens industry one easy additional step lens insertion possible keep 10% wearers contact lenses ongoing basis improve symptoms others 50 60% a single drop put lens insertion considerably change contact lens experience subjects report ocular dryness noncompliance new step certainly big issue instilling one drop lens insertion rated easy large majority subjects 3 4 would recommend wearers relatives it well accepted easy way improve contact lens wear another important aspect study positive outcome use sh drops insertion verified multiple types care systems habitually recommended wearers even subjects using hydrogen peroxide system found positive effect use sh drop lens insertion practitioners therefore hesitate recommend step symptomatic wearers order improve overall contact lens experience sodium hyaluronate drop insertion contact lenses improves overall comfort clinical effect highly significant end wearing period evening its use helps eliminate significantly reduce discomfort symptoms related contact lens wear improve contact lens experience sh drops also help maintain stable vision throughout day possible say use sh based drops blink contacts insertion addresses two major reasons contact lens wearers drop market the use sh drops insertion considered improve overall contact lens performance symptomatic contact lens wearers

purpose . this study aimed to evaluate outcomes from the use of a sodium hyaluronate ( sh)-based comfort drop , instilled before the insertion of contact lenses , in a population of symptomatic contact lens wearers . methods . this was a cross - over , open - label , multi - sites study . subjects were fitted with silicone hydrogel lenses and followed for two months . before insertion of the lenses , sh drops was instilled in the lens for half of the group . the other half did the same on the second month . objectives and subjective outcomes were measured and compared before from baseline with the ones collected after usage of sh drops .

grateful financial support industrial source technology development program ministry knowledge economy 10044909 next generation biogreen 21 program ssac grant pj01111803 rda kribb initiative program south korea

abstractbacterial volatiles protect plants either by directly inhibiting a pathogenic fungus or by improving the defense capabilities of plants . the effect of bacterial volatiles on fungal growth was dose - dependent . a low dosage did not have a noticeable effect on botrytis cinerea growth and development , but was sufficient to elicit induced resistance in arabidopsis thaliana . bacterial volatiles displayed negative effects on biofilm formation on a polystyrene surface and in in planta leaf colonization of b. cinerea . however , bacterial volatile - mediated induced resistance was the major mechanism mediating protection of plants from b. cinerea . it was responsible for more than 90% of plant protection in comparison with direct fungal inhibition . our results broaden our knowledge of the role of bacterial volatiles in plant protection .

surgical intervention corneal perforation indicated anterior chamber reform within short period time herein report successful management small paracentral corneal perforation using autologous iris incarceration tissue adhesive a 41-year old man developed small paracentral corneal perforation 0.5 mm size right eye treating physician attempted remove residual rust ring removal piece metallic foreign body the eye initially managed bandage soft contact lens ameliorate aqueous leakage however without success iatrogenic iris incarceration wound first induced followed application cyanoacrylate tissue adhesive perforated site result anterior chamber immediately reformed maintained cyanoacrylate tissue adhesive iatrogenic incarceration autologous iris effective treating type small corneal perforation this technique simple potentially useful small paracentral corneal perforations outside visual axis without good apposition corneal perforation caused small corneal punctures sometimes managed bandage soft contact lens patching and/or tissue adhesive 2 3 4 however surgical intervention usually indicated anterior chamber reform within short period time herein report successful experience managing small paracentral corneal perforation without good apposition could tamponaded soft bandage lens iatrogenic iris incarceration followed cyanoacrylate tissue adhesive a 41-year old man noted irritation right eye diagnosed piece metallic foreign body right cornea iatrogenic corneal perforation encountered attending physician attempted remove residual rust ring corneal burr slit lamp examination revealed paracentral corneal penetration diameter approximately 0.5 mm fig the anterior chamber flat active aqueous leakage confirmed positive seidel test fluorescein intravenous antibiotics cefozopran hydrochloride initiated conjunction prophylactic topical antibiotic 0.5% levofloxacin three times daily after 48 h treatment significant improvement right eye concerns raised possible complications secondary angle closure glaucoma prolonged hypotony since puncture located paracentrally near visual axis simple primary closure wound corneal patch graft deemed undesirable as wound completely punched injected viscoelastic materials anterior chamber came wound made quite difficult dry surface cornea application tissue adhesive therefore provide necessary support gluing amniotic membrane plugging tried slippery fixed wound a paracentesis port created blunt 27-gauge needle 2-mm bent tip attached viscoelastic injecter was gently inserted anterior chamber paracentesis using bent tip small tug iris care taken touch injure corneal endothelium anterior capsule lens meticulous drying wound incarcerated iris performed tiny amount ethyl-2-cyanoacrylate adhesive aron alpha sankyo co. ltd attention paid ensure glue spread surrounding cornea the corneal puncture thus successfully closed without sutures anterior chamber remained well formed postoperatively patient treated topical 0.1% fluorometholone 0.5% levofloxacin three times daily pupillary distortion observed iris dilated however patient felt visual disturbance herein reported case iatrogenic paracentral corneal perforation without good apposition successfully treated autologous iris incarceration cyanoacrylate adhesive conventionally small corneal perforations treated stitches without corneal patch graft tissue adhesives 2 3 4 recently amniotic membrane fibrin glue the indications use bandage soft contact lens treat corneal laceration include small wounds good apposition good alignment wound edges incarceration prolapse uvea lens corneal puncture wounds less 2.0 mm size usually heal without sutured case wound relatively small less 1 mm apposition wound optimal due sharply demarcated punch made restoring anterior chamber extremely difficult using bandage soft contact lens cases good apposition a small amount viscoelastic materials perforation site often enough allow safe application tissue adhesive without gluing intraocular contents the technique described case appears useful secondary minimal residual scarring related astigmatism a second advantage technique iris introduced perforation site inside eye controlled manner prevent excessive iris prolapse provide stable support tissue adhesive this adequate use amniotic membrane slippery difficult fixed wound furthermore technique requires foreign tissue thereby eliminating risk immunological tissue reactions the tissue adhesive may also theoretically circumvent potential risks epithelial growth preventing corneal epithelium growing directly iris this technique safe effective paracentrally located corneal perforation however significant synechiae involving angle could prove problematic perforation peripheral very small descemet membrane transplantation instead iris incarceration may option situation conclusion application cyanoacrylate adhesive following iatrogenic incarceration iris seems simple effective managing small paracentral corneal perforations without good apposition

backgroundsurgical intervention for corneal perforation is indicated when the anterior chamber does not reform within a short period of time . herein , we report the successful management of a small paracentral corneal perforation using autologous iris incarceration and tissue adhesive.casea 41-year - old man developed a small paracentral corneal perforation ( 0.5 mm in size ) in the right eye , while the treating physician attempted to remove the residual rust ring after removal of a piece of metallic foreign body.observationsthe eye was initially managed with a bandage soft contact lens to ameliorate the aqueous leakage ; however , without success . iatrogenic iris incarceration of the wound was first induced , followed by application of cyanoacrylate tissue adhesive to the perforated site . as a result , the anterior chamber was immediately reformed and maintained . complete corneal epithelialization of the perforation was achieved in 2 months without visual compromises.conclusionscyanoacrylate tissue adhesive with iatrogenic incarceration of the autologous iris was effective in treating this type of small corneal perforation . this technique is simple and potentially useful for small paracentral corneal perforations outside the visual axis and without good apposition .

long recognized psychiatric disorders symptoms aggregate families evidence substantial role genetic factors incontrovertible genetic epidemiological studies autism bipolar disorder schizophrenia show risk developing one specific psychiatric illnesses proportional amount genetic material shared affected individual heritability estimated least 80% disorders 2 4 put context equivalent type i diabetes 80% greater breast cancer parkinson disease the majority psychiatric disorders like common conditions genetically complex psychiatry genetic complexity compounded phenotypic complexity psychiatric diagnosis made basis biological investigation validated common pathogenesis psychiatric disorders autism schizophrenia bipolar disorder therefore effectively groups symptoms making syndromes define groups patients show broadly similar outcomes respond similarly treatment such diagnostic categories therefore likely heterogeneous boundaries somewhat arbitrary autism schizophrenia bipolar disorder traditionally considered separate disease entities although share common behavioral characteristics cognitive deficits the distinction schizophrenia bipolar disorder justified many years reference family studies showing disorders seem breed true however view challenged recent large scale study shown relatives individuals affected schizophrenia increased risks bipolar disorder vice versa definitive genetic epidemiological studies genetic relationship autism disorders lacking although evidence shared genetic factors recent years new molecular genetic findings particularly application genome wide association studies ( gwass genomic technologies 11 14 implicated risk factors disorders allowed possibility genetic relationship explored directly current orthodoxies challenged 8 10 autism spectrum disorders asds autism asperger syndrome rett syndrome developmental psychiatric disorders high heritability past years genetic studies asds consistently identified rare de novo point mutations large structural variants present genes encoding interacting synaptic proteins such studies reported co segregation putative high risk alleles deletions point mutations asds performed called burden analysis different alleles particular gene aggregated frequency cases compared controls initial studies asds using small samples found rare missense point structural mutations copy number variants cnvs x linked neuroligin-3 neuroligin-4 nlgn4 genes 17 19 neuroligins family post synaptic proteins bind trans synaptically family pre synaptic proteins called neurexins although findings interesting incomplete penetrance mutations lack power made results equivocal however missense mutations subsequently identified neurexin-1 nrxn1 gene high frequency individuals autism these suggestive findings augmented results recent genomic studies discussed traditional karyotyping gwass comparative genome hybridization cgh analyses used identify large chromosomal structural losses deletions gains duplications individuals asds 21 24 burden analysis study approximately 200 affected individuals identified two translocation events nrxn1 separate samples one disrupting coding sequence lying 5 gene study 1,000 pedigrees using approximately 10,000 single nucleotide polymorphisms snps identified 300 kb deletion coding exons nrxn1 co segregating autism genome wide analyses also implicated related interacting synaptic protein coding genes etiology asds first study 427 asd cases using approximately 500,000 snps identified 6 mb de novo deletion encompassing nlgn4 270 kb deletion shank3 second burden analyses revealed high frequency point mutations shank3 asd cases third cntnap2 encodes member neurexin family resides juxtaparanodal region myelinated neurons shows evidence common allele association asds well increased burden rare protein coding mutations large de novo deletions the evidence implicating synaptic cell adhesion molecules related proteins asds strong data implicating schizophrenia arguably even stronger whole genome screen large chromosomal abnormalities using array cgh performed 93 individuals schizophrenia identified hemizygous loss nrxn1 one case the deletion exon 1 also present affected sibling deletions nrxn1 observed 372 controls suggesting allele may pathogenic the study also found large 1.4 mb de novo duplication event individual asd spanned apba2 intriguing result given gene encodes protein mint2 binds intracellular domains neurexins further independent cgh study identified rare deletions nrxn1 monozygotic twins diagnosed early onset schizophrenia higher density lower cost genome wide screens using gwas technologies made feasible screen many thousands individuals smaller copy number variations using 300,000 probes across genome assayed approximately 3,000 european cases 10 times many european controls burden analysis revealed many deletions nrxn1 locus the authors reported significant excess protein coding deletions present cases finding replicated similar analysis performed independent sample approximately 3,000 cases european descent 3,000 controls also two studies using smaller sample sizes so far data implicating neuroligins shanks schizophrenia reported however hemizygosity cntnap2 gene encodes member neurexin family contactin associated protein like 2 caspr2 reported schizophrenia also individuals mental retardation although available data provide relatively strong evidence disruption neurexin-1 locus nrxn1 risk factor schizophrenia asds evidence relation bipolar disorder lacking this might reflect relative paucity studies addressing hypothesis could result discontinuity bipolar disorder schizophrenia relation role cnvs see the recent application genome wide technologies shown burden large rare cnvs increased schizophrenia compared controls implicates specific loci studies magnitude yet performed asds although evidence involvement specific cnvs in contrast evidence global burden duplications deletions bipolar disorder substantially less schizophrenia asds specific deletions associated schizophrenia include 22q11.2 1q21.1 15q13.3 also found association mental retardation autism attention deficit hyperactivity disorder 36,41,46 50 15q13.3 also implicated idiopathic generalized epilepsy therefore nrxn1 deletions apparent large cnvs confer risk range neurodevelopmental phenotypes including autism mental retardation schizophrenia however similar evidence lacking bipolar disorder suggestion cnvs might less prominent role phenotype the advent gwas allowed common snp variation human genome tested association first wave studies reported schizophrenia bipolar disorder 54 56 autism several loci implicated genome wide levels statistical significance schizophrenia including znf804a encoding protein zinc finger nucleic acid binding domains major histocompatibility complex mhc region these studies also provided strong evidence genetic overlap schizophrenia bipolar disorder however associations implicate common alleles small effects findings gwass yet clearly suggest specific biological process so far systematic comparisons gwas data asds schizophrenia bipolar disorder however intriguing associations reported voltage gated calcium channel genes across phenotypes 56,58 60 furthermore recent reports association common alleles several gaba receptor genes subtype bipolar disorder schizophrenia implicate loci also reported associated asds 23,63 65 the evidence involvement neurexins nrxn1 neuroligins nlgn4 related proteins shanks shank3 mint2 a2bp1 caspr2 cntnap2 asds substantial growing there also strong evidence implicating genes schizophrenia although examined given overlap possible genes may exerting effects biological pathway common disorders the neurexins family transmembrane proteins extracellular membranous intracellular domains neurexins divided two groups neurexins encoded three genes the neurexins primarily expressed neurons known pre synaptic heterophilic adhesion molecules typically bind across synapse neuroligins the neuroligins represent similar class proteins neurexins binding two types molecule controlled alternative splicing the intracellular domains neurexins neuroligins bind scaffolding proteins assemble large molecular complexes known link synaptic systems receptors ion channels vesicle release machinery neurexins best known ability promote cell adhesion synaptogenesis neuroligins present neighboring cell even non neuronal cell lines it seems neurexins neuroligins necessary excitatory inhibitory synaptogenesis possibly functional synapse maturation it hypothesized neurexins neuroligins involved promiscuous generation many synapses activity dependent pruning however multiple neurexin gene knockout studies mice seem contradict suggest neurexin neuroligin complex essential synapse formation synapse function deletions -neurexin result increased lethality normal synapse number gross anatomy severely impaired synaptic functioning pattern strikingly similar neuroligin gene knockouts such biological roles fit hypotheses etiology autism schizophrenia neurodevelopmental insult adult imbalance excitatory inhibitory neurotransmission occur absence overt macro pathology shank3 implicated autism several lines evidence 23,25,26,68 70 functions post synaptic scaffolding protein binds indirectly neuroligins forming potentially functional circuit neurexin neuroligin shank dysregulated asds the involvement -neurexins pre synaptic neurotransmission suggests functional link voltage gated calcium channels integral pre synaptic function plasticity implicated involved autism schizophrenia bipolar disorder 56,58 60,73 therefore evidence asds schizophrenia bipolar disorder suggests convergence specific processes involved development regulation synaptic transmission further work biology neurexins neuroligins related proteins certainly required seems likely pathogenic roles proteins illuminated human genetic studies whole genome studies many thousands affected individuals uncovering evidence genetic overlap autism schizophrenia bipolar disorder studies cnvs rare alleles found overlap autism schizophrenia whereas common snp variants shown overlap schizophrenia bipolar disorder these findings suggest schizophrenia autism neurodevelopmental disorders may share underlying pathogenic mechanisms challenges view completely unrelated diagnostic entities the findings also support view schizophrenia stronger neurodevelopmental component bipolar disorder suggest lies gradient decreasing neurodevelopmental impairment syndromes mental retardation autism one hand bipolar disorder the identification rare common alleles predisposing prototypically distinct psychiatric disorders provides challenges ways disorders diagnosed researched we argued basis recent genetic data findings point common pathophysiological mechanisms important area future research we based conclusion fact several rare cnvs including deletions nrxn1 associated mental retardation autism schizophrenia overlap common risk alleles seen schizophrenia bipolar disorder 11 13 we propose disorders phenomenologically accept may many genetic environmental risk factors shared phenotypes it clear much future work required equally clear constrained current categorical diagnostic systems such studies explore relationship genes biological variables dimensional measures key domains psychopathology across current diagnostic categories we previously argued need undertake endeavors across functional psychoses schizophrenia bipolar disorder however recent data point need consider broader clinical spectrum includes also autism mental retardation cognitive impairment asd autism spectrum disorder cnv copy number variant cgh comparative genome hybridization gwas genome wide association study nlgn4 neuroligin-4 nrxn1 neurexin-1 snp single nucleotide polymorphism

there is strong evidence that genetic factors make substantial contributions to the etiology of autism , schizophrenia and bipolar disorders , with heritability estimates being at least 80% for each . these illnesses have complex inheritance , with multiple genetic and environmental factors influencing disease risk ; however , in psychiatry , complex genetics is further compounded by phenotypic complexity . autism , schizophrenia and bipolar disorder are effectively syndromic constellations of symptoms that define groups of patients with broadly similar outcomes and responses to treatment . as such the diagnostic categories are likely to be heterogeneous and the boundaries between them somewhat arbitrary . recent applications of whole - genome technologies have discovered rare copy number variants and common single - nucleotide polymorphisms that are associated with risk of developing these disorders . furthermore , these studies have shown an overlap between the genetic loci and even alleles that predispose to the different phenotypes . the findings have several implications . first , they show that copy number variations are likely to be important risk factors for autism and schizophrenia , whereas common single - nucleotide polymorphism alleles have a role in all disorders . second , they imply that there are specific genetic loci and alleles that increase an individual 's risk of developing any of these disorders . finally , the findings suggest that some of the specific genetic loci implicated so far encode proteins , such as neurexins and neuroligins , that function in synaptic development and plasticity , and therefore may represent a common biological pathway for these disorders .

fever common reason seeking medical attention accounts 20% emergency department visits.1 many mothers caregivers perceived phobia associated child fever reasons include serious infection seizure brain damage death.2 paediatricians regularly advised parents seek immediate medical attention children developed fever fever great diagnostic importance especially developing countries infectious diseases malaria pneumonia prevalent.3 developing countries relatively low level literacy financial constraint procuring reliable thermometers many parents rely palpation assess children fever.4 objective use thermometer detect presence fever often limited health care settings resource poor settings an accurate determination absence fever child assures parents saves cost preventing unnecessary investigations medication ownership thermometer seems poor although study environment found support study new york usa 78% caregivers owned thermometers nearly half 48% still utilised tactile method assess fever.4 another study india contrast 15% owned thermometer 23.8% knowing use it.5 underscores fact use palpation means assessing fever mothers common universal majority mothers care givers environment still utilise tactile assessment determine children fever controversies reliability tactile assessment fever variously reported study found accurate enough least younger infants,4 others reported overestimates presence fever.67 reliability use palpation detect fever still remains uncertain this study carried determine reliability tactile assessment fever nigerian mothers under-5 children also determine surface hand used influence accuracy the study ascertained palpation single multiple anatomical sites influence accuracy tactile assessment fever children age 5 years presenting care mothers recruited study ethical approval obtained research ethics committee federal medical centre owerri imo state nigeria commencement study the subjects recruited children outpatient clinic children emergency room hospital information age sex child mother age highest educational qualification heq captured using profoma thereafter eligible child tactile assessment fever mother the response mother regard presence absence fever documented following child axillary temperature measured documented axillary temperature measured using mercury glass thermometer left axilla 5 minutes reading frequencies percentages mean analysed appropriate sensitivity specificity positive predictive values ppvs negative predictive values npvs tactile assessment calculated using axillary temperature 37.5c cut fever the different hand surfaces used palpation well single versus multiple sites palpation also compared there 60 males 53 females giving male female ratio 1.1:1 seven 6.2% mothers primary school education 38 33.6% 68 60.2% secondary tertiary education respectively fever detected touch reported 81 71.6% children mothers 65 57.5% mothers able correctly predict presence fever children there statistical significant difference ability mothers correctly predict fever respect heq 2.66 p 0.265 comparison fever detection palpation mothers highest educational qualification heq irrespective part hand used palpation sensitivity detection fever palpation 82.4% specificity 37.1% the use palmer surface hand better sensitivity 95.2% dorsum hand 69.2% table 2 shows sensitivity specificity ppv npv part hand used palpation sensitivity specificity positive predictive value ppv negative predictive value npv part hand used palpation total 71 62.8% mothers palpated one site remaining 42 37.2% used single site table 3 shows different sites used mothers palpation table 4 compares sensitivity specificity ppv npv use single site versus multiple sites palpation the use multiple sites better sensitivity 86.7% use single site 76.2% sites used mothers palpate fever palpation single site versus multiple sites the present study shows mothers trusted good extent assess presence fever children palpation a sensitivity 82.4% obtained study similar reported okposio abhulimhen iyoha benin nigeria well graneto soglin chicago usa documented sensitivity 89.2% 84% respectively.89 studies nigeria reported higher sensitivity value 96.3% 94.6% respectively.610 specificity 37.1% obtained present study quite poor lower specificities reported workers ranging 64.3% 82%.489 however higher 23% reported akinbami et al.6 heterogeneity methodology studies may responsible differences obtained present study earlier studies for instance akinbami et al.,6 used rectal temperature 38c cut fever wammanda onazi10 adopted axillary temperature 37.2c cut fever present study okposio abhulimhen iyoha8 used axillary temperature 37.5c definition fever although specificity present study poor relatively high sensitivities obtained studies still make maternal palpation presence fever considerably useful method assessment childhood fever although palpation overestimates fever elicitation fever mothers would encourage early presentation documented concern fever strong motivational factor seeking medical care.2 educational qualification mother influence accuracy palpation presence fever it therefore expected mother irrespective educational background able appreciate fever palpation npv 71.9% if mother says child fever history subjective assessment fever without use thermometer 72% reliable the likelihood child adjudged febrile mother truly febrile measured ppv present study 51.9% this higher 39% reported wheybrew co workers.7 higher ppv reported study may influenced prevalence fever present study present study 57.5% study subjects fever study wheybrew et al.,7 27% children fever the palmer surface hand commonly used hand surface mothers palpate fever the sensitivity specificity npv ppv palmer surface higher dorsal surface use surfaces this may attributed fact receptors detection heat temperature concentrated palmer surface finger tips.11 use surfaces may confuse mother sensation obtained likely vary hence reduced sensitivity obtained study compared palmer surface alone the forehead common site used tactile assessment fever study this corroborates reports previous studies also found forehead common anatomical site used tactile assessment fever.4 may possibly explained fact head easily accessible compared parts body it observed palpation multiple sites sensitive use single site this agreement earlier report okposio abhulimhen iyoha well singhi sood.812 implies fever assessed palpation without use thermometer palpation multiple body sites utilised palpation palmer surface hand using multiple sites improves reliability tactile assessment fever clinicians discountenance mothers complaint fever children detected palpation without use thermometer mothers taught palpate multiple sites palmer surface palpating presence fever children improve accuracy

background : many mothers still rely on palpation to determine if their children have fever at home before deciding to seek medical attention or administer self - medications . this study was carried out to determine the accuracy of subjective assessment of fever by nigerian mothers in under-5 children.patients and methods : each eligible child had a tactile assessment of fever by the mother after which the axillary temperature was measured . statistical analysis was done using spss version 19 ( ibm inc . chicago illinois , usa , 2010).result : a total of 113 mother / child pairs participated in the study . palpation overestimates fever by 24.6% . irrespective of the surface of the hand used for palpation , the sensitivity , specificity , positive predictive value ( ppv ) and negative predictive value ( npv ) of tactile assessment were 82.4% , 37.1% , 51.9% and 71.9% , respectively . the use of the palmer surface of the hand had a better sensitivity ( 95.2% ) than the dorsum of the hand ( 69.2% ) . the use of multiple sites had better sensitivity ( 86.7% ) than the use of single site ( 76.2%).conclusion : tactile assessment of childhood fevers by mothers is still a relevant screening tool for the presence or absence fever . palpation with the palmer surface of the hand using multiple sites improves the reliability of tactile assessment of fever .

pancreatic ductal adenocarcinoma pdac represents fourth leading cause cancer mortality worldwide incidence approximately 217,000 new cases year nearly matched 213,000 deaths parkin et al 2001 several frequent genetic events underlying initiation progression human pancreatic cancer identified hezel et al 2006 maitra hruban 2008 these include activating mutations kras proto oncogene occur 90% pdac caldas kern 1995 considered key driver pancreatic carcinogenesis mutations inactivating tp53 gene occur 5075% patients redston et al 1994 moreover several lines evidence implicate mutations inactivating brca2 tumour suppressor estimated 520% familial pdac hahn et al 2003 couch et al 2007 germline carriers deleterious brca2 mutations commonly truncate encoded protein exhibit increased lifetime risk developing pdac addition well known predisposition cancers breast ovary breast cancer linkage consortium 1999 within high risk pancreatic cancer kindreds inherited mutations brca2 represent frequently encountered germline genetic alteration hahn et al 2003 the incidence germline brca2 mutations apparently sporadic pancreatic cancers may high breast ovarian cancer goggins et al 1996 more recently palb2 encodes brca2-interacting protein also essential homology directed dna repair emerged pancreatic cancer susceptibility allele jones et al 2009 three new transgenic models pancreatic adenocarcinoma associated brca2 inactivation recently described skoulidis et al one models incorporate activation kras oncogene feldmann et al 2011 in contrast two models skoulidis et al 2010 rowley et al 2011 use conditional gene targeted allele developed tuveson jacks colleagues jackson et al 2001 ; johnson et al 2001 tissue specific activation oncogenic krasg12d driven single allele loxp cre mediated recombination mimicking genetic event frequently triggers kras activation human cancers cre recombinase expression controlled pdx1 promoter expressed e8.5 required organogenesis pancreas whereby loss gene associated absence pancreatic formation jonsson et al 1994 offield et al the expression pdx1-cre transgene therefore occurs throughout pancreatic cellular compartment albeit stochastic manner trigger kras activation hingorani et al patients carry germline mutations affecting brca2 harbour germline mutant allele somatic tissues whereas second brca2 allele wildtype wooster et al 1994 it widely believed loss second wild type brca2 allele nascent cancer cells termed loss heterozygosity loh necessary emergence tumours germline mutation carriers pancreatic cancer model developed laboratory skoulidis et al 2010 ) it carries somatic tissues truncated allele murine brca2 brca2 truncates gene evolutionarily conserved functionally critical region encoded exon 11 resembling deleterious germline mutations found human carriers friedman et al 1998 the second brca2 allele brca2 jonkers et al 2001 conditionally disrupted remove exon 11 loxp cre recombination pancreas this event driven pdx1-cre therefore occurs tissues undergo kras activation there evidence brca2 brca2 alleles express truncated protein product patel et al 1998 ; notably models developed rowley et al feldmann et al both exclusively use strain homozygous conditional brca2 allele thus germline heterozygosity brca2 modelled experiments all three new pancreatic cancer models incorporate conditional alleles inactivate tp53 pancreas mimic frequent loss tumour suppressor human pancreatic cancers somewhat different tp53 alleles used group important distinction given nature tp53 mutations thought affect pancreatic cancer development olive et al 2004 two studies employ gain function mutants affecting single allele these either structural mutant tp53 feldmann et al 2011 contact mutant tp53 skoulidis et al 2010 both tp53 mutants associated development carcinomas olive et al 2004 in contrast third study uses null allele tp53 wherein exons 210 deleted pdx1-cre activation rowley et al 2011 manner less faithfully represents human cancer associated mutations thus clear foregoing three recently published models pancreatic carcinogenesis associated brca2 inactivation harbour important differences tissue specificity nature timing mutant tp53 brca2 alleles also presence mutant kras we believe distinctions vital understanding marked differences pancreatic carcinogenesis observed studies key findings highlighted table 1 brca2 believed follow classical two hit paradigm tumour suppression smith et al 1992 collins et al initial studies soon discovery brca2 reported consistent inactivation wild type brca2 allele loss heterozygosity loh breast ovarian cancer cells mutation carriers collins et al 1995 gudmundsson et al 1995 engendering widely accepted view brca2 loh essential event carcinogenesis a notes dissent emerged later studies king et al 2007 willems et al 2008 gained widespread attention context unexpectedly reveal brca2 heterozygosity promotes pancreatic cancer development mice men tp53 wildtype tp53 cohorts murine model heterozygosity brca2 brca2 genotype acts kras accelerate progression development pdac a similar conclusion reached studies small number human pancreatic cancer samples carriers icelandic founder mutation brca2 allele brca2 5 bp deletion exon 9 causes frame shift leading expression short unstable protein product mikaelsdottir et al 2004 one possibility genotype causes mutator phenotype owing defects dna repair arising known role brca2 homologous dna recombination patel et al 1998 ; however previous studies murine embryo fibroblasts mefs heterozygous brca2 reveal statistically significant effects sensitivity genotoxic agents patel et al neither brca2 mice friedman et al 1998 strains heterozygous brca2 truncation mutants connor et al 2001 yan et al 2004 exhibit cancer predisposition notably lacz mutation reporter gene boerrigter et al 1995 incorporated germline mice heterozygous brca2 truncation similar identical brca2 tutt et al 2002 reveals evident mutator phenotype hand mefs strain showed mild alteration dna repair kinetics recovery 4gy ionizing radiation thus little convincing evidence heterozygosity brca2 mutant alleles creates dna repair defect could explain heightened cancer predisposition although possibility yet conclusively excluded connection important note cellular approaches yet account cooperative effect mutant kras pancreatic carcinogenesis associated brca2 heterozygosity suggested murine model developed skoulidis et al even subtle increase mutational load induced brca2 heterozygosity mutant kras expressing cells might undetectable cellular experiments significant vivo could plausibly accelerate progression pre malignant pancreatic intra epithelial panin lesions occur frequently even apparently normal pancreatic parenchyma hruban et al 2008 overt malignancy whether different brca2 alleles behave manner similar brca2 clear like brca2 however instability truncated protein encoded brca2 mikaelsdottir et al 2004 suggests haploinsufficiency brca2 opposed trans dominant effect mutant brca2 protein accounts phenotypic effects heterozygosity patients carry icelandic founder mutation describe heterozygous effect brca2 cohorts despite presence mutant kras interpretation difference straightforward since brca2 allele engenders brca2 loss cells expressing pdx1-cre unlike brca2 expressed somatic cells this raises possibility non cell autonomous effects brca2 heterozygosity example stromal cells rather nascent cancer cells may account cancer predisposing effect brca2 allele mitotic functions also ascribed brca2 interestingly defects g2 checkpoint function menzel et al 2011 mitotic checkpoint enforcement choi et al 2012 completion cell division cytokinesis daniels et al 2004 ; whether roles brca2 may explain effect heterozygosity tumour development yet explored heterozygosity brca2 allele enough trigger cytokinetic defects mefs daniels et al 2004 unclear whether mitotic functions perturbed brca2 heterozygosity importantly recent data human studies support brca2 heterozygosity enough promote carcinogenesis breast cancers incomplete loss remaining wild type allele observed using techniques sensitive applied original studies king et al 2007 importantly large scale unbiased genomic sequencing high grade serous ovarian carcinomas highlighted retention wild type allele end stage disease 25% germline brca2 carriers atlas 2011 furthermore detailed study prostate tumour progression brca2 germline mutation carriers uncovered loh high grade prostatic intraepithelial neoplasias considered precursor lesions development prostate adenocarcinoma 55% malignant tumours analysed willems jones et al 2012 collectively data suggest cancers arising germline brca2 mutation carriers frequently fail exhibit loss wildtype allele failure exhibit loh occurs brca2-mutant cancers several different tissues interestingly conclusions set emerging backdrop ongoing studies tissue samples patients familial forms pancreatic cancer study 58 pancreatic intra epithelial neoplasms intraductal papillary mucinous neoplasms reveals somatic losses brca2 copy number infrequent hong et al 2012 however definitive evidence addressing extent lessons gemms brca2-deficient pancreatic cancers applied human neoplasia awaits results extensive genome sequencing studies pancreatic cancer samples patients harbouring germline brca2 mutations murine pancreatic cancers emerging brca2 strains brca2 alleles inactivated pdx1-cre expressing cells exhibit preponderance acinar cell carcinoma histology correspondingly 3 four human pancreatic cancers brca2 mutation carriers exhibited loh also acinar type skoulidis et al 2010 normally accounts 12%% human pancreatic cancers hruban 2007 this raises possibility genotypes promote evolution acinar cell carcinomas rather pdac ( 2011 also observe differences histopathological spectrum pancreatic malignancies mice brca2 well tp53 inactivated compared tp53 deficiency alone these observations raise possibility nature brca2 mutations timing coincidence alterations tp53 may alter histopathological evolution pancreatic cancers mice however observations remain limited allow firm conclusions drawn draw attention simply emphasize need studies we others shown patel et al 1998 lee et al 1999 tutt et al 1999 genome wide dna damage follows homozygous inactivation brca2 leads checkpoint activation cell cycle arrest rather unrestrained cellular proliferation typical cancer we previously proposed venkitaraman 2009 checkpoint inactivation may therefore essential pre requisite homozygous brca2 inactivation loh carcinogenesis the work skoulidis et al provides strong vivo evidence hypothesis supported observations rowley et al murine models bi allelic brca2 inactivation leads loss exocrine pancreatic parenchyma concomitant increase adipose tissue progressive loss organ functionality further demonstrate pancreatic insufficiency preceded widespread occurrence dna double strand breakage marked h2ax staining cells lacking copies brca2 moreover skoulidis et al rowley et al find concomitant inactivation tp53 function prevents pancreatic insufficiency allows rapid pdac development pancreas mice carrying bi allelic mutations inactivating brca2 when observations studies synthesized picture emerges wherein brca2 heterozygosity germline mutation carriers may suffice allow development kras driven pdac later inactivation tp53 checkpoint genes may allow eventual loss second brca2 allele although loh obligate step may promote emergence advanced cancers indeed inferences small study 5 samples human pancreatic cancer patients support scenario although remains firmly established results suggest brca2 heterozygosity suffices pdac formation driven mutant kras mice men however rationale use targeted agents parp1 inhibitors parp1i brca2-deficient cancers contingent upon bi allelic brca2 inactivation tumour cells bryant et al therefore confirmed work skoulidis et al 2010 pdac cells retain functional brca2 allele resistant parpi astrazeneca compound olaparib thus parp1 inhibitors reserved clinical use brca2 loh verified tumour assessment emerges critical requirement design human clinical trials treatment brca2-deficient cancers these findings exemplify new generation gemms pdac may represent valuable surrogate models preclinical tests therapeutic efficacy patients importantly models allow vivo proof new therapeutic concepts may also provide platform assess pharmacodynamic pharmacokinetic properties new agents although species specific differences may limit interpretations the models also provide flexible method assess impact therapy tumour progression using adapted multimodal imaging drug bioavailability including tissue drug penetrance analyses an important feature determines particular gemm useful preclinical platform model recapitulates similar clinical response standard therapy agents clinical use human for instance kpc mouse model relatively unaffected gemcitabine similar small clinical benefit agent advanced pancreatic cancer setting humans olive et al 2009 each gemm likened patient particular tumour type hence enrolled preclinical trial novel agents eklund et al 2013 guerra barbacid 2013 such trials facilitated use adapted imaging techniques monitor tumour development progression such utility beginning impact clinical setting humans early phase clinical trials shown promise combination nanoparticle albumin linked paclitaxel nab paclitaxel gemcitabine advanced pdac frese colleagues used kpc mouse model pancreatic cancer provide mechanistic understanding synergistic effect combination paclitaxel appears inhibit breakdown gemcitabine modulation degradative enzyme cytidine deaminase reactive oxygen species dependent pathway frese et al 2012 such mechanistic analyses may help rationalize clinical strategy using drug combination for instance nab paclitaxel used inducting agent followed gemcitabine enhance tumouricidal effect latter rational clinical trials man likely benefit incorporation vivo component provides relatively rapid feedback predicted response new agents preclinical assessment using gemms either used screen potentially useful therapeutic agents progressing clinical trials alternatively critically assess mechanisms action vivo agent found effective small scale trial progressing larger phase iii clinical trial one potential advantage gemms unlike human trials allow sequential sampling appropriate tumour tissues assess pharmacodynamic impact particular agent pancreatic cancer several novel agents targeting diverse range molecular pathways tested gemms complement early phase clinical trials olive et al 2009 plentz et al 2009 cook et al the results trials due course affirm refute value pdac gemms predictive tool clinical efficacy human cancers the potential value pdac gemms surrogates preclinical testing new therapies critically dependent closely models mimic human pdac several studies hingorani et al 2003 tuveson hingorani 2005 olive et al 2009 plentz et al 2009 cook et al 2012 emphasized similarities histopathology cancer progression clinical behaviour even drug pharmacodynamics pdac gemms human pdacs initial observations suggest murine kpc pdacs bear resemblance human disease insofar exhibit high degree genomic instability evident multiple non reciprocal chromosomal translocations hingorani et al 2005 however emerging data large scale sequencing human pdac tumours biankin et al 2012 imperative validate gemms genomic level compare genomic landscapes murine human tumours pdac gemms incorporate high penetrance genetic events initiating oncogenes inactivated tumour suppressor genes early stage large number susceptible cells resulting stereotypy malignancies arising therein may reflect heterogeneity likely present human cancers importantly genetic heterogeneity human cancers may give rise differing therapeutic responses particular agent due differing genetic epigenetic signatures constituent cells it conceivable individual tumours take differing genetic routes achieve tumoural progression depending type initiating genetic lesions secondary genetic hits occur stochastically it presumed inactivation genes involved maintaining genomic stability e.g. brca2 models pancreatic cancer may promote stochastic acquisition genetic consequent morphologic heterogeneity due expected increase mutation rate however point remains established future studies also important implications potential value gemm models testing new therapeutic approaches pdac

chromosomal instability is a hallmark of human cancer cells , but its role in carcinogenesis remains poorly resolved . insights into this role have emerged from studies on the tumour suppressor brca2 , whose inactivation in human cancers causes chromosomal instability through the loss of essential functions of the brca2 protein in the normal mechanisms responsible for the replication , repair and segregation of dna during cell division . humans who carry heterozygous germline mutations in the brca2 gene are highly predisposed to cancers of the breast , ovary , pancreas , prostate and other tissues . here , we review recent studies that describe genetically engineered mouse models ( gemms ) for pancreatic cancer associated with brca2 mutations . these studies not only surprisingly show that brca2 does not follow the classical knudson two hit paradigm for tumour suppression , but also highlight features of the interplay between tp53 inactivation and carcinogenesis in the context of brca2 deficiency . thus , the models reveal novel aspects of cancer evolution in carriers of germline brca2 mutations , provide new insights into the tumour suppressive role of brca2 , and establish valuable new preclinical settings for testing approaches to pancreatic cancer therapy ; together , these features emphasize the value of gemms in cancer research .

central venous catheters cvc often used intensive care unit icu monitoring treatment critically ill patients provide long term venous access however use may result nosocomial catheter related bloodstream infection crbsi the estimated number crbsi approximately 250,000 annually united states 24 incidence approximately 1.65 infections per 1000 central line days reported 6.8 infections per 1000 central line days asia indeed crbsi associated 2.27-fold increased risk mortality icu host factors chronic illness bone marrow transplantation immune deficiency malnutrition total parenteral nutrition previous history crbsi old age skin trauma increase risk crbsi addition catheter factors also play role crbsi onset duration catheterization type catheter conditions insertion insertion site care skill person inserted catheter 1013 cases crbsi empiric antibiotic therapy must started soon possible must based clinical characteristics patients catheter removal necessary cases severe sepsis hemodynamic instability endocarditis erythema bacteremia persisting 72 hours antibiotic therapy once responsible organism identified tailored antibiotic therapy may started usually lead better outcomes however little known risk factors early crbsi onset data available clinical manifestations mortality crbsi china therefore aim present study assess risk factors catheter characteristics 73 cases nosocomial crbsi icu january 2010 december 2012 we analyzed clinical features treatment outcomes provide evidence early diagnosis treatment crbsi well risk factors early crbsi onset this study performed taizhou hospital integrated traditional chinese western medicine zhejiang zhejiang wenlin china patients hospitalized icu january 2010 december 2012 the ethics committee hospital approved study waived requirement individual consent patients included 1 developed bacteremia fungemia cvc within 48 h cvc removal 2 fever 38c shivering hypotension 3 apparent source infection in addition least one following criteria met 1 pathogenic agent isolated catheter tip peripheral venous blood least detected semi quantitative method catheter fragment contained 15 bacteria cfu quantitative method catheter fragment contained 10 bacteria cfu 2 intra catheter peripheral blood culture positive bacterial colony count intra catheter blood least 3 times greater compared peripheral blood 3 time report positive intra catheter blood culture least 2 h earlier positive peripheral blood culture patients excluded 1 clinical manifestation bloodstream infection 2 causes bloodstream infection could excluded the clinical data patients nosocomial crbsi collected analyzed accordance 2009 clinical practice guidelines diagnosis management intravascular catheter related infection infectious diseases society america idsa patients age sex reason icu admission underlying diseases time position indwelling cvc type cvc pathogenic identification treatment prognosis mortality attributable crbsi reviewed timely removal catheter defined cvc removal within 12 h fever onset appropriate antibiotic use defined empirical use antibiotics confirmed covered pathogenic bacteria drug sensitive test later patients timely cvc removal appropriate antibiotic use assigned group a. patients timely cvc removal inappropriate antibiotic use assigned group b. patients untimely cvc removal appropriate antibiotic use assigned group c. finally patients untimely cvc removal inappropriate antibiotic use assigned group d. continuous data expressed means standard deviation sd inter group comparison measured data performed using analysis variance anova tests used post hoc analysis categorical data presented proportions compared using chi square tests using segmentation approach compare group pairs the risk factors early crbsi evaluated using univariate multivariate logistic regression analyses this study performed taizhou hospital integrated traditional chinese western medicine zhejiang zhejiang wenlin china patients hospitalized icu january 2010 december 2012 the ethics committee hospital approved study waived requirement individual consent patients included 1 developed bacteremia fungemia cvc within 48 h cvc removal 2 fever 38c shivering hypotension 3 apparent source infection in addition least one following criteria met 1 pathogenic agent isolated catheter tip peripheral venous blood least detected semi quantitative method catheter fragment contained 15 bacteria cfu quantitative method catheter fragment contained 10 bacteria cfu 2 intra catheter peripheral blood culture positive bacterial colony count intra catheter blood least 3 times greater compared peripheral blood 3 time report positive intra catheter blood culture least 2 h earlier positive peripheral blood culture patients excluded 1 clinical manifestation bloodstream infection 2 causes bloodstream infection could excluded the clinical data patients nosocomial crbsi collected analyzed accordance 2009 clinical practice guidelines diagnosis management intravascular catheter related infection infectious diseases society america idsa patients age sex reason icu admission underlying diseases time position indwelling cvc type cvc pathogenic identification treatment prognosis mortality attributable crbsi reviewed timely removal catheter defined cvc removal within 12 h fever onset appropriate antibiotic use defined empirical use antibiotics confirmed covered pathogenic bacteria drug sensitive test later patients timely cvc removal appropriate antibiotic use assigned group a. patients timely cvc removal inappropriate antibiotic use assigned group b. patients untimely cvc removal appropriate antibiotic use assigned group c. finally patients untimely cvc removal inappropriate antibiotic use assigned group d. inter group comparison measured data performed using analysis variance anova tests used post hoc analysis categorical data presented proportions compared using chi square tests using segmentation approach compare group pairs the risk factors early crbsi evaluated using univariate multivariate logistic regression analyses the 73 crbsi patients included 42 males 31 females mean age 63.221.3 years range 4387 years the mean time cvc indwelling 20.89.2 days range 739 days 64.3% participants cvc indwelling 14 days there 27 20 15 11 patients assigned groups b c respectively there 21 patients 28.8% 3 types underlying diseases 43 patients 4 types underlying diseases 9 patients 12.3% 5 types underlying diseases there 23 cases 31.5% 3 high risk factors crbsi 50 cases 68.5% 4 high risk factors univariate logistic regression analysis revealed acute physiology chronic health evaluation ii apache ii score 23 parenteral nutrition transfusion blood products age 65 years diabetes 3 types underlying diseases renal insufficiency multiple organ dysfunction syndrome mods immunosuppression chemoradiotherapy correlated occurrence crbsi within 14 days cvc indwelling table 2 the factors statistically significant p<0.05 univariate analyses included multivariate analysis apache ii score 20 3 types underlying diseases independent risk factors crbsi occurring within 14 days cvc indwelling table 3 the types cvc position cvc indwelling listed table 1 duration cvc indwelling crbsi occurrence shown table 4 femoral cvc showed shortest time crbsi onset 14.25.1 days followed internal jugular cvc 20.98.0 subclavian cvc 33.83.9 p<0.05 time crbsi onset longer single lumen cvc compared dual lumen cvc 33.73.9 vs. 16.96.3 days p<0.05 the common pathogens gram positive bacteria followed gram negative bacteria fungi candida strains infection associated methicillin resistant staphylococcus aureus mrsa methicillin resistant coagulase negative staphylococci mrcons observed 32 cases infection extended spectrum -lactamase esbl)-producing bacteria observed 15 cases vancomycin resistant enterococci vre infection seen 5 cases carbapenem resistant gram negative bacteria crgnb detected 13 cases table 5 all 73 crbsi patients manifested fever body temperature ranging 38.1 40.3c including 20 cases 27.3% body temperature 38.139c 53 cases 77.7% body temperature 39c routine blood test showed mean white blood cell count 19.69.310/l range 12.130.210/l mean neutrophile count 87.38.5% range 8291.2% / l subjects 39 cases 53.4% crp levels 165 mg l abnormal liver function mild moderate elevation alt ast levels detected 17 cases 23.2% abnormal renal function except 6 cases renal failure enrollment study found 27 cases 36.9% highest serum creatinine level 671 mol l circulation failure observed 23 cases 31.5% 27 cases 50.9% found complicated mods redness swelling site indwelling catheter found 14 cases 19.1% including 7 cases 9.7% local purulent secretions the major treatment patients crbsi cvc removal use effective antibiotics we observed 47 cases cvc removed within 12 h blood sampling showed rapid drop body temperature following cvc removal seventeen cases cvc removed report positive blood culture cvc removed 9 cases empirical antibiotic therapy administered subjects confirmation positive blood culture continued positive blood culture confirmed replaced another antibiotic the survivors showed mean time defervescence 6.92.1 days mean time negative conversion blood culture 6.42.4 days table 6 the 73 crbsi patients included 42 males 31 females mean age 63.221.3 years range 4387 years the mean time cvc indwelling 20.89.2 days range 739 days 64.3% participants cvc indwelling 14 days there 27 20 15 11 patients assigned groups b c respectively there 21 patients 28.8% 3 types underlying diseases 43 patients 4 types underlying diseases 9 patients 12.3% 5 types underlying diseases there 23 cases 31.5% 3 high risk factors crbsi 50 cases 68.5% 4 high risk factors univariate logistic regression analysis revealed acute physiology chronic health evaluation ii apache ii score 23 parenteral nutrition transfusion blood products age 65 years diabetes 3 types underlying diseases renal insufficiency multiple organ dysfunction syndrome mods immunosuppression chemoradiotherapy correlated occurrence crbsi within 14 days cvc indwelling table 2 the factors statistically significant p<0.05 univariate analyses included multivariate analysis apache ii score 20 3 types underlying diseases independent risk factors crbsi occurring within 14 days cvc indwelling table 3 the types cvc position cvc indwelling listed table 1 duration cvc indwelling crbsi occurrence shown table 4 femoral cvc showed shortest time crbsi onset 14.25.1 days followed internal jugular cvc 20.98.0 subclavian cvc 33.83.9 p<0.05 time crbsi onset longer single lumen cvc compared dual lumen cvc 33.73.9 vs. 16.96.3 days p<0.05 the common pathogens gram positive bacteria followed gram negative bacteria fungi candida strains infection associated methicillin resistant staphylococcus aureus mrsa methicillin resistant coagulase negative staphylococci mrcons ) infection extended spectrum -lactamase esbl)-producing bacteria observed 15 cases vancomycin resistant enterococci vre infection seen 5 cases carbapenem resistant gram negative bacteria crgnb detected 13 cases table 5 all 73 crbsi patients manifested fever body temperature ranging 38.1 40.3c including 20 cases 27.3% body temperature 38.139c 53 cases 77.7% body temperature 39c routine blood test showed mean white blood cell count 19.69.310/l range 12.130.210/l mean neutrophile count 87.38.5% range 8291.2% / l subjects 39 cases 53.4% crp levels 165 mg l abnormal liver function mild moderate elevation alt ast levels detected 17 cases 23.2% abnormal renal function except 6 cases renal failure enrollment study found 27 cases 36.9% highest serum creatinine level 671 mol l circulation failure observed 23 cases 31.5% 27 cases 50.9% found complicated mods redness swelling site indwelling catheter found 14 cases 19.1% including 7 cases 9.7% local purulent secretions the major treatment patients crbsi cvc removal use effective antibiotics we observed 47 cases cvc removed within 12 h blood sampling showed rapid drop body temperature following cvc removal seventeen cases cvc removed report positive blood culture cvc removed 9 cases empirical antibiotic therapy administered subjects confirmation positive blood culture continued positive blood culture confirmed replaced another antibiotic the survivors showed mean time defervescence 6.92.1 days mean time negative conversion blood culture 6.42.4 days table 6 the aim present study investigate clinical characteristics treatment prognosis crbsi intensive care unit icu chinese center well risk factors early crbsi among 73 crbsi patients enrolled present study major risk factors included advanced age long term catheter indwelling parenteral nutrition diabetes apache ii score 23 3 types underlying diseases multivariate analysis showed acute physiology chronic health evaluation ii apache ii score 20 3 types underlying diseases independent factors associated crbsi occurring within 14 days cvc indwelling advanced age long term catheter indwelling parenteral nutrition diabetes apache ii score 23 3 types underlying diseases well known risk factors crbsi patients advanced age multiple underlying diseases diabetes usually show decreased immune function our findings showed significantly subjects catheter indwelling 14 days compared catheters indwelling 14 days the prolongation catheterization one day reported associated 1.8-fold increased risk crbsi early crbsi may serious implications patients since interfere patient treatment the results present study showed poor patient condition indicated apache ii score high number disease types independently associated early crbsi onset therefore prevention crbsi priority critically ill patients complicated diseases indeed mortality rate crbsi reported 8.8% subjects aged 65 years 3.8% aged 65 years indeed large proportion patients high apache ii scores large number concomitant diseases accordingly previous study showed mortality rate due crbsi icu 28.1% critically ill patients the present study demonstrated position cvc indwelling closely associated time crbsi onset the longest mean duration catheterization found subclavian vein crbsi onset followed internal jugular vein the shortest duration observed femoral vein observed previous studies the skin subclavian vein flat easier clean disinfect resulting late crbsi onset accordingly subclavian vein recommended first choice site catheterization idsa guidelines followed internal jugular vein femoral vein in addition crbsi occurred later using single lumen catheter double lumen catheter indeed significantly higher occurrence crbsi observed double lumen catheters compared single lumen catheters present study common pathogens causing crbsi gram positive bacteria followed gram negative bacteria fungi cons predominant gram positive bacteria followed mrsa 85.7% staphylococci resistant methicillin 41.7% enterococci resistant vancomycin the common gram negative bacteria acinetobacter baumannii followed pseudomonas aeruginosa esbls producing bacteria 80.0% 68.4% respectively resistant carbapenem antibiotics the high prevalence antibiotic resistant bacteria may associated wide use broad spectrum antibiotics icu candida albicans constituted 42.9% candida infections systematic review eligible studies related crbsi associated parenteral nutrition published 1970 march 2012 major pathogens staphylococcus spp followed gram negative bacillus candida spp consistent current study findings however distribution causal agents present study slightly different observed united states therefore choice empiric treatment made according possible geographical differences empirical selection antibiotics based patient clinical profile recent history antibiotic use drug resistance allergy underlying diseases clinical epidemiology health facility in addition adequate anti infective therapy crbsi administered prevent complications inflammatory deep venous thrombosis infectious endocarditis the number patients small single center no control group included preventing us studying risk factors crbsi china however present study first identify risk factors early crbsi onset apache ii score 20 presence 3 types diseases associated earlier crbsi onset chinese center critically ill patients icu particularly monitored appearance crbsi

backgroundcatheter - related bloodstream infection ( crbsi ) is a life - threatening condition encountered in patients with long - term central venous catheter ( cvc ) indwelling . the objective was to investigate the clinical characteristics , treatment , and prognosis of crbsi in the intensive care unit ( icu ) in a chinese center , as well as the risk factors for early crbsi.material/methodsa total of 73 crbsi patients were retrospectively studied in relation to patients clinical and epidemiological data , microbiological culture , and treatment . patients were treated at the taizhou hospital of integrated traditional chinese and western medicine in zhejiang ( zhejiang wenlin , china ) between january 2010 and december 2012.resultsin this chinese center , the most common pathogens were gram - positive cocci , followed by gram - negative bacilli and fungi . a high prevalence of antibiotic - resistant pathogens was detected , and a higher percentage of non - candida albicans spp . was observed . multivariate analysis showed that an acute physiology and chronic health evaluation ii ( apache ii ) score > 20 and > 3 types of underlying diseases were independent factors associated with crbsi occurring within 14 days of cvc indwelling . untimely cvc removal and/or inappropriate use of antibiotics led to significantly longer time to defervescence and time to negative conversion of blood culture ( all p<0.05).conclusionsin this chinese center , gram - positive bacteria are predominantly detected in crbsi . apache ii score > 20 and the presence of > 3 types of diseases were associated with earlier crbsi onset . timely removal of cvc and appropriate use of antibiotics resulted in improved outcomes .

critical need develop potent selective therapeutic agents capable targeting malignant tissue without compromising normal cell viability while chemotherapeutic agents e.g. doxorubicin docetaxel remain widely used clinic lack inherent selectivity desired limit toxicity normal cells addition administration chemotherapeutic agents induce drug resistance resulting disease progression thus development targeted therapies could circumvent nonspecific interactions potentially overcome drug resistance cancer therapy intriguing studies currently exploring new methods engage biomolecular targets high affinity specificity including generation multivalent heterobifunctional constructs advances chemical synthesis techniques cross coupling conjugation strategies enabled chemists decorate plethora molecular species targeting moieties providing access elaborate molecular architectures tailored occupy distinct binding sites within one multiple biomacromolecules although types compounds fall outside molecular weight range typical drug compounds 5003000 da increasing interest developing new chemical entities modulate biomolecular targets novel ways address selectivity requirements emerging date limited examples evaluating potential targeting androgen receptor ar steroidal conjugates the ar important drug target treatment prostate cancer subject research several decades a large number bioactive compounds targeting ar identified via screening efforts review we begin providing rationale continued studies prostate cancer pharmacology targeting ar particular focus placed examining current approaches specifically engage modulate ar activity steroid conjugates utilizing rational design principles androgens class steroid hormones consist 19-carbon derivatives cholesterol synthesized testis adrenal glands they also precursors estrogens female sex hormones produced hydroxylation elimination aromatization androgens enzyme aromatase functioning primarily ar ligand dependent transcription factor androgens play fundamental role development survival male reproductive tissues prostate influencing gene expression levels the body maintains control testosterone abundant androgen men levels within normal reference range 240800 ng dl low levels testosterone resulting zinc deficiency aging lead fatigue erectile dysfunction by contrast high levels testosterone linked variety diseases including prostate cancer prostate cancer remains common cancer among men globally estimated affect 900 000 patients every year second leading cause cancer related deaths men 258 000/year approximately one every six men diagnosed prostate cancer u.s if detected early arsenal therapeutic options currently provide promising chance long term survival however 40% patients develop castration resistant prostate cancer crpc arising drug resistance vida infra associated poor survival rates the ar 110 kda protein shares sequence homology nuclear hormone receptors superfamily including progesterone receptor pr glucocorticoid receptor gr estrogen receptor er the ar consists four basic elements n terminal domain dna binding domain hinge region ligand binding domain lbd the first domain 559 amino acid long intrinsically disordered n terminal domain contains ligand independent activation function 1 af-1 activation function sites encode signature motifs containing lxxll fxxlf sequences recruit co regulatory proteins essential transcription the highly conserved region within nuclear hormone receptors including ar centrally located dna binding domain consisting two zinc finger domains recognize specific dna consensus sequences known androgen response elements figure 1a the third domain dubbed hinge region connects dna binding domain ligand binding domain figure 1b ligand binding domain lbd contains ligand dependent activation function 2 af-2 forms ligand binding pocket mediates interactions ar heat shock proteins figure 1b importantly af-2 interact fxxlf binding motif located within n terminal domain feature unique ar x ray crystal structure androgen receptor ar dna binding domain ribbon red complex androgen response elements sticks pdb code 1r4i b ar ligand binding domain ribbon gray portion hinge region ribbon blue complex native ligand sticks green pdb code 1i37 ( c amino acids residues establish high affinity binding native ligand dht pdb code 2ama the crystal structure ar lbd bound native ligand dht reveals amino acid residues critical maintaining high binding affinity figure 1c although van der waals forces contribute binding affinity hydrogen bonds establish stronger interactions native ligand arg752 forms hydrogen bond o3 atom ketone steroid ligand mutagenesis arg752 shown compromise binding affinity suggesting importance interaction achieving high affinity in addition asn705 thr877 form hydrogen bonds 17- hydroxyl group steroid ligand mutagenesis asn705 thr877 also resulted reduced binding affinity specificity establishing importance maintaining high affinity it important note modifications 17- hydroxyl group result diminished binding affinity even large substituent modifications 17- position often retain strong binding interactions the ar ligand dependent transcription factor stabilized cytoplasm chaperone proteins figure 2 competitive displacement chaperones dihydrotestosterone dht androgen biosynthesized testosterone enzyme 5-reductase activates ar upon activation conformational change brings n- c termini proximity facilitates ar dimerization upon translocation nucleus ar binds palindromic 5-tgttct-3 consensus sequences androgen response elements promoter regions target genes this event stimulates recruitment necessary cofactors including lxxll fxxlf motif containing proteins components transcriptional machinery regulate gene expression abbreviations dht dihydrotestosterone hsp heat shock protein p phosphorylation site fxxlf coactivator protein the ar mediates variety androgen dependent diseases including benign prostatic hypertrophy bph prostatic intraepithelial neoplasia pin prostate cancer it proposed prostate cancer often originates high grade prostatic intraepithelial neoplasia hgpin process subtle alterations shape size prostate cells occur more importantly progression prostate cancer linked elevated expression ar malignant tissue suggesting ar plays central role prostate cancer cell biology although many hypotheses regarding involvement ar prostate cancer progression postulated precise molecular mechanisms fully understood patients diagnosed localized metastatic prostate cancer usually undergo androgen deprivation therapy reduction circulating androgen levels chemical castration gonadotropin releasing hormone agonists surgical castration unfortunately methods completely eliminate circulating levels androgens tumor capable local androgen synthesis due expression androgen biosynthetic enzymes this led numerous research efforts focusing development inhibitors interfere key enzymes cytochrome p450 17a1 cyp17a1 androgen biosynthesis exemplified recent fda approval abiraterone zytiga the standard treatment approach prostate cancer involves androgen deprivation therapy conjunction small molecule anti androgens block ar signaling figure 3a anti androgens compete dht binding ar thus inhibiting ar transactivation variety mechanisms including disruption nuclear localization interruption dna binding interference coactivator recruitment unfortunately patients receiving anti androgen therapy eventually develop drug resistance indicated rising levels serum prostate specific antigen psa gene regulated ar leading lethal disease state termed castration resistant prostate cancer crpc small molecule inhibitors targeting ar anti androgens b activation function 2 inhibitors c allosteric bf3 site regulators n terminal domain inhibitors purple denotes approved therapies androgen dependent prostate cancer orange represents approved therapies castration resistant prostate cancer current mechanisms proposed advancement crpc include following:(1)alterations ar co regulatory protein balance;(2)somatic gain function mutations within ar majority lbd resulting activation steroid hormones anti androgens;(3)generation new fusion gene products;(4)ar ligand independent activation via cross talk signaling pathways.these mechanisms garnered significant attention ability reactivate ar disease progression provide conceptual underpinning guide development new therapeutic interventions nevertheless currently crpc primarily treated chemotherapeutic agents immunotherapy abiraterone vida supra alterations ar co regulatory protein balance somatic gain function mutations within ar majority lbd resulting activation steroid hormones anti androgens generation new fusion gene products ar ligand independent activation via cross talk signaling pathways ar identified via chemical screening efforts include compounds act af-2 figure 3b bf3 site figure 3c ar regulate activity the bf3 site hydrophobic binding pocket located adjacent af-2 surface ar allosterically regulate binding interactions ar coactivator proteins the development noncompetitive modulators compete dht ligand binding could circumvent drug resistance ar pharmacology while promising noncompetitive approaches yet yield candidates clinical implementation likely high concentrations required suppress ar activity future may important utilize structure based design generate potent af-2 bf3 inhibitors in contrast continuing interest anti androgen drug development led fda approval enzalutamide targets ar ligand binding domain treatment crpc figure 3a unfortunately recent evidence suggests drug resistance enzalutamide emerge point mutations within ar lbd f876l additionally drug resistance proposed arise constitutively active ar splice variants lacking ar ligand binding domain this led researchers focus innovative ways antagonize ar splice variants development n terminal domain inhibitors figure 3d it important note however structural information exists ar n terminal domain complicating design n terminal domain antagonists although tempting speculate ar splice variants mainly responsible drug resistance enzalutamide precise molecular mechanisms remain unknown evidence suggests full length ar required signaling although different sets studies demonstrate er splice variants constitutively active absence ligand also intriguing report similarly suggested gr become constitutively active absence lbd future research may illuminate whether nuclear hormone receptors exhibit similar modes action bioactive hit compounds typically identified screening efforts often lack potency selectivity required translation clinical setting reason compounds iterative rounds synthesis rigorous bioassays strategy remains widely utilized academic industrial research programs rational design therapeutic agents aims streamline issues initially identifying potent selective compounds below describe different strategies used target ar steroid conjugates along preliminary evaluation potential applications ar pharmacology the atp dependent ubiquitin proteasome pathway quality control mechanism conducts programmed metabolic degradation proteins ubiquitin protein ligase e3 associates ubiquitin conjugating enzyme e2 providing subsequent tagging ubiquitin chains protein substrates results degradation proteasome rational design strategies aimed toward selectively targeting proteins degradation e3 could establish approach diminish levels aberrantly functioning proteins crews lab pioneered general strategy modulate levels selective proteins engagement ubiquitin system use conjugates dubbed proteolysis targeting chimeric molecules protacs first steroid conjugate selectively induce ar degradation developed protacs consist three components targeting moiety dht linker recognition element e3 the modular synthesis protacs establishes significant pharmacological advantage protacs particularly amenable chemical modification permitting control physicochemical features products initial ex vivo studies aimed toward degrading ar yielded protac-5 figure 4a protac-5 outfitted peptide sequence alapyip e3 recognition domain induce ubiquitination upon hydroxylation central proline residue assess biological activity protac-5 administered human embryonic kidney cells hek293 stably expressed ar fluorescent fusion protein protein degradation quantified reduction fluorescence signal concentration 25 protac-5 successfully degraded ar without compromising normal cell viability control studies vehicle treated cells maintain fluorescence suggesting protac-5 engages ar cell induces degradation to confirm results cells treated protac-5 immunoblotted ar significant decrease ar protein level proteolysis targeting chimeric molecules protacs ar synthesis protac-5 b chemical structure protac aa c chemical structure small molecule e3 recognition element left cocrystal structure small molecule e3 recognition element blue sticks e3 orange surface rendered right pdb code 3zrc figure adapted refs 53 55 56 more recently derivative protac-5 dubbed protac aa figure 4b administered ar expressing prostate cancer cell line lncap evaluate effects cell proliferation protac aa contains shorter hydroxylated recognition element e3 slightly modified arginine tail enhance cell permeability the arginine tail enhances cell permeability uptake mechanism mimicking antennapedia hiv tat proteins protac aa inhibited cell growth inhibitory concentration ic50 value 3.8 72 h 0.217 144 h. control protac lacking arginine tail displayed ic50 values 12.5 72 h 1.5 144 h. western blot analysis performed establish ar protein levels reduced taken together results suggest arginine tail enhances biological activity maintaining specificity importantly prostate cancer cell lines express ar pc-3 du-145 cells protac aa significant effect cell viability establishing selective activity protacs remain promising candidates applications ar pharmacology difficulties large scale production may impede rapid translation clinic current efforts focused developing druglike protac molecules recent discovery first small molecule targeting e3 figure 4c ic50 value 4.1 competitive fluorescence polarization data indicated small molecule binds e3 confirmed cocrystal structure subsequent optimization led first submicromolar small molecule targeting e3 ic50 0.90 future may begin see small molecule protacs targeting ar may include example enzalutamide tethered similar small molecules capable recruiting e3 apoptosis programmed cell death physiological cell suicide mechanism critical cellular homeostasis inadequate activation apoptotic pathway play role development cancer autoimmune diseases inhibitors apoptosis proteins iaps play fundamental role regulating apoptosis cellular processes iaps contain ring domain possesses e3 activity establishing ability induce proteasomal degradation tagging proteins ubiquitin chains the hashimoto lab developed specific nongenetic iaps dependent protein erasers snipers relative protacs snipers consist targeting moiety dht linker recognition element iaps the targeted ubiquitination proteins snipers relies small molecule iap recognition elements figure 5 the biological activity ar targeting compound sniper-13 evaluated western blot human mammary tumor mcf-7 ) cells express ar sniper-13 decreased ar protein levels concentration 30 high concentration required induce degradation may attributed hydrolytically unstable ester oxime linkages chemical structure specific nongenetic iaps dependent protein eraser 13 sniper-13 the modular assembly snipers allows incorporation virtually targeting moiety this characteristic ability recruit e3 small molecule establishes versatile molecular platform address many protein targets future research efforts may focus generating stable linkages targeting moiety iap recognition element altering linker lengths optimize activity sniper conjugates various protein targets metallo based cytotoxic agents cisplatin remain viable option treatment cancer mechanistic standpoint these compounds exert biological activities binding nucleobases dna inducing damage dna ultimately triggers apoptosis although widely used clinic agents generally nonspecific exhibit shortcomings include severe side effects resulting compromised normal cell viability drug resistance this led exploration metallo based chemotherapeutic agents target specific organs tumors minimize adverse side effects conjugation targeting moiety metallo based cytotoxic agents could potentially circumvent nonspecific interactions selectivity targeting cells overexpress particular proteins establishing delivery vector localize effects new therapeutic agents recent studies hannon group discovered first metallo based chemotherapeutic conjugates target ar the authors developed efficient protocol readily synthesize array steroid conjugates act delivery vehicles ethisterone 17-ethynyl homologue testosterone conjugated pyridines quinolines isoquinolines utilizing sonogashira cross coupling conditions subsequent coordination platinum(ii complexes yielded metallo based bifunctional agents figure 6 initial evaluation cytotoxic effects two promising metallo based bifunctional agents cell lines express ar t47d cells revealed promising biological activity cis conjugate ic50 15.9 trans conjugate ic50 63 suggesting geometry platinum(ii complexes plays critical role additionally cis conjugate exhibited greater potency cisplatin ic50 32 cell uptake studies reveal targeting moiety enhances drug delivery suggesting hydrophobic character ethisterone facilitates molecular transport across cellular membrane importantly presence targeting moiety cis conjugate relative control compounds lacking steroid moieties led significant structural effects dna the distortion dna upon binding cis conjugate greater observed cisplatin suggesting steric bulk ethisterone promotes greater unwinding dna accommodate binding complex metallo based conjugates significant utility platform targeted drug delivery the work outlined suggests metallo based conjugates crafted exert toxic effects preferentially cell types overexpress ar demonstration coadministration competitive ligand dht abrogates activity metallo conjugate would support hypothesis targeting mediated specifically binding ar ultimately similar strategies could potentially elaborated targeting additional metallo conjugates range malignant cell types mitigating cytotoxic effects tissues alkylating agents act dna damaging mechanisms commonly used cancer therapy crafting alkylating therapies specifically target malignant cells could minimize cytotoxic effects normal cells lead development potent anticancer agents effort block dna repair enzymes the essigmann group developed heterobifunctional dna damaging agents specifically target prostate cancer cells figure 7 the alkylating agent n n bis-2-chloroethylaniline linked steroid hormone targets ar allowing conjugate simultaneously bind ar dna this strategy results blockade dna repair enzymes prostate cancer cells overexpress ar subsequently leading disruption ar mediated transcription signaling using radiometric competitive binding assays relative binding affinity n bis-2-chloroethylaniline determined 20% ar 4.2% pr this result establishes conjugate selective ar pr addition n n bis-2-chloroethylaniline negative control n n bis-2-methoxyethylaniline covalently modified dna administration alkylating agent concentration 10 induced apoptosis determined flow cytometry cleavage poly adp ribose polymerase parp western blot analysis as expected negative control induce apoptosis equivalent concentration more importantly xenograft studies immunocompromised mice revealed 90% inhibition tumor growth intraperitoneal injection daily dose 30 mg kg these results demonstrate effectiveness using targeted alkylating agents selectively suppress prostate cancer tumor growth an emerging avenue molecular pharmacology development multivalent therapeutic agents multivalency used establish enhanced binding affinity termed avidity specificity corresponding biomolecular targets multisite binding contacts displaying ligands targeting moieties modular oligomer frameworks allows chemists precisely craft architectures capable inhibiting highly specific protein in addition ability create monodisperse molecular scaffolds enables control important physicochemical features products including solubility cellular uptake peptoids class peptidomimetics composed n substituted glycine units joined tertiary amide linkages peptoids recently exploited multivalent platforms design conjugates capable targeting different nucleic acids protein receptors peptoids stable proteases display enhanced cell permeability profiles incorporation 200 different peptoid side chains enabled numerous applications chemistry biology including enantioselective catalysis molecular recognition antimicrobial activity intracellular delivery antitumor activity vivo peptoids compatible solid phase synthesis techniques assembled sequence specific manner afford monodisperse products additionally conformation peptoid oligomers controlled though macrocyclic constraints side chain interactions utilizing peptoids versatile chemical platform kirshenbaum lab designed multivalent ethisterone conjugates specifically target ar ldb modulate ar activity via different mechanisms action ethisterone conjugated 17- position peptoid scaffold via highly stable triazole linakges initial efforts evaluated effects valency spacing conformational ordering ar activity figure 8) fluorescence polarization assays conducted determine conjugates compete dht binding ar ligand binding domain results studies revealed hexavalent 4 spaced divalent conjugates 5 6 compete binding mono- di- trivalent conjugates 13 cyclic divalent conjugate 7 compete ar binding control peptoid conjugate outfitted pr ligands activate ar luciferase reporter assay suggesting ethisterone conjugates selective ar cell proliferation studies model castration resistant prostate cancer lncap abl cells conjugates 6 7 exhibited potent antiproliferative properties as expected anti androgen bicalutamide vide supra failed suppress proliferation resistant cell line importantly cytotoxic effects conjugates 6 7 observed cell lines express ar pc-3 hek293 cells establishing conjugates selectively target ar figure adapted ref 90 follow investigation authors used confocal microscopy time resolved fluorescence resonance energy transfer chromatin immunoprecipitation flow cytometry microarray analysis gain insight mechanism action conjugates 6 7 upon administration conjugates 6 7 hek293 cells transfected ar fluorescent fusion protein conjugate 6 promote ar nuclear localization conjugate 7 suggesting competitive noncompetitive mechanisms action respectively ar coactivator recruitment assays revealed conjugate 6 promote binding ar coactivator proteins conjugate 7 partially recruited coactivator proteins dna binding experiments conjugates 6 7 reduced occupancy ar psa enhancer vida supra conjugate 7 conjugate 6 induced arrest g0/g1 phase cell cycle displayed contrasting patterns global gene expression intriguingly conjugate 6 7 share extensive chemical similarities indicating disposition ligand presentation scaffolds exert significant influence mechanism action conjugate 6 promote ar nuclear localization coactivator binding inhibited dna binding in contrast conjugate 7 promoted ar nuclear localization induced cell cycle arrest noncompetitive mode action the modularity peptoid synthesis establishes versatile chemical platform generate array three dimensional architectures target modulate activity different biomolecular targets generation peptidomimetic conjugates capable antagonizing ar via distinct mechanisms action could circumvent drug resistance ar pharmacology peptoids offer chemical platform utilized optimize biological activity hold significant promise next generation therapeutics prostate cancer the estrogen receptor er well characterized mechanism action it known native ligand estradiol binding er ligand binding domain induces conformational rearrangement promotes dimerization determined site specific mutational analysis additionally x ray crystal structures er dimers presence ligand er modulators reported establishing template molecular design elucidating structural parameters er dimer complex pioneering work katzenellenbogen lab probed er function various bivalent conjugates tethered different linkers using high resolution structural information first steroidal constructs aimed toward targeting er dimer initial studies focused developing correlation linker length binding affinity the authors concluded bivalent conjugates incorporating 35 linker suitable enhancing er binding affinity steroidal bivalent conjugates modulate estrogen receptor er activity bivalent binding interactions crystal structure er ligand binding domain gray ribbon pdb code 1ere bound native ligand estradiol red spheres nonsteroidal bivalent conjugates induce agonistic antagonistic er conformations figure 10 designed synthesized order distinguish intra- intermolecular binding events bivalent agonist conjugates displayed weak binding affinity presumably due burial hydrophilic linker within protein interior in contrast determined antagonist conjugates incorporating 14.4 linker induced intramolecular binding event i.e. one targeting moiety optimized competitive binding binding additional hydrophobic pockets activation function 2 figure 11 additionally 29 linker found induce intermolecular binding event increases linker length 29 resulted reducing binding affinities presumably due unfavorable entropic effects importantly antagonistic nonsteroidal bivalent conjugates potent inhibiting cell proliferation breast cancer cells mcf7 monovalent pharmacophore control er conformation dependent upon ligand binding er bound agonist conformation gray ribbon diethylstilbestrol colored spheres helix-12 orange coactivator peptide red pdb code 3erd b er bound antagonist conformation gray ribbon hydroxytamoxifen colored spheres helix-12 orange pdb code 3ert diagram depicting intra- intermolecular er binding events dependent linker length a critical objective molecular design approach ability induce different er conformations dependent upon ligand binding as discussed conformation induced upon agonist antagonist ligand binding er figure 10 plays critical role biological outcome antagonist conformation intra- intermolecular binding event occur two distinct targeting moieties an open question whether structure based design utilized generate heterobifunctional conjugates target two distinct binding sites ar i.e. one targeting moiety optimized competitive binding ligand binding domain binding additional hydrophobic pocket af-2 bf3 over past decade targeting canonical membrane associated ar heterobifunctional multivalent constructs displaying anti androgen drug ligands emerged potential family therapeutics these compound classes hold great promise effective therapeutic agents due ability modulate ar activity unique mechanisms action large number reports we highlight representative examples promising strategies used target ar nonsteroidal conjugates recently oyelere lab reported nonsteroidal heterobifunctional conjugate outfitted histone deacetylase inhibitors figure 12a histone deacetylase inhibitors show great promise preclinical cancer models inability selectively target malignant tissue restricted therapeutic development conjugating histone deacetylase inhibitors nonsteroidal anti androgen ligands selective modulation ar activity concentrations lower clinical anti androgens achieved these results introduce novel method antagonize ar pave way next generation therapeutics nonsteroidal conjugates targeting ar chemical structure heterobifunctional conjugate displaying histone deacetylase inhibitor linked nonsteroidal antiandrogen ligand b chemical structure heterobifunctional conjugate displaying doxorubicin linked nonsteroidal antiandrogen ligand c schematic depiction multivalent gold nanoparticle displaying nonsteroidal antiandrogen ligands target membrane associated ar figure obtained refs 98 99 101 similar studies koch lab reported nonsteroidal heterobifunctional conjugate containing doxorubicin nonselective cytotoxic therapeutic dna intercalator used clinic figure 12b enhance selectivity doxorubicin conjugated nonsteroidal anti androgen ligand salicylamide linker hydrolyzed t1/2 57 min physiological conditions yield doxorubicin formaldehyde schiff base the anti androgen conjugate successfully delivered doxorubicin formaldehyde schiff base cells overexpressing ar highlighting ability approach enhance selectivity releasing pharmacophore prostate cancer cells lastly el sayed lab introduced first nonsteroidal multivalent conjugates selectively target membrane associated ar figure 12c bicalutamide conjugated gold nanoparticles generating architectures display approximately 2.25 0.02 10 ligands particle the multivalent compounds enhanced potency 1 order magnitude comparison monovalent ligand prostate cancer cells these results establish conjugation numerous copies known pharmacophore molecular scaffold significantly increase antiproliferative effects may overcome resistance arises monovalent treatment there growing appreciation design potent selective therapeutic agents targeting ar prostate cancer patients targeted drug therapy beginning play pivitol role new drug discovery efforts classically small molecules identified via chemical screening efforts considered offer relatively straightforward path clinical implementation certain cases extensive high resolution structural information enables structure activity relationship profiles utilized optimization facilitating translation clinic unfortunately therapeutic responses short lived acquired resistance the studies highlighted review indicate new chemical entities designed engage ar high potency additional preclinical studies required validate potential clinical translation many cases necessary evaluate critical parameters selectivity vivo potency binding affinity as discussed chemical modifications certain positions steroid core result diminished binding affinities potentially limiting utility ar pharmacology these molecular architectures demonstrated elicit potent biological responses importantly target ar novel ways future may begin see examples monodisperse homo- heterogeneous bivalent multivalent displays high resolution structural data enable evaluation structure activity relationships propelled many small molecule drug discovery efforts more importantly heterobifunctional displays likely designed target two distinct binding sites ar enhancing potency establishing new modes ar antagonism these constructs could potentially address challenge overcoming resistance prostate cancer patients

the androgen receptor ( ar ) is a major therapeutic target in prostate cancer pharmacology . progression of prostate cancer has been linked to elevated expression of ar in malignant tissue , suggesting that ar plays a central role in prostate cancer cell biology . potent therapeutic agents can be precisely crafted to specifically target ar , potentially averting systemic toxicities associated with nonspecific chemotherapies . in this review , we describe various strategies to generate steroid conjugates that can selectively engage ar with high potency . analogies to recent developments in nonsteroidal conjugates targeting ar are also evaluated . particular focus is placed on potential applications in ar pharmacology . the review culminates with a description of future prospects for targeting ar .

patients acute coronary syndrome acs key increased survival preservation myocardial function rapid diagnosis followed appropriate early intervention enable timely management strategies intervention carried delays level management prehospital setting emergency department ed subsequently cardiac catheterisation laboratory must minimised the diagnosis acute myocardial infarction requires combination good prompt clinical history 12-lead electrocardiogram ecg also appropriate cardiac markers cases st elevation ami stemi results cardiac markers required decisions management intervention 15 prehospital 12-lead ecgs performed transmitted eds save precious time diagnosis institutions set targets door ecg time exceed 10 min door needle time must within 30 min door intervention time within 90 min arrival 611 the acquisition 12-lead ecg important step assess cardiac status routine emergency situations cardiac emergency the time accuracy information important elements critical effect patient outcomes accurate ecg measurements essential diagnostic purposes well serial comparison evaluate changes cardiac status help prediction future events clinical practice relevance ecg depends upon reproducibility individual points mind the application single lead electrodes torso standard 12-lead ecg acquisition takes time a relatively new praecordial v1v6 v quick patch developed provides one piece template placement six praecordial leads makes application easy the person performing 12-lead ecg need reach one single item applied patient torso instead six separate pieces items traditional case the patch characteristics enable conform variations thoracic dimensions retaining ability providing accurate electrode placement training use new patch requires minimal amount time template provides cues user placement performed the v quick patch potential used anywhere setting 12-lead ecg carried this study assess time required obtain 12-lead ecg using v quick patch system versus standard single electrode systemthe agreement 12-lead ecgs acquired techniques assessed two assessors time required obtain 12-lead ecg using v quick patch system versus standard single electrode system agreement 12-lead ecgs acquired techniques assessed two assessors null hypothesis would difference times taken level agreement quality ecgs acquired two different techniques one hundred fifty healthy male female volunteers least 18 years age various body builds weights enrolled any volunteer incidentally found significant ecg changes given appointment cardiologist necessary evaluation preparation consisted cleaning skin alcohol wipes site lead placement electrodes placed routine standard positions single electrode 12-lead ecg following removal standard electrodes v quick template attached praecordium acquisition next 12-lead ecg this done using unique identification number number ended even digit would v quick technique ecg performed first the volunteers may experience minor dermatological effects applications removal electrodes the time measured stopwatch instance lead application commenced techniques v quick technique would moment staff tears pack open standard method would time staff picks card single electrode adhesive the stopwatch stopped last electrode applied satisfactorily ecg printout complete the two ecgs acquired different techniques volunteer performed staff the two ecgs compared blinded fashion two senior emergency physicians both inter assessor consistency two readers ecgs intra assessor interpretation ecgs volunteer agreement analysed the following cases excluded known electrolytes abnormalities ingestion medication potential cause electrolyte imbalance e.g. diuretics potassium supplements)congestive heart failurehistory renal hepatic impairmentacute illness day ecgs acquiredingestion medication could affect heart rate rhythmthe presence dermatological disease may affect skin sites electrode applicationimplanted pacemaker automated implanted cardiac defibrillator aicd known electrolytes abnormalities ingestion medication potential cause electrolyte imbalance e.g. diuretics potassium supplements congestive heart failure history renal hepatic impairment acute illness day ecgs acquired ingestion medication could affect heart rate rhythm presence dermatological disease may affect skin sites electrode application implanted pacemaker automated implanted cardiac defibrillator aicd patients excluded first time using v quick device singapore prior literature available use asian patients also long going take nurses obtain 12-lead ecg despite null hypothesis difference techniques decided first perform trial normal healthy cohort volunteers in next phase plan include patients requiring ecg presenting ed an agilent pagewriter 300pi hewlett packard pagewriter xli used obtain ecgs these machines utilise computerised analysis system ones used ed the v quick patch system packaged one guide template four replaceable limb lead electrodes the electrodes pre gelled simple peel stick application the guide template flexibility accommodate different body sizes geometries allowing movement praecordial electrode along guide attain best suit recommended position figs 1 2 2the standard single lead electrocardiogram ecg system v quick patch placement system standard single lead electrocardiogram ecg system spss version 10.0 spss inc p values confidence intervals analysed using student test collection data some information gathered included bra cup size ladies thought good subjective assessment breast size volumeshirt size men would give approximate indication shoulder widthdistance leads v2v4 v4v6 measurements may indicate broad chest expanse chest area ecg electrodes placed bra cup size ladies thought good subjective assessment breast size volume shirt size men would give approximate indication shoulder width distance leads v2v4 v4v6 measurements may indicate broad chest expanse chest area ecg electrodes placed ecgs analysed compared blinded fashion two assessors the following examined heart rate rhythmspecial patterns bundle branch block ventricular hypertrophyr wave amplitude r wave progressions wave depthpresence q wave morphology heart rate rhythm special patterns bundle branch block ventricular hypertrophy r wave amplitude r wave progression presence q wave morphology study approved institutional review board volunteer given written informed consent form sign participation study one hundred fifty healthy male female volunteers least 18 years age various body builds weights enrolled any volunteer incidentally found significant ecg changes given appointment cardiologist necessary evaluation preparation consisted cleaning skin alcohol wipes site lead placement electrodes placed routine standard positions single electrode 12-lead ecg following removal standard electrodes v quick template attached praecordium acquisition next 12-lead ecg this done using unique identification number number ended even digit would v quick technique ecg performed first the volunteers may experience minor dermatological effects applications removal electrodes the time measured stopwatch instance lead application commenced techniques v quick technique would moment staff tears pack open standard method would time staff picks card single electrode adhesive the stopwatch stopped last electrode applied satisfactorily ecg printout complete the two ecgs acquired different techniques volunteer performed staff the two ecgs compared blinded fashion two senior emergency physicians both inter assessor consistency two readers ecgs intra assessor interpretation ecgs volunteer agreement analysed the following cases excluded known electrolytes abnormalities ingestion medication potential cause electrolyte imbalance e.g. diuretics potassium supplements)congestive heart failurehistory renal hepatic impairmentacute illness day ecgs acquiredingestion medication could affect heart rate rhythmthe presence dermatological disease may affect skin sites electrode applicationimplanted pacemaker automated implanted cardiac defibrillator aicd known electrolytes abnormalities ingestion medication potential cause electrolyte imbalance e.g. diuretics potassium supplements congestive heart failure history renal hepatic impairment acute illness day ecgs acquired ingestion medication could affect heart rate rhythm presence dermatological disease may affect skin sites electrode application implanted pacemaker automated implanted cardiac defibrillator aicd patients excluded first time using v quick device singapore prior literature available use asian patients also long going take nurses obtain 12-lead ecg despite null hypothesis difference techniques decided first perform trial normal healthy cohort volunteers in next phase plan include patients requiring ecg presenting ed an agilent pagewriter 300pi hewlett packard pagewriter xli used obtain ecgs these machines utilise computerised analysis system ones used ed the v quick patch system packaged one guide template four replaceable limb lead electrodes the electrodes pre gelled simple peel stick application the guide template flexibility accommodate different body sizes geometries allowing movement praecordial electrode along guide attain best suit recommended position figs 1 2 2the standard single lead electrocardiogram ecg system v quick patch placement system standard single lead electrocardiogram ecg system p values confidence intervals analysed using student test collection data information gathered included bra cup size ladies thought good subjective assessment breast size volumeshirt size men would give approximate indication shoulder widthdistance leads v2v4 v4v6 measurements may indicate broad chest expanse chest area ecg electrodes placed bra cup size ladies thought good subjective assessment breast size volume shirt size men would give approximate indication shoulder width distance leads v2v4 v4v6 measurements may indicate broad chest expanse chest area ecg electrodes placed ecgs analysed compared blinded fashion two assessors the following examined heart rate rhythmspecial patterns bundle branch block ventricular hypertrophyr wave amplitude r wave progressions wave depthpresence q wave morphology heart rate rhythm special patterns bundle branch block ventricular hypertrophy r wave amplitude r wave progression presence q wave morphology study approved institutional review board volunteer given written informed consent form sign participation study the mean ages male female volunteers 4310.8 5212.7 years respectively female volunteers higher body mass index bmi 26.54.2 versus 24.35.8 p=0.0003 table 1 the race distribution also shown table 1 table 1demographic comparison data male female volunteers male n=150)female n=150)age range19682372mean age4310.85212.7racechinese10895malay2015indian1827other413bmi range18.939.517.538.8mean24.35.826.54.2standard methodmean time s)58.610.866.012.2range44.968.554.082.1v quick methodmean time s)40.56.750.25.6range35.145.941.353.9v2v4 distance cm)12.49.2v4v6 distance cm)10.69.8range shirt size1524 range bra cup size 20b 47c 69d 14 demographic comparison data male female volunteers comparing mean time taken techniques v quick method proved faster male 40.56.7 versus 58.610.8 p<0.0001 female volunteer 50.25.7 versus 66.012.2 p<0.0001 groups table 2 even male female volunteers taken together i.e. 300 volunteers compared two techniques v quick method also shown significantly faster 45.43.8 versus 61.93.5 p<0.0001 table 2 table 2comparison methods p values standard methodv quick methodp values 95% ci)mean time 300 volunteers s)61.93.545.43.8p<0.0001 15.922 17.078)mean time 150 female volunteers s)66.012.250.25.7p<0.0001 13.636 17.964)mean time 150 male volunteers s)58.610.840.56.7p<0.0001 16.058 20.142 comparison methods p values different methods compared genders table 3 v quick method shown significantly faster male volunteers compared female volunteers the results also noted standard method faster acquire 12-lead ecg standard method male volunteers table 3comparison genders two methods malefemalep values 95% ci)mean time standard method s)58.610.866.012.2p<0.0001 10.018 4.782)mean time v quick method s)40.56.750.25.7p<0.0001 11.113 8.287 comparison genders two methods blinded analyses ecgs 100% agreement two assessors comparing ecgs person male female volunteers acquired two different techniques intra assessor agreement ecg read two assessors separately inter assessor agreement the ecg continues critical component evaluation patients emergency cardiac symptoms this tool hundred years old standard clinical practice half century the applications new signal processing techniques expansion use additional leads innovative techniques offered information cardiac electrical activity even though one common tests performed today hardly published studies time taken acquire often ecgs acquired ecg technicians nurses years become versatile familiar importance accurate lead placement according thoracic landmarks there also developments aim making praecordial lead placement easier faster the v quick technique showed favourable result study terms timing well agreement standard 12-lead ecg 300 normal volunteers the technique simple skill relatively easy acquire one already knows concept 12-lead ecg the fact template manufactured fit average standard size adult torso make adjustment rarely necessary however said onus still lie operator carrying ecg strive accurate placement besides accuracy timing important factor try meet targets door ecg time institutions it important highlight multiple factors affect timing busy department particular point time first contact time triage time manpower staffing issues well availability ecg machines throughout ed among factors it thus crucial institution look wider picture trying address timing issues meet key performance indicators kpis female volunteers longer time required explained presence breast tissue requires attention paid standard landmarks whether leads placed breast tissue comparatively one template deal still faster apply v quick system rather apply leads singly praecordium shown study study the v quick system gave stable baseline ecg recordings unlike techniques ecg belt common problem praecordial electrode position important variability placement may result ecg changes nurses performing ecgs every individual marked points application skin marker reduce variability placement two techniques increasing practice place limb leads torso thought reduce application time however shown significant amplitude waveform changes compared ecg acquired limb leads placed usual recommended limb positions 19 20 this trial first kind conducted singapore using v quick patch system looking results also past experience utilisation system appears useful consideration use prehospital environment conditions unpredictable stressful relatively easy apply using whilst moving ambulance also less challenge compared application small single adhesives paramedics would apply six different sites praecordium many prehospital patients also cold clammy sweaty often adhesives small electrodes work well this one single larger template v quick system would plus point we future consider trial using v quick system patients present ed also prehospital group patients requiring 12-lead ecg the v quick patch manufactured us based company likely standardisation template average american torso size study the template system applied asian volunteers application adjustments made template thus creating this may due slightly smaller chest expanse average asian compared caucasian counterpart data collection distances v2v4 v4v6 were noted thought useful simple measure chest thoracic expanse the shirt size may reflection may less constant objective would much affected personal choice the goes female volunteers bra cup size may reflect breast tissue size volume if similar studies conducted caucasian population would interesting see results compare trial the nurses performing ecgs blinded would impossible achieve it would optimal except fact logistically rather challenging would incurred extra cost videotaping in study 300 male female volunteers v quick patch system proved significantly faster single electrode standard 12-lead system acquisition electrocardiograms ecgs the time taken also faster male compared female volunteers reasons discussed

introductionthe v - quick patch template system is compared with the standard 12-lead electrocardiogram ( ecg ) acquisition technique in this paper . the objectives of the study were : ( a ) to study and compare the time taken to produce the printed 12-lead ecg and ( b ) to look at the level of agreement when the ecgs were compared by two blinded , independent assessors.methodsone hundred and fifty each of male and female volunteers signed an informed consent form to participate in the clinical study . nurses were put through a 4-h training session to familiarise themselves with the v - quick patch system . the timings were measured with a stopwatch with the specific start and end points defined . the final ecg printouts were then compared by two blinded , independent assessors for several set criteria.resultsthe v - quick patch system was proved to be significantly faster than the standard 12-lead system in the acquisition of the ecg in both male and female volunteers . the time taken in male volunteers was also noted to be significantly faster than in female volunteers.conclusionthe two assessors shared a 100% agreement level when comparing the ecgs acquired by both techniques in the same individual ( intra - assessor agreement ) and when each ecg was read by the two assessors separately ( inter - assessor agreement ) .

studies phrynoderma conducted early middle last century therefore clinical features phrynoderma association nutritional deficiency signs studied elucidate relationship phrynoderma nutritional status patients a cross sectional descriptive study 125 consecutive phrynoderma patients attending outpatient department opd dermatology conducted tertiary care hospital period two years patients detailed history particular reference age gender seasonal variations socioeconomic status family history disease and cutaneous examination findings distribution sites involvement morphology lesions signs nutritional deficiencies noted phrynoderma diagnosed clinically patient presents discrete brown skin colored acuminate keratotic papules central keratin plug predominantly distributed elbows knees extensor extremities and/or buttocks all patients phrynoderma examined investigator irrespective age gender included study the proportion patients phrynoderma attending opd 0.51% among 125 patients 79 63.2% male 46 36.8% female the age patients range 3 26 years mean 10 4.3 years table 1 a majority patients 88% low socioeconomic group remaining 12% middle socioeconomic group phrynoderma common students 94.4% followed lactating mothers 4.8% laborers 0.8% the disease recurrent 17 13.6% patients calendar year 8.33% patients presented summer 46.7% rainy 45% winter season age distribution phrynoderma cases lesions asymptomatic 114 91.2% patients mild itching present 11 8.8% patients the disease localized elbows knees extensor extremities and/or buttocks 106 84.8% patients generalized trunk and/or face 19 15.2% patients elbows buttocks alone affected 16 12.8% 2 1.6% patients respectively table 2 the site onset elbows 106 84.8% patients followed knees 8% buttocks 4% extensor extremities 4% in 100% patients lesions discrete keratotic acuminate brown skin colored papules central keratinous plug figure 1 the surrounding skin dry scaly 44 35.2% patients pigmented 72 57.6% patients various diseases conditions sites involvement phrynoderma discrete brown skin colored keratotic follicular acuminate papules central keratinous plug localized elbows associated diseases conditions phrynoderma histopathological changes phrynoderma phrynoderma disease occurring children adolescents aged 5 15 years the disease uncommon 5% children 5 years age present study thus increased nutritional demand childhood lactation lack good nutrition due low socioeconomic status may responsible occurrence phrynoderma patients the proportion phrynoderma cases present study less compared studies found 1.3% 3% 5% these studies conducted middle last century decline proportion phrynoderma patients past decades therefore nutrition seems play important role pathogenesis phrynoderma many studies including present one family history phrynoderma low 0% 3.57% 5% the absence disease siblings also taking diet suggests role factors manifestation phrynoderma the distribution site onset lesions indicate importance pressure friction development lesions the incidence phrynoderma higher cooler months year documented present study the flare disease duirng winter may due follicular prominence generally occurs otherwise normal children cold weather the typical case phrynoderma presents bilaterally symmetrical discrete keratotic follicular brown skin colored acuminate papules central keratinous plug localized elbows knees buttocks extensor extremities generalized disease these distinct clinical features help differentiating phrynoderma common follicular keratotic disorders keratosis pilaris lichen spinulosis pityriasis rubra pilaris follicular lichen planus although occurrence phrynoderma related nutritional status patient present study signs symptoms nutritional deficiency the ocular manifestations vit deficiency patients phrynoderma reported 5% it also demonstrated incidence phrynoderma less prevalence signs vit deficiency high vice versa extremely low levels serum vit 0.1 mol l normal 1.4 4 mol l reported secondary vit malabsorption following small bowel bypass surgery obesity colectomy pancreatic insufficiency diet survey serum vit levels apparently healthy phrynoderma patients normal controls revealed statistically significant difference two groups the vit deficiency cause phrynoderma largely based therapeutic response cod liver oil considered source vit a. however later also demonstrated source efa the patients presenting signs vit b complex deficiency less number compared studies 47.6% 50% 65% 92% 25% none patients present study presented nutritional deficiency signs efa vit e vit c protein calorie malnutrition regarding efa intake india computation based revised figures fat content cereals pulses shown invisible fat present even poorest indian diet provides minimum amount linoleic acid needed ie 3% total energy the ratio eicosatrienoic acid arachidonic acid 0.4 considered accurate indicator efa deficiency the studies showing efa deficiency phrynoderma patients used alkaline isomerization ai method estimation fatty acid levels ai the fatty acids estimated according number double bonds chain length taken consideration thereby overestimating dienoic trienoic acids underestimating tetraenoic pentaenoic acids this results high trienoic tetraenoic acid ratio gas exchange chromatography gc method fatty acids estimated according double bonds chain length thus ratio obtained gc method reflects efa deficiency phrynoderma patients the levels linoleic acid phospholipids sensitive indicators efa deficiency ratio linoleic arachidonic acid indicator efficient conversion linoleic acid arachidonic acid also normal phrynoderma patients the cutaneous manifestations riboflavin pyridoxine niacin efa deficiency states number similarities riboflavin required pyridoxine metabolism turn required tryptophan niacin metabolism niacin necessary fatty acid synthesis the combination therapy vit b complex efa vit b complex vit e shown better results compared single drug therapy the serum levels linoleic acid vit e also increased significantly combination therapy vitamins b complex group used alone failed show therapeutic response in addition deficiency single vitamin b complex group rare poor nutritional diets malabsorption often associated multiple nutritional deficiencies impaired balance threshold levels nutrients without deficiency state seem alter intersecting biochemical pathways local milieu nutrients resulting phrynoderma this compared formation comedones acne low level linoleic acid sebum attributed pathogenesis follicular hyperkeratinization follicular hyperkeratosis keratin plugging along epidermal hyperkertosis acanthosis characteristic features phrynoderma the clinical histopathological skin changes closely resembling phrynoderma noted rats fed fat deficient diet hence abnormality efa metabolism pilosebaceous unit caused vit b complex appears important step pathogenesis phrynoderma the main drawback present study lack biochemical evidence nutritional status serum levels nutrients patients these investigations done non availability resource poor set normal serum levels nutrients vit vit b complex efa reported phrynoderma patients however available literature findings hypothesized phrynoderma may multifactorial disease involving multiple nutrients local factors like pressure friction environmental factors manifesting setting increased nutritional demand further prospective case control studies investigating levels vit b complex vit vit e efa blood skin also relation treatment required delineate exact pathogenesis phrynoderma

background : phrynoderma is a type of follicular hyperkeratosis . various nutritional deficiency disorders have been implicated in the etiology of phrynoderma.aim:to determine clinical features of phrynoderma and its association with nutritional deficiency signs.materials and methods : a cross - sectional descriptive study of 125 consecutive patients with phrynoderma attending the outpatient department ( opd ) of dermatology was conducted in a tertiary care hospital . in all patients , a detailed history was taken and cutaneous examination findings such as distribution , sites of involvement , morphology of the lesions , and signs of nutritional deficiencies were noted.results:the proportion of patients with phrynoderma attending the opd was 0.51% . there were 79 males and 46 females . age of the patients was in the range of 3 - 26 years with a mean of 10 4.3 years . the lesions were asymptomatic in 114 ( 91.2% ) patients . the distribution of lesions was bilateral and symmetrical in 89 ( 71.2% ) patients . the disease was localized ( elbows , knees , extensor extremities , and/or buttocks ) in 106 ( 84.8% ) patients . the site of onset was elbows in 106 ( 84.8% ) patients . the lesions were discrete , keratotic , follicular , pigmented or skin colored , acuminate papules in all patients . signs of vitamin a and vitamin b - complex deficiency were present in 3.2% and 9.6% patients , respectively . epidermal hyperkeratosis , follicular hyperkeratosis , and follicular plugging were present in the entire biopsy specimen.conclusion:phrynoderma is a disorder with distinctive clinical features and can be considered as a multifactorial disease involving multiple nutrients , local factors like pressure and friction , and environmental factors in the setting of increased nutritional demand .

dieulafoy lesion aberrant submucosal vessel lies close contact mucous membrane may lead exposure causing massive gastrointestinal gi bleeding.1 cause approximately 6% upper gi bleeding.2 commonly found proximal stomach especially within 6 cm gastroesophageal junction predominantly lesser curvature however occur anywhere gi tract small bowel.3 due difficult access diagnosis small bowel bleeding often delayed therefore multidisciplinary approach needed obtain proper diagnosis treatment we experienced two cases ileal dieulafoy lesion bleeding presented massive hematochezia instability vitals the two cases diagnosed treated successfully using single balloon enteroscopy sbe hemoclips we discuss clinical features well useful diagnostic therapeutic modalities ileal dieulafoy lesion computer assisted search english language literature a 47-year old man admitted hospital hematochezia 8 hours he denied previous medical history well taking medication non steroidal anti inflammatory drugs initial physical examination initial blood hemoglobin level 10.2 g dl values within normal limits we tried perform colonoscopy failed due poor bowel preparation fresh blood second day admission developed hypovolemic shock due massive hematochezia blood hemoglobin level dropped 6.2 g dl he underwent abdominal computed tomography ct showed contrast media filled bowel lumen terminal ileum the contrast media leaked vessel supplied branch right ileocolic artery superior mesenteric artery bowel lumen around terminal ileum filled contrast media fig 1 however failed control bleeding artery small embolization treatment he received transfusions 10 units packed red blood cells prepared emergency surgery vital signs became stabilized underwent colonoscopy we found fresh blood throughout entire colon terminal ileum could find bleeding focus small bowel evaluation performed sbe retrograde approach found dieulafoy lesion distal ileum approximately 20 cm ileocecal valve fig for evaluation small bowel lesions performed capsule endoscopy could find abnormal lesion except previously placed hemoclips fig she obstructive coronary artery disease took aspirin antiplatelet agent admission blood hemoglobin level 8.7 g dl day admission underwent egd colonoscopy could find bleeding focus she underwent abdominal ct specific lesion gi tract suspicious bleeding we recommended enteroscopy evaluation obscure bleeding focus refused melena ceased vital signs stabilized discharged home ninth admission day six days discharge revisited hospital due recurrent fresh hematochezia time blood pressure dropped 88/56 mm hg pulse 107 beats min as underwent egd colonoscopy performed sbe retrograde approach we found fresh blood clot adherent mucosa proximal ileum saline irrigation detected active bleeding focus narrow point normal appearing mucosa fig she discharged home evidence recurrent gi bleeding 2 month follow visit a 47-year old man admitted hospital hematochezia 8 hours he denied previous medical history well taking medication non steroidal anti inflammatory drugs initial physical examination initial blood hemoglobin level 10.2 g dl values within normal limits we tried perform colonoscopy failed due poor bowel preparation fresh blood second day admission developed hypovolemic shock due massive hematochezia blood hemoglobin level dropped 6.2 g dl he underwent abdominal computed tomography ct showed contrast media filled bowel lumen terminal ileum the contrast media leaked vessel supplied branch right ileocolic artery superior mesenteric artery bowel lumen around terminal ileum filled contrast media fig 1 however failed control bleeding artery small embolization treatment he received transfusions 10 units packed red blood cells prepared emergency surgery vital signs became stabilized underwent colonoscopy we found fresh blood throughout entire colon terminal ileum could find bleeding focus small bowel evaluation performed sbe retrograde approach found dieulafoy lesion distal ileum approximately 20 cm ileocecal valve fig for evaluation small bowel lesions performed capsule endoscopy could find abnormal lesion except previously placed hemoclips fig she obstructive coronary artery disease took aspirin antiplatelet agent admission blood hemoglobin level 8.7 g dl day admission underwent egd colonoscopy could find bleeding focus she underwent abdominal ct specific lesion gi tract suspicious bleeding we recommended enteroscopy evaluation obscure bleeding focus refused melena ceased vital signs stabilized discharged home ninth admission day six days discharge revisited hospital due recurrent fresh hematochezia time blood pressure dropped 88/56 mm hg pulse 107 beats min as underwent egd colonoscopy performed sbe retrograde approach we found fresh blood clot adherent mucosa proximal ileum saline irrigation detected active bleeding focus narrow point normal appearing mucosa fig she discharged home evidence recurrent gi bleeding 2 month follow visit dieulafoy lesion associated massive life threatening hemorrhage accounts 1% 2% cases major gi bleeding.4 dieulafoy lesion used describe finding large submucosal artery otherwise histologically normal lying close contact mucous membrane the etiology abnormally tortuous submucosal artery unknown.5 incidence varies 0.5% 14% depending upon selection criteria.2 documented diagnosis dieulafoy lesion based upon classical histologic features however histologic evaluation usually unavailable case recently vascular lesions treated nonsurgical modalities.6 endoscopists rely entirely endoscopic features make diagnosis critical review many published articles endoscopic criteria diagnosis dieulafoy lesion 1 active arterial spurting micropulsatile streaming minute less 3 mm mucosal defect normal surrounding mucosa 2 visualization protruding vessel without active bleeding within minute mucosal defect normal surrounding mucosa 3 fresh densely adherent clot narrow point attachment minute mucosal defect normal appearing mucosa.7 although endoscopic features could proven true dieulafoy lesion historical criteria mentioned endoscopic finding remains convincing dieulafoy lesion one study authors classified vascular lesions small bowel following six groups 1 type 1a punctulate erythema 1 mm regardless oozing 2 type 1b patchy erythema mm regardless oozing 3 type 2a punctulate lesions 1 mm pulsatile bleeding 4 type 2b pulsatile red protrusion without surrounding venous dilatation 5 type 3 pulsatile red protrusion surrounding venous dilatation 6 type 4 unclassified lesions being different size one another types 1a 1b considered angioectasia type 2a 2b considered dieulafoy lesion type 3 represented arteriovenous malformation.6 according classification first case considered type 2b second case 2a angioectasia venous capillary lesion thus likely treated endoscopic cauterization however dieulafoy lesion arteriovenous malformation may cause arterial bleeding require endoscopic treatment clip laparotomy large lesions the authors concluded classification would useful selecting hemostatic procedure.6,8 small bowel bleeding continues difficult visualize directly routine endoscopy therefore diagnosis bleeding small bowel often delayed localize source small bowel bleeding multidisciplinary approaches abdominal ct angiography radionuclide scan capsule endoscopy needed development endoscopy push enteroscopy performed diagnose treat bleeding source push enteroscopy two types sbe double balloon enteroscopy dbe two approaches anterograde per oral retrograde per anal studies sbe effective dbe appropriate diagnosis management obscure gi bleeding cases identifying bleeding source providing means treatment high percentage patients.9,10 however studies dbe higher total enteroscopy rate sbe accompanied higher diagnostic yield.11,12 enteroscopy low risk complications however acute pancreatitis enteroscopy frequent complication taken consideration written informed consent.13 several cases ileal dieulafoy lesion bleeding reported most treated surgically diagnosed histology.14 16 however endoscopic treatment replace surgery significant portion small bowel dieulafoy lesion cases since widespread availability endoscopic angiographic treatment surgery play minor role often left last therapeutic option rebleeding lesions failed nonsurgical treatment.16 summary appropriate evaluation small bowel bleeding aggressive multidisciplinary approach radiologic intervention enteroscopy surgery performed advances endoscopic techniques enteroscopy could play major role diagnosis treatment ileal dieulafoy lesion bleeding

ileal dieulafoy lesion is an unusual vascular abnormality that can cause gastrointestinal bleeding . it can be associated with massive , life - threatening hemorrhage and requires urgent angiographic intervention or surgery . ileal dieulafoy lesion is hard to recognize due to inaccessibility and normal - appearing mucosa . with advances in endoscopy , aggressive diagnostic and therapeutic approaches including enteroscopy have recently been performed for small bowel bleeding . we report two cases of massive ileal dieulafoy lesion bleeding diagnosed and treated successfully by single balloon enteroscopy with a review of the literature .

obesity stigma negative stereotypes obese people widespread damaging health dignity human rights quality life obese individuals standard media biomedical depictions obese individuals contribute stigmatization positing obesity incidence nearly entirely dependent individualistic actions furthermore obese individuals may occupy numerous intersecting social roles identities based gender class race social positions biomedical media depictions invariably refer obesity crisis epidemic obesity multifactorial multilevel etiology reduced energy balance model causation inadequately explains weight trajectories despite oft reported inefficacy weight loss dieting public health interventions may unsuccessful due narrow focus weight loss overly simplistic notion obese individuals live experience bodies contexts inhabit opportunities available seeking wellness happiness full life furthermore health posited ultimate goal projects frequently focus weight deem fatness higher weights necessarily pathological this especially important given populations often targeted campaigns may differ culturally socioeconomically dominant groups these diverse factors may affect lifeways priorities health conceptualizations manners may require depth exploration produce truly beneficial sensitive programming qualitative social scientists anthropologists sociologists trained methods ethnography may uniquely suited explore lived experiences obese individuals they may aid developing public health strategy suited priorities lifestyles individuals implemented manner consistent salutogenic positive holistic understanding health promotion this paper discusses potential depth qualitative social science research concretely contribute program delivery even within expanding fields critical obesity research warin gunson note compiling actual obese people experiences perspectives limited actual collaboration disciplines rather public health researchers programmers qualitative social scientists operating separate silos importantly rather reliance stereotypes approach would facilitate compassionate evidence based policy programming understanding patients providers view health related topics actors must negotiate views care setting context critical planning effective respectful public health care delivery biomedical research nearly invariably posits obesity health crisis despite evidence obese individuals may metabolically healthy overweight people live longer normal weight persons obese individuals chronic disease populations fit obese persons compared unfit normal weight persons 2 6 weight bias moderate high among healthcare professionals trainees including specializing obesity nutrition related practice 711 a recent systematic review physicians views treating adult obesity found physicians believed important treat obesity they confident skills respecting obesity treatment although obesity knowledge actually limited physicians believed largely unsuccessful treating obesity attributed patient noncompliance lack motivation coincides general view overweight obese individuals lazy similar results attained foster colleagues 2 nationally representative american surveys n 5000 physicians felt treatment obesity ineffective held negative views obese patients appearance compliance attributed obesity causation lifestyle factors sought greater compensation delivering obesity treatment similar studies physicians new york state expressed frustration attempting treat obesity this frustration based extent obesity contributing factors outside control low sense self efficacy treating obesity perceived lack reimbursement however also feel unsuccessful treating obesity largely result noncompliance lack motivation patients reimbursement appeared less issue regarding physicians views childhood obesity studies also presented nuanced views clinicians attitudes relating obesity treatment for example physician bmi may mediate physicians likelihood counseling obese patients among primary care physicians weight loss discussions likely initiated physicians believed clients higher bmi normal weight physicians also likely feel confident administering said advice feel physicians responsible serving normal weight role models doubt patients would trust weight related advice overweight obese clinicians another study conducted new york city involved chart review patient survey results suggest little focus obesity practice it found physicians relatively unlikely enter official diagnosis overweight obesity patient chart advice weight loss refer dietitian patients generally wanted lose weight receive physician advice referral dietitian qualitative study german physicians patients views obesity management found doctors concerned potential overemphasis obesity both physicians patients emphasized need multidisciplinary approaches obesity management excess burden primary care centres emphasized respectful trusting relationships practitioners patients the need services professional involvement delivered physicians providers either separate within primary care setting also referred groups a recent debate canadian family physician journal highlighted practitioners may developing critical view orthodoxy advocating weight loss every obese patient bosomworth presents review possible negative mortality morbidity quality life outcomes weight loss it suggested metabolically healthy obese individuals strive remain weight stable gain lose weight an accompanying editorial encourages promoting self acceptance healthy lifestyles obese patients weight loss nearly impossible havrankova presents argument weight loss public health goal futile contends focus preventing obesity 21 22 garrel posits obesity prevention largely outside practitioner purview argues instead obesity treatment 23 24 based guidelines garrel supports urging weight loss obese individuals comorbid conditions physicians work obese individuals without comorbid conditions prevent weight gain 23 24 treatment obese individuals comorbid conditions would involve treating comorbidities setting realistic weight goals patients warning unsafe weight loss methods it allows possibility obese patients may healthy benefit treatment physicians likely refer dietitians gastric bypass surgeons prescribe medication dietitians agree primacy obesity treatment sample 514 canadian dietitians about 90% felt obesity contributed ill health large majority felt obese individuals encouraged lose weight however also emphasized importance health measures weight obesity treatment majority advised clients weighing indeed many dietitians positively disposed toward weight neutral health every size haes approach however variation plans adopt structured eating plans abandoning weight loss goal some also argued certain larger sizes exceeded healthy limits examining clinician perspectives obesity appears essential therefore is establishment trusting respectful relationships clinicians obese patients designing interventions obese persons these relationships must sustained light obesity likely intractability potential nonpathological nature greater understanding clinicians obese patients health perspectives perceptions priorities life course paramount achieving aims better understanding experience visiting healthcare providers larger patient may also provide valuable insight sensitive care delivery health weight)-centric treatment approaches greater awareness stigma obese individuals encounter day day life well healthcare system particular importance improve health quality life examining issues depth essential rectify social justice issue immense health quality life implications significant population ethnographers applied social science researchers may especially adept exploring issue learning obese persons oppression may endure this discrimination occurs interpersonal encounters institutional settings social situations places employment health care settings this discrimination particularly notable among heavier individuals bmi 35 kg 40% reported experiencing discrimination american 1995 96 national midlife development survey women younger adults also considerably higher relative risk experiencing weight stigma these findings coincide well reports high levels internationally stigmatizing attitudes toward obese persons the public emphasizes presumed causal role individual developing obesity single strongest predictor possessing stigmatizing attitude the pervasiveness obesity stigma health care settings particular importance obese individuals health public health planning 7 8 awareness sentiments may make especially difficult obese individuals find adequate medical care social scientists could explore issues seek deploy depth exploratory perspective obese individuals emic perspective the depiction obesity biomedical media popular accounts contributes manifestation degree obesity stigma present among public gard wright identified obesity research communicated public way erases inherent uncertainty imprecision epidemiological studies presents obesity necessarily health crisis despite nondefinitive evidence as work generated within biomedical institution presumed unquestionably objective lacking moralizing political ideological thrust such frame allows dismissal alternate views fallacious whether originate alternate epistemological scholarly arenas lay perspectives embodied experience it also disqualifies concerns research may affect affected antifat bias these portrayals may inadvertently silence source important understanding public health programming needs obese individuals obese individuals presenting homogenizing blaming view individuals lifestyles experiences these views likely reliant assumptions rather data obese persons lived experiences bringing forth obese individuals perspectives learning embodied knowledge multidisciplinary research may enhance public health programming efforts media commentators particular moral values help establish component life choose implicate causing obesity often values related presumed unique modern life diminished quality family time slothful nature current generation children similar accounts evolutionary explanations obesity stressed media may present inaccurate understanding biological archaeological evidence humanity evolutionary past regardless validity claims arguments invoke golden era nonobesity health continue deploy moralistic frame denigrates modern lifestyles decadent slothful glutinous particularly adult commentators often direct ire toward children embodying presumed deterioration societal values lifestyles child health prevention programs reliant framing may risk stigmatizing disempowering harming children rather trying understand children perspectives these portrayals posit individuals responsible cause cure obesity this depiction contributes stigmatizing attitudes toward obese individuals come viewed embodying remediable social health burden borne part others it discounts obese individuals may healthy may choose emphasize holistic less weight centric conceptualizations health may lives preclude investment engagement self care interestingly since 2003 lawrence detected shift american media coverage obesity personal responsibility framing dominates however increasing focus placed environmental factors may contribute obesity particularly fast food industry unfortunately emphasis significantly affected antifat attitudes 28 29 while environmental risks obesity acknowledged individuals assumed willingly incur risks thus still largely culpable body size worthy disapprobation this discrimination spread previously nonstigmatizing nations rather castigating obese individuals size qualitative social science researchers may position rewrite common stereotypical assumptions obese individuals lives working collaboratively better understand health experiences often maligned group addressing health concerns relevant in addition referring obesity epidemic crisis 2 31 even extreme figurative device employing military metaphors this discourse effectively constructs obese individuals targets war even domestic terrorists terms like contagious also used describing obesity epidemiological studies using methods social network analysis 33 34 this frame seems particularly detrimental potentially health enhancing stress reducing benefits social support networks individuals developing obesity is extremely feared 35 36 may isolating obese individuals friendship portrayed risk developing obesity it suggested stigma may serve motivator weight loss among obese individuals 1 38 however evidence provides far bleaker picture stigmatizing obese individuals ineffective tactic reducing obesity rates rather stimulating healthful behaviors stigma likely produce poor eating habits inactivity thus may actually augment obesity prevalence disordered eating 1 3942 additionally catastrophic rhetoric used describe obesity epidemic suggested potential contributor rising rates eating disorders interventions designed ameliorate childhood obesity also implicated development eating disorders 4449 given adverse psychological outcomes produced weight discrimination stigma may also compromise physical psychological health stress induced neuroendocrine dysregulation 1 50 muennig colleagues found difference ideal weight actual weight stronger effect mentally physically unhealthy days bmi american adults suggesting body dissatisfaction may potent impact health objective fatness the health effects stigma induced stress likely exacerbated healthcare discrimination 7 8 consequent inadequate care subsequent avoidance healthcare providers obesity may increase risk poverty downward social mobility subsequent ill health prejudice discrimination affecting education employment income housing healthcare opportunities thus weight stigma may far health damaging previously thought far damaging excess weight for example formerly obese adolescent girls continued suffer lower self esteem characteristic chronically obese adolescents additionally qualitative evidence weight loss efforts concomitant lifestyle dynamic emotional changes may result dissolutions friendships this deterioration social support may unacknowledged mechanism weight stigma affects health may previously erroneously attributed weight in stigmatizing context individuals may utilize variety stigma management techniques these methods may include attempting lose weight act contrition minimize fatness obese individuals oppressed 5458 monaghan conducted ethnography male members united kingdom slimming club explored effects stigma lives one method managing stigma utilized individuals varied acceptance obesity discredited state involved accounts related concerning weight issues 56 58 some accounts entailed offering excuses mitigated individual responsibility obesity appeals genetic condition environmental issues other individuals justified size appeals enjoyment food pride powerful size individuals rejected discredited nature obesity emphasized natural body diversity deemphasized importance weight regarding health 56 58 while one account may suit particular context different account may useful different situations 56 58 cope stigmatizing environments being aware everyday effects stigma individuals lives lifestyles health essential factor planning salutogenic engaging beneficial inclusive public health strategies for example understanding obese persons decisions avoid particularly stigmatizing physical fitness venues may allow public health planners design atmosphere conducive supporting individuals sizes engage enjoyable health conducive movement the literature thus suggests obese individuals experience substantial stigmatization exist environment saturated nonproblematized information concerning health risks excess weight this information often presented manner assumes control health resides individual possibility health obesity mutually exclusive achieving wellness constitutes moral imperative greater lesser extent individuals appear adopted discourse allowed influence perspectives lifestyles individuals health perceptions may influenced pervasive mainstream weight discourse also mediated somatic understandings wellbeing as evidence suggests obese individuals may healthy obese individuals may absorb popular obesity related messaging based somatic signaling knowledge lifestyles individuals may also differing priorities regarding wellness supersede weight concerns emphasis experiencing pleasure mitigating income related food insecurity regardless health risks associated obesity greater understanding obese individuals feel concerning health quality life obese individuals regard priorities concerning health feel would benefit quality life wellness necessary these views may also alter time different weight trajectories experienced essential consider given chronicity obesity crafting public health messaging programs policies is considering potentially multifaceted effects stigma obese individuals lives 59 60 this includes incorporating stake actors local social world within social dimensions stigma 60 p. 1525 for example may involve stigma impeding life chances financial life opportunities fulfillment individual familial role functions 59 60 this view stigma also considers manner stigma sociosomatic psychobiological moral somatic moral emotional pathways stigma may direct physiological consequences these data obese people perspectives health add rich work conducted critical obesity fat studies theoretical understandings body qualitative research done discursive bodily health perspectives individuals sizes example 2 32 3648 5658 6268 recent movements emerged critical weight centric public health model one undertaking health every size haes approach haes advocates critical weight loss dieting low rates sustained weight loss potentially negative effects physical mental health effects include lowered self esteem heightened stress weight cycling bone loss furthermore haes advocates critical overestimation excess weight effects morbidity mortality discounting existence healthy obese individuals ethics promoting weight loss given low levels success possible harms instead haes movement promotes benefits engaging enjoyable physical activity body acceptance haes practitioners advice eating nutritionally according intuitive eating model based intuitive cues hunger satiety clinical trials haes lifestyle interventions demonstrated improvements psychosocial clinical physiological behavioral measures independent weight loss critically participants experience negative consequences including weight gain results compared favorably diet focused intervention groups 3 4 the inclusion social scientists public health strategizing would facilitate inclusion true insiders i.e. obese individuals perceptions advantages disadvantages novel developments these scholars often outside health fields raised critiques concerning dominant obesity discourse studies critiquing biological epidemiological underpinnings prevailing view obesity major health concern product individual behavior published a variety issues raised scholars include use terms epidemic crisis referring obesity moralization presumed health issue resultant ethical stigmatizing implications interventions messaging dominant obesity discourse effects individuals bodily understandings 2 32 3648 5658 6268 findings similar studies would benefit intervention planners better understanding daily lives obese persons obese individuals perspectives health plan beneficial interventions anthropology traditional focus subaltern become cognizant reflective power relations research past colonially complicit eras anthropology a focus today stigmatized populations obese persons would draw upon emerging strengths anthropology including utilization anthropology social critique development critical ethnography while contemporary social climate obese individuals often stigmatized obese individuals allies also marshaled resistance dominant obesity messaging monaghan conducted ethnography lives male members united kingdom slimming club 56 58 this provided invaluable information experience management stigma individuals lives further investigations experience weight stigma men women children necessary grapple source discrimination life health damaging attracted limited attention censure indeed such forms stigma may operate largely unconsciously participant observation may valuable tool detecting stigma effects providing empirical support effecting change further ethnography obese individuals focus health applied aims would immeasurably useful health care providers public health professionals general public sizes thus collaboration ethnographers health researchers planning health interventions would serve interests disciplines importantly needs obese individuals ethnographers could study individuals experiences health care settings fitness centers employment educational settings weight bias reported documenting manner in obese individuals live choices make priorities wellness quality life contributions society potential experiences others biases applied social scientists could put human face obesity work toward producing positive life affirming inclusive public health focused reducing stigma blame helping people achieve high quality life applied social science enhance obese individuals quality life providing greater understanding problematization taken granted assumptions regarding obese individuals health lives lifestyles this epitomizes rabinow views particularly salient novel area anthropological exploration the problematization serious speech acts practices things classifications anthropological experimentation areas could move aspects culture viewed natural contingent finally reflexive perspective 70 p. 67 the problematization taken granted social attitudes processes particularly relevant studying weight stigma largely considered unremarkable even acceptable current sociocultural climes prevailing attitudes suggest obese individuals necessarily unhealthy lazy self indulgent lacking power internalization accounts may make individuals feel unhealthy unworthy immoral disempowered based body size innocuous lifestyle choices emic understandings obese individuals lives ethnography similar depth research methods could lead less stigmatizing healthful view obese individuals navigate everyday lives this depth understanding may contribute invaluably public health approaches future a greater understanding would available constitutes health obese individuals prioritize terms wellbeing experiences seeking live well lives this perspective would help counter potentially traumatic past experiences dieting healthcare discrimination produce patient centered approach congruent obese individuals lives wishes help establish trusting relationships bonds obese individuals health care providers public health officials applied social scientists particularly access health practitioner policy related audiences may especially valuable research endeavor respect disseminating findings facilitating progress these individuals may circulate critique key stakeholders health related fields position effect change specific opportunities qualitative researchers could involve participant observation obese persons interactions healthcare system identify persons find challenging promising aspects care settings critical ethnography could also conducted within existing prevention programs identify successful sensitive problematic components existing programs multisited ethnography could allow obese persons identify sites stigmatization places support undertaking self care needless say challenges would arise attempting critical ethnography lived experience obese individuals investment orthodox obesity understandings entrenched among biomedical public health lay audiences seeking conduct disseminate research this may particularly evident trying demonstrate value largely unaware obesity stigma pervasiveness effects internalized negative stereotypes obese persons researchers would also reflexive concerning biases truly accurately reflect findings research participants order best stimulate change researchers would also strive create positive open minded collaborative relationships variety biomedical public health lay audiences order exchange findings manner conducive cooperation reform otherwise researchers risk merely circulating critique among already critical dominant obesity discourse incapable initiating reform public health interventions messaging facilitating the collaboration partnerships patient providers policy makers would great value this would allow policy makers learn patients experiences desires rewrite public health messaging programs clinical guidelines better address obese persons needs priorities terms healthcare system allocation address systemic interpersonal discrimination establish weight industry regulations keeping respect consumers truth advertising these possibilities along numerous others would allow researchers public health planners learn health priorities pertinent needlessly patholigized stigmatized population it may open doors previously nonconsidered structural reforms provision holistic local health needs these individuals inundated daily messages concerning risk embody solely virtue size these risks thought extend beyond health health others sustainability health care system even society future furthermore society deems culpable presumed poor health straining health care system within environment exposure messaging renders obese individuals population worthy special consideration planning public health programs avoid reinforcing demeaning stereotypes what lacking despite oppressive focus obesity perspectives obese individuals concerning health goals wishes quality life furthermore limited attempts made bridge communicative gaps physicians obese patients gain thorough view physicians perceptions obesity given applied social scientists dedication understanding emic perspectives expertise depth exploratory methods ethnography may particularly suited aid pursuit the outcome may beneficial inclusive nonstigmatizing public health programs individuals would greatly benefit

obesity is viewed as a major public health concern , and obesity stigma is pervasive . such marginalization renders obese persons a special population . weight bias arises in part due to popular sources ' attribution of obesity causation to individual lifestyle factors . this may not accurately reflect the experiences of obese individuals or their perspectives on health and quality of life . a powerful role may exist for applied social scientists , such as anthropologists or sociologists , in exploring the lived and embodied experiences of this largely discredited population . this novel research may aid in public health intervention planning . through these studies , applied social scientists could help develop a nonstigmatizing , salutogenic approach to public health that accurately reflects the health priorities of all individuals . such an approach would call upon applied social science 's strengths in investigating the mundane , problematizing the taken for granted and developing emic ( insiders ' ) understandings of marginalized populations .

celiac disease cd common autoimmune disorder estimated affect approximately 1% us population many studies evaluated methods identifying high risk populations patients need proactive screening case identification many chronic diseases quality improvement approaches applied populations patients cd standardize care track outcomes effectively identifying tracking outcomes cohorts patients however development cd patient registries often requires significant resources including practitioners informatics teams administrative support our center developed maintained patient registry including newly diagnosed patients since early 2012 prior studies evaluated reliability international classification diseases-9 icd-9 searches identify patients cd moderate success yielding 17% sensitivity contrast experiments done using manually defined keywords classify high risk cd cases generating much accurate results 73% sensitivity few efforts combining different aspects available information improve classification accuracy including use data visits laboratory tests pathology reports described pathology reports previously used building natural language processing nlp systems automated chart review patients define nlp broadly methods process textual data one study found pathology reports insufficient detecting breast cancer using nlp approach compared manual approaches others found use nlp analyzing pathology reports effective detection colon cancer manifestation prostatectomy details currently lack research exists investigating usefulness pathology reports automated cd detection based subset patients prescreened using icd-9 code 579.0 evaluated effectiveness using natural language features pathology reports improve identification cd patients this paper among first adopt machine learning approach increase accuracy cd patient identification based combination pathological clinical metrics in contrast prior research conducted contrast experiments using natural language features clinical features different machine learning configurations celiac identification usa clinical system patients visited hospital 2012 2015 icd-9 code 579.0 assigned indicating concern cd our subsequent analysis therefore based patients concerns rather general patient population a laboratory test result marked nominal value either high 20 eu ml normal 20 eu ml an upper endoscopy egd may also performed resulted text based pathology report could used nlp analysis the registry developed confirmed diagnosis cd based confirmation abnormal laboratory serology testing biopsy confirmation detailed chart review patients registry cd most confirmed cd patients confirmed experts cd one pathology report false positive cd patients confirmed experts cd either one zero pathology report twenty nine patients two pathology reports excluded experiment presented challenge analysis inconsistent information cd diagnoses existed different periods time patients multiple ttg laboratories the elevated laboratory result obtained within past 6 months egd chosen represent laboratory status cd patient variables used celiac classification listed table 1 report category the total number pathology reports used one indicating patients egd done 0 otherwise v1 user define phrases high risk phrases v2 and automatically extracted phrases n gram phrases generated machine learning program v3 chosen evaluate effectiveness utilizing pathology reports for laboratory category laboratory results v4 total number labs done used v5 icd-9 category the total number cd icd-9 codes assigned v6 used classification variables celiac disease classification evaluate effectiveness using different combinations variables feature selection methods developed two additional set variables based aforementioned six sets variables v1v6 combining first six categories variables created variable set 7 v7 automatically selecting features v6 using a feature selection algorithm resulted variable set 8 v8 table 1 expert knowledge driven feature selection identified list nine key phrases described clinicians commonly used pathology reports indicators high risk cd these nine key phrases converted lower cases included brunner gland hyperplasia flattening villi intraepithelial lymphocytes marsh gland stage marsh lesion marsh s3 lesion shortened villi villous blunting villous atrophy each key phrase first converted binary variable value one indicating phrase existed pathology report 0 otherwise automatically extracted n gram features phrases generated using weka open source java based machine learning program developed university waikato new zealand the maximum number words included n gram feature chosen three resulting large set text features consist uni- bi- tri grams we collected phrases trigrams length longest user provided high risk phrase also three we kept first 4000 text features stop words i.e. list common words skipped process converting documents n gram features term frequency inverse document frequency tf idf calculated n gram feature tf idf data transformation method commonly used text classification show importance word document collection documents it found useful several recent classification information retrieval studies using medical documents finally n grams converted lowercase avoid duplications tf idf transformation therefore chose three representative classifiers four major categories experiment paper the four classifier categories included bayes function based lazy model tree classifiers category previous studies classifying medical documents showed performance variation among different models could relatively small within category compared across categories this another main reason chose experiment three classifiers rather available classifiers category selected classification models experiments different ways configure classifier cases experiments default configuration chosen long setting compatible type data classify for example libsvm standard algorithm regularized c support vector classification selected well default radial kernel type table 2 for one twelve classification models table 2 also experimented use eight different variable configurations v1v8 table 1 find best variable configuration produce best possible classifier each variable configuration used inputs one twelve classification models resulting twelve experiments variable configuration classification model best performance overview architectural design experiments feature selection variable selection critical optimizing classification model appropriately selected features could minimize chance overfitting well reduce feature redundancy training time face high dimensional variables a typical feature selection process included two separate steps available weka tool used the first step define feature evaluation method using criteria based features selected second step define search method features searched selected final feature set there single rule thumb choosing best possible feature selection method performing several experiments found default feature selection method weka worked best us the feature selection model used implemented correlation based feature evaluation model called cfssubseteval the cfssubseteval feature evaluation model ranks features based correlation class minimizing redundancy variables the underlying feature search model best first feature search model searches attribute space allowing backtracking detailed discussion cfssubseteval best first beyond scope paper recommend researchers refer original work original authors each classifier validated using 10-fold cross validation method entire dataset confirmed cases controls the best model highest area curve auc variable configuration reported auc commonly used metric machine learning based evolution chance corrected measure independent class distributions chi square test conducted test statistical association variables celiac status in addition auc also reported precision recall f1 kappa precision recall f1 classic machine learning measures evaluating overall accuracy 1 best 0 worst predicting positive cases the kappa score measured level agreement user classifier 1 complete agreement 0 complete disagreement for expert knowledge driven feature selection identified list nine key phrases described clinicians commonly used pathology reports indicators high risk cd these nine key phrases converted lower cases included brunner gland hyperplasia flattening villi intraepithelial lymphocytes marsh gland stage marsh lesion marsh s3 lesion shortened villi villous blunting villous atrophy each key phrase first converted binary variable value one indicating phrase existed pathology report 0 otherwise automatically extracted n gram features phrases generated using weka open source java based machine learning program developed university waikato new zealand the maximum number words included n gram feature chosen three resulting large set text features consist uni- bi- tri grams we collected phrases trigrams length longest user provided high risk phrase also three we kept first 4000 text features stop words i.e. list common words etc skipped process converting documents n gram features term frequency inverse document frequency tf idf ) tf idf data transformation method commonly used text classification show importance word document collection documents it found useful several recent classification information retrieval studies using medical documents finally n grams converted lowercase avoid duplications tf idf transformation therefore chose three representative classifiers four major categories experiment paper the four classifier categories included bayes function based lazy model tree classifiers category previous studies classifying medical documents showed performance variation among different models could relatively small within category compared across categories this another main reason chose experiment three classifiers rather available classifiers category selected classification models experiments different ways configure classifier cases experiments the default configuration chosen long setting compatible type data classify for example libsvm standard algorithm regularized c support vector classification selected well default radial kernel type table 2 for one twelve classification models table 2 also experimented use eight different variable configurations v1v8 table 1 find best variable configuration produce best possible classifier each variable configuration used inputs one twelve classification models resulting twelve experiments variable configuration the classification model best performance chosen best model underlying variable configuration used overview architectural design experiments feature selection variable selection critical optimizing classification model appropriately selected features could minimize chance overfitting well reduce feature redundancy training time face high dimensional variables a typical feature selection process included two separate steps available weka tool used the first step define feature evaluation method using criteria based features selected second step define search method features searched selected final feature set there single rule thumb choosing best possible feature selection method performing several experiments found default feature selection method weka worked best us the feature selection model used implemented correlation based feature evaluation model called cfssubseteval the cfssubseteval feature evaluation model ranks features based correlation class minimizing redundancy variables the underlying feature search model best first feature search model searches attribute space allowing backtracking detailed discussion cfssubseteval best first beyond scope paper recommend researchers refer original work original authors therefore chose three representative classifiers four major categories experiment paper the four classifier categories included bayes function based lazy model tree classifiers category previous studies classifying medical documents showed performance variation among different models could relatively small within category compared across categories this another main reason chose experiment three classifiers rather available classifiers category selected classification models experiments different ways configure classifier cases experiments the default configuration chosen long setting compatible type data classify for example libsvm standard algorithm regularized c support vector classification selected well default radial kernel type table 2 for one twelve classification models table 2 also experimented use eight different variable configurations v1v8 table 1 find best variable configuration produce best possible classifier each variable configuration used inputs one twelve classification models resulting twelve experiments variable configuration the classification model best performance chosen best model underlying variable configuration used appropriately selected features could minimize chance overfitting well reduce feature redundancy training time face high dimensional variables a typical feature selection process included two separate steps available weka tool used the first step define feature evaluation method using criteria based features selected second step define search method features searched selected final feature set there single rule thumb choosing best possible feature selection method performing several experiments found default feature selection method weka worked best us the feature selection model used implemented correlation based feature evaluation model called cfssubseteval the cfssubseteval feature evaluation model ranks features based correlation class minimizing redundancy variables the underlying feature search model best first feature search model searches attribute space allowing backtracking detailed discussion cfssubseteval best first beyond scope paper recommend researchers refer original work original authors each classifier validated using 10-fold cross validation method entire dataset confirmed cases controls the best model highest area curve auc variable configuration reported auc commonly used metric machine learning based evolution chance corrected measure independent class distributions chi square test conducted test statistical association variables celiac status in addition auc also reported precision recall f1 kappa precision recall f1 classic machine learning measures evaluating overall accuracy 1 best 0 worst predicting positive cases the kappa score measured level agreement user classifier 1 complete agreement 0 complete disagreement our data extraction resulted identification 1498 unique patients icd-9 codes suggesting possible cd we found 363 confirmed cases positive celiac cases table 3 matching previously establish celiac registry 1135 false positive cases negative celiac cases table 3 demographic data shown table 3 chi square test found statistically significant associations age gender race celiac status classification variables visit laboratory pathology report counts laboratory results found significant differentiating cd patients study mainly focused using clinical variables celiac classification include demographic variables building classifiers table 3 patient characteristics association celiac disease table 4 listed results summarizing best classification model variable configuration models ranked based auc score highest lowest among twelve classification models experimented libsvm was found top performer five experiments experiments 3 4 5 6 8) followed logistic model two experiments experiments 1 7 nave bayes one experiment experiment 2 the best performing model logistic model using available features filtered automatic feature selection process it achieved auc 0.94 kappa 0.78 f1 0.92 table 4 statistics best classification models variable configuration among experiments using single category variables experiment 2 48 nave bayes model based automatically selected n gram features generated best results auc 0.92 kappa 0.73 f1 0.90 also overall second best performing model also conducted feature selection executing nave bayes model aimed find best possible performance using text features despite slightly lower performance the nave bayes model much faster models terms model building model evaluation time case took nave bayes seconds build evaluate model 10-fold cross validation took minutes libsvm logistic model finish case using 4000 features using high risk phrases number laboratories number icd-9 experiment 6 7 8) resulted lowest model accuracy using laboratory resulted higher accuracy previous three it also intriguing using number pathology reports alone slightly underperformed using report text features alone however additional features resulted higher accuracy given results based combination different features table 5 listed automatically selected n gram phrases contrast user provided high risk phrases it found automatically created n gram features covered majority user provided high risk phrases also potential accurate specific finding relevant information for example automatically generated n grams celiac abdominal pain directly relevant features cd initially clinicians list comparison user defined high risk phrases automated key phrases provided high risk features included automatically selected feature list phrase marsh lesion converted lower case processing we found may due extreme low frequency usage phrases pathology reports 1 363 confirmed cases exact phrase table 5 italicized phrases highlighted overlaps two lists user provided automatically extracted the automatically extracted list represented subset relevant n gram features table 5 our machine learning approach could quickly effectively choose relevant variables various clinical sources automated cd classification the level accuracy achieved higher compared previous studies topic gram features performed well cd detection experiment agreed improved results previous studies using text features in contrast previous studies automated feature generation selection approaches efficient finding relevant classification features short period time effective user provided high risk phrases this result showed automated list effective providing comprehensive list relevant keywords manual methods the number laboratories done effective actual lab results reported case cd classification auc 0.71 vs. 0.83 this could increased number labs done fact laboratory ordered may suggest higher likelihood abnormal laboratory result our experiments also agreed previous studies fact icd-9 code alone produced poor results kappa 0.28 f1 0.75 auc 0.63 therefore suggested researchers use icd-9 code prescreening tool subsetting patients study in addition results added evidence previous studies nave bayes model generated accurate results even pathology report features used kappa 0.73 f1 0.90 auc 0.92 this indicates pathology report important pierce synoptic reporting celiac status contains invaluable information celiac classification in addition combining clinical text features altogether classification accuracy kappa score could improved kappa 0.78 f1 0.92 auc 0.94 this suggests complementary effect using structured data unstructured data celiac classification finally feature selection found crucial step celiac detection improving efficiency accuracy classification feature selection dramatically removed redundant noninformative terms 4000 60 although new task cd classification feature selection methods found widely used disease classification studies including alzheimer disease asthma case variables i.e. number pathology reports lab results could individually predict celiac reasonably high accuracy however result may problem dependent studies implementing metrics may may result similar performance even still observed performance gain adding nlp features accuracy agreement increased reasonable amount the automated tool developed could speed process refining subset population initially identified based icd-9 code 579.0 this refining process automatically confirm patient either positive negative cd case based knowledge patient obtained laboratories pathology notes the classification accuracy 90% correctness f1 case greatly eliminated need often time consuming process manual review first since cd classification task performed subset patients prescreened icd-9 code rather general patient population performance final classification results may depend accuracy initial icd-9 assignment example actual cd patients assigned icd-9 code 579.0 initially chance identified machine learning system it therefore important make sure initial icd-9 code assignment include many high risk cases possible second although feature selection method found critical yielding high performance conducted systematic experiments possible feature selection methods provided weka machine learning system given experience likely methods may improve accuracy others may even lower accuracy finally machine learning algorithms experimented default configuration used classification it likely different configurations parameter setting may lead different results accuracy therefore researchers encouraged experiment different classifier settings achieve best possible results tasks since cd classification task performed subset patients prescreened icd-9 code rather general patient population performance final classification results may depend accuracy initial icd-9 assignment for example actual cd patients assigned icd-9 code 579.0 initially chance identified machine learning system it therefore important make sure initial icd-9 code assignment include many high risk cases possible second although feature selection method found critical yielding high performance conducted systematic experiments possible feature selection methods provided weka machine learning system given experience likely methods may improve accuracy others may even lower accuracy finally machine learning algorithms experimented default configuration used classification it likely different configurations parameter setting may lead different results accuracy therefore researchers encouraged experiment different classifier settings achieve best possible results tasks in paper compared results 96 machine learning experiments cd identification based 12 classification model variations eight feature set variations the logistic model used combination clinical pathology report features generated best results kappa 0.78 f1 0.92 auc 0.94 our results agreement previous studies insufficiency using icd-9 codes alone merits using pathology report features cd identification this study provides new evidence adopting feature selection techniques improve classification efficiency accuracy based subpopulation prescreened patients using icd-9 code this study demonstrated viable computational approach automatically reviewing confirming prescreened cd patients based icd-9 code state art accuracy much improved previous research topic

background : celiac disease ( cd ) is a common autoimmune disorder . efficient identification of patients may improve chronic management of the disease . prior studies have shown searching international classification of diseases-9 ( icd-9 ) codes alone is inaccurate for identifying patients with cd . in this study , we developed automated classification algorithms leveraging pathology reports and other clinical data in electronic health records ( ehrs ) to refine the subset population preselected using icd-9 code ( 579.0).materials and methods : ehrs were searched for established icd-9 code ( 579.0 ) suggesting cd , based on which an initial identification of cases was obtained . in addition , laboratory results for tissue transglutaminse were extracted . using natural language processing we analyzed pathology reports from upper endoscopy . twelve machine learning classifiers using different combinations of variables related to icd-9 cd status , laboratory result status , and pathology reports were experimented to find the best possible cd classifier . ten - fold cross - validation was used to assess the results.results:a total of 1498 patient records were used including 363 confirmed cases and 1135 false positive cases that served as controls . logistic model based on both clinical and pathology report features produced the best results : kappa of 0.78 , f1 of 0.92 , and area under the curve ( auc ) of 0.94 , whereas in contrast using icd-9 only generated poor results : kappa of 0.28 , f1 of 0.75 , and auc of 0.63.conclusion:our automated classification system presented an efficient and reliable way to improve the performance of cd patient identification .

possible presence infection cervical smear tests usually reported cytologists based cytological criteria therefore non specific cervicitis inflammatory changes smear report common these findings usually unclear clinical approaches appropriate guideline management patients it known women recalled cultures treatment inflammatory change considered minor changes balance normal vaginal flora cause overgrowth pathogens lead discharge although common complain among sexually active women still gaps knowledge bacterial vaginosis bv vaginal candidiasis vc trichomoniasis three infections commonly associated vaginal discharge 1 a number studies indicated association bv postoperative complications gynecologic surgery well pregnancy complications 25 trichomonas vaginalis tv also another common cause vaginal discharge studies found association pregnancy complications 3 indicated tv pregnancy predisposing factor preterm delivery delivering low birth weight infants 6 pap smear test detection bv showed sensitivity 88.2% specificity 98.6% positive predictive value ppv 96.8% 7 sensitivity specificity ppv pap test tv diagnosis reported 98% 96% 88% respectively 8) also recent studies demonstrated co infection vaginal pathogenic organisms pap tests 2 9 10 this study aimed investigate prevalence pathogenic vaginal microorganisms presence inflammation pap smear among iranian women sample evaluate possible co infection organisms this cross sectional study carried pap smear samples women referred gynecological clinic taleghani hospital tehran iran october 2008 march 2009 exclusion criteria pregnancy smoking use oral contraception and/or corticosteroids regular use vaginal douche women chronic systemic diseases systemic immunosuppression prior data collection objectives study explained community meetings all women received laboratory results written form results explained consultation the study protocol also approved institutional review boards department gynecology obstetrics data obtained privacy using information form applied one investigator followed gynecological examination specimen collection done one gynecologist cervical smear samples based conventional method prepared spatula fixed glass slides stained papanicolaou technique tv defined presence trophozoites pap smears vc diagnosed fungal hyphae budding yeasts present pap smears the presence inflammation smears divided mild moderate severe categories mild inflammation defined less 30 inflammatory cells high power field moderate inflammation defined 30 100 inflammatory cells high power field severe inflammation defined 100 inflammatory cells high power field the frequency pathogenic microorganism included bv tv vc determined specimens statistical significance analyzed using statistical package social sciences spss software version 17 the comparison data performed pearson chi square fisher exact test mild inflammation noted 136 26.1% moderate 155 29.4% severe 117 22.2% table 1 frequency pathogenic vaginal microorganisms inflammation among pap smear samples among samples bv presence clue cells common pathogenic microorganism seen 91 samples 17.2% followed vc n 56 10.6% tv n 2 0.4% shown low prevalence among patients table 1 the overall prevalence pathogenic microorganisms greater among reproductive age participants meanwhile prevalence bv well vc significantly different p 0.042 p 0.006 respectively non- post- menopausal women inflammation detected 76% n 402 pap smears 80.1% n 322 reported among reproductive age women however severity inflammation significantly different non- post- menopausal women overall presence inflammation significantly associated vc p 0.002 severity correlated infection although significant relation presence inflammation bv severity inflammation specimen significantly increased association bv p 0.001 in study prevalence bv among pap smear specimens women living tehran 17% similar azargoon study 16% population semnan another city center iran 3 higher rate hamadan western city iran reported 28.5% 11 comparison asian countries india but 38% african study botswana 703 pregnant women 13 some predisposing factors bv include sexual relationship one partner early onset sexual activity orogenital sexual contacts coitus menstruation use iud smoking 2 14 differences sexual behavior risk factors different societies justify differences bv prevalence mentioned studies reproductive age women indicated prevalence bv muslim women lower non muslims although prevalence tv pap samples study much less several studies similar reports demonstrated depuydt et al flanders detected real time pcr 0.37% 15 however rate 18% study hamedan 11 8.5% among indian women 16 19% african study 13 a probable reason variations could differences pap sampling quality cytologists skill several studies show increased vaginal ph 5 bv tv infection 3 5 17 demirezen et al turkey 600 pap tests detected tv 6% women bv 44% tv cases they also found significant relationship tv bv infection might due hypothesis tv phagocytosis vaginal lactobacilli increases vaginal ph generates anaerobic environment providing appropriate condition growth anaerobic microorganisms 18 19 also confirmed heller study 400 pap samples concluded bv diagnosis considered presence tv pap smears 2 study found significant association bv tv conventional pap smear sampling might low prevalence tv samples the effect habits behaviors vaginal flora among different population evaluated assessments study presence candida pap smear samples associated report inflammation therefore reporting inflammation pap smear test might considered candidiasis evaluation treatment several studies investigated association infection existence inflammation cervical smears 2023 some found inflammation pap smear relatively low predictive value presence infection 21 they stated inflammation exactly consequence infection causes might responsible appearance 20 in contrast others indicated significant association inflammatory smear reproductive tract infections 22 24 as burke et al 22 study 256 women found evidence inflammation 9.7% smears genital tract infection 29.2% participants overall they found infection 48% women inflammatory changes smear test vs. 27.3% women whose smear tests showed evidence inflammation they discovered prevalence infection chlamydia trachomatis candida bacteroides gardnerella vaginalis higher inflammatory smear group they concluded women inflammatory smear likely mask infection women whose smear shows evidence inflammation 22 prevalence bv infection 17% study similar studies true tv 1% the use diagnostic methods may probably valuable conventional pap smear evaluation tv among iranian women needs investigations report inflammation samples high use newer pap smear techniques thinprep liquid base preparation studies recommended based results inflammation reported pap smears demands evaluation vc proper treatment

objectivenon - specific cervicitis or inflammatory changes in a smear report are common which are usually unclear for clinical approaches . to investigate the frequency of inflammation and pathogenic vaginal microorganisms in cervical smears among an iranian population sample.materials and methodsthis cross - sectional study was carried out on pap smear samples of women referred to gynecological clinic of taleghani hospital in tehran , iran , between october 2008 and march 2009 . this study was conducted on 528 conventional papanicolaou cervical smears . the frequency and severity of inflammation and prevalence of bacterial vaginosis ( bv ) , trichomonas vaginalis ( tv ) , and vaginal candidiasis ( vc ) was determined in the samples . also co - infection of the microorganisms in pap samples was evaluated . percentage , meanstandard deviation of the outcome parameters were calculated . the comparison between data was performed with the pearson 's chi square or fisher 's exact test.resultsthe prevalence of bv , vc , and tv in pap samples was 17% , 11% , and 0.4% respectively . overall , the prevalence of these microorganisms in women of reproductive age was higher than menopausal women . there was a significant association between vc and the presence of inflammation in our samples.conclusionbased on our results , inflammation in the pap smears can suggest an infection of vc and the patients should be considered for proper vc treatment .

brucellosis highly contagious zoonosis caused bacteria genus brucella listed class b animal epidemics world organization animal health oie the genus six classic species b. abortus b. melitensis b. suis b. canis b. ovis b. neotomae recently marine species b. ceti b. pinnipedialis b. microti b. inopinata included genus foster et al the incidence human brucellosis estimated oie 500,000 new cases per year worldwide however official statistics widely acknowledged underestimated considerable number cases origin infection identified the majority human cases worldwide attributed b. melitensis pappas et al 2005 in general b. melitensis b. suis virulent humans b. abortus b. canis 2006 species cause infection humans rarely diaz aparicio 2013 the disease prevalent western parts asia india middle eastern southern european latin american countries the transmission brucella infection prevalence region depend several factors dietary habits methods processing milk milk products social customs husbandry practices climatic conditions socioeconomic status environmental hygiene mantur amarnath 2008 as infectious dose low infections occupational risk farmers veterinarians abattoir workers schneider et al 2013 laboratory personnel others work animals consume products pepin et al 1997 human brucellosis transmitted inhalation animal contact consumption dairy products undercooked meat products the consumption traditional dishes raw liver cause human infection malik 1997 however estimated even 10 100 microorganisms sufficient cause disease humans pappas et al 2006 thus contaminated meat meat products could represent source infection especially come animals slaughtered acute phase disease fao 1986 consumed raw undercooked the handling preparation infected meat offal may give rise contamination foodstuffs kitchen utensils cds epr/2006.7 2006 tests carried us carcasses bovine swine slaughtered infected revealed 1.2% bovine carcasses 3.5% swine carcasses contaminated brucella spp ( sadler 1960 similar study conducted india 100 carcasses goats two 700 neck muscle samples analysed tested positive brucella melitensis randhawa karla 1970 other studies shown offal slaughtered ruminants constitutes risk transmission infection fatma mahdey 2010 sekulovski 2008 in orderto evaluate prevalence contamination meats assess risk infection consumers professionally exposed workers investigation 307 carcasses bovines sheep goats slaughtered resulted positive serological testing brucella spp performed the study carried within framework national programmes eradication monitoring animal diseases prevention zoonoses italy approved decisions 2011/807/ue 2012/761/ue european commission 2011,2012 the study lasted 12 months carried 10 abattoirs southern italy brucellosis infection still widespread cattle sheep goat farms after slaughter swab samples taken 307 carcasses 40 cattle 60 sheep 207 goats these animals came 24 different farms slaughtered proved positive serological tests brucella spp previous studies randhawa karla 1970 fatma mahdey 2010 brucella spp contamination carcasses slaughtered animals evaluated means microbiological tests carried samples muscle organ tissue present study swab samples taken accordance procedure indicated decision european commission 2001 duplicate samples taken means swabs dry moistened sterile aqueous solution wiped external internal surfaces carcasses immediately slaughter storage refrigerators a total 608 swab samples taken transport medium deemed necessary samples promptly consigned laboratory underwent polymerase chain reaction pcr analysis brucella spp accordance following procedure dna extraction commercially available kit qiaamp dna mini kit qiagen valencia ca amplification carried means nested pcr technique based published protocol romero et al 1995 ; the second step utilised pair oligonucleotides internal used first step produce smaller fragment prove functional result first pcr specific ( 5-tcgagcgcccgcaagggt gagcgg-3 r2 5-aaccatagtgtctc cactaacc-3 allowing amplification product 905 bp second step pcr primer pairs r0 ( 5-tagctagttg gtggggtaaaggc-3 r1 5-caggcttgcgcccattgtcc-3 used allowing amplification product 144 bp prepare reaction mixture first pcr 12.5 l commercially available pcr master mix pcr master mix promega corp madison wi usa used 1x concentration sample the reaction mixture contains 50 units ml taq polymerase 400 mm datp dgtp dctp dttp 3 mm mgcl2 reaction mixture 0.5 l primer f4 r2 concentration 0.2 6.5 l h2o added final reaction volume 25 l positive control pcr the negative control made master mix water prepare reaction mixture second pcr the thermal profile first pcr involved initial denaturation 95c 4 followed 35 cycles denaturation 94c 1 annealing 54c 1 extension 72c 1 final extension 72c 7. thermal profile second pcr first one except cycle number consisted 30 cycles second pcr accordance italian law aim eradicating brucellosis cattle sheep goats the carcasses infected animals may freely marketed outcome post slaughter examination favourable internal organs udders must destroyed parts organs therefore removed subjected culture tests bacteriological identification typing brucella spp means technique described manual office international des epizooties ch this involves streaking brucella agar medium added selective supplement horse serum incubation 37c atmosphere enriched 5 10% co2 observation 10 days subsequent species typing isolated strains carried national reference centre brucellosis teramo italy means pcr restriction fragment length polymorphism technique accordance procedure prescribed vol the biomolecular tests revealed presence brucella spp 25/307 carcasses 8% 1 bovine carcass 2.5% 9-year old cow 9 carcasses sheep 15% aged 18 months 5 years farm 15 carcasses goats 7.2% aged 12 months 6 years coming 5 different farms figure 1 the 25 carcasses found contaminated brucella spp belonged 25 animals slaughtered 6 different slaughterhouses the bacteriological tests carried organs 307 animals examined allowed isolating brucella spp 136/307 cases i.e. 44% the typing analysis allows detecting type 3 brucella abortus cattle organs type 3 brucella melitensis sheep goats organs the organs specifically lymph nodes carcasses tested positive pcr swabs also proved positive bacteriological testing type 3 brucella abortus bovine carcass type 3 brucella melitensis sheep goat carcasses the results obtained show carcasses i.e. meat animals slaughtered due brucella spp this situation seen case pathogens like salmonella spp specific laboratory tests prescribed measures taken present study the percentage meats proved contaminated far higher studies quoted sandler 1960 randhawa karla 1970 contamination brucella spp was detected higher percentage sheep carcasses cattle goat carcasses bacteriological tests conducted organs detected highest percentage contamination among goats the brucella serotypes isolated organs type 3 brucella abortus cattle type 3 brucella melitensis sheep goats these serotypes isolated recent years geographical area considered casalinuovo et al 2011 de massis et al 2014 ) thus environments facilities slaughterhouses may also severely contaminated thereby constituting risk contagion staff involved butchery meat handling risk contamination carcasses animals infected brucellosis indeed positivity carcasses involved 6/10 slaughterhouses our samples taken end butchery process carcasses ready storage refrigerators delivery retail outlets this means contaminated carcass could turn contaminate equipment utensils meats foodstuffs consequently even domestic kitchens contamination could involve equipment utensils food products indeed brucella spp survives well temperatures reached refrigerators freezers refrigerated frozen foods may become contaminated pcr testing swab samples taken carcasses animals tested positive brucella proved useful this practice could applied systematically since noninvasive sampling technique damage commercial meat cuts economically advantageous requires far less time 24 48 h detection means culture media carcasses resulted positive pcr might sequestered potentially submitted different confirmation tests annex section iv chapter ix f regulation ce n. 854/2004 european commission 2004 deals done meat infected animals lays preventive measures taken regard specific risks brucellosis indeed regulation requires animals proving positive uncertain brucellosis slaughtered separately precautions taken avoid risk contaminating carcasses butchery process abattoir staff udders genital organs blood animals classified unfit human consumption meat freely marketed postmortem examination reveals lesions attributable acute infection brucella spp otherwise meat animals like meat healthy animals allowed enter food chain these measures justified need avoid destruction large amounts meat valuable food source nevertheless results present study show carcasses consequently meat animals positive brucellosis may contaminated brucella spp this means consumers may exposed risk infection sufficiently informed regard origin meat time purchase

a study was conducted in order to evaluate the contamination by brucella spp . of meat from animals slaughtered because they had resulted positive for brucellosis at some time during their life . after slaughter and before delivery to market outlets , swab samples were taken from 307 carcasses of infected animals : 40 cattle , 60 sheep and 207 goats . the swabs were subsequently analysed by means of polymerase chain reaction ( pcr ) tests . in addition , bacteriological tests were carried out on the lymph nodes and internal organs of the same animals . brucella spp . was detected by means of pcr in 25/307 carcasses ( 8% ) : 1 bovine ( 2.5% ) , 9 sheep ( 15% ) and 15 goats ( 7.2% ) and was isolated by means of a cultural method in 136/307 carcasses ( 44% ) . moreover , additional analysis , performed on lymph nodes from the same carcasses that had proved positive by pcr , allowed highlighting type 3 brucella abortus in the bovine carcass and type 3 brucella melitensis in the sheep and goat carcasses . the study shows that cattle , sheep and goats meat of animals slaughtered because they had tested positive for brucellosis may be contaminated by brucella spp . as this could constitute a real risk of transmission to both butchery personnel and consumers , the meat of animals infected by brucella spp . should be analysed before being marketed . in this respect , pcr technique performed on swabs proved to be more useful , practical and faster than the traditional bacteriological method .

behet disease bd chronic inflammatory disease characterized recurrent oral genital ulceration uveitis1 clinical manifestations include skin lesions arthritis thrombosis neurologic symptoms intestinal ulceration polychondritis known rare complication bd case bd combined polychondritis reported firestein et al reported five patients bd relapsing polychondritis rp 19852 we recently experienced case 40-year old male patient bd polychondritis report herein unusual case along review literature a 40-year old man referred hospital evaluation fever oral ulcers skin rashes lasted 10 days he previously suffered intermittent oral ulceration uveitis treated oral prednisolone intraocular injections triamcinolone another hospital 1 year prior admission physical examination his blood pressure 130/80 mmhg pulse rate 80 per minute body temperature 38.4. conjunctival injections eyes several aphthous ulcers tongue figure 1a buccal mucosa a small round ulcer tenderness found penile root figure 1b several round erythematous skin lesions present chest thigh figure 2a his left knee joint swollen figure 2b tender needle aspiration revealed turbid inflammatory synovial fluid the laboratory findings follows hemoglobin 14.8 g dl white blood cell count 10,400/l platelet count 291,000/l erythrocyte sedimentation rate 104 mm hr c reactive protein 205 mg l normal range 0~3.4 mg l the renal liver function tests normal tests rheumatoid factor antinuclear antibody negative the urinalysis normal microorganisms grown blood urine cultures his chest radiography showed abnormal findings mild splenomegaly observed abdominal sonography skin biopsy performed histologic examination showed perivascular mononuclear cell infiltrations extravasation red blood cells fibrinoid necrosis blood vessel walls findings compatible cutaneous vasculitis combined bd figure 3 5th day admission ear auricles became swollen red tender figure 4 he remembered similar auricular inflammation 2 years ago improved taking non steroidal anti inflammatory drugs administered colchicine 1.2 mg day prednisolone 30 mg day clinical symptoms improved discharged tenth day admission polychondritis inflammation various catilagenous structures auricle nose trachea also known involve eye joints when polychondritis relapsing called relapsing polychondritis rp)8 rp various cutaneous manifestations also seen including palpable purura erythema nodosum panniculitis livedo reticularis urticaria there case reports polychondritis combined rheumatologic diseases systemic lupus erythematosus sle rheumatoid arthritis ra systemic vasculitis9 11 however reports polychondritis combined bd firestein et al first reported five patients features characteristic bd rp authors termed mouth genital ulcers inflamed cartilage magic syndrome2 polychondritis still known rare complication bd five cases bd combined polychondritis reported since firestein report3 7 patient bd diagnosed based upon recurrent oral genital ulcers skin vasculitis arthritis past history uveitis the chondritis developed bilateral auricles eye joints acute exacerbation period bd resolved improvement symptoms bd this suggests development polychondritis may associated bd case treatment combined cases bd rp colchicine steroid dapsone skin involvement bd includes various lesions erythema nodosum pustules papules pseudofolliculitis acneiform folliculits sweet syndrome pyoderma gangrenosum12 the histopathology papulopustular lesions usually shows vasculopathy characterized perivascular infiltration mononuclear cells infiltrate mainly composed lymphocytes the erythema nodosum like lesion known characterized extravascular neutrophilic infiltration panniculitis followed lymphocyte infiltration evolution skin lesions14 case main histopathylogic finding skin lesions perivascular mononuclear cell infiltration fibrinoid necrosis vessel walls characteristic findings vasculopathy although cases bd combined polychondritis rarely reported bd polychondritis seem separate entities compared frequency various clinical features bd rp table 1 showed manifestations considered unique one disease could found disease2 these similarities clinical manifestations bd rp imply seems something common pathogenesis bd polychondritis the pathogenesis bd rp associated autoimmunity patients rp the antibodies collagen type ii ix xi elevated sera15 16 frequency human leukocyte antigen hla)-dr4 increased17 additionally rp associated autoimmune diseases ra sle9 10 patients bd autoantibodies antiendothelial cell antibody antibody -tropomyosin found18 20 postulated cross reactivity molecular mimicry peptides streptococcal proteins viral heat shock proteins hsps homologues human hsps mucosal antigens result selection autoreactive cells21 have proposed autoimmunity components cartilage type ii collagen proteoglycans elastic tissue could common mechanism bd rp2 in particular elastic tissue ubiquitous human mucosa mainly involved bd cartilage major target rp however rarity combined cases yet investigations common pathogenesis two diseases additional research clearly needed although reports polychondritis combined bd think combination diseases might overlooked far closer scrutiny would reveal cases

polychondritis is an inflammatory disorder that affects various catilagenous structures , and the clinical features include auricular , nasal and respiratory tract chondritis . it also involves the eyes , audiovestibular apparatus , joints and vascular structures . polychondritis can be associated with several rheumatologic diseases such as systemic lupus erythematosus , rheumatoid arthritis and systemic vasculitis . however , polychondritis is a rare complication of behet 's disease ( bd ) and only ten cases with combined bd and polychondritis have been reported on around the world . in this report , we describe a 40-year - old korean man with bd who suffered from polychondritis that manifested as bilateral auricular chondritis , conjunctivitis and arthritis .

discussing methodology detailed understanding finite element method significance essential basic idea finite element method find solution complicated problem replacing simpler one finite element method actual continuum body matter these elements considered interconnected specified points called nodes nodal points a variety element types shapes available provide users required flexibility meet compatibility completeness requirements it useful solving complex structural problems dividing complex structures many simpler smaller segments the technical improvement computers finite element software improved accuracy speed analysis general field medicine microsoft windows 98 se operating system)computer aided designing cad modeling software proe-2001msc microsoft windows 98 se operating system computer aided designing cad modeling software proe-2001 msc the creation geometric model mesh generation boundary conditions applied case postprocessing simulating creation geometric model developing finite element model system geometric model generated using cad software the three dimensional model create maxillary four incisors surrounding structures alveolar bone periodontal ligament the morphological dimensions teeth used given wheeler dental anatomy ( 6 edition standard dimensions bounding points cross section space x z coordinates determined plotted cad graphics screen bounding points called key points smooth lines drawn connecting key points simulating profile natural tooth sketch model volume section body is created solid modeling figures 1 2 similar manner periodontal ligament surrounding tooth alveolar bone as difficult obtain hourglass shape periodontal ligament study assumed 0.25 mm thickness around radicular portion although thickness varies alveolar crest apex the alveolar bone modeled rectangular block structure labial thickness 3 mm apically 5 mm distally existing tooth figure 4 sectioned natural tooth sectioned natural tooth periodontal ligament surrounding alveolar bone tooth modeled alveolar bone modeled rectangular block finite element model generation mesh generation forms backbone finite element analysis stage geometric model converted finite element model the cad model meshed divided several three dimensional tetrahedral shaped finite elements table 1 tetrahedral elements used order precisely mesh curved irregular regions present tooth figure 5 the mechanical properties material bone periodontal ligament tooth taken previously published values table 2 number elements nodes model mechanical properties tooth periodontal ligament alveolar bone superior surface bone model boundary conditions areas model restrained movements assigned follows superior surface bone model area representing palatal surface area constrained bases avoid overall rigid body motion 3 figure 5 the three dimensional model oriented way mesiodistal plane represented x axis vertical plane axis labiolingual plane z axis evaluate center resistance the force applied distance 5.2 mm incisal edge modeled tooth labiolingual direction along z axis the magnitude force given 15 g intrusive force 120 g retractive force tooth determination center resistance center resistance tooth point pure force acts produce linear movement without rotation since geometric property finite element analysis easily locate microsoft windows 98 se operating system)computer aided designing cad modeling software proe-2001msc microsoft windows 98 se operating system computer aided designing cad modeling software proe-2001 msc the creation geometric model mesh generation boundary conditions applied case postprocessing simulating tooth movement determination center resistance done creation geometric model developing finite element model system geometric model generated using cad software the three dimensional model create maxillary four incisors surrounding structures alveolar bone periodontal ligament the morphological dimensions teeth used given wheeler dental anatomy ( 6 edition standard dimensions bounding points cross section space x z coordinates determined plotted cad graphics screen bounding points called key points smooth lines drawn connecting key points simulating profile natural tooth sketch model volume section body is created solid modeling figures 1 2 similar manner periodontal ligament surrounding tooth alveolar bone as difficult obtain hourglass shape periodontal ligament study assumed 0.25 mm thickness around radicular portion although thickness varies alveolar crest apex the alveolar bone modeled rectangular block structure labial thickness 3 mm apically 5 mm distally existing tooth figure 4 sectioned natural tooth sectioned natural tooth periodontal ligament surrounding alveolar bone tooth modeled alveolar bone modeled rectangular block finite element model generation mesh generation forms backbone finite element analysis stage geometric model converted finite element model the cad model meshed divided several three dimensional tetrahedral shaped finite elements table 1 tetrahedral elements used order precisely mesh curved irregular regions present tooth figure 5 the mechanical properties material bone periodontal ligament tooth taken previously published values table 2 number elements nodes model mechanical properties tooth periodontal ligament alveolar bone superior surface bone model boundary conditions areas model restrained movements assigned follows superior surface bone model area representing palatal surface area constrained bases avoid overall rigid body motion 3 figure 5 the three dimensional model oriented way mesiodistal plane represented x axis vertical plane axis labiolingual plane z axis evaluate center resistance force was applied distance 5.2 mm incisal edge modeled tooth labiolingual direction along z axis the magnitude force given 15 g intrusive force 120 g retractive force tooth determination center resistance center resistance tooth point pure force acts produce linear movement without rotation since geometric property finite element analysis easily locate creation geometric model developing finite element model system geometric model generated using cad software the three dimensional model create maxillary four incisors surrounding structures alveolar bone periodontal ligament the morphological dimensions teeth used given wheeler dental anatomy ( 6 edition standard dimensions bounding points cross section space x z coordinates determined plotted cad graphics screen bounding points called key points smooth lines drawn connecting key points simulating profile natural tooth sketch model volume section body is created solid modeling figures 1 2 similar manner the periodontal ligament surrounding tooth alveolar bone also modeled figure 3 as difficult obtain hourglass shape periodontal ligament study assumed 0.25 mm thickness around radicular portion although thickness varies alveolar crest apex the alveolar bone modeled rectangular block structure labial thickness 3 mm apically 5 mm distally existing tooth figure 4 sectioned natural tooth sectioned natural tooth periodontal ligament surrounding alveolar bone tooth modeled alveolar bone modeled rectangular block finite element model generation mesh generation forms backbone finite element analysis stage geometric model converted finite element model the cad model meshed divided several three dimensional tetrahedral shaped finite elements table 1 tetrahedral elements used order precisely mesh curved irregular regions present tooth figure 5 the mechanical properties material bone periodontal ligament tooth taken previously published values table 2 number elements nodes model mechanical properties tooth periodontal ligament alveolar bone superior surface bone model boundary conditions areas model restrained movements assigned follows superior surface bone model area representing palatal surface area constrained bases avoid overall rigid body motion 3 figure 5 the three dimensional model oriented way mesiodistal plane represented x axis vertical plane axis labiolingual plane z axis evaluate center resistance the force applied distance 5.2 mm incisal edge modeled tooth labiolingual direction along z axis the magnitude force given 15 g intrusive force 120 g retractive force tooth determination center resistance center resistance tooth point pure force acts produce linear movement without rotation since geometric property finite element analysis easily locate the location center resistance studied years numerous model systems studies include analytical models direct measurement vivo photo elastic technique strain gauge technique laser reflection technique holographic method etc although model systems provided insight displacement characteristics tooth applied forces studies obvious limitations direct measurement vivo distinct disadvantage invasive furthermore difficult apply controlled force variables human subjects anatomic constraints the major limitation studies used physical models synthetic substances used simulate periodontal ligament many materials physical properties exactly duplicating periodontal ligament analytical techniques root morphology approximated parabolid conical wedge shape leading different results overcome the essence method predetermined force idealized assemblage separated finite regions elements these considered interconnected specific points called nodes common boundaries.[5 the results obtained study accordance study matsui et al

aim : the aim of this study is to evaluate the center of resistance of maxillary incisors during simultaneous intrusion and retraction.subjects and methods : in this study , the following steps were employed namely , ( 1 ) preprocessing - the creation of geometric model , mesh generation and boundary conditions . ( 2 ) postprocessing - the tooth movement and determination of center of resistance.results:the center of the mid - sagittal plane approximately 6 mm apical and 4 mm posterior to a line perpendicular to the occlusal plane from the labial alveolar crest of the central incisor resistance for the maxillary four incisors was located within the.conclusion:finite element is a sound mechanical method of analysis as it was sufficient enough to provide insight into interactions between orthodontic forces , and dental tissues and reliability of this study . further by using this study clinically , the center of resistance can be precisely located in single rooted tooth during orthodontic treatment .

study protocol approved animal care use committee juntendo university male c57bl/6 mice 7 weeks old purchased oriental yeast tokyo japan housed specific pathogen deficient apoe mice 6 weeks old purchased jackson laboratory bar harbor housed specific pathogen mice maintained 12-h light dark cycle fed standard rodent diet clea japan nihon bioresearch provided water ad libitum except noted mice treated either high dose 24 nmol kg body wt day low dose 300 pmol kg body wt day exendin-4 sigma aldrich tokyo japan saline mini osmotic pump alzest model 1004 durect cupertino ca delivered solution continuously 28 days age 8 weeks osmotic pump implanted skin back mouse local anesthesia islets isolated standard collagenase digestion method described previously 16 mouse aortic vascular endothelial cells isolated cultured described previously 17 von willebrand factor antibody dako carpinteria ca negative immunostaining anti-smooth muscle actin sigma aldrich mouse aortic vascular smooth muscle cells isolated cultured described previously 18 the cultured cells verified smooth muscle cells immunostaining using anti-smooth muscle actin peritoneal macrophages harvested mice cold pbs 3 days intraperitoneal injection 3% thioglycolate media the pooled macrophages mouse cultured rpmi 1640 supplemented 0.2% fcs 10 mmol l hepes 1 mmol l sodium pyruvate 2 mmol l l glutamine 100 units ml penicillin 100 g ml streptomycin 50 mol l 2-mercaptoethanol 95% relative humidity 5% co2 37c allow cell adhesion human peripheral blood mononuclear cells isolated whole blood collected overnight fasted healthy volunteers mono poly resolving medium ds pharma biomedical osaka japan heparinization monocytes isolated peripheral blood mononuclear cells positive selection using macs cd14 microbeads miltenyi biotec bergisch gladbach germany we confirmed 95% isolated cells cd14 monocytes flow cytometry analysis cells cultured rpmi 1640 supplemented 2% fcs 10 mmol l hepes 1 mmol l sodium pyruvate 2 mmol l l glutamine 100 units ml penicillin 100 g ml streptomycin 50 mol l 2-mercaptoethanol 37c humidified atmosphere 5% co2 thp-1 cells cultured cultured rpmi 1640 supplemented 10% fcs 10 mmol l hepes 1 mmol l sodium pyruvate 2 mmol l l glutamine 100 units ml penicillin 100 g ml streptomycin 50 mol l 2-mercaptoethanol 95% relative humidity 5% co2 37c human umbilical vein endothelial cells huvecs cultured 500-ml bottle endothelial cell basal medium-2 following growth supplements 0.2 ml hydrocortisone 2 ml human fibroblast growth factor basic 0.5 ml vascular endothelial growth factor 0.5 ml r3-igf-1 0.5 ml ascorbic acid 0.5 ml heparin 10 ml fbs 0.5 ml human epidermal growth factor 0.5 ml ga-1000 cambrex bioscience walkersville charles city ia 95% relative humidity 5% co2 37c all samples sonicated ice centrifuged 15,000 g 4c 20 min supernatants collected western blot analysis performed using anti glp-1r antibody ab3907 abcam cambridge u.k rabbit anti glyceraldehyde-3-phosphate dehydrogenase antibody cell signaling technology beverly described previously 16 the intraperitoneal glucose tolerance test ipgtt performed age 12 weeks 4 weeks implantation osmotic pump briefly 1.0 g kg body wt glucose injected intraperitoneally overnight fasting blood glucose level measured glucometer one touch ultra life scan burnaby canada plasma insulin levels measured using elisa kit morinaga kanagawa japan the insulin tolerance test performed age 12 weeks 0.75 units kg body wt insulin humalin ; blood samples collected retro orbital venous plexus awake mice measure blood glucose plasma insulin concentrations after mice killed intraperitoneal injection sodium pentobarbital 1 mg kg abbott laboratories tissue preparation performed systemic perfusion saline 10% buffered formalin fixation performed immersion isolated thoracic aorta 10% buffered formalin 4c en face immunohistochemistry endothelial surface thoracic aorta cut open longitudinally along ventral side scissors placed glass slide then immunohistochemistry performed using anti mouse mac-2 monoclonal antibody dako cederlane burlington canada next specimen placed slide glass intimal side covered coverslip specimens viewed microscope e800 nikon tokyo japan connected xyz controller digital camera sony tokyo japan count number endothelium adherent monocytes set rectangular area sides twice length long short diameters vessel opening intracostal arteries respectively centered opening the total number mac-2immunopositive cells within entire rectangular areas counted aorta cell density area was calculated cell count determined examiner blinded treatment regimen divided total area 1921 fluorescent staining the samples embedded optimal cutting temperature compound sectioned air dried washed pbs after immersion blocking solution 10% goat serum pbs 30 min room temperature sections incubated overnight 4c humidified chamber labeling rabbit polyclonal anti glp1 receptor antibody 1:50 ls a1205 mbl international woburn rabbit anti mac-2 monoclonal antibody 1:200 dako the specimens placed appropriate goat secondary antibody conjugated alexa fluor dyes invitrogen carlsbad ca diluted 1:300 pbs 30 min room temperature specimen placed glass slide dapi containing mounting medium vector laboratories burlingame ca added tissue covered cover glass samples viewed confocal laser scanning microscopy fluoview fv1000 olympus tokyo japan the heart aorta flushed normal saline followed 10% buffered formalin described previously 20 quantitative analysis arteriosclerotic lesions aortic sinus the heart cut two halves top half embedded optimal cutting temperature compound cross sectioned 4-m thickness 50-m interval cryostat twelve consecutive sections taken sequentially aortic valve throughout aortic sinus allowed dry room temperature 30 min the mean lesion area 12 sections calculated expressed square millimeters the isolated macrophages washed incubated without 0.03 0.3 3 nmol l exendin-4 10 mol l forskolin adenylate cyclase activator sigma aldrich 1 h followed incubation without lipopolysaccharide lps 1 g ml sigma aldrich 1 h. inhibit exenatide signal macrophages incubated 5 mol l mdl-12330a sigma aldrich specific adenylate cyclase inhibitor 10 mol l pki14 22 sigma aldrich protein kinase pka inhibitor 30 min adding exendin-4 control macrophages incubated vehicle dmso final concentration 0.1% treatment nuclear protein extracts isolated peritoneal macrophages content nuclear factor-b nf-b p65 determined using specific elisa kit using method recommended manufacturer imgenex san diego ca 22 total rna extracted peritoneal macrophages using rna easymicro kit qiagen tokyo japan instructions provided manufacturer first strand cdna synthesized using 1 g total rna oligo dt primers superscript reverse transcriptase invitrogen described previously 23 the resulting cdnas amplified using sybr green pcr kit applied biosystems foster city ca quantitative pcr performed abi prism 7700 sequence detection system perkin elmer life sciences boston the relative abundances mrnas calculated comparative cycle threshold method tata box after collection stabilization 6 h monocytes cultured presence absence different concentrations exendin-4 24 h. culture supernatants removed cells washed pbs remove nonadherent cells the fc receptors blocked clear back human fc receptor blocking reagent mbl nagoya japan 5 min room temperature then cells stained phycoerythrin labeled antibodies anti human cd11b corresponding isotype control nonspecific mouse igg phycoerythrin bd biosciences cologne germany incubated 4c 15 min bonferroni multiple comparison test used comparisons among multiple treatment groups control group the study protocol approved animal care use committee juntendo university male c57bl/6 mice 7 weeks old purchased oriental yeast tokyo japan housed specific pathogen deficient apoe mice 6 weeks old purchased jackson laboratory bar harbor housed specific pathogen mice maintained 12-h light dark cycle fed standard rodent diet clea japan nihon bioresearch provided water ad libitum except noted mice treated either high dose 24 nmol kg body wt day low dose 300 pmol kg body wt day exendin-4 sigma aldrich tokyo japan saline mini osmotic pump alzest model 1004 durect cupertino ca delivered solution continuously 28 days age 8 weeks osmotic pump implanted skin back mouse local anesthesia islets isolated standard collagenase digestion method described previously 16 mouse aortic vascular endothelial cells isolated cultured described previously 17 von willebrand factor antibody dako carpinteria ca negative immunostaining anti-smooth muscle actin sigma aldrich mouse aortic vascular smooth muscle cells isolated cultured described previously 18 the cultured cells verified smooth muscle cells immunostaining using anti-smooth muscle actin peritoneal macrophages harvested mice cold pbs 3 days intraperitoneal injection 3% thioglycolate media the pooled macrophages mouse cultured rpmi 1640 supplemented 0.2% fcs 10 mmol l hepes 1 mmol l sodium pyruvate 2 mmol l l glutamine 100 units ml penicillin 100 g ml streptomycin 50 mol l 2-mercaptoethanol 95% relative humidity 5% co2 37c allow cell adhesion human peripheral blood mononuclear cells isolated whole blood collected overnight fasted healthy volunteers mono poly resolving medium ds pharma biomedical osaka japan heparinization monocytes isolated peripheral blood mononuclear cells positive selection using macs cd14 microbeads miltenyi biotec bergisch gladbach germany we confirmed 95% isolated cells cd14 monocytes flow cytometry analysis cells cultured rpmi 1640 supplemented 2% fcs 10 mmol l hepes 1 mmol l sodium pyruvate 2 mmol l l glutamine 100 units ml penicillin 100 g ml streptomycin 50 mol l 2-mercaptoethanol 37c humidified atmosphere 5% co2 thp-1 cells cultured cultured rpmi 1640 supplemented 10% fcs 10 mmol l hepes 1 mmol l sodium pyruvate 2 mmol l l glutamine 100 units ml penicillin 100 g ml streptomycin 50 mol l 2-mercaptoethanol 95% relative humidity 5% co2 37c human umbilical vein endothelial cells huvecs cultured 500-ml bottle endothelial cell basal medium-2 following growth supplements 0.2 ml hydrocortisone 2 ml human fibroblast growth factor basic 0.5 ml vascular endothelial growth factor 0.5 ml r3-igf-1 0.5 ml ascorbic acid 0.5 ml heparin 10 ml fbs 0.5 ml human epidermal growth factor 0.5 ml ga-1000 cambrex bioscience walkersville charles city ia 95% relative humidity 5% co2 37c all samples sonicated ice centrifuged 15,000 g 4c 20 min the supernatants collected western blot analysis performed using anti glp-1r antibody ab3907 abcam cambridge u.k rabbit anti glyceraldehyde-3-phosphate dehydrogenase antibody cell signaling technology beverly described previously 16 the intraperitoneal glucose tolerance test ipgtt performed age 12 weeks 4 weeks implantation osmotic pump briefly 1.0 g kg body wt glucose injected intraperitoneally overnight fasting blood glucose level measured glucometer one touch ultra life scan burnaby canada plasma insulin levels measured using elisa kit morinaga kanagawa japan the insulin tolerance test performed age 12 weeks 0.75 units kg body wt insulin humalin eli lilly indianapolis 6 h fasting blood samples collected retro orbital venous plexus awake mice measure blood glucose plasma insulin concentrations after mice killed intraperitoneal injection sodium pentobarbital 1 mg kg abbott laboratories tissue preparation performed systemic perfusion saline 10% buffered formalin fixation performed immersion isolated thoracic aorta 10% buffered formalin 4c en face immunohistochemistry endothelial surface the thoracic aorta cut open longitudinally along ventral side scissors placed glass slide then immunohistochemistry performed using anti mouse mac-2 monoclonal antibody dako cederlane burlington canada next specimen placed slide glass intimal side covered coverslip specimens viewed microscope e800 nikon tokyo japan connected xyz controller digital camera sony tokyo japan count number endothelium adherent monocytes set rectangular area sides twice length long short diameters vessel opening intracostal arteries respectively centered opening the total number mac-2immunopositive cells within entire rectangular areas counted aorta cell density area was calculated cell count determined examiner blinded treatment regimen divided total area 1921 fluorescent staining samples embedded optimal cutting temperature compound sectioned air dried washed pbs after immersion blocking solution 10% goat serum pbs 30 min room temperature sections incubated overnight 4c humidified chamber labeling rabbit polyclonal anti glp1 receptor antibody 1:50 ls a1205 mbl international woburn rabbit anti mac-2 monoclonal antibody 1:200 dako the specimens placed appropriate goat secondary antibody conjugated alexa fluor dyes invitrogen carlsbad ca diluted 1:300 pbs 30 min room temperature the specimen placed glass slide dapi containing mounting medium vector laboratories burlingame ca added tissue covered cover glass samples viewed confocal laser scanning microscopy fluoview fv1000 olympus tokyo japan the heart aorta flushed normal saline followed 10% buffered formalin described previously 20 quantitative analysis arteriosclerotic lesions aortic sinus heart cut two halves top half embedded optimal cutting temperature compound cross sectioned 4-m thickness 50-m interval cryostat twelve consecutive sections taken sequentially aortic valve throughout aortic sinus allowed dry room temperature 30 min the mean lesion area 12 sections calculated expressed square millimeters the isolated macrophages washed incubated without 0.03 0.3 3 nmol l exendin-4 10 mol l forskolin adenylate cyclase activator sigma aldrich 1 h followed incubation without lipopolysaccharide lps 1 g ml sigma aldrich 1 h. inhibit exenatide signal macrophages incubated 5 mol / l mdl-12330a sigma aldrich specific adenylate cyclase inhibitor 10 mol l pki14 22 sigma aldrich protein kinase pka inhibitor 30 min adding exendin-4 control macrophages incubated vehicle dmso final concentration 0.1% nuclear protein extracts isolated peritoneal macrophages content nuclear factor-b nf-b p65 determined using specific elisa kit using method recommended manufacturer imgenex san diego ca 22 total rna extracted peritoneal macrophages using rna easymicro kit qiagen tokyo japan instructions provided manufacturer first strand cdna synthesized using 1 g total rna oligo dt primers superscript reverse transcriptase invitrogen described previously 23 the resulting cdnas amplified using sybr green pcr kit applied biosystems foster city ca quantitative pcr performed abi prism 7700 sequence detection system perkin elmer life sciences boston the relative abundances mrnas calculated comparative cycle threshold method tata box after collection stabilization 6 h monocytes cultured presence absence different concentrations exendin-4 24 h. culture supernatants removed cells washed pbs remove nonadherent cells the fc receptors blocked clear back human fc receptor blocking reagent mbl nagoya japan 5 min room temperature then cells stained phycoerythrin labeled antibodies anti human cd11b corresponding isotype control nonspecific mouse igg phycoerythrin bd biosciences cologne germany incubated 4c 15 min bonferroni multiple comparison test used comparisons among multiple treatment groups control group as first step elucidate antiatherosclerotic effects exendin-4 first investigated expression glp-1 receptor cells associated atherosclerogenesis similar lung pancreatic -cells 24 mice peritoneal macrophages vascular smooth muscle cells abundantly expressed glp-1 receptor protein expression level higher freshly isolated endothelial cells similar expression level macrophages glp-1 receptor abundantly expressed thp-1 cells derived human monocytes freshly isolated human monocytes in contrast freshly isolated endothelial cells abundant expression glp-1 receptor detected huvecs fig in addition immunohistochemical staining showed glp-1 receptor expression cells expressed mac-2 marker macrophages located atherosclerotic lesions aortic valve apoe mice fig these results may suggest glp-1 directly act monocytes macrophages affect progression atherosclerosis a expression glp-1 receptor murine lung liver isolated murine islets isolated murine macrophages ms cultured murine endothelial cells ecs cultured murine smooth muscle cells smcs human monocyte derived line thp-1 cells huvecs c immunohistochemical staining glp-1 receptor green mac-2 marker macrophages red atherosclerotic lesions apoe mice ( a high quality digital representation figure available online issue investigate effect glp-1 receptor activation atherosclerosis c57bl/6 mice received continuous infusion 300 pmol kg day low dose 24 nmol kg day high dose exendin-4 28 days treatment period neither dose affected body weight fig 2a after 24-day treatment doses exendin-4 improved glucose tolerance without affecting insulin secretion fig the results insulin tolerance test similar two groups fig treatment high dose exendin-4 low dose slightly increased total cholesterol hdl cholesterol compared control group table 1 density monocytes adhered endothelial cells thoracic aorta markedly suppressed low- high dose treatment groups compared control fig exendin-4 reduced monocytic adhesion endothelium c57bl/6 mice changes body weight treatment exendin-4 c57bl/6 mice n 6 b blood glucose concentrations ipgtt 24-day treatment exendin-4 n 6 c plasma insulin levels ipgtt 24-day treatment exendin-4 n 6 d results insulin tolerance test group 24-day treatment exendin-4 n 6 e density adherent mac-2positive cells endothelial cells branching areas group mice 28-day treatment n 6 representative en face views immunohistologic staining mac-2 antibody data mean sem p 0.05 versus high dose group p 0.05 versus low dose group ( high quality digital representation figure available online issue ) results laboratory tests c57bl/6 mice apoe mice 28-day treatment exendin-4 data mean se blood samples collected c57bl/6j mice n 6 apoe mice n 13 except hba1c n 6 hba1c fasting state 28-day treatment exendin-4 * p 0.05 vs. control group p 0.05 low ex4 vs. high ex4 low ex4 low dose exendin-4 high ex4 high dose exendin-4 tg triglycerides cm chylomicron sd ldl small dense ldl hba1c a1c na applicable explore role glp-1 receptor activation progression atherosclerosis treatment high dose exendin-4 modestly reduced body weight gain glucose tolerance decreased serum total cholesterol level without affecting ldl cholesterol level fig on hand treatment low dose exendin-4 modestly reduced glucose level 30 min glucose injection without affecting parameters investigated fig the density monocytes adhered endothelial cells thoracic aorta significantly lower low- high dose groups control group fig quantification mrna expression thoracic aorta showed exendin-4 treatment significantly downregulated intercellular adhesion molecule-1 icam-1 tended downregulate vcam-1 fig the oil red positive area aortic valve level significantly reduced high dose group compared control group fig the area atherosclerotic lesions low dose group also tended smaller control group however difference significant the metabolic effect exendin-4 apoe mice changes body weight exendin-4 treatment apoe mice n 13 b blood glucose concentrations ipgtt 24-day treatment exendin-4 n 6 c plasma insulin levels ipgtt 24-day treatment exendin-4 n 6 d results insulin tolerance test group 24-day treatment exendin-4 n 6 data mean sem p 0.01 versus high dose group p 0.01 versus low dose group exendin-4 reduced monocyte adhesion endothelium atherosclerotic lesions apoe mice en face immunohistochemical staining mac-2 antibody aorta group density adherent mac-2positive cells endothelium branching areas group mice 28-day treatment n 7 representative en face views immunohistologic staining mac-2 antibody b aortas harvested group mice 28-day treatment used isolation total rna the mrna expression levels icam-1 vcam-1 determined quantitative rt pcr relative gene expression displayed level expression test mice relative control group set 1.0 n 57 c representative histologic sections aortic sinuses stained oil red 28-day treatment the mean area oil red positive lesions determined n 20 ( high quality digital representation figure available online issue data obtained apoe mice c57bl/6 mice suggested exendin-4 could beneficial effects atherosclerosis without affecting metabolic parameters could potentially prevent progression atherosclerosis direct action cells involved atherosclerogenesis the abundant expression glp-1 receptor monocytes macrophages inhibitory effects exendin-4 monocyte adhesion endothelial cells encouraged us investigate effects exendin-4 inflammatory response indeed incubation 1 g ml lps 1 h induced 10-fold increases expression levels tnf- monocyte chemoattractant protein-1 mcp-1 representative cytokine chemokine isolated macrophages respectively data shown thus investigated effects various concentrations exendin-4 0.033 nmol l counteracting response exendin-4 concentrations significantly suppressed lps induced increases expression levels tnf- mcp-1 macrophages fig glp-1 receptor well known gs protein coupled receptor thus activation glp-1 receptor results increased camp concentration due activation adenylate cyclase 25 explore mechanism exendin-4induced suppression tnf- mcp-1 expression macrophages preincubated peritoneal macrophages mdl-12330a specific adenylyl cyclase inhibitor forskolin adenylyl cyclase activator the addition mdl-12330a completely suppressed inhibitory effect exendin-4 expression levels tnf- mcp-1 fig on hand forskolin significantly suppressed lps induced tnf- mcp-1 expression macrophages levels suppression forskolin similar exendin-4 fig these results suggest inhibitory effects exendin-4 expression tnf- mcp-1 largely dependent activation adenylate cyclase next investigated downstream pathway camp using pki14 22 specific pka inhibitor similar mdl-12330a inhibitory effect exendin-4 significantly reversed pki14 22 fig 5d suggesting involvement pka anti inflammatory effect exendin-4 exendin-4 reduced inflammatory response camp signaling pathway macrophages reduced expression cd11b human monocytes peritoneal macrophages isolated 8-week old c57bl/6 mice were incubated various concentrations exendin-4 0.033 nmol l 1 h followed treatment lps 1 g ml 1 h. macrophages used isolation total rna the mrna expression levels tnf- mcp-1 determined quantitative rt pcr relative gene expression displayed level expression peritoneal macrophages without addition exendin-4 set 1.0 n 45 b peritoneal macrophages preincubated 5 mol l mdl-12330a 30 min addition 0.3 nmol l exendin-4 incubated lps 1 g ml 1 h. macrophages used isolation total rna n 46 / l exendin-4 10 mol l forskolin 1 h followed lps 1 g ml 1 h. macrophages used isolation total rna n 45 mol l pki14 22 30 min addition 0.3 nmol l exendin-4 incubated lps 1 g ml 1 h. macrophages used isolation total rna n 45 mol l mdl-12330a 30 min addition 0.3 nmol l exendin-4 incubated lps 1 g ml 1 h. macrophages used isolation nuclear protein extracts the nuclear level nf-b p65 determined enzyme linked immunosorbent assay elisa n 34 f human monocytes isolated healthy volunteers incubated without various concentrations exendin-4 0.033 nmol l 24 h. surface expression cd11b assessed flow cytometry although nf-b major regulator expression tnf- mcp-1 vasoactive intestinal peptide pituitary adenylate cyclase activating polypeptide known inhibit nf-b dependent gene activation activation pka cultured monocytic cell line thp-1 26 thus investigated effect exendin-4 lps induced nuclear translocation nf-b p65 macrophages without stimulation nuclear nf-b p65 detected peritoneal macrophages however stimulation lps robustly induced nuclear translocation nf-b p65 data shown such translocation markedly suppressed exendin-4 inhibitory effect completely abolished mdl-12330a fig these results indicate exendin-4 inhibits nuclear translocation nf-b p65 activating camp parallel expression tnf- mcp-1 finally investigated effect exendin-4 human monocytes thus investigated effects exendin-4 counterreceptors cd11b isolated human peripheral monocytes exposure 0.3 3 nmol l 0.03 nmol l exendin-4 24 h significantly reduced surface expression cd11b assessed flow cytometry fig these results suggest activation glp-1 receptor antiatherogenic effects human circulating monocytes as first step elucidate antiatherosclerotic effects exendin-4 first investigated expression glp-1 receptor cells associated atherosclerogenesis similar lung pancreatic -cells 24 mice peritoneal macrophages vascular smooth muscle cells abundantly expressed glp-1 receptor protein expression level higher freshly isolated endothelial cells similar expression level macrophages glp-1 receptor abundantly expressed thp-1 cells derived human monocytes freshly isolated human monocytes in contrast freshly isolated endothelial cells abundant expression glp-1 receptor detected huvecs fig in addition immunohistochemical staining showed glp-1 receptor expression cells expressed mac-2 marker macrophages located atherosclerotic lesions aortic valve apoe mice fig these results may suggest glp-1 directly act monocytes macrophages affect progression atherosclerosis a expression glp-1 receptor murine lung liver isolated murine islets isolated murine macrophages ms cultured murine endothelial cells ecs cultured murine smooth muscle cells smcs human monocyte derived line thp-1 cells huvecs c immunohistochemical staining glp-1 receptor green mac-2 marker macrophages red atherosclerotic lesions apoe mice ( a high quality digital representation figure available online issue ) to investigate effect glp-1 receptor activation atherosclerosis c57bl/6 mice received continuous infusion 300 pmol kg day low dose 24 nmol kg day high dose exendin-4 28 days treatment period neither dose affected body weight fig 2a after 24-day treatment doses exendin-4 improved glucose tolerance without affecting insulin secretion fig the results insulin tolerance test similar two groups fig treatment high dose exendin-4 low dose slightly increased total cholesterol hdl cholesterol compared control group table 1 the density monocytes adhered endothelial cells thoracic aorta markedly suppressed low- high dose treatment groups compared control fig exendin-4 reduced monocytic adhesion endothelium c57bl/6 mice changes body weight treatment exendin-4 c57bl/6 mice n 6 b blood glucose concentrations ipgtt 24-day treatment exendin-4 n 6 c plasma insulin levels ipgtt 24-day treatment exendin-4 n 6 d results insulin tolerance test group 24-day treatment exendin-4 n 6 e density adherent mac-2positive cells endothelial cells branching areas group mice 28-day treatment n 6 representative en face views immunohistologic staining mac-2 antibody data mean sem p 0.05 versus high dose group p 0.05 versus low dose group ( high quality digital representation figure available online issue ) results laboratory tests c57bl/6 mice apoe mice 28-day treatment exendin-4 data mean se blood samples collected c57bl/6j mice n 6 apoe mice n 13 except hba1c n 6 hba1c fasting state 28-day treatment exendin-4 * p 0.05 vs. control group p 0.05 low ex4 vs. high ex4 low ex4 low dose exendin-4 high ex4 high dose exendin-4 tg triglycerides cm chylomicron sd ldl small dense ldl hba1c a1c na applicable to explore role glp-1 receptor activation progression atherosclerosis treated apoe mice low- high dose exendin-4 treatment high dose exendin-4 modestly reduced body weight gain glucose tolerance decreased serum total cholesterol level without affecting ldl cholesterol level fig 3 table 1 hand treatment low dose exendin-4 modestly reduced glucose level 30 min glucose injection without affecting parameters investigated fig the density monocytes adhered endothelial cells thoracic aorta significantly lower low- high dose groups control group fig quantification mrna expression thoracic aorta showed exendin-4 treatment significantly downregulated intercellular adhesion molecule-1 icam-1 tended downregulate vcam-1 fig the oil red positive area aortic valve level significantly reduced high dose group compared control group fig the area atherosclerotic lesions low dose group also tended smaller control group however difference significant the metabolic effect exendin-4 apoe mice changes body weight exendin-4 treatment apoe mice n 13 b blood glucose concentrations ipgtt 24-day treatment exendin-4 n 6 c plasma insulin levels ipgtt 24-day treatment exendin-4 n 6 d results insulin tolerance test group 24-day treatment exendin-4 n 6 * p 0.01 versus high dose group p 0.01 versus low dose group exendin-4 reduced monocyte adhesion endothelium atherosclerotic lesions apoe mice en face immunohistochemical staining mac-2 antibody aorta group density adherent mac-2positive cells endothelium branching areas group mice 28-day treatment n 7 representative en face views immunohistologic staining mac-2 antibody b aortas harvested group mice 28-day treatment used isolation total rna the mrna expression levels icam-1 vcam-1 determined quantitative rt pcr relative gene expression displayed level expression test mice relative control group set 1.0 n 57 c representative histologic sections aortic sinuses stained oil red 28-day treatment the mean area oil red positive lesions determined n 20 ( high quality digital representation figure available online issue ) the data obtained apoe mice c57bl/6 mice suggested exendin-4 could beneficial effects atherosclerosis without affecting metabolic parameters could potentially prevent progression atherosclerosis direct action cells involved atherosclerogenesis the abundant expression glp-1 receptor monocytes macrophages inhibitory effects exendin-4 monocyte adhesion endothelial cells encouraged us investigate effects exendin-4 inflammatory response indeed incubation 1 g ml lps 1 h induced 10-fold increases expression levels tnf- monocyte chemoattractant protein-1 mcp-1 representative cytokine chemokine isolated macrophages respectively data shown thus investigated effects various concentrations exendin-4 0.033 nmol l counteracting response exendin-4 concentrations significantly suppressed lps induced increases expression levels tnf- mcp-1 macrophages fig glp-1 receptor well known gs protein coupled receptor thus activation glp-1 receptor results increased camp concentration due activation adenylate cyclase 25 explore mechanism exendin-4induced suppression tnf- mcp-1 expression macrophages preincubated peritoneal macrophages mdl-12330a specific adenylyl cyclase inhibitor forskolin adenylyl cyclase activator the addition mdl-12330a completely suppressed inhibitory effect exendin-4 expression levels tnf- mcp-1 fig on hand forskolin significantly suppressed lps induced tnf- mcp-1 expression macrophages levels suppression forskolin similar exendin-4 fig these results suggest inhibitory effects exendin-4 expression tnf- mcp-1 largely dependent activation adenylate cyclase next investigated downstream pathway camp using pki14 22 specific pka inhibitor similar mdl-12330a inhibitory effect exendin-4 significantly reversed pki14 22 fig 5d suggesting involvement pka anti inflammatory effect exendin-4 exendin-4 reduced inflammatory response camp signaling pathway macrophages reduced expression cd11b human monocytes peritoneal macrophages isolated 8-week old c57bl/6 mice were incubated various concentrations exendin-4 0.033 nmol l 1 h followed treatment lps 1 g ml 1 h. macrophages used isolation total rna the mrna expression levels tnf- mcp-1 determined quantitative rt pcr relative gene expression displayed level expression peritoneal macrophages without addition exendin-4 set 1.0 n 45 b peritoneal macrophages preincubated 5 mol l mdl-12330a 30 min addition 0.3 nmol l exendin-4 incubated lps 1 g ml 1 h. macrophages used isolation total rna n 46 c peritoneal macrophages incubated 0.3 nmol l exendin-4 10 mol l forskolin 1 h followed lps 1 g ml 1 h. macrophages used isolation total rna n 45 mol l pki14 22 30 min addition 0.3 nmol l exendin-4 incubated lps 1 g ml 1 h. macrophages used isolation total rna n 45 e peritoneal macrophages preincubated 5 mol l mdl-12330a 30 min addition 0.3 nmol l exendin-4 incubated lps 1 g ml 1 h. macrophages used isolation nuclear protein extracts the nuclear level nf-b p65 determined enzyme linked immunosorbent assay elisa n 34 f human monocytes isolated healthy volunteers incubated without various concentrations exendin-4 0.033 nmol l 24 h. surface expression cd11b assessed flow cytometry although nf-b major regulator expression tnf- mcp-1 vasoactive intestinal peptide pituitary adenylate cyclase activating polypeptide known inhibit nf-b dependent gene activation activation pka cultured monocytic cell line thp-1 26 thus investigated effect exendin-4 lps induced nuclear translocation nf-b p65 macrophages without stimulation nuclear nf-b p65 detected peritoneal macrophages however stimulation lps robustly induced nuclear translocation nf-b p65 data shown such translocation markedly suppressed exendin-4 inhibitory effect completely abolished mdl-12330a fig these results indicate exendin-4 inhibits nuclear translocation nf-b p65 activating camp parallel expression tnf- mcp-1 finally investigated effect exendin-4 human monocytes thus investigated effects exendin-4 counterreceptors cd11b isolated human peripheral monocytes exposure 0.3 3 nmol l 0.03 nmol l exendin-4 24 h significantly reduced surface expression cd11b assessed flow cytometry fig these results suggest activation glp-1 receptor antiatherogenic effects human circulating monocytes in present study provide evidence exendin-4 glp-1 receptor agonist prevents progression atherosclerosis apoe mice without major effects metabolic parameters our data suggest exendin-4 markedly reduced accumulation monocytes macrophages vascular wall least part suppressing inflammatory response macrophages activation camp pka pathway the results showed exendin-4 decreased monocyte adhesion endothelial cells two nondiabetic mice c57bl6 apoe mice mouse strains exendin-4 reduced glucose level ipgtt however two strains nondiabetic effect exendin-4 glucose level play minor effect antiatherosclerogenetic properties study confirmed expression glp-1 receptor endothelial cells smooth muscle cells macrophages monocytes because cells play critical roles progression atherosclerosis glp-1 receptor activation cells may involved atherosclerogenesis indeed several groups reported beneficial effects glp-1 receptor activation endothelial cells 1013 hand confirmed present study direct anti inflammatory effect glp-1 monocytes macrophages indeed treatment exendin-4 concentrations observed treatment humans 27,28 reduced expression inflammatory mediators tnf- mcp-1 activated macrophages tnf- mcp-1 among important cytokines chemokines whose atherogenic effect established tnf- and furthermore forced expression tnf- mcp-1 leukocytes promotes advanced atherosclerotic lesions 29,31 thus addition effect glp-1 endothelial cells effect monocytes macrophages may also major impact attenuation atherosclerosis it reported previously main effects glp-1 mediated activation adenylate cyclase production camp 25 using adenylate cyclase inhibitor activator also demonstrated study stimulation camp exendin-4 critical attenuated production proinflammatory mediators macrophages this result consistent previous studies increased intracellular levels camp inhibited tnf- production transcription macrophage 3234 these data suggest exendin-4 regulates inflammatory response macrophages via camp pka pathway inhibits proinflammatory cytokine production reported recently 35,36 our results showed pka activation inhibition nf-b p65 translocation mediate overexpression inflammatory cytokines increased camp level elicited glp-1 receptor activation the adhesion circulating monocytes intimal endothelial cells thought one earliest events complex pathologic process atherosclerogenesis 14,15 this mediated interaction specific adhesion molecules vascular endothelial cells integrin counterreceptors monocytes activation monocytes cytokines chemokines hypercholesterolemia hyperglycemia leads increased expression integrin increased monocyte expression cd11b correlates adhesion cells endothelium patients hypercholesterolemia 37 our results showed potential suppressive effect exendin-4 surface expression cd11b human monocytes hand demonstrated exendin-4 decreased expression icam-1 interacts cd11b monocytes apoe mice these effects may contribute least part reduced monocyte adhesion endothelium vivo however additional experiments required clarify mechanism exendin-4induced inhibition cd11b expression conclusion data suggest glp-1 receptor activation significantly reduced accumulation monocytes macrophages vascular wall eventually inhibited atherosclerogenesis regulating inflammation macrophages via camp pka pathway integrin related gene expression monocytes these unique effects glp-1 receptor activation may help design new therapies cardiovascular disease patients type 2 diabetes

objectiveexogenous administration of glucagon - like peptide-1 ( glp-1 ) or glp-1 receptor agonists such as an exendin-4 has direct beneficial effects on the cardiovascular system . however , their effects on atherosclerogenesis have not been elucidated . the aim of this study was to investigate the effects of glp-1 on accumulation of monocytes / macrophages on the vascular wall , one of the earliest steps in atherosclerogenesis.research design and methodsafter continuous infusion of low ( 300 pmol kg1 day1 ) or high ( 24 nmol kg1 day1 ) dose of exendin-4 in c57bl/6 or apolipoprotein e deficient mice ( apoe/ ) , we evaluated monocyte adhesion to the endothelia of thoracic aorta and arteriosclerotic lesions around the aortic valve . the effects of exendin-4 were investigated in mouse macrophages and human monocytes.resultstreatment with exendin-4 significantly inhibited monocytic adhesion in the aortas of c57bl/6 mice without affecting metabolic parameters . in apoe/ mice , the same treatment reduced monocyte adhesion to the endothelium and suppressed atherosclerogenesis . in vitro treatment of mouse macrophages with exendin-4 suppressed lipopolysaccharide - induced mrna expression of tumor necrosis factor- and monocyte chemoattractant protein-1 , and suppressed nuclear translocation of p65 , a component of nuclear factor-b . this effect was reversed by either mdl-12330a , a camp inhibitor or pki14 - 22 , a protein kinase a specific inhibitor . in human monocytes , exendin-4 reduced the expression of cd11b.conclusionsour data suggested that glp-1 receptor agonists reduced monocyte / macrophage accumulation in the arterial wall by inhibiting the inflammatory response in macrophages , and that this effect may contribute to the attenuation of atherosclerotic lesion by exendin-4 .

disability prevalence increases advanced age disability elderly population major public health problem this especially crucial chinese 88 million persons aged 65 china alone number projected increase 341 million 2050 importantly disability prevalence chinese elderly population increasing 35 contrast declining trend developed countries 6 7 there big gap scale problem quantity quality available information the first population based report china published 16 years ago countable number publications 35 812 exist literature study large population based data shanghai biggest city china singapore city country southeast asia majority residents descendents immigrants south china aimed examine independent contributions physical cognitive health disability chinese older adults explore potential influences environment participants present study identified two big population based studies shanghai survey alzheimer disease dementia ssadd singapore longitudinal ageing study slas the ssadd conducted 1987 based probability sample 5055 community dwelling chinese older adults jing'an district shanghai city the slas conducted among total population sample 2808 older adults south east region singapore baseline assessments slas completed september 2003 december 2005 details ssadd 8 13 and selected 4639 participants ssadd sample 2397 participants slas cohort all participants complete data demographic information physical health cognitive function obtained mini mental state examination mmse total score least 21 points face face interviews conducted psychiatrists psychiatric nurses shanghai trained research nurses singapore data collected wide range variables present analysis extracted following variables databases age sex functional status chronic diseases self rated health status mmse total score functional status assessed participant level dependency performing 8 activities daily living adl eating grooming dressing transferring walking toileting bathing climbing stairs the participants asked following illnesses conditions present time slas list 14 medical conditions covered interview the participants asked history medical problem medical conditions included list recorded problems selected ten chronic diseases data available samples hypertension diabetes heart diseases singapore defined heart attack heart failure atrial fibrillation stroke shanghai effects stroke kidney disorder singapore kidney failure chronic obstructive lung disease shanghai emphysema bronchitis asthma arthritis shanghai arthritis rheumatism mental illness cancer singapore identified problems statistical analysis the number chronic diseases used objective measure physical health singapore self rated health status assessed using single question general would say health excellent good good fair poor shanghai question asked four choices excellent good fair poor excellent good together created new variable self rated health status 1 excellent good 2 good 3 fair 4 poor this used continuous variable multivariate analyses score ranged 1 4 higher value representing poorer health status the test validated shanghai singapore respectively summed scores mmse range 0 30 higher values denoting better cognitive functioning chi square test dichotomous variables independent sample test continuous variables used compare difference regarding various characteristics two study samples multiple logistic regressions used examine relationship health factors disability influence environment analysis based combined sample age sex number chronic diseases self rated health status mmse total score environment singapore versus shanghai included regression model the set variables except environment used stratified analysis the prevalence disability much higher shanghai sample 5.0% singapore sample 1.8% however shanghai participants also older chronic diseases poorer self rated health status obtained lower mmse total score compared singapore counterparts table 1 a scrutiny eight adl items revealed difference significant bathing 3.5% versus 0.4% climbing stairs 3.1% versus 1.2% multiple logistic model based combined sample number chronic diseases self rated health status mmse total score environment the odds ratio 1.35 95% ci 1.221.50 number chronic diseases 2.85 95% ci 2.363.43 self rated health status continuous 0.89 95% ci 0.850.94 mmse total score 0.68 95% ci 0.480.96 environment singapore versus shanghai respectively results stratified analysis showed strength associations health variables disability differed two samples table 3 for example shanghai sample one point increase 4-point self rated health status scale associated 3.13 times 95% ci 2.553.85 higher odds disability odds ratio 1.73 95% ci 1.072.81 singapore sample mmse total score the corresponding odds ratio 0.91 95% ci 0.860.96 shanghai sample 0.83 95% ci 0.730.94 singapore sample based data two population based studies found number chronic diseases self rated health status cognitive function unmeasured environment factors represented study sample significantly associated functional disability among chinese older adults the contributions physical health 8 18 cognitive function 19 20 functional disability documented well replicated analysis large population based data firstly quantified strength differential contributions self reported physical health self rated health status cognitive function disability chinese elderly population the findings relevant important policymakers medical practitioners academics participants singapore sample 32% less likely disability compared participants shanghai sample participants studies chinese older adults living big city number important covariates adjusted multiple regression models it less likely observed difference caused unmeasured factors individual level however careful examination two difficult adl tasks bathing climbing stairs provided us plausible explanations 1987 families shanghai bathroom shower bathing considered complicated task in contrast majority slas participants lived public houses standard bathroom shower easy climb stairs our study provided fresh evidence role environmental factors disablement process 2123 as pointed verbrugge jette disability personal characteristic instead gap personal capability environmental demand disability diminished swiftly markedly physical mental demands given task reduced our findings suggest environment affects prevalence disability also modifies strength associations disability health variables strengths study include concurrent measuring various health factors simultaneous inclusion factors multivariate model however causal relationship could drawn given cross sectional nature study design in conclusion physical health cognitive function significantly associated disability among chinese older adults living asian metropolises all authors contributed substantially design analysis interpretation data participated drafting revising paper

objective . we aimed to examine the independent contributions of physical health and cognitive function to disability among chinese older adults living in two asian metropolises and explore the potential influences of environment . design and participants . cross - sectional analysis based on data from two population - based studies : the shanghai survey of alzheimer 's disease and dementia ( n = 4639 ) and the singapore longitudinal ageing study ( n = 2397 ) . disability was defined as needing help in at least one activity of daily living . results . the prevalence of functional disability was higher in shanghai sample ( 5% ) than that in singapore sample ( 1.8% ) . number of chronic diseases , self - rated health status , cognitive function ( measured by the mini - mental state examination ) , and environment ( singapore versus shanghai ) significantly contributed to functional disability independent of each other . the adjusted odds ratio was 1.35 ( 95%ci 1.221.50 ) , 2.85 ( 95% ci 2.363.43 ) , 0.89 ( 95% ci 0.850.94 ) , and 0.68 ( 95% ci 0.480.96 ) , respectively . the strength of associations between health variables and disability appeared to be influenced by environment . conclusion . physical health and cognitive function independently contributed to functional disability . the associations are modulated by environmental factors .

conventional nontargeted chemotherapeutics antimetabolites microtubule inhibitors dna intercalating alkylating agents effective killing cancer cells due indiscriminate penetration nearly cells also damage healthy cells causing toxicities myelosuppression alopecia mucositis peripheral neuropathy cardiotoxicity minimize collateral damage healthy tissues physicians must often either reduce dosage decrease frequency drug administration leading incomplete elimination diseased tissue on basis considerations recent approaches cancer therapy focused developing methods specifically target chemotherapeutic agents cancer cells allowing improved tumor suppression fewer adverse events the common approach drug targeting relied specificity monoclonal antibody tumor specific antigen conjugation highly cytotoxic drug tumor specific antibody tumor selective drug delivery examples tumor targeted antibody drug conjugates adcs include trastuzumab emtansine brentuximab vedotin although several adcs shown significant success preclinical clinical settings questions ability penetrate solid tumors raised a related strategy achieve tumor selective drug delivery involves use low molecular weight targeting ligands similarly deliver attached drugs specifically cancer cells drug conjugates figure 1a also target receptors overexpressed malignant cells much smaller sizes may permit thorough tumor penetration ( general representation ligand conjugated cytotoxic payload via peptide linker the circle represents cholecystokinin 2 receptor cck2r binding ligand whereas linker represented oval cytotoxic drug payload the solid black line represents covalent bond ligand linker dotted line symbolizes cleavable self immolative bond ( b chemical structures cck2r ligand crl conjugated cytotoxic antimicrotubule agents desacetyl vinblastine hydrazide tubulysin b hydrazide via hydrophilic peptide linker paper z-360 low molecular weight ligand cholecystokinin 2 receptor cck2r modified deliver two potent antimicrotubule agents currently available desacetyl vinblastine monohydrazide tubulysin b hydrazide cck2r transmembrane receptor primarily found epithelial cells gi tract brain bind gastrin amidated peptides gastrin cholecystokinin family cck2r overexpressed many cancers lung pancreas liver gi tract esophagus colon gastrointestinal stromal tumors cck2r brain inaccessible drugs pass blood brain barrier restricted expression pattern renders cck2r possible candidate ligand targeted drug delivery we report design synthesis biological evaluation two highly potent cck2r targeted chemotherapeutic agents demonstrate efficacy tolerability treating cck2r expressing human tumor xenografts nu nu mice h cys trt)-2-cl trt resin obtained novabiochem san diego ca diisopropylethylamine dipea piperidine dimethylformamide dmf isopropyl alcohol proh reagents purchased sigma aldrich tubulysin b desacetyl vinblastine hydrazide activated derivatives kind gift endocyte inc dulbecco modified eagle medium dmem roswell park memorial institute medium rpmi fetal bovine serum fbs g418 geneticin purchased gibco grand island ny the hek 293 cell line transfected wild type cck2r generous gift dr cells cultured monolayers dmem supplemented 10% fetal bovine serum g418 disulfate 400 g ml 1% 2 mm glutamine 1% penicillin streptomycin 37 c 5% co2:95% humidified air atmosphere kb cells cultured frozen stocks rpmi supplemented concentrations fetal bovine serum penicillin streptomycin glutamine hek 293 cells the cck2r ligand z-360 synthesized previously described abbreviated crl consistent previous publications the peptide spacer prepared using fmoc protected solid phase peptide synthesis outlined scheme 1 supporting information si figure 1 named l1 as shown scheme 1 si figure 1 crl coupled peptide spacer solid phase cleaved resin using standard cleavage cocktail solution crude crl l1 purified preparative rp hplc 2 mm ammonium acetate buffer ph 5.0 b ch3cn solvent gradient 5% b 80% b 25 min yield requisite product lrms lc ms z h calcd c72h110n12o27s 1607.79 found 1608 a solution saturated sodium bicarbonate 2 ml hplc grade water bubbled argon continuously 10 min crl l1 36 mg 0.0226 mmol dissolved argon purged hplc grade water 2.0 ml ph reaction mixture increased 7 using argon purged sodium bicarbonate a solution disulfide activated davbh 11.10 mg mmol thf 2.0 ml added reaction mixture si figure 2 the progress reaction monitored using analytical lrms lcms stirring 20 min reaction found reach completion crude crl l1-davbh purified preparative rp hplc 20 mm ammonium acetate ph 7.2 b ch3cn solvent gradient 5% b 80% b 30 min yielding desired product lrms lc ms z h calcd c118h168n18o36s2 2478 found 2478 crl l1-tubbh synthesized activated tubulysin b hydrazide following procedure used synthesis crl l1-davbh si figure 2 removing thf reduced pressure crl l1-tubbh purified preparative rp hplc 2 mm ammonium acetate buffer ph 7.0 b ch3cn solvent gradient 5% b 80% b 25 min yield requisite product lrms lc ms z h calcd c117h177n19o38s3 2553.96 found 2554 l1-davbh l1-tubbh synthesized activated davbh tubbh respectively following procedure outlined synthesis crl l1-davbh si figure 1 3 each compound purified reverse phase hplc 2 mm ammonium acetate buffer ph 7.0 b ch3cn solvent gradient 5% b 80% b 25 min yield requisite product lrms lc ms z h calcd l1-davbh c89h134n14o32s2 1976.22 found 1976 z h calcd l1-tubbh c88h143n15o34s3 2051.35 found 2051 conjugates found stable saline least four weeks monitored lc ms cck2r transfected hek 293 cells 100,000 cells well seeded amine coated 24-well plates allowed form monolayers the spent medium well replaced fresh medium containing increasing concentrations test agents incubating 2 h 37 c cells rinsed 3 fresh medium incubated additional 66 h 37 c fresh medium spent medium well replaced fresh medium 0.5 ml containing h thymidine 1 ci ml cells incubated additional 4 h. washing cells 3 medium dissolved 0.5 ml 0.25 naoh thymidine incorporation determined counting cell associated radioactivity using scintillation counter packard packard instrument company the ic50 value derived plot percent h thymidine incorporation versus log concentration using graph pad prism 4 tablecurve 2d software hek 293 cells expressing cck2r 5.0 10 50% hc matrigel injected shoulders 56 week old female nu nu mice an age matched group animals similarly implanted 1 10 kb cells 100 l cell culture medium kb cells overexpress cck2r kb xenograft model served negative control tumors measured two perpendicular directions 3 per week vernier calipers volumes calculated 0.5 x l x w l longest axis millimeters w axis perpendicular l millimeters solutions administered either intraperitonealy crl l1-davbh l1-davbh intravenously crl l1-tubbh l1-tubbh crl l1 each mouse received 2 mol kg test control agent 100 l saline per injection injections given 3 per week 3 weeks mice weighed dosing measure gross toxicity all animal work performed guidance purdue laboratory animal program reviewed purdue animal care use committee tumors excised fixed formalin embedded paraffin sectioned stained hematoxylin eosin purdue histology phenotyping laboratory proper design ligand targeted chemotherapeutic agent requires selection high affinity ligand good selectivity cancer enriched receptor ii identification therapeutic agent sufficient potency kill cancer cells captured cancer specific receptor iii construction linker enable delivery release attached drug preferentially within targeted cells cholecystokinin receptor ligand crl shown exhibit high affinity 0.47 nm strong selectivity cck2r 600-fold specificity cck1r selected exploration targeting ligand drug delivery cck2r expressing cancer cells figure 1b avoid nonspecific adsorption cck2r negative cells incorporated water soluble peptide spacer referred l1 ligand therapeutic payload figure 1b previous results lab shown slight loss affinity effect specificity crl conjugated payload via hydrophilic linkers present study two highly potent microtubule inhibitors desacetyl vinblastine hydrazide davbh tubulysin b hydrazide tubbh attached crl l1 peptide spacer self immolative disulfide linker this linker allows selective release cytotoxic agent upon entry reducing environment cancer cells detailed schemes synthesis crl conjugates davbh tubbh described si figures 2 3 complete chemical structures conjugates found si figures 47 determine cytotoxicity targeting specificity crl l1-davbh conjugate incubated crl l1-davbh free davbh nontargeted l1-davbh cck2r transfected hek 293 cells 2 h followed incubation cells drug free medium 66 h. cell viability measured via incorporation h thymidine shown figure 2 potency free davbh crl l1-davbh 9 29 nm respectively whereas potency nontargeted conjugate l1-davbh markedly reduced factor 1000 ic50 value 50 importantly crl found show cytotoxicity toward hek 293-cck2r cells si figure 8) demonstrating aforementioned cytotoxicity due targeted therapeutic agent rather obstruction cck2r crl vitro cytotoxicity davbh derivatives the cytotoxicity free davbh circles nontargeted l1-davbh triangles targeted crl l1-davbh squares conjugates hek 293 cells transfected cck2r pulsed 2 h 37 c washed culture medium three times incubated additional 66 h. cells incubated h thymidine 4 h washed three times final wash cells dissolved 0.5 ml 0.25 naoh viability cells assessed analyzing incorporation h thymidine injected 2 mol kg crl l1-davbh intraperitoneally mice bearing cck2r transfected hek 293 xenografts frequency 3x per week 3 weeks n 5 this dose selected based previous studies showing significant therapeutic benefit similar folate- dupa targeted chemotherapeutics administered concentration shown figure 3a crl l1-davbh was found markedly delay tumor growth lead complete tumor eradication determine whether residual lesion comprised predominately cancer stromal cells h&e staining was performed excised tumors day 33 unlike saline control which comprised almost entirely cancer cells figure 4a crl l1-davbh treated tumors exhibited greatly decreased ratio cancer stromal cells figure 4b expected nontargeted l1-davbh exerted little impact tumor growth demonstrating critical role ligand targeting crl l1-davbh efficacy figure 3a importantly body weights crl l1-davbh treated group remained essentially constant course study suggesting targeted therapy grossly toxic animals figure 3b vivo therapeutic efficacy gross toxicity davbh conjugates ( volume tumors b percent weight change nu nu mice subcutaneously injected cck2r transfected hek 293 cells 5.0 10 50% hc matrigel day 0 mice randomized different treatment groups n 5 began treatment day 15 tumor volume approximately 100 mm saline diamonds nontargeted l1-davbh triangles targeted crl l1-davbh squares conjugates administered intraperitoneally following tiw schedule 3 weeks excised tumors mice treated saline b crl l1-davbh sectioned stained hematoxylin eosin arrows show abundance cancer cells panel reduced number cancer cells panel b. complete tumor remission achieved dose 2 g kg crl l1-davbh elected develop potent cck2r targeted conjugate for purpose tubulysin b hydrazide tubbh microtubule inhibitor 10 potency davbh conjugated crl via l1 linker figure 1 si figures 2 3 as shown figure 5a free tubulysin b hydrazide found potent vitro exhibiting ic50 2.7 nm hek 293-cck2r cells similarly targeted crl l1-tubbh conjugate yielded ic50 2 nm whereas nontargeted l1-tubbh displayed anticipated significant drop potency ic50 310 nm due membrane impermeability lack targeting ensure that cytotoxicity crl l1-tubbh receptor mediated cells incubated crl l1-tubbh presence 100-fold excess crl l1 as expected ic50 value competed crl l1-tubbh nearly identical nontargeted l1-tubbh conjugate ic50 340 310 nm respectively vitro cytotoxicity tubbh derivatives the cytotoxicity free tubbh circles nontargeted l1-tubbh triangles targeted crl l1-tubbh squares targeted crl l1-tubbh presence 100-fold greater concentration competing crl l1 diamonds conjugates hek 293 cells transfected cck2r pulsed 2 h 37 c washed culture medium three times incubated additional 66 h. cells incubated h thymidine 4 h washed three times after final wash cells dissolved 0.5 ml 0.25 naoh viability cells assessed analyzing incorporation h thymidine error bars represent standard deviation determine efficacy crl l1-tubbh vivo followed treatment regimen used davbh conjugates except tubbh conjugates injected intravenously crl l1-tubbh showed efficient antitumor activity eliminating detectable malignant lesions five mice figure 6a prolonged survival si figure 9 expected nontargeted l1-tubbh showed difference saline treated control group identical concentration crl l1-tubbh targeted conjugate tested moreover crl l1-tubbh competed 100-fold excess crl l1 antitumor effect targeted tubbh negated figure 6a this indicates antitumor activity observed crl l1-tubbh group entirely receptor mediated importantly crl l1-tubbh treated mice displayed visible signs gross toxicity weight loss observed figure 6c vivo therapeutic efficacy gross toxicity tubbh conjugates ( b volume tumors c percent weight change nu nu mice subcutaneously injected 5.0 10 cck2r transfected hek 293 cells cck2r(+ cells 50% hc matrigel c 1.0 10 kb cells cck2r( cells 50% hc matrigel day 0 mice randomized different treatment groups n 5 began treatment day 17 tumor volume 100 mm saline diamonds nontargeted l1-tubbh triangles targeted crl l1-tubbh squares targeted crl l1-tubbh presence 100-fold greater concentration competing crl l1 circles conjugates administered via lateral vein injection following tiw schedule 3 weeks finally thoroughly establish necessity receptor mediated targeting crl l1-tubbh efficacy targeted conjugate tested using protocol cck2r negative kb xenograft model vitro crl l1-tubbh conjugate yielded ic50 value 269 nm si figure 9 similar ic50 values observed nontargeted competed targeted conjugates cck2r expressing hek cells as shown figure 6b crl l1-tubbh showed difference tumor growth vehicle control because free tubulyisn b hydrazide known cytotoxic kb cells lack reduction tumor volume kb xenograft must derive absence cck2r intrinsic resistance tubbh again none mice study exhibited signs gross toxicity substantial weight loss observed figure 6d conjugation cytotoxic drugs ligands selectively target cancer cells emerged promising method improve drug efficacy reduce drug toxicity although low molecular weight ligand targeted cancer drugs tested vivo date results suggest potential design targeted therapeutics human malignancies promising folate receptor targeted chemotherapeutic agents demonstrated capacity treat malignancies ovary lung kidney breast endometrium psma targeted drugs shown promise treating cancers prostate lhrh receptor- v3-targeted therapeutics expand list include cancers ovary breast date ligands available target cancers gi tract study attempted obtain ligand would deliver attached drugs gastroinstestinal malignancies on basis literature demonstrating overexpression cck2r gastrointestinal stromal tumors colon stomach esophageal pancreatic cancers crl conjugates described paper potential address deficiency one interesting results emerge study high specificity displayed crl l1-davbh crl l1-tubbh cck2r expressing cancer cells crl davbh tubbh found hydrophobic predicting conjugate ligand drug would extremely lipophilic resulting strong proclivity associate nonspecifically cell membranes thus increase hydrophilicity water soluble linker l1 utilized conjugate crl cytotoxic drugs cck2r dependent binding crl l1-davbh crl l1-tubbh established absence toxicity crl deleted ii conjugate binding competed excess ligand iii cck2r absent targeted cancer cell drug conjugates rendered tumor specific judicious choice hydrophilic linker due linker impeding diffusion hydrophobic drug cell membranes thus reducing nonspecific activity however effect somewhat concentration dependent conjugates diffuse cells therefore high concentrations nonreceptor mediated activity occur case linking crl davbh tubbh the insertion bulky water soluble linker ligand drug converted otherwise nonspecific drug conjugate highly receptor targeted drug conjugate physiologically relevant concentrations our crl tubbh conjugates also tested xenograft tumors similar exposed davbh conjugates except additional treatment group included crl l1-tubbh competed excess crl l1 similar davbh conjugates nontargeted l1-tubbh conjugate appear effect tumor growth the lack efficacy l1-conjugated cytotoxic compounds vivo likely due hydrophilic linker allow cytotoxic compound readily diffuse cell membrane whereas targeted crl l1-davbh crl l1-tubbh showed efficacy vivo somewhat surprisingly crl l1-davbh conjugate regress tumor volume even though similar concentrations davbh conjugated folate dupa exhibit tumor regression this result likely due lower number cck2r receptors present cell surface compared folate psma this resulting lack tumor regression vivo encouraged testing potent cytotoxic agent similar mechanism action when potent crl l1-tubbh conjugate tested halt tumor growth even regressed tumor indistinguishable level importantly crl l1-tubbh treatment group competed crl l1 showed effect inhibiting tumor growth similar vehicle control nontargeted conjugate assess necessity receptor mediated targeting tested xenografts receptor negative kb cell line targeted crl l1-tubbh conjugate unlike cck2r positive hek 293 xenografts the kb xenografts showed neither regression delay tumor growth treated conjugate taken together results strongly support necessity receptor mediated targeting conjugates vivo efficacy summary current study demonstrates cck2r targeting ligands used deliver drugs selectively cck2r positive tumors generate dramatic receptor specific antitumor effect without observable gross toxicity healthy tissues because current treatments colon lung pancreatic related cancers commonly cause hair loss bone marrow suppression weight loss forth prospects developing less toxic treatment malignant diseases offer optimism management cancers future

as the delivery of selectively targeted cytotoxic agents via antibodies or small molecule ligands to malignancies has begun to show promise in the clinic , the need to identify and validate additional cellular targets for specific therapeutic delivery is critical . although a multitude of cancers have been targeted using the folate receptor , psma , bombesin receptor , somatostatin receptor , lhrh , and v3 , there is a notable lack of specific small molecule ligand / receptor pairs to cellular targets found within cancers of the gi tract . because of the selective gi tract expression of the cholecystokinin 2 receptor ( cck2r ) , we undertook the creation of conjugates that would deliver microtubule - disrupting drugs to malignancies through the specific targeting of cck2r via a high affinity small molecule ligand . the cytotoxic activity of these conjugates were shown to be receptor mediated in vitro and in vivo with xenograft mouse models exhibiting delayed growth or regression of tumors that expressed cck2r . overall , this work demonstrates that ligands to cck2r can be used to create selectively targeted therapeutic conjugates .

often new physics faculty member faced duty renovating introductory physics labs we provide list experiments equipment needed convert half traditional labs 1-year introductory physics lab microcomputer based laboratories mbls our student body consists mostly science majors take algebra based course but lab renovation described could used calculus based group well we would suggest adjusting lab manuals nowadays mbls usually choice thinking renovation they effectively demonstrated advantage learning process years.[14 moreover science students labs important reinforcing concepts learned class it common hear lab sections students commenting grasp concept learned class due experiment performed the overall 1-year lab experience follows guidelines provided american association physics teachers it contains brief theoretical description procedures followed day lab lab day students come prepared ready start without additional instructions the instructor circles around stations guide answer appropriate questions needed lab day students handed lab report it contains data analysis part discovery questions ends summary conclusion part report completed student session we find appropriate version half meet overall goal therefore kept traditional labs list experiments chosen based lecture material one concerns always able cover theory lab performed student one year introductory microcomputer based laboratory experiment list understanding motion free fall projectile motion atwood machine boyle law electrical equivalent heat heat transfer electrostatic charge ohm law rc circuit magnetic induction a suggestion beginner try experiments hand get really familiar sensors software work most time lack understanding use apparatus assuming defective course we list total equipment needed per station implement 1-year lab described it expected laboratory printer shared among groups each station consists laptop science workshop 750 interface ci-7650 datastudio software ci-6870 g lab station science workshop interface laptop motion sensor sensors used interface data measurement motion sensor ci-6742a photogate pulley system me-6838 accessory photogate me-9204b time flight accessory me-6810 pressure sensor ci-6532a temperature sensor ci6605a power amplifier ci-6552a charge sensor ci-6555 voltage sensor ci-6503 photogate head me-9498a figure 2 displays sensors left right pressure sensor charge sensor photogate head datastudio software collects analyzes data it easy use interface allowing students explore data for instance left screenshot ure 3 displays graph voltage versus time the data collected using voltage sensor magnet dropped coil the students select region graph software calculates area curve the screenshot right figure 3 displays curve fitting feature datastudio left screenshot induction lab using graph area curve calculation the pressure column filled measurements taken using pressure sensor boyle law lab using table display additional equipment needed pasco perform experiments picket fence me-9377a projectile mini launcher me-6825a photogate mounting bracket me-6821a extension cable pi-8117 thermodynamics kit ci-6514a charge producers ci-6555 faraday ice pail es-9057b ac dc electronics lab em-8656 bar magnet em-8620 general lab supplies needed include pair scissors goggles one digital balance ohaus sp-601 one meterstick one thermometer tongs handling hot bottles gloves handle hot containers braided physics string se-8050 500 ml glass container 90c water banana plug cord red black 5 set se-9750 se-975 masses hanger set me-8979 universal table clamp me-9376b calorimetry cups td-8825a hot plates se-8830 oftentimes task hard accomplish solo faculty small institution we provided list experiments equipments needed upgrade half experiments mbls 1-year introductory physics lab we would like add although used pasco comparable systems market the intention paper help others lab renovation order better fulfill purpose

nowadays , data acquisition software and sensors are being widely used in introductory physics laboratories . this allows the student to spend more time exploring the data that is collected by the computer hence focusing more on the physical concept . very often , a faculty is faced with the challenge of updating or introducing a microcomputer - based laboratory ( mbl ) at his or her institution . this article will provide a list of experiments and equipment needed to convert about half of the traditional labs on a 1-year introductory physics lab into mbls .

octane - enhancing constituents of gasoline pose a number of health hazards . this paper considers the relative risks of metallic ( lead , manganese ) , aromatic ( e.g. , benzene ) , and oxygenated additives in both industrialized and developing countries . technological advances , particularly in industrialized countries , have allowed the progressive removal of lead from gasoline and the increased control of exhaust emissions . the developing world , by contrast , has relatively lax environmental standards and faces serious public health problems from vehicle exhaust and the rapid increase in automobile use . financial obstacles to the modernization of refineries and vehicle fleets compound this problem and the developing world continues to import large quantities of lead additives and other hazardous materials . progress in decreasing environmental health problems depends both on the adoption of international public health standards as well as efforts to decrease dependence on the private automobile for urban transport.imagesfigure 1.figure 2 .

type 2 diabetes mellitus t2 dm growing epidemic united states america alone nearly 25.8 million people approx 8.3% total population disease.1 estimates placed global prevalence disease around 217 million.2 consequence individual prolonged exposure hyperglycemia marked increase risk mortality morbidity associated reduction life expectancy around 1213 years.3 diagnosis usually occurs time development disease often already experienced occult pathology time patients receive diagnosis early adoption aggressive approach disease management improves patient outcome marked reductions morbidity mortality.46 first line approach treatment lifestyle modification.7 however t2 dm progressive nature lifestyle changes sufficient halt disease difficult achieve intense efforts patients healthcare team frequently fail similarly conventional antidiabetic drugs eg metformin sulfonylureas etc often fail slow progression t2 dm despite availability broad range agents employing different mechanisms action.79 progressive nature disease overwhelms available therapies highlighted ukpds study suggesting fewer half patients actually achieve adequate levels disease control.4,5 recognized need new treatment options t2 dm characterization mechanisms facilitating glucose resorption kidney raised possibility novel treatment diabetes inhibition type 2 sodium glucose transporter sglt2 672 amino acid high capacity low affinity transmembrane protein promotes reabsorption glucose glomerular filtrate passes nephrons.10 several candidate molecules currently development may soon available use treatment diabetes we provide brief review sglt2 inhibitors possible role treatment t2 dm most 99% plasma glucose entering kidney filters nephrons though glomeruli normal circumstances the reabsorptive capacity early part nephron proximal tubule sufficient clear filtered glucose load luminal fluid enters loop henl normal individuals around 180 g glucose passes proximal tubules day almost completely reabsorbed.11,12 plasma glucose concentrations increase filtered glucose load increases linear manner when rate glucose entering nephron rises 260350 mg min/1.73 example patients diabetes excess glucose outstrips resorptive capacity appears urine glycosuria).13 healthy adult equates blood glucose concentration approximately 11 mmol l 200 mg dl).14 much 90% filtered glucose load extracted s1 segment remaining 10% removed distal straight tubules s2 s3 segments figure 1 recently the mechanisms behind glucose reabsorption poorly understood although proposed early 1960 glucose transmembrane flux could achieved coupling glucose transport sodium.15figure 1summary glucose reabsorption lumen nephrons glucose concentration glomerular filtrate reflects plasma concentration normal healthy subjects the majority glucose reabsorption believed occur early part proximal tubule glucose concentration glomerular filtrate reflects plasma concentration normal healthy subjects the majority glucose reabsorption believed occur early part proximal tubule since start 20 century phlorizin toxic 2-glucoside phloretin known increase glycosuria used study renal function.16,17 1930s phlorizin used non invasive human experiments revealed fundamental mechanisms renal hemodynamics metabolic transport.18 1950s studies delineated phlorizin mechanism action inhibition glucose transport kidney small intestine cellular molecular levels renal micropuncture studies conducted phlorizin 1970s showed transporter located brush border proximal tubule sodium required renal absorption glucose.11,19,20 studies performed since confirmed phlorizin competitive inhibitor glucose transport binding affinity transporter 1000- 3000-fold greater glucose.21 rabbit homolog human type 1 sodium glucose transporter sglt1 coded slc5a gene first mammalian cotransporter carrier protein identified cloned sequenced.22 family slc5a gene sodium dependent transporters since sequenced identified broad range tissues.23,24 sglt1 sglt2 perhaps slc5a family members received greatest coverage within literature high affinity low capacity slgt1 main gastrointestinal glucose transporter the relatively widespread distribution sglt1 contrasted almost exclusive expression luminal surface proximal tubules mainly renal cortex low glucose affinity high capacity sglt2 responsible renal tubular glucose reabsorption.2226 cellular glucose sodium uptake occurs 1:1 ratio figure 2 the sodium potassium adenosine triphosphatase atp pump transports sodium across basolateral surface intracellular fluid maintaining physiological levels sodium cell cellular glucose concentrations maintained facilitative glucose outflow transporters basolateral membrane cell after binding intracellular glucose transporters undergo conformational change subsequently moderates movement glucose concentration gradient back blood figure 3 figure 2representation 1:1 transport sodium glucose across luminal membrane epithelial cells early part proximal tubule facilitated sglt2figure 3summary glucose transport cascade brush border epithelial cells proximal tubule demonstrating process driven sodium gradient maintained na k atpase basal membrane representation 1:1 transport sodium glucose across luminal membrane epithelial cells early part proximal tubule facilitated sglt2 summary glucose transport cascade brush border epithelial cells proximal tubule demonstrating process driven sodium gradient maintained na k atpase basal membrane the antidiabetic properties phlorizin investigated 1980s partially pancreatectomized rats phlorizin increased glucose secretion urine associated normalizing plasma glucose without inducing hypoglycemia.17 despite promising vitro properties phlorizin fit profile come expect modern therapeutic agent phlorizin also potentially toxic non selective inhibiting sglt1 sglt2 transporters last decade several alternative candidate molecules targeted specifically inhibit sglt2 investigated pre clinical clinical settings.27 aim take advantage potential turning glucose reabsorption new therapeutic target treatment t2 dm first reports devised sglt2 inhibitors started emerge scientific literature second half 1990s developed view overcoming shortcomings phlorizin sglt2 inhibitors represented new mechanism manage hyperglycemia acted independently insulin irrespective patients glycemic status first indications suggest mechanism action independent insulin reduces glycemia used combination traditional antidiabetic treatments results early compounds promising terms specificity transporter compound t-1095 inhibitory capacity sglt2 4-fold greater sglt1.25 pharmacodynamic studies demonstrated attenuated hyperinsulinemia hypertriglyceridemia kk rats following oral administration t-1095.26 lowering insulin resistance hba1c levels along normalized hepatic glucose production glucose utilization rate also observed streptozotocin induced diabetic rats26,28 zucker diabetic fatty rats28,29 following oral administration t-1095 long term administration t-1095 restored impaired insulin secretion pancreatic -cells goto kakizaki gk rats30 suppressed diabetic complications c57bl ksj db db mice gk rats.31,32 however retained co inhibition sglt1 t-1095 led development compound discontinued 2003 reached phase ii clinical trials various sglt2 inhibitors based glucoside structure phlorizin since proposed table 1 narratives discovery pathway different inhibitors recently published.27,33 glucoside moiety phlorizin binds sglt2 transporters o-linked phenolic distal ring responsible inhibitory properties.34 structure activity analysis parent molecule shows addition lipophilic groups distal ring augments inhibition sglt2 transporter increases selectivity sglt2 sglt1.25 however linkage metabolic target -glucosidase enzymes curtail activity sglt2 inhibitors vivo address possible limitation therapeutic utility candidate sglt2 inhibitors both o- c glucosides appear bind single site sglt2 transporter the aromatic heteroaromatic c glucosides metabolically stable glucosides due relative resistance hydrolysis alternative candidate sglt2 inhibitors also considered include modified sugar rings n glucosides recently bridged ketal ring.35table 1candidate sglt2 inhibitorsmanufacturers johnson johnson tanabe seiyaku co. ltd japan t-1095 sanofi aventis ave-2268 glaxosmithkline remogliflozin kgt 1681/sergliflozin wyeth way-123783 astellas pharma inc asp 1941/ym-543 boehringer ingelheim gmbh bi-10773/bi-44847 boehringer ingelheim gmbh ajinomoto canagliflozin johnson johnson tanabe mitsubishi ta-7284/jnj 28431754 bristol myers squibb co astrazeneca dapagliflozin lexicon lx 4211 isis pharmaceuticals isis 388626 roche chugai r7201/csg452 pfizer pf-04971729 candidate molecules also registered kissei taisho theracos daiichi sankyo candidate sglt2 inhibitors manufacturers johnson johnson tanabe seiyaku co. ltd japan t-1095 sanofi aventis ave-2268 glaxosmithkline remogliflozin kgt 1681/sergliflozin wyeth way-123783 astellas pharma inc asp 1941/ym-543 boehringer ingelheim gmbh bi-10773/bi-44847 boehringer ingelheim gmbh ajinomoto canagliflozin johnson johnson tanabe mitsubishi ta-7284/jnj 28431754 bristol myers squibb co astrazeneca dapagliflozin lexicon lx 4211 isis pharmaceuticals isis 388626 roche chugai r7201/csg452 pfizer pf-04971729 candidate molecules also registered kissei taisho theracos daiichi sankyo administration synthesized strands nucleic acid targeted specifically bind sglt2 messenger rna blocks transporter translation protein production expression cells proximal tubule a summary status inhibitor development provided table 2.3654table 2clinical status sglt2 moleculescompoundphasecompanyclintrial.gov studies completed totalliterature citationsc glucosidesdapagliflozin bms-512148)iiibristol myers squibb co./astrazeneca38/47 canagliflozin ta-7284 jnj 28431754)iiijohnson johnson tanabe mitsubishi7/26 asp-1941iiiastellas pharma inc./kotobuki18/27 bi-10773iiiboehringer ingelheim16/31-lx-4211iilexicon pharmaceuticals4/4-dsp-3235 kga-3235 gsk 1614235 1614235)iglaxosmithkline dainippon sumitomo license kissei pharmaceuticals)1/1-o glucosidessergliflozin gw869682)iiglaxosmithkline3/3 remogliflozin kgt 1681)iiglaxosmithkline kissei sanofi aventis16/16 ave-2268-sanofi aventis1/1-ym-543iiaastellas pharmaceutical inc kotobuki1/1-otherisis sglt2rx isis-388626)iisis pharmaceuticals1/1-pf04971729iipfizer7/8- clinical status sglt2 molecules as discussion suggests several hypothetical reasons sglt2 transporter represents opportune target managing blood glucose however challenge establish therapeutic utility demonstrating acceptable safety profile a detailed summary clinical findings recently published.55 mechanism action sglt2 inhibitors predicts beneficial effect long term glucose lowering capacity clinical setting may impart significant reductions hba1c modest hba1c lowering region 0.5%0.9% may predicted early clinical studies would comparable achieved currently marketed oral agents.55 remains seen whether promoting glucose excretion result long term benefits patient terms returning metabolic balance even weight loss clearly blocking glucose reabsorption permits clearance glucose body thus must eventually serve reduce levels plasma glucose the amount glucose available excretion dependent amount entering nephrons turn depends blood glucose concentration glomerulus thus amount glucose excreted greater blood plasma glucose concentrations highest effect glucose removal might expected greatest times needed glucose levels highest post prandial hyperglycemia the benefit patients treatment provided mild moderate glycemic control might questioned potential glucose excretion would relatively low nevertheless patients achieve moderate glycemic control may exposed clinically relevant post prandial glucose excursions impart disproportionate effects hba1c possibly morbidity mortality associated t2dm.56 patient population sglt2 inhibitors might attenuate impact post prandial glucose spikes nevertheless clinical experience agents meglitinides target post prandial glucose control suggest clinical benefit approach disappointing treatments targeting post prandial glucose levels provide little modest improvements hba1c little evidence long term outcome benefits patients.57 sglt2 may responsible much 90% glucose reabsorption kidney clinical potential much 160 g glucose excreted day following effective sglt2 inhibition.23 however appears actual glucose loss achieved clinical studies half predicted.38 clear whether consequence compensating mechanisms undertaking tubular reabsorption incomplete inhibition transporter thus far safety profile sglt2 inhibitors reported clinical studies appears fulfill expectations.33,34,55,40,58 sglt2 inhibitors designed target highly specific membrane transporter almost exclusively expressed within renal tubules clearly compared less specific molecules potential for it also unlikely sglt2 inhibitors induce hypoglycemia since plasma glucose levels low amount glucose excreted also low.59 prediction appears confirmed clinical studies reported thus far show apparent increases hypoglycemic episodes sglt2 inhibitors.50,60 even sglt2 blocked completely degree renal glucose recovery maintained via relatively unhindered sglt1 transporter one aspect sglt2 inhibition raised potential issue safety concern glycosuria could predispose patients increased urinary tract infections uti there reports infection clinical studies.60,61 however study reviewed risk factors developing utis women diabetes observed glucosuria significant contributing factor.62 interestingly rare group individuals express sglt2 transporter functionality partially completely lost due genetic mutation autosomal recessive dominant pattern inheritance reported these people appear suffer ill consequences suggesting blockade transporter per se t2 dm patients would offer immediate risk patients expressing mutations decreased renal tubular reabsortion glucose lumen absence hyperglycemia signs tubular dysfunction it clear whether familial renal glucosuria due sglt2 mutation protects t2 dm although sglt2 deletion animal models appears improve glucose homeostasis preserve pancreatic -cell function.63 find recorded evidence increased disposition urinary tract vulvovaginal infections although identification study subjects difficult due rarity disease clearly clinical development programs need address concern possible increased risk uti increased glucose content urine following sglt2 inhibition likely serve increase urinary flow consequence osmotic diuretic effect lumen nephron this could result modest possibly beneficial reductions blood pressure raises additional safety concerns associated possible loss fluid solutes this may particular concern elderly patients capacity maintain fluid balance however noted effect considerably lower seen frequently used loop diuretics apparent change glomerular filtration rate would indicative direct effect renal function simple instructions maintaining state hydration regular drinks may serve overcome concerns urinary infection fluid imbalance the mechanism action sglt2 inhibitors predicts beneficial effect long term glucose lowering capacity clinical setting may impart significant reductions hba1c modest hba1c lowering region 0.5%0.9% may predicted early clinical studies would comparable achieved currently marketed oral agents.55 remains seen whether promoting glucose excretion result long term benefits patient terms returning metabolic balance even weight loss clearly blocking glucose reabsorption permits clearance glucose body thus must eventually serve reduce levels plasma glucose the amount glucose available excretion dependent amount entering nephrons turn depends blood glucose concentration glomerulus thus amount glucose excreted greater blood plasma glucose concentrations highest effect glucose removal might expected greatest times needed glucose levels highest post prandial hyperglycemia the benefit patients treatment provided mild moderate glycemic control might questioned potential glucose excretion would relatively low nevertheless patients achieve moderate glycemic control may exposed clinically relevant post prandial glucose excursions impart disproportionate effects hba1c possibly morbidity mortality associated t2dm.56 patient population sglt2 inhibitors might attenuate impact post prandial glucose spikes nevertheless clinical experience agents meglitinides target post prandial glucose control suggest clinical benefit approach disappointing treatments targeting post prandial glucose levels provide little modest improvements hba1c little evidence long term outcome benefits patients.57 sglt2 may responsible much 90% glucose reabsorption kidney clinical potential much 160 g glucose excreted day following effective sglt2 inhibition.23 however appears actual glucose loss achieved clinical studies half predicted.38 clear whether consequence compensating mechanisms undertaking tubular reabsorption incomplete inhibition transporter thus far safety profile sglt2 inhibitors reported clinical studies appears fulfill expectations.33,34,55,40,58 sglt2 inhibitors designed target highly specific membrane transporter almost exclusively expressed within renal tubules clearly compared less specific molecules potential for it also unlikely sglt2 inhibitors induce hypoglycemia since plasma glucose levels low amount glucose excreted also low.59 prediction appears confirmed clinical studies reported thus far show apparent increases hypoglycemic episodes sglt2 inhibitors.50,60 even sglt2 blocked completely degree renal glucose recovery maintained via relatively unhindered sglt1 transporter one aspect sglt2 inhibition raised potential issue safety concern glycosuria could predispose patients increased urinary tract infections uti there reports infection clinical studies.60,61 however study reviewed risk factors developing utis women diabetes observed glucosuria significant contributing factor.62 interestingly rare group individuals express sglt2 transporter functionality partially completely lost due genetic mutation autosomal recessive dominant pattern inheritance reported these people appear suffer ill consequences suggesting blockade transporter per se t2 dm patients would offer immediate risk patients expressing mutations decreased renal tubular reabsortion glucose lumen absence hyperglycemia signs tubular dysfunction it clear whether familial renal glucosuria due sglt2 mutation protects t2 dm although sglt2 deletion animal models appears improve glucose homeostasis preserve pancreatic -cell function.63 find recorded evidence increased disposition urinary tract vulvovaginal infections although identification study subjects difficult due rarity disease clearly clinical development programs need address concern possible increased risk uti increased glucose content urine following sglt2 inhibition likely serve increase urinary flow consequence osmotic diuretic effect lumen nephron this could result modest possibly beneficial reductions blood pressure raises additional safety concerns associated possible loss fluid solutes this may particular concern elderly patients capacity maintain fluid balance however noted effect considerably lower seen frequently used loop diuretics apparent change glomerular filtration rate would indicative direct effect renal function simple instructions maintaining state hydration regular drinks may serve overcome concerns urinary infection fluid imbalance the question arises sglt2 inhibitors might fit current cascade treatments management t2 dm while treatment t2 dm follows prescribed guidelines many approaches permutations application clinical practice although sglt2 inhibitors mechanism action would make suitable initial monotherapy patients early stage t2 dm unlikely would considered stage most treatments currently initiated metformin relatively inexpensive good historical safety profile efficacious clearly first launched sglt2 inhibitors able compete metformin purely issue cost however explained earlier advantage may minimal patients achieving degree glycemic control promoting escape mechanism glucose sglt2 inhibitors introduce new mode control t2 dm exception -glucosidase inhibitors block glucose uptake gut currently available antidiabetic therapies directly indirectly modulate insulin manipulate endogenous glucose utilization despite modest effect hba1c predicted sglt2 inhibitors the introduction novel means reducing hyperglycemia increases treatment options available physicians disease frequently requires use multiple agents achieve control targets.57 expected favorable safety profile insulin independent mechanism action appear support use sglt2 inhibitors combination antidiabetic drugs insulin dependent therapies become less effective development insulin resistance and/or deterioration -cell function particularly patients low insulin resistance high glucose poorly controlled disease the insulin independent action sglt2 inhibitors suggests potential synergistic effect scenarios the insulin independent action sglt2 inhibitors also means may use type 1 diabetes perhaps means moderating post prandial glucose excursions increasing excretion glucose sglt2 inhibitors offer opportunity increase calorie loss t2 dm patients overweight the continual loss 8090 g glucose per day 300400 calories significant loss calories work synergistically weight reduction programs.64 short term studies animals man appear confirm predicted weight reducing property this contrasts several drug therapies including sulfonylureas insulin thiazolidinediones generally associated weight gain.65,66 currently data confirm whether rate calorie loss continues chronic therapy clinical setting would easy overcome benefits patients may derive sglt2 inhibition perceived drug means acclimatized higher levels plasma glucose would experience marked change appetite substantial loss glucose calories.67 one final issue drugs work need delivered epithelial luminal surface nephron requiring cleared glomerulus consequently efficacy may affected instances functioning kidney impaired example diabetic nephropathy most patients t2 dm eventually succumb progressive nature disease point require multiple therapies attain treatment targets half patients t2 dm achieve hba1c target less 7% despite many different treatment options currently available with sglt2 inhibitors introducing alternative means managing glucose gain treatment option may increase ability control t2 dm experience lead better understanding patients likely respond best circumstances

there is a recognized need for new treatment options for type 2 diabetes mellitus ( t2 dm ) . recovery of glucose from the glomerular filtrate represents an important mechanism in maintaining glucose homeostasis and represents a novel target for the management of t2 dm . recovery of glucose from the glomerular filtrate is executed principally by the type 2 sodium - glucose cotransporter ( sglt2 ) . inhibition of sglt2 promotes glucose excretion and normalizes glycemia in animal models . first reports of specifically designed sglt2 inhibitors began to appear in the second half of the 1990s . several candidate sglt2 inhibitors are currently under development , with four in the later stages of clinical testing . the safety profile of sglt2 inhibitors is expected to be good , as their target is a highly specific membrane transporter expressed almost exclusively within the renal tubules . one safety concern is that of glycosuria , which could predispose patients to increased urinary tract infections . so far the reported safety profile of sglt2 inhibitors in clinical studies appears to confirm that the class is well tolerated . where sglt2 inhibitors will fit in the current cascade of treatments for t2 dm has yet to be established . the expected favorable safety profile and insulin - independent mechanism of action appear to support their use in combination with other antidiabetic drugs . promotion of glucose excretion introduces the opportunity to clear calories ( 8090 g [ 300400 calories ] of glucose per day ) in patients that are generally overweight , and is expected to work synergistically with weight reduction programs . experience will most likely lead to better understanding of which patients are likely to respond best to sglt2 inhibitors , and under what circumstances .

goal defibrillation df testing verify detection ventricular tachyarrhythmias implantable cardioverter defibrillators icds ascertain df efficacy all clinical trials demonstrated benefit icd therapy included kind df testing according food drug administration the instructions use icds include recommendation df testing time implantation the absence df testing may medico legal implications whether device fails terminate ventricular fibrillation vf follow nevertheless df testing deliberately omitted majority patients enrolled contemporary icd studies despite stochastic nature df ineffective shocks vf induction result system modifications 5% patients system modifications repositioning electrode reversal polarity use high energy device use dual instead single coil electrode implantation additional subcutaneous electrode may unnecessary even harmful since many patients successful retesting original system configuration no single system modification ever shown improve outcome current devices programming an inadequate safety margin occurs infrequently incidence ventricular tachyarrhythmias requiring shocks low 46% per year df testing never shown improve survival associated increased albeit small risk major adverse events 0.4% omitting routine df testing the approach icd implantation likely change considerably procedures shorter performed without technical support anaesthesia similar simple trial hypothesized current high energy icd devices omission df testing implantation result inferior shock efficacy follow compared standard df testing as opposed simple trial used composite outcome arrhythmic death failed appropriate shock primary end point study shock efficacy true ventricular tachycardia vt vf episodes occurring patient follow the nordic icd trial nct01282918 prospective randomized parallel group multi centre non inferiority trial conducted 48 centres five european countries designed investigate effect df testing time icd implantation first shock efficacy fse follow briefly patients indication icd implantation according current european society cardiology guidelines included patients hypertrophic arrhythmogenic right ventricular cardiomyopathy excluded special considerations necessary icd implantation one thousand seventy seven patients randomized 1 1 first time icd implantation without df testing procedure patients followed least 12 months regular site visits remotely via home monitoring hm biotronik se co. kg berlin germany all true ventricular tachyarrhythmias treated icd shock patient included primary analysis the study conducted accordance declaration helsinki country specific regulatory requirements the protocol approved institutional review board ethics committee participating centre a standard df testing protocol used participating centres described previously briefly initial shock df programmed 15 j. successful df testing terminated unsuccessful second shock programmed 24 j delivered confirmed if unsuccessful system revision recommended df testing procedure repeated the primary end point fse terminate true episodes vt vf follow a blinded two member clinical event committee reviewed diagnostic information vt vf treated icd shocks safety end points included procedural serious adverse events vt vf conversion rate cause cardiac arrhythmic mortality follow all adverse events reviewed adjudicated data safety monitoring board dsmb consisting three members all patient deaths adjudicated dsmb according classification originally described epstein et al the goal nordic icd trial demonstrate non inferiority df testing compared df testing respect primary endpoint fse this margin chosen variation fse published trials range 8392% after pre specified blinded assessment sample size increased 1080 patients recruited period 28 months follow least 12 months the intention treat itt population included randomized patients evaluated safety population included randomized patients received icd per protocol pps population included randomized patients received icd major protocol violation however time dependent outcomes patients included date protocol violation the primary analysis performed per protocol episode data set p pps included sufficiently documented shocked episodes pps patients baseline characteristics presented group wise itt population mean sd frequency compensate dependence structure induced recurrent episodes similar outcome patients random effects logit model patients random factor group fixed factor used calculation two sided 95% confidence interval ci difference fsenodftest fsedftest state non inferiority ci lie completely pre specified non inferiority margin 10% without use model takes correlation structure account patients extreme numbers shocks would dominated results procedural safety outcomes compared student test test fisher exact test whichever appropriate hazard rate reduction assessed using cox proportional hazards model two tailed p value 0.05 considered statistically significant data analysed using sas version 9.4 stata version 13.1 independent statistical institute department medical biometry epidemiology university medical centre hamburg eppendorf germany a.s k.w the first draft manuscript written first author revised co authors the authors take full responsibility accuracy completeness findings well fidelity study protocol the nordic icd trial nct01282918 prospective randomized parallel group multi centre non inferiority trial conducted 48 centres five european countries designed investigate effect df testing time icd implantation first shock efficacy fse follow briefly patients indication icd implantation according current european society cardiology guidelines included patients hypertrophic arrhythmogenic right ventricular cardiomyopathy excluded special considerations necessary icd implantation one thousand seventy seven patients randomized 1 1 first time icd implantation without df testing procedure patients followed least 12 months regular site visits remotely via home monitoring hm biotronik se co. kg berlin germany all true ventricular tachyarrhythmias treated icd shock patient included primary analysis the study conducted accordance declaration helsinki country specific regulatory requirements the protocol approved institutional review board ethics committee participating centre a standard df testing protocol used participating centres described previously briefly initial shock df programmed 15 j. successful df testing terminated unsuccessful second shock programmed 24 j delivered confirmed if shocks successful twice df testing terminated unsuccessful system revision recommended df testing procedure repeated the primary end point fse terminate true episodes vt vf follow a blinded two member clinical event committee reviewed diagnostic information vt vf treated icd shocks safety end points included procedural serious adverse events vt vf conversion rate cause cardiac arrhythmic mortality follow all adverse events reviewed adjudicated data safety monitoring board dsmb consisting three members all patient deaths adjudicated dsmb according classification originally described epstein et al the goal nordic icd trial demonstrate non inferiority df testing compared df testing respect primary endpoint fse this margin chosen variation fse published trials range 8392% after pre specified blinded assessment sample size increased 1080 patients recruited period 28 months follow least 12 months the intention treat itt population included randomized patients evaluated safety population included randomized patients received icd per protocol pps population included randomized patients received icd major protocol violation however time dependent outcomes patients included date protocol violation the primary analysis performed per protocol episode data set p pps included sufficiently documented shocked episodes pps patients baseline characteristics presented group wise itt population mean sd frequency compensate dependence structure induced recurrent episodes similar outcome patients random effects logit model patients random factor group fixed factor used calculation two sided 95% confidence interval ci difference fsenodftest fsedftest state non inferiority ci lie completely pre specified non inferiority margin 10% without use model takes correlation structure account patients extreme numbers shocks would dominated results procedural safety outcomes compared student test test fisher exact test whichever appropriate mortality analysed itt population using kaplan meier approach log rank test hazard rate reduction assessed using cox proportional hazards model two tailed p value 0.05 considered statistically significant data analysed using sas version 9.4 stata version 13.1 independent statistical institute department medical biometry epidemiology university medical centre hamburg eppendorf germany a.s k.w the first draft manuscript written first author revised co authors the authors take full responsibility accuracy completeness findings well fidelity study protocol the baseline clinical characteristics cardiovascular medications 1077 patients comprising intention treat population without df 537 df 540 presented table 1 seven hundred one patients 65.1% ischaemic cardiomyopathy 873 81.8% received icd primary prevention sudden cardiac death ten patients six df group four without df group receive icd leaving 1067 patients safety evaluation figure 1 both groups well matched baseline demographics clinical characteristics well use dual 51.6% single coil 48.4% electrode table 2 table 1baseline demographic clinical characteristics subjects treatment group intention treat)characteristictotal n 1077)without df test n 537)with df test n 540)age year)64.8 10.9)64.7 11.2)64.9 10.6)male sex n 873 81.1)430 80.1)443 82.0)ischaemic disease n 701 65.1)341 63.5)360 66.7)hypertension n total n 509/700 72.7)249/349 71.3)260/351 74.1)diabetes mellitus n 368 34.2)183 34.1)185 34.3)renal insufficiency n 303 28.1)144 26.8)159 29.4)nyha class n i59 5.5)31 5.8)28 5.2 ii464 43.1)226 42.1)238 44.1 iii487 45.2)254 47.3)233 43.1 iv14 1.3)5 0.9)9 1.7 unknown53 4.9)21 3.9)32 5.9)nyha class n ii523 48.6)257 47.9)266 49.3 iii501 46.5)259 48.2)242 44.8 unknown53 4.9)21 3.9)32 5.9)estimated bmi n 1054)28.2 4.9)28.1 4.8)28.2 5.1)lvef n 20%101 9.4)53 9.9)48 8.9 2030%566 52.6)278 51.8)288 53.3 30%404 37.5)204 38.0)200 37.0 done6 0.6)2 0.4)4 0.7)af enrolment n 85 7.9)45 8.4)40 7.4)indication implantation n total n primary prevention873/1067 81.8)434/531 81.7)439/536 81.9 secondary prevention194/1067 18.2)97/531 18.3)97/536 18.1)hospital stay days n 1066)4.0 4.0)4.0 3.6)4.0 3.8)medication n ace inhibitors receptor blockers n 986 91.6)501 93.3)485 89.8 -blockers n 1007 93.5)500 93.1)507 93.9 ca antagonists n 140 13.0)74 13.8)66 12.2 spironolactones n 618 57.4)302 56.2)316 58.5 diuretics n 794 73.7)395 73.6)399 73.9 nitrates n 75 7.0)38 7.1)37 6.9 digitalis n 105 9.7)54 10.1)51 9.4 lipid lowering agents n 763 70.8)377 70.2)386 71.5 amiodarone n 116 10.8)61 11.4)55 10.2 dronedarone n 5 0.5)4 0.7)1 0.2 sotalol n 9 0.8)5 0.9)4 0.7 anti arrhythmics n 22 2.0)9 1.7)13 2.4 platelet aggregation inhibitor n 753 69.9)370 68.9)383 70.9 anti coagulants n 350 32.5)183 34.1)167 30.9 cardiovascular medication n 255 23.7)121 22.5)134 24.8)values means sd there significant differences p 0.05 groups except ace inhibitors p 0.039 t test metric variables -test categorical fisher exact test f appropriate.ace inhibitors angiotensin converting enzyme inhibitors af atrial fibrillation receptor blockers angiotensin receptor blockers bmi body mass index df defibrillation nyha new york heart association itt intention treat lvef left ventricular ejection fraction table 2procedural characteristics according treatment group successful primary implantable cardioverter defibrillator implantation evaluated safety)characteristictotal n 1067)without df test n 533)with df test n 534)p valueimplanted lead type n single coil516 48.36)262 49.16)254 47.57)0.603 dual coil551 51.64)271 50.84)280 52.43)type n single chamber icd466 43.67)236 44.28)230 43.07)0.648 dual chamber icd245 22.96)116 21.76)129 24.16 crt d356 33.36)181 33.96)175 32.77)left position n 1058 99.16)527 98.87)531 99.44)0.341 f)vegm signal amplitude mv n 1057)12.42 5.30)12.72 5.37)12.12 5.21)0.065pacing threshold v pulse duration 0.4 ms n 527)0.51 0.20)0.49 0.17)0.53 0.22)0.040pacing threshold v pulse duration 0.5 ms n 533)0.45 0.23)0.45 0.27)0.44 0.17)0.613patients received df test n 527 49.39)7 1.31)520 97.38)delivered icd shocks per patient0.71 1.16)0.01 0.11)1.41 1.31)<0.001df energy final position j n 526)16.74 4.47)16.67 4.38)patients intra procedural system revisions icd programming n total n 25/527 4.74)25/520 4.81)patients electrode repositionings n total n 8/527 1.52)8/520 1.54)patients changes shock polarity n total n 20/527 3.80)20/520 3.85)patients modification lead system shock pathway total n 5/527 0.95)5/520 0.96)patients revisions programming n total n 7/527 1.33)7/520 1.35)procedure duration min n 1060)64.54 39.88)63.11 40.84)65.97 38.88)0.243fluoroscopy exposure duration min n 1056)8.02 11.43)8.00 11.44)8.05 11.43)0.943crt cardiac resynchronization therapy defibrillator df defibrillation efs evaluated safety vegm ventricular electrogram icd implantable cardioverter-defibrillator.one patient inducible itt full analysis set patients randomized efs evaluated safety set patients randomized successful primary icd implantation pps per protocol set patients randomized compliant study protocol p pps randomized patients least one day risk protocol deviation includes sufficiently documented shocked episodes time point protocol deviation baseline demographic clinical characteristics subjects treatment group intention treat values means sd there significant differences p 0.05 groups except ace inhibitors p 0.039 t test metric variables -test categorical fisher exact test f appropriate ace inhibitors angiotensin converting enzyme inhibitors af atrial fibrillation receptor blockers angiotensin receptor blockers bmi body mass index df defibrillation nyha new york heart association itt intention treat lvef left ventricular ejection fraction procedural characteristics according treatment group successful primary implantable cardioverter defibrillator implantation evaluated safety crt cardiac resynchronization therapy defibrillator df defibrillation efs evaluated safety vegm ventricular electrogram icd implantable cardioverter defibrillator itt full analysis set patients randomized efs evaluated safety set patients randomized successful primary icd implantation pps per protocol set patients randomized compliant study protocol p pps randomized patients least one day risk protocol deviation includes sufficiently documented shocked episodes time point protocol deviation 534 patients randomized df arm successful first icd implantation figure 1 testing performed 97.4% one patient vt vf inducible four hundred ninety four 519 inducible patients 95.2% passed test without system reconfiguration 440 patients 84.8% vt vf terminated 15 j shock 25 patients the initial df test successful system reconfigurations performed 20 5 table 2 all patients subsequently passed test successfully still higher energy level final position 22.2 7.4 j vs. 16.4 4.0 j p 0.001 significantly shocks 5.7 3.0 vs. 1.2 0.7 p 0.001 494 patients passed test immediately in addition duration procedure 90.2 56.2 min vs. 63.5 36.1 min p 0.001 fluoroscopy exposure 11.6 14.2 min vs. 7.5 10.7 min p 0.064 increased median follow 22.7 22.9 months respectively 211 true vt vf episodes least one appropriate shock occurred 8.6% per protocol patients df testing 218 true vt vf episodes occurred 8.8% patients without df testing table 3 figure 1 all true vt vf episodes terminated appropriate icd shock without df test group whereas 96.7% vt vf terminated group df test thus conversion efficacy 100.0% without df testing 96.7% df testing the model based shock efficacy 97.1% without df testing 94.1% df testing the difference fse 3.0% ci 3.0% 9.0% favour without df test group remained pre defined 10% margin primary end point pnon inferiority 0.001 table 3 figure 2 table 3primary analysis shock efficacy per protocol episode data set)totalwithout df testwith df testall patients least one delivered appropriate shock true vt vf episode n total n 91/1046 8.7)46/523 8.8)45/523 8.6)all true vt vf episodes least one delivered appropriate icd shock vt vf n429218211delivered appropriate shocks per true vt vf episode mean sd))1.18 0.69)1.09 0.37)1.28 0.90)true vt vf episodes terminated first appropriate icd shock n total n 384/429 89.5)204/218 93.6)180/211 85.3)true vt vf episodes terminated appropriate icd shock n total n 422/429 98.4)218/218 100.0)204/211 96.7)true vt vf episodes terminated appropriate icd shock n total n 7/429 1.6)0/218 0.0)7/211 3.3)conversion efficacy proportion 95% ci)]0.98 0.97;0.99)1.00 0.98;1.00)0.97 0.93;0.99)first shock efficacy model based proportion 95% ci)]0.96 0.89;0.98)0.97 0.91;0.99)0.94 0.83;0.98))difference groups without df testing vs. df testing 0.03 0.03;0.09).df defibrillation icd implantable cardioverter defibrillator p pps per protocol episode data set vt vf ventricular tachycardia ventricular fibrillation primary analysis shock efficacy per protocol episode data set difference groups without df testing vs. df testing 0.03 0.03;0.09 df defibrillation icd implantable cardioverter defibrillator p pps per protocol episode data set vt vf ventricular tachycardia ventricular fibrillation a total 201 serious adverse events related study procedure occurred 168 patients 15.7% 89 events occurred 74 patients without df testing 112 events occurred 94 patients df testing within 30 days follow p 0.095 the significant difference single serious adverse events intra operative hypotension manifested often patient df testing 1.7% without df testing 0.0% p 0.004 lead related complications occurred 15 patients without df testing 2.8% compared 24 events 21 patients df testing 3.9% p 0.311 one sudden death occurred study group within 30 days follow mean length hospitalization 4.0 days study groups table 1 the proportion patients inappropriate shocks follow low 3.7% similar groups forty four 52 patients without df testing died respectively p 0.377 table 4 differences respect non cardiac cardiac mortality statistically significant six deaths 5 df 1 without df testing adjudicated arrhythmic due repetitive ventricular tachyarrhythmias vt clusters p 0.100 table 4 table 4mortality intention treat)outcomeeventsincidencelog rank95% ciwithout df test n 537)with df test n 540)without df testwith df testp valuehrlowerupperall cause mortality44 8.2%)52 9.6%)0.0440.0530.3770.8350.5591.248non cardiac mortality24 4.5%)23 4.3%)0.0240.0230.9071.0350.5841.833unknown primary organ cause)6 1.1%)10 1.9%)0.0060.010.3060.5930.2161.632cardiac mortality14 2.6%)19 3.5%)0.0140.0190.3500.7210.3611.438arrhythmic mortality1 0.2%)5 0.9%)0.0010.0050.1000.1980.0231.694non arrhythmic mortality13 2.4%)13 2.4%)0.0130.0130.9480.9750.4522.103unknown cardiac mortality0 0.0%)1 0.2%)00.001df defibrillation hr hazard ratio itt intention treat.incidence referring person years without df test 1001.5 df test 985.9 mortality intention treat df defibrillation hr hazard ratio itt intention treat incidence referring person years without df test 1001.5 df test 985.9 the baseline clinical characteristics cardiovascular medications 1077 patients comprising intention treat population without df 537 df 540 presented table 1 seven hundred one patients 65.1% ischaemic cardiomyopathy 873 81.8% received icd primary prevention sudden cardiac death ten patients six df group four without df group receive icd leaving 1067 patients safety evaluation figure 1 both groups well matched baseline demographics clinical characteristics well use dual 51.6% single coil 48.4% electrode table 2 table 1baseline demographic clinical characteristics subjects treatment group intention treat)characteristictotal n 1077)without df test n 537)with df test n 540)age year)64.8 10.9)64.7 11.2)64.9 10.6)male sex n 873 81.1)430 80.1)443 82.0)ischaemic disease n 701 65.1)341 63.5)360 66.7)hypertension n total n 509/700 72.7)249/349 71.3)260/351 74.1)diabetes mellitus n 368 34.2)183 34.1)185 34.3)renal insufficiency n 303 28.1)144 26.8)159 29.4)nyha class n i59 5.5)31 5.8)28 5.2 ii464 43.1)226 42.1)238 44.1 iii487 45.2)254 47.3)233 43.1 iv14 1.3)5 0.9)9 1.7 unknown53 4.9)21 3.9)32 5.9)nyha class n ii523 48.6)257 47.9)266 49.3 iii501 46.5)259 48.2)242 44.8 unknown53 4.9)21 3.9)32 5.9)estimated bmi n 1054)28.2 4.9)28.1 4.8)28.2 5.1)lvef n 20%101 9.4)53 9.9)48 8.9 2030%566 52.6)278 51.8)288 53.3 30%404 37.5)204 38.0)200 37.0 done6 0.6)2 0.4)4 0.7)af enrolment n 85 7.9)45 8.4)40 7.4)indication implantation n total n primary prevention873/1067 81.8)434/531 81.7)439/536 81.9 secondary prevention194/1067 18.2)97/531 18.3)97/536 18.1)hospital stay days n 1066)4.0 4.0)4.0 3.6)4.0 3.8)medication n ace inhibitors receptor blockers n 986 91.6)501 93.3)485 89.8 -blockers n 1007 93.5)500 93.1)507 93.9 ca antagonists n 140 13.0)74 13.8)66 12.2 spironolactones n 618 57.4)302 56.2)316 58.5 diuretics n 794 73.7)395 73.6)399 73.9 nitrates n 75 7.0)38 7.1)37 6.9 digitalis n 105 9.7)54 10.1)51 9.4 lipid lowering agents n 763 70.8)377 70.2)386 71.5 amiodarone n 116 10.8)61 11.4)55 10.2 dronedarone n 5 0.5)4 0.7)1 0.2 sotalol n 9 0.8)5 0.9)4 0.7 anti arrhythmics n 22 2.0)9 1.7)13 2.4 platelet aggregation inhibitor n 753 69.9)370 68.9)383 70.9 anti coagulants n 350 32.5)183 34.1)167 30.9 cardiovascular medication n 255 23.7)121 22.5)134 24.8)values means sd there significant differences p 0.05 groups except ace inhibitors p 0.039 t test metric variables -test categorical fisher exact test f appropriate.ace inhibitors angiotensin converting enzyme inhibitors af atrial fibrillation receptor blockers angiotensin receptor blockers bmi body mass index df defibrillation nyha new york heart association itt intention treat lvef left ventricular ejection fraction table 2procedural characteristics according treatment group successful primary implantable cardioverter defibrillator implantation evaluated safety)characteristictotal n 1067)without df test n 533)with df test n 534)p valueimplanted lead type n single coil516 48.36)262 49.16)254 47.57)0.603 dual coil551 51.64)271 50.84)280 52.43)type n single chamber icd466 43.67)236 44.28)230 43.07)0.648 dual chamber icd245 22.96)116 21.76)129 24.16 crt d356 33.36)181 33.96)175 32.77)left position n 1058 99.16)527 98.87)531 99.44)0.341 f)vegm signal amplitude mv n 1057)12.42 5.30)12.72 5.37)12.12 5.21)0.065pacing threshold v pulse duration 0.4 ms n 527)0.51 0.20)0.49 0.17)0.53 0.22)0.040pacing threshold v pulse duration 0.5 ms n 533)0.45 0.23)0.45 0.27)0.44 0.17)0.613patients received df test n 527 49.39)7 1.31)520 97.38)delivered icd shocks per patient0.71 1.16)0.01 0.11)1.41 1.31)<0.001df energy final position j n 526)16.74 4.47)16.67 4.38)patients intra procedural system revisions icd programming n total n 25/527 4.74)25/520 4.81)patients electrode repositionings n total n 8/527 1.52)8/520 1.54)patients changes shock polarity n total n 20/527 3.80)20/520 3.85)patients modification lead system shock pathway total n 5/527 0.95)5/520 0.96)patients revisions programming n total n 7/527 1.33)7/520 1.35)procedure duration min n 1060)64.54 39.88)63.11 40.84)65.97 38.88)0.243fluoroscopy exposure duration min n 1056)8.02 11.43)8.00 11.44)8.05 11.43)0.943crt cardiac resynchronization therapy defibrillator df defibrillation efs evaluated safety vegm ventricular electrogram icd implantable cardioverter-defibrillator.one patient inducible itt full analysis set patients randomized efs evaluated safety set patients randomized successful primary icd implantation pps per protocol set patients randomized compliant study protocol p pps randomized patients least one day risk protocol deviation includes sufficiently documented shocked episodes time point protocol deviation baseline demographic clinical characteristics subjects treatment group intention treat values means sd there significant differences p 0.05 groups except ace inhibitors p 0.039 t test metric variables -test categorical fisher exact test f appropriate ace inhibitors angiotensin converting enzyme inhibitors af atrial fibrillation receptor blockers angiotensin receptor blockers bmi body mass index df defibrillation nyha new york heart association itt intention treat lvef left ventricular ejection fraction procedural characteristics according treatment group successful primary implantable cardioverter defibrillator implantation evaluated safety crt cardiac resynchronization therapy defibrillator df defibrillation efs evaluated safety vegm ventricular electrogram icd implantable cardioverter defibrillator itt full analysis set patients randomized efs evaluated safety set patients randomized successful primary icd implantation pps per protocol set patients randomized compliant study protocol p pps randomized patients least one day risk protocol deviation includes sufficiently documented shocked episodes time point protocol deviation of 534 patients randomized df arm successful first icd implantation figure 1 testing performed 97.4% one patient vt vf inducible four hundred ninety four 519 inducible patients 95.2% passed test without system reconfiguration 440 patients 84.8% vt vf terminated 15 j shock 25 patients the initial df test successful system reconfigurations performed 20 5 table 2 all patients subsequently passed test successfully still higher energy level final position 22.2 7.4 j vs. 16.4 4.0 j p 0.001 significantly shocks 5.7 3.0 vs. 1.2 0.7 p 0.001 494 patients passed test immediately in addition duration procedure 90.2 56.2 min vs. 63.5 36.1 min p 0.001 fluoroscopy exposure 11.6 14.2 min vs. 7.5 10.7 min p 0.064 increased during median follow 22.7 22.9 months respectively 211 true vt vf episodes least one appropriate shock occurred 8.6% per protocol patients df testing 218 true vt vf episodes occurred 8.8% patients without df testing table 3 figure 1 all true vt vf episodes terminated appropriate icd shock without df test group whereas 96.7% vt vf terminated group df test thus conversion efficacy 100.0% without df testing 96.7% df testing the model based shock efficacy 97.1% without df testing 94.1% df testing the difference fse 3.0% ci 3.0% 9.0% favour without df test group remained pre defined 10% margin primary end point pnon inferiority 0.001 table 3 figure 2 table 3primary analysis shock efficacy per protocol episode data set)totalwithout df testwith df testall patients least one delivered appropriate shock true vt vf episode n total n 91/1046 8.7)46/523 8.8)45/523 8.6)all true vt vf episodes least one delivered appropriate icd shock vt vf n429218211delivered appropriate shocks per true vt vf episode mean sd))1.18 0.69)1.09 0.37)1.28 0.90)true vt vf episodes terminated first appropriate icd shock n total n 384/429 89.5)204/218 93.6)180/211 85.3)true vt vf episodes terminated appropriate icd shock n total n 422/429 98.4)218/218 100.0)204/211 96.7)true vt vf episodes terminated appropriate icd shock n total n 7/429 1.6)0/218 0.0)7/211 3.3)conversion efficacy proportion 95% ci)]0.98 0.97;0.99)1.00 0.98;1.00)0.97 0.93;0.99)first shock efficacy model based proportion 95% ci)]0.96 0.89;0.98)0.97 0.91;0.99)0.94 0.83;0.98))difference groups without df testing vs. df testing 0.03 0.03;0.09).df defibrillation icd implantable cardioverter defibrillator p pps per protocol episode data set vt vf ventricular tachycardia ventricular fibrillation figure 2primary outcome per protocol population primary analysis shock efficacy per protocol episode data set difference groups without df testing vs. df testing 0.03 0.03;0.09 df defibrillation icd implantable cardioverter defibrillator p pps per protocol episode data set vt vf ventricular tachycardia ventricular fibrillation a total 201 serious adverse events related study procedure occurred 168 patients 15.7% 89 events occurred 74 patients without df testing 112 events occurred 94 patients df testing within 30 days follow p 0.095 the significant difference single serious adverse events intra operative hypotension manifested often patient df testing 1.7% without df testing 0.0% p 0.004 lead related complications occurred 15 patients without df testing 2.8% compared 24 events 21 patients df testing 3.9% p 0.311 one sudden death occurred study group within 30 days follow mean length hospitalization 4.0 days study groups table 1 the proportion patients inappropriate shocks follow low 3.7% similar groups forty four 52 patients without df testing died respectively p 0.377 table 4 differences respect non cardiac cardiac mortality statistically significant six deaths 5 df 1 without df testing adjudicated arrhythmic due repetitive ventricular tachyarrhythmias vt clusters p 0.100 table 4 table 4mortality intention treat)outcomeeventsincidencelog rank95% ciwithout df test n 537)with df test n 540)without df testwith df testp valuehrlowerupperall cause mortality44 8.2%)52 9.6%)0.0440.0530.3770.8350.5591.248non cardiac mortality24 4.5%)23 4.3%)0.0240.0230.9071.0350.5841.833unknown primary organ cause)6 1.1%)10 1.9%)0.0060.010.3060.5930.2161.632cardiac mortality14 2.6%)19 3.5%)0.0140.0190.3500.7210.3611.438arrhythmic mortality1 0.2%)5 0.9%)0.0010.0050.1000.1980.0231.694non arrhythmic mortality13 2.4%)13 2.4%)0.0130.0130.9480.9750.4522.103unknown cardiac mortality0 0.0%)1 0.2%)00.001df defibrillation hr hazard ratio itt intention treat.incidence referring person years without df test 1001.5 df test 985.9 mortality intention treat df defibrillation hr hazard ratio itt intention treat incidence referring person years without df test 1001.5 df test 985.9 defibrillation testing icd implantation matter debate last two decades best knowledge the nordic icd trial first prospective randomized trial investigated effect df testing actual fse follow single primary end point this contrast recently published simple trial used composite outcome arrhythmic death failed appropriate shock terminate spontaneous episode vt vf determine non inferiority testing approach nordic icd also provides detailed information intra operative df testing outcome 9196% icd implants system modifications reported 2.212.0% patients high energy devices used nordic icd study 4.8% inducible patients inadequate safety margin required one two system reconfigurations 3.8 1.0% respectively this agreement results sudden cardiac death heart failure trial scd heft 97.8% patients df threshold 20 j. following system revisions patients passed test successfully significantly higher energy level require revision 22.2 j vs. 16.4 j p 0.001 this agreement canadian survey insufficient safety margin initial system configuration could improved 56% patients while device revisions provided benefit patients follow significantly increased total procedure time 30 min fluoroscopy exposure 40% the mean number shocks needed find acceptable safety margin also increased four fold however trial variable use devices shock efficacy similar observed nordic icd trial the observed lower rate arrhythmic death patients without df testing 0.2 vs. 0.9% corresponds sudden cardiac death rate reported another trial 0.9 1.2% patients without df testing similar follow period simple trial use specific model multiple shock events patient the shock efficacy compared level raw shock efficacy nordic icd simple trials a higher raw shock efficacy observed group without df vs. df testing nordic icd 93.6 vs. 85.3% simple 92.0 vs. 88.5% hand higher first shock energy used 40 j vs. 31 j simple associated higher fse irrespective testing the high level fse consistent relatively low mean df energy 16.7 j testing 95% inducible patients undergoing successful testing without system reconfiguration a 15.7% serious adverse events rate 17.6% testing 13.9% without testing p 0.095 found within 30 days following successful icd implantation this much higher described recent reports complication rates ranged 2.3 2.7% patients without adequate safety margins df testing compared simple trial results regarding adverse events included pre defined primary safety outcome 5.6% without df testing vs. 6.5% df testing it noteworthy nordic icd trial collected data protocol specified complications also procedure- patient related adverse events might explain higher event rate the 30-day procedure related mortality rate 0.2% consisted two sudden deaths range recent reports the stroke rate 0.8% patients without 0.2% patients df testing p 0.217 agreement 0.4% rate reported credit registry the two types adverse events increased patients df testing intra operative hypotension 1.7% vs. 0.0% p 0.004 lead related complications 3.9 vs. 2.8% p 0.311 recent article summarized implant related complications directly indirectly related df testing procedure the rate haemodynamic complications 1% lead dislodgement listed frequent single complication rates 1 6% both nordic icd simple trials showed trend towards lower procedural complication rates patients without df testing despite different definitions methods used analysing events nordic icd 13.9 vs.17.6% p 0.095 simple 5.6 vs. 6.5% p 0.33 the rates cause cardiac non cardiac mortality similar patients without df testing agreement non randomized trials reported accordance simple trial statistical difference cause mortality patients without df testing however annual mortality rates nordic icd lower compared large randomized icd trials also lower simple trial the nordic icd trial supports hypothesis 40 j devices used df testing improve df efficacy follow it suggests df testing various measures improve df efficacy lengthen procedure may even harmful the nordic icd trial one two large prospective randomized trials support routine use df testing left sided first time icd implantation this work supported biotronik se co. kg berlin germany funding pay reports receiving grants personal fees form medtronic st jude medical biosense biotronik boston scientific conduct study grants personal fees biosense biotronik outside submitted work reports receiving grants personal fees non financial support biotronik conduct study personal fees non financial support boehringer ingelheim outside submitted work reports personal fees biotronik conduct study grants st jude medical personal fees boston scientific outside submitted work

aimsthis trial was designed to test the hypothesis that shock efficacy during follow - up is not impaired in patients implanted without defibrillation ( df ) testing during first implantable cardioverter - defibrillator ( icd ) implantation.methods and resultsbetween february 2011 and july 2013 , 1077 patients were randomly assigned ( 1 : 1 ) to first time icd implantation with ( n = 540 ) or without ( n = 537 ) df testing . the intra - operative df testing was standardized across all participating centres , and all icd shocks were programmed to 40 j irrespective of df test results . the primary end point was the average first shock efficacy ( fse ) for all true ventricular tachycardia and fibrillation ( vt / vf ) episodes during follow - up . the secondary end points included procedural data , serious adverse events , and mortality . during a median follow - up of 22.8 months , the model - based fse was found to be non - inferior in patients with an icd implanted without a df test , with a difference in fse of 3.0% in favour of the no df test [ confidence interval ( ci ) 3.0 to 9.0% , pnon - inferiority < 0.001 for the pre - defined non - inferiority margin of 10% ) . a total of 112 procedure - related serious adverse events occurred within 30 days in 94 patients ( 17.6% ) tested compared with 89 events in 74 patients ( 13.9% ) not tested ( p = 0.095).conclusiondefibrillation efficacy during follow - up is not inferior in patients with a 40 j icd implanted without df testing . defibrillation testing during first time icd implantation should no longer be recommended for routine left - sided icd implantation .

bladder cancer seventh common malignancy world men third common poland women cancer occurs less frequently 17 world 15 poland contrast men increasing trend morbidity observed 15 poland 2010 6296 incidents bladder cancer noticed 4919 men 1377 women mortality cancer high reaching 50.2% men 46.5% women approximately 1520% bladder cancers infiltrate bladder muscle layer cases treatment choice radical cystectomy connects necessity urine diversion bladder removal 2010 1260 patients required urinary diversion europe year 140 000 new cases bladder cancer noticed gives 25 000 patients urinary diversion urinary diversion divided incontinent continent formed orthotopically non orthotopically incontinent ileocutaneostomy commonly used among surgeons type urinary diversion greatest chances constructed using tissue engineering methods use ileal segment beside advantages maintaining ureter continuity together urine outflow using autologous material well developed blood supply number disadvantages especially long term follow 1012 this method also associated necessity additional surgical procedure conduction ileum the use tissue engineering techniques gives opportunities construct artificial conduits de novo laboratory without affecting ileum the first attempt incontinent urinary diversion carried 1851 london john simon conducted two ureterosigmoidal fistulas using specially designed silver catheter ernst kster performed first time radical cystetctomy patient suffering localized urinary bladder cancer robert coffey developed new method ureter implantation bowel wall used clinical practice charles mayo 1912 coffey mayo technique this method used bowel submucosal tunnel protects urine outflow reflux modifications frank hinman henry m. weyrauch analyzed 740 urinary diversion procedures performed using 60 different methods the effective incontinent urinary diversion method highest survival rate years cuteneous ureterostomy the first attempt unilateral cuteneous ureterostomy performed 1889 jean f. a. le dentu 1892 ludwik rydygier performed first bilateral cuteneous ureterostomy 1905 rowsing modified rydygier technique constructing nipple ureter exteriorization sliver urine reservoir placed other incontinent urinary diversion techniques included ureter anastomosis urethral groove eduard sonnenburg 1881 ureter implantation vagina karl pawlick 1888 the use ileal segment conduit urine diversion performed first time 1911 zaayer two patients the first patient died cancer second peritonitis 1950 eugene m. bricker presented 307 incontinent urinary diversion procedures using ileal segment reaching 12.4% mortality 3.4% directly involved urinary diversion this method considered gold standard 35 years modification used even past 65 years the first attempt artificial urinary conduit construction using tissue engineering methods performed drewa 2007 experiment small intestine submucosa sis acellular seeded 3t3 fibroblast cell line used rat models the experiment performed 6 animals patent conduits observed three rats end follow no differences cell layer regeneration observed seeded unseeded groups additionally acellular matrices induced less severe inflammatory responses five years later two groups conducted urinary conduit construction porcine model animals divided 2 groups acellular matrices n 4 matrices seeded urothelial cells n 6 patent urostomy obtained 5 animals differences seeded unseeded matrices they used polyglicolic acid coated poly(lactide co glicolide scaffold pga plga unseeded seeded smooth muscle cells derived different origin bladder adipose tissue blood in contrast previously presented experiments study bladder removed ureters transplanted conduit obtained results showed use smooth muscle cells different origins led regeneration neo organ resembling native bladder tissue composed urothelium smooth muscle layers the use acellular scaffold resulted fibrous connective tissue development small number smooth muscle cells acellular matrices used 6 animals scaffolds seeded urothelial cells used 24 rabbits study bladder also removed ureters transplanted conduit group matrices seeded up conduit lumen covered multilayer urothelium severe complications observed unseeded group 4 animals died one month end follow two remaining animals fistulas lack urothelium regeneration scaffold seeded urothelial cells protected scar kidney stone formation atresia hydronephrosis study compared two acellular matrices form different origins autologous naturally derived acellular aortic arch synthetic policaprolactone plcl produced using electrospining method the experiment conducted 12 wistar rats divided 2 equal groups six rats tested scaffold obtained results indicated acellular aortic arch unsuitable scaffold urinary conduit construction rat model animals acellular aortic arch conduit in plcl scaffold used 3 cases constructed conduits patent end follow 4 weeks one case intense urine flow without presence pus urinary tract observed autologous acellular aortic arch despite easy accessibility proper extracellular matrix composition unsuitable urinary conduit construction small diameter elastic structure leads atresion one week surgical procedure plcl rigid diameter regulated electrospining method better results obtained using scaffold unsatisfactory results plcl probably caused open urinary tract follow use urostomy bags rat models impossible such complications observed ureter segment regenerated using scaffolds use plcl resulted ureter segment reconstruction 4 cases n 6 confirmed urography group all currently performed attempts experimental artificial urinary conduit construction using tissue engineering techniques presented figure 1 different approaches currently used experimental urinary conduit construction using tissue engineering techniques experiment carried one ureter ; b two ureters anastomosed artificial conduit urinary bladder removed 21 22 23 plcl caprolactone pga plga polyglicolic acid coated poly lactide co glicolide scaffold sis small intestine submucosa bam bladder acellular matrix literature there still small number works describing use tissue engineering urinary conduit construction paper sloff et al experiments describing use tissue engineering urinary diversion construction conducted far evaluated they analyzed 8 works 5 related urinary conduit 3 neo bladder construction this shows little known ureter segment regeneration including artificial urinary conduit construction continues unresolved problem synthetic polymers promising properties materials produced de novo different shapes porosity degradation time obtained best solution for a patient would use acellular scaffold without necessity cell seeding such approach eliminate invasive collection tissue cell isolation long vitro culture 2 weeks construction artificial urinary conduit without cell seeding makes ideal shelf product could purchased pharmacy directly surgical procedure scaffold protects seeded cells toxic influence urine important issue vitro culture study urine acted cytotoxic agent urothelial cells bone marrow mesenchymal stem cells 26 27 urothelial cells build inner layer ureter protecting urine components reabsorption urothelium self regenerate scaffold surface transplantation migration surrounding tissue 2830 epithelial regeneration potentially easier case ureter segment regeneration urothelial cells migrate two edges compared urinary conduit data available literature gives contrary results use urothelial cells tissue engineering applications some studies showed differences seeded unseeded group hand researches obtained positive results scaffold seeded urothelial cells 22 23 they concluded 4 week follow epithelial layer regenerated 0.5 cm scaffold clinically important segment longer scaffolds regeneration observed anastomotic edges dense fibrosis throughout grafts previous experiments only one scaffold type analyzed work compared two scaffold types natural synthetic obtained results showed better regeneration urothelium synthetic biodegradable polymer compared natural derived acellular blood vessel matrix it noted material artificial urinary conduit construction produced components protect fibrosis calcification urine substrates scaffold surface the crucial point regeneration smooth muscle layer restoration peristaltic waves reconstructed segment the urinary conduit must constructed rigid material order protect scaffold occlusion site anastomosis skin such properties result peristaltic wave arrest site ureter anastomosis scaffold lead urine reflux development hydronephrosis quick regeneration smooth muscle layer prevent development side effects mentioned despite fact acellular scaffold might best solution papers indicate scaffold pre seeded cells autologous form bladder biopsy mesenchymal stem cells different origin showed better smooth muscle layer regeneration compared unseeded controls the best type cells seems mesenchymal stem cells fat tissue bone marrow promising sources like amniotic fluid hair follicles 13 33 use differentiated autologous cells bladder biopsy limited due risk cancer development case bladder cancer patients main candidates urinary conduit construction the number cells seeded scaffold also important conception seems simple cells cm scaffold better results achieved the achievement large cell numbers challenging issue average bladder cancer patient 65 years old mesenchymal stem cells proliferation capacity decreases age patient increasing passage numbers 36 37 hand authors suggested stem cell proliferation capacity dependent donors age increase chances tissue engineering therapy use artificial urinary conduit construction despite large number cells necessary regeneration hard obtain costly price culture media containing appropriate growth factors that efficient cell culture method developed provide success procedure many years atypical smooth muscle cells asmcs localized proximal regions renal pelvis considered peacemaker peristaltic waves responsible urine passage bladder recent studies indicated interstitial cells cajal like cells icc lc expressing c kit gene sparsely distributed within lamina propria muscle layer upper urinary tract play important role promoting pyeloureteric peristalsis contraction waves generated renal pelvis probably propagated coordinated modulated upper urinary tract icc lc 41 42 iccs thanks automatism able replace discontinued atypical smcs impulses maintain peristaltis lower ureter parts 40 43 vitro isolation culture icc lc urinary tract yet established taking consideration previous works experience urinary conduit construction addition cells coculture smooth muscle cells potentially accelerate restoration peristaltic waves reconstructed segment could prevent development hydronephrosis the first attempt incontinent urinary diversion carried 1851 london john simon conducted two ureterosigmoidal fistulas using specially designed silver catheter the patient died one year procedure due peritonitis 1891 ernst kster performed first time radical cystetctomy patient suffering localized urinary bladder cancer robert coffey developed new method ureter implantation bowel wall used clinical practice charles mayo 1912 coffey mayo technique this method used bowel submucosal tunnel protects urine outflow reflux modifications frank hinman henry m. weyrauch analyzed 740 urinary diversion procedures performed using 60 different methods the effective incontinent urinary diversion method highest survival rate years cuteneous ureterostomy the first attempt unilateral cuteneous ureterostomy performed 1889 jean f. a. le dentu 1892 ludwik rydygier performed first bilateral cuteneous ureterostomy 1905 rowsing modified rydygier technique constructing nipple ureter exteriorization sliver urine reservoir placed other incontinent urinary diversion techniques included ureter anastomosis urethral groove eduard sonnenburg 1881 ureter implantation vagina karl pawlick 1888 the use ileal segment conduit urine diversion performed first time 1911 zaayer two patients the first patient died cancer second peritonitis 1950 eugene m. bricker presented 307 incontinent urinary diversion procedures using ileal segment reaching 12.4% mortality 3.4% directly involved urinary diversion this method considered gold standard 35 years modification used even past 65 years effective method incontinent urinary diversion developed the first attempt artificial urinary conduit construction using tissue engineering methods performed drewa 2007 experiment small intestine submucosa sis acellular seeded 3t3 fibroblast cell line used rat models the experiment performed 6 animals patent conduits observed three rats end follow five years later two groups conducted urinary conduit construction porcine model animals divided 2 groups acellular matrices n 4 matrices seeded urothelial cells n 6 patent urostomy obtained 5 animals differences seeded unseeded matrices they used polyglicolic acid coated poly(lactide co glicolide scaffold pga plga unseeded seeded smooth muscle cells derived different origin bladder adipose tissue blood in contrast previously presented experiments study bladder removed ureters transplanted conduit obtained results showed use smooth muscle cells different origins led regeneration neo organ resembling native bladder tissue composed urothelium smooth muscle layers the use acellular scaffold resulted fibrous connective tissue development small number smooth muscle cells acellular matrices used 6 animals scaffolds seeded urothelial cells used 24 rabbits study the bladder also removed ureters transplanted conduit group matrices seeded up conduit lumen covered multilayer urothelium severe complications observed unseeded group 4 animals died one month end follow two remaining animals fistulas lack urothelium regeneration scaffold seeded urothelial cells protected scar kidney stone formation atresia hydronephrosis study compared two acellular matrices form different origins autologous naturally derived acellular aortic arch synthetic policaprolactone plcl produced using electrospining method the experiment conducted 12 wistar rats divided 2 equal groups six rats tested scaffold obtained results indicated acellular aortic arch unsuitable scaffold urinary conduit construction rat model in animals acellular aortic arch conduit atresion observed second group in plcl scaffold used 3 cases constructed conduits patent end follow 4 weeks one case intense urine flow without presence pus urinary tract observed autologous acellular aortic arch despite easy accessibility proper extracellular matrix composition unsuitable urinary conduit construction small diameter elastic structure leads atresion one week surgical procedure plcl rigid diameter regulated electrospining method better results obtained using scaffold unsatisfactory results plcl probably caused open urinary tract follow use urostomy bags rat models impossible such complications observed ureter segment regenerated using scaffolds use plcl resulted ureter segment reconstruction 4 cases n 6 confirmed urography group all currently performed attempts experimental artificial urinary conduit construction using tissue engineering techniques presented figure 1 different approaches currently used experimental urinary conduit construction using tissue engineering techniques experiment carried one ureter b two ureters anastomosed artificial conduit urinary bladder removed 21 22 23 poly l lactide co caprolactone pga plga polyglicolic acid coated poly lactide co glicolide scaffold sis small intestine submucosa bam bladder acellular matrix in literature still small number works describing use tissue engineering urinary conduit construction paper sloff et al experiments describing use tissue engineering urinary diversion construction conducted far evaluated they analyzed 8 works 5 related urinary conduit 3 neo bladder construction this shows little known ureter segment regeneration including artificial urinary conduit construction continues unresolved problem synthetic polymers promising properties materials produced de novo different shapes porosity degradation time obtained best solution for a patient would use acellular scaffold without necessity cell seeding such approach eliminate invasive collection tissue cell isolation long vitro culture 2 weeks construction artificial urinary conduit without cell seeding makes ideal shelf product could purchased pharmacy directly surgical procedure scaffold protects seeded cells toxic influence urine important issue vitro culture study urine acted cytotoxic agent urothelial cells bone marrow mesenchymal stem cells 26 27 urothelial cells build inner layer ureter protecting urine components reabsorption urothelium self regenerate scaffold surface transplantation migration surrounding tissue 2830 epithelial regeneration potentially easier case ureter segment regeneration urothelial cells migrate two edges compared urinary conduit data available literature gives contrary results use urothelial cells tissue engineering applications some studies showed differences seeded unseeded group hand researches obtained positive results scaffold seeded urothelial cells 22 23 they concluded 4 week follow epithelial layer regenerated 0.5 cm scaffold clinically important segment longer scaffolds regeneration observed anastomotic edges dense fibrosis throughout grafts previous experiments only one scaffold type analyzed work compared two scaffold types natural synthetic obtained results showed better regeneration urothelium synthetic biodegradable polymer compared natural derived acellular blood vessel matrix it noted material artificial urinary conduit construction produced components protect fibrosis calcification urine substrates scaffold surface the crucial point regeneration smooth muscle layer restoration peristaltic waves reconstructed segment the urinary conduit must constructed rigid material order protect scaffold occlusion site anastomosis skin such properties result peristaltic wave arrest site ureter anastomosis scaffold lead urine reflux development hydronephrosis quick regeneration smooth muscle layer prevent development side effects mentioned despite fact acellular scaffold might best solution some papers indicate scaffold pre seeded cells autologous form bladder biopsy mesenchymal stem cells different origin showed better smooth muscle layer regeneration compared unseeded controls the best type cells seems mesenchymal stem cells fat tissue bone marrow promising sources like amniotic fluid hair follicles 13 33 use differentiated autologous cells bladder biopsy limited due risk cancer development case bladder cancer patients main candidates urinary conduit construction the number cells seeded scaffold also important conception seems simple cells cm scaffold better results achieved the achievement large cell numbers challenging issue average bladder cancer patient 65 years old mesenchymal stem cells proliferation capacity decreases age patient increasing passage numbers 36 37 hand authors suggested stem cell proliferation capacity dependent donors age increase chances tissue engineering therapy use artificial urinary conduit construction despite large number cells necessary regeneration hard obtain costly price culture media containing appropriate growth factors that efficient cell culture method developed provide success procedure many years atypical smooth muscle cells asmcs localized proximal regions renal pelvis considered peacemaker peristaltic waves responsible urine passage bladder recent studies indicated interstitial cells cajal like cells icc lc expressing c kit gene sparsely distributed within lamina propria muscle layer upper urinary tract play important role promoting pyeloureteric peristalsis contraction waves generated renal pelvis probably propagated coordinated modulated upper urinary tract icc lc 41 42 iccs thanks automatism able replace discontinued atypical smcs impulses maintain peristaltis lower ureter parts 40 43 vitro isolation culture icc lc urinary tract yet established taking consideration previous works experience urinary conduit construction addition cells coculture smooth muscle cells potentially accelerate restoration peristaltic waves reconstructed segment could prevent development hydronephrosis in conclusion success ureter conduit construction using tissue engineering techniques depends finding proper scaffold we believe implementation procedure possible urinary conduit first commercially available product clinical practice constructed using regenerative medicine confirmation opinion registered clinicaltrial.gov study incontinent urinary conduit construction using pga plga scaffold seeded autologous smooth muscle cells derived adipose tissue biopsy

introductionincontinent urinary diversion using an ileal conduit is the most popular method used by urologists after bladder cystectomy resulting from muscle invasive bladder cancer . the use of gastrointestinal tissue is related to a series of complications with the necessity of surgical procedure extension which increases the time of surgery . regenerative medicine together with tissue engineering techniques gives hope for artificial urinary conduit construction de novo without affecting the ileum.material and methodsin this review we analyzed history of urinary diversion together with current attempts in urinary conduit construction using tissue engineering methods . based on literature and our own experience we presented future perspectives related to the artificial urinary conduit construction.resultsa small number of papers in the field of tissue engineered urinary conduit construction indicates that this topic requires more attention . three main factors can be distinguished to resolve this topic : proper scaffold construction along with proper regeneration of both the urothelium and smooth muscle layers.conclusionsartificial urinary conduit has a great chance to become the first commercially available product in urology constructed by regenerative medicine methods .

~1.81 million chronic kidney disease patients globally undergoing hemodialysis hd treatment estimated 90,000 5% thereof treated hemodiafiltration hdf).1 online hdf suggested possible cost effective alternative compared standard hd mainly due superior middle molecule clearance resulting combination diffusion convection solute removal mechanisms several studies suggest hdf may lead better clinical results high flux hd hf hd).2,3 according recent survey hdf started prevent dialysis complications increase efficiency dialysis uremic accumulates treat dialysis related hypotension dialysis related amyloidosis intractable pruritus restless leg syndrome arthralgia related dialysis related amyloidosis.4 however date randomized studies conclusively supported superiority hdf regarding clinical outcomes.57 addition concerns raised cost utility,8 leading investigators conduct cost analyses assess differential expenditures two therapies.9 recent editorial vanholder et al concluded expensive therapies reimbursed evidence cost utility defined ratio treatment cost treatment outcome taking consideration also quality life qol sufficiently robust.10 cost hdf per se decreased recent years due 1 increased market penetration subsequent economy scale production equipment disposables 2 pressure health care costs due economic crises prevailing since 2008 3 safety reliability online method reduced demand microbiological testing monitoring.11,12 furthermore publication study canaud et al,2 hdf treatment effectiveness increasingly understood positively associated magnitude convection volume used resulting trends toward application higher hdf convection volumes applied earlier cost analyses accordingly need new cost effectiveness evaluation aim study assess cost effectiveness hdf compared hd simulation using existing study data these data facilitate understanding whether longer survival better health related quality life hrqol able offset possibly higher costs hdf thus whether worth investing innovative therapy the analysis comprised simulation1315 based results combined previous observational studies randomized clinical trials meta analysis following steps 1 estimation survival function hf hd patients membrane permeability outcome study16 dataset b estimation survival function hdf patients using risk reduction estimates due treatment effect meta analysis mostovaya et al17 even though includes also studies comparing hdf low flux hd considered best proxy respect alternative meta analyses available literature 2 simulation survival sample patients allocated hf hd hdf using three state markov models 3 application state specific hrqol coefficients differential costs materials testing consumption water derived literature in addition secondary subgroup analysis considered eshol5 estimations related diabetics nondiabetics this done among studies considered eshol study provides results related particular subgroups patients two mirror image markov models figure 1 developed aim generate simulation comparing survival sample patients treated hf hd hdf three possible states considered alive therapy dead due disease therapy dead cause ie considering overall mortality based age- sex adjusted life tables the probabilities defining transition patients states calculated previously estimated hazard functions specified defined time frame cycle the model run specified number cycles see hypothetical cohort patients moved states the length cycle fixed 1 year number cycles ten total 10 years accordingly total dialysis costs divided 1 direct health care costs staff material vascular access routine diagnostics hospitalization drugs medications 2 direct nonhealth care costs cost transport cost informal care 3 indirect nonhealth care costs productivity losses due disability premature death it reasonable assume direct nonhealth care costs indirect nonhealth care costs two therapies also direct health care costs vary treatment consequence the comparison incremental costs hdf respect hf hd focused costs equipment disposables ultrapure water testing water consumption.18 order obtain generalizable results analyzed two alternative cost settings involving involving use ultrapure water the first based assumption since dialyzers used perform hdf hf hd designed better solute clearances wider range uremic toxins higher hydraulic permeability reasonable argue water dialysis fluid microbiological purity necessary therapies due backfiltration phenomena leading similar costs water testing consequence differences total water consumption negligible possible limit analysis costs disposables blood lines dialyzers equipment in prospective observational study oates et al,9 patients hf hd switched hdf others remained hf hd the additional cost disposables related hdf 1.32 per session 207 per annum p.a standard case use cuvette relative blood volume monitoring the alternative cost setting based analysis lebourg et al12 addition cost variability due disposables monitors difference cost two different therapies caused water analysis water consumption considered the additional hdf cost per session ranged minimum value 1.29 202 p.a maximum value 4.86 730 p.a once relevant costs determined corresponding estimated discounted costs differential cost taken literature allocated state model contradicting results reported difference qol related hf hd hdf few studies addressed qol evaluation none provided hrqol coefficients specific comparison hf hd hdf fundamental purpose analysis mazairac et al8 estimated higher scores euroqol 5d patients hdf compared hd this preference based measure yields set weights quality adjusted life years qalys calculations based study as data qol differences hdf hf hd literature coefficients used simulation particular hrqol coefficients linked age selected model otherwise fundamental component benefit measurement would neglected recommended economic analysis guidelines,19 annual discount rate 3.5% costs benefits selected changes results function different values parameters considered parameter uncertainty included model probabilistic sensitivity analysis taking intrinsic imprecision estimate parameters survival model account consequently quality quantity information available reflected probability distributions assigned input parameter model.20 several monte carlo simulations performed also patients different risk profiles age patients aged 40 50 60 years sex diabetic status.21,22 sake simplicity cohort simulated 50-year old male patients selected reference population analysis discussed next section found similar results age subgroups identical conclusions terms cost effectiveness therefore used cohort briefly recap main results analysis the characteristics cohort coherent results edta registry reported van de luijtgaarden et al:23 last 20 years indeed mean age patients started hd treatment 5560 years average 60% patients male output analyses included scatter plots simulations cost effectiveness plane estimation incremental cost effectiveness ratios icers computation cost effectiveness acceptability curves ceacs the results 1,000 monte carlo simulations cohort 50-year old male patients shown cost effectiveness scatter plot figure 2 mean values simulations specific cohort patients the icer 6,982/qaly basis simulations possible derive probability cost effectiveness given different thresholds shown figure 3 the ceac corresponding hdf starts 0 meaning possibility alternative therapy cost saving threshold must least 2,000 per qaly probability cost effectiveness 0 the probability cost effectiveness increased 50% threshold 7,000/qaly 70% threshold 15,000/qaly 81% commonly accepted threshold 40,000/qaly the probability cost effectiveness always stayed 84% threshold 40,000/qaly this asymptotic value results fact fraction simulations represent cases alternative therapy hdf causes higher costs provided fewer benefits this means even budget constraint probability alternative therapy cost effective traditional one hf hd preferred analyses carried subgroups 40- 50- 60-year old male female patients table 1 hdf appears cost effective younger patients this message reinforced figure 4 shows given threshold value associated higher probability hdf cost effective 40- 50-year old patients 60-year old ones moreover 60-year old groups probability around 28% females males hdf cost effective even extreme values threshold regarding discount rates costs benefits results robust even considering alternative scenarios lower higher discount rates by applying 0% 5% discount rate costs effects 50-year old male patient icer decreased 6,676/qaly increased 6,960/qaly respectively confirming robustness estimations a analysis performed investigate importance qualitative component qalys qol compared quantitative one life years the effect using overall hrqol coefficients rather hrqol coefficients linked patient age cohort 50-year old male patients shown figure 5 the mean icer increased 19,423/qaly importantly almost 37% simulations left qaly zero value less effectiveness costs accordingly uncertainty around decision whether invest new therapy much higher even infinite value threshold would lead probability cost effectiveness 65% data shown this caused uncertain values overall hrqol coefficients leading cases one period spent hf hd worth even period hdf this shows crucial consider role qol evaluations previously cost inputs point estimates whereas costs vary range assessed lebourg et al.12 ceac subgroup 50-year old male patients shows 21.5% simulations cost saving figure 6 the probability cost effective reaches 80% commonly accepted threshold 40,000/qaly increases 2.4 percentage points even threshold 70,000/qaly hdf seems little expensive instance icers younger patients rise 5,878/qaly 5,732/qaly 7,748/qaly 7,724/qaly female male patients respectively table 2 this subgroup analysis confirmed previous results icer increases age cohort diabetic patients follow different survival function subject different treatment effects alternative therapy compared nondiabetic patients nevertheless comparison cohort 50-year old diabetic male patients cohort 50-year old nondiabetic male patients reveal significant differences figure 7 given commonly accepted threshold 40,000/qaly study demonstrated compared hf hd hdf cost effective treatment patients dialysis this particularly true patients 60 years irrespective sex diabetic status either way variability results due different characteristics patients affect conclusions cost effectiveness hdf these results quite different reported mazairac et al8 cost utility analysis hdf versus conventional low flux hd based unadjusted results convective volume convective transport study contrast).7 estimated incremental cost per quality adjusted life year hdf compared low flux hd 287,679 certainly 140,000 favorable assumptions eg applying convection volume 20.3 l they argued hdf characterized higher cost better health cost effective given currently accepted cost utility thresholds however mentioned analysis performed patients treated montreal canada according protocol yielded different positive results.24 analysis hand reports hdf cost effective probability ~81% this conclusion based considered commonly accepted threshold health policy decisions,25 ~40,000 30,000 gbp 50,000 usd per qaly even threshold difficult quantify and is often based rule thumb commonly applied estimate forgone health resulting services displaced accommodate additional costs new technology note magnitude difference two studies may related different comparison groups low flux hd contrast study hf hd model the icer estimates show good value money doubts cost effectiveness hdf hf hd remain due residual ~20% probability costs higher and/or benefits lower hdf this line reasoning also shared mcbrien manns26 commentary mazairac study argued hdf characterized higher procedural costs proven health benefit our study specifies survival difference important factor especially considering lack agreement aspect differences terms qol fundamental importance must measured this rationale conducting value based study incorporating hrqol main outcome hdf versus hf hd convective treatments hdf indeed reduce intradialytic hypotension affects qol possibly limit poor appetite nutritional state patients chronic kidney disease however believe research warranted assess impact hdf specifically nutritional state end stage renal disease patients therefore study recommends prioritizing research evaluates qol related hdf it must stressed purpose economic decision model synthesize relevant evidence order make sense inform decision adoption new technology uncertain context.15 indeed identified relevant studies involving therapies assessment included uncertainty parameter estimates without neglecting relevant information literature our result therefore based published data accuracy sensitive decisions forced take model construction phase the first decision use survival estimates hf hd arm membrane permeability outcome16 randomized clinical trial this trial based incident patients whereas studies hdf mainly performed prevalent european dialysis patients additionally low crude mortality rate observed membrane permeability outcome study may produce results completely transferrable ordinary nontrial participating patients indeed stressed palmer et al,20 relative risk reductions applied low baseline risks produce low absolute reduction event rates low gains health it possible low overall mortality rate affected lack difference diabetic nondiabetic patients as previously stressed one main requirements economic evaluation include relevant evidence regard effectiveness data concept change meta analysis is usual technique employed synthesize available evidence date five meta analyses aggregating results main convective dialysis therapy studies17,2730 have published however huge heterogeneity interventions included meta analyses meta analysis mostovaya et al17 one focused trials hdf convective therapy appropriate definition convective volume nevertheless still great variety patients treatment modalities characteristics among studies indeed instance high- low flux modalities included comparison consequence a relative risk based random effects model used makes estimate pooled effect conservative takes account clinical heterogeneity among single studies note meta analysis includes also contrast study cost effectiveness analysis include relevant randomized clinical trials contrary instance economic evaluation mazairac based contrast study subjective decision complete cost effectiveness analysis instead take consideration relevant available evidence previously pointed regard subanalysis diabetic patients eshol5 estimates risk reduction used as eshol study provided positive results favor hdf concerns overestimation reduction mortality could raised nevertheless model show difference two groups patients result highlights impact mortality less important effect qol the estimations used study mainly derived meta analysis basically 23 years follow time appropriate time horizon would patient lifetime especially true case treatment chronic diseases initiation intervention middle aged patients may cost effect implications rest lives.31 important role decision model therefore bridge gap observed trials would expected happen long term forcing one make assumptions long term effects consequences treatment modalities additionally lack qol coefficients related hf hd forced use data comparison hdf low flux hd.8 uncertainty around results high typical kind analyses neglecting available evidence uncertainty would contrast comprehensive nature economic model this study contributes also show scarcity heterogeneity data available literature jeopardize economic evaluation hdf compared hf hd new studies needed assess precisely differential benefits therapies value information analysis might help identify specifically parameters would worth investigated a comprehensive evaluation performed randomized clinical trial required order include relevant cost components eg pharmaceuticals hospitalizations prevention long term dialysis related complications beta-2 microglobulin amyloidosis transplantation access analysis possible savings resulting reduction cost ancillary pharmacological therapy hospitalization considered current analysis

backgroundclinical studies suggest that hemodiafiltration ( hdf ) may lead to better clinical outcomes than high - flux hemodialysis ( hf - hd ) , but concerns have been raised about the cost - effectiveness of hdf versus hf - hd . aim of this study was to investigate whether clinical benefits , in terms of longer survival and better health - related quality of life , are worth the possibly higher costs of hdf compared to hf-hd.methodsthe analysis comprised a simulation based on the combined results of previous published studies , with the following steps : 1 ) estimation of the survival function of hf - hd patients from a clinical trial and of hdf patients using the risk reduction estimated in a meta - analysis ; 2 ) simulation of the survival of the same sample of patients as if allocated to hf - hd or hdf using three - state markov models ; and 3 ) application of state - specific health - related quality of life coefficients and differential costs derived from the literature . several monte carlo simulations were performed , including simulations for patients with different risk profiles , for example , by age ( patients aged 40 , 50 , and 60 years ) , sex , and diabetic status . scatter plots of simulations in the cost - effectiveness plane were produced , incremental cost - effectiveness ratios were estimated , and cost - effectiveness acceptability curves were computed.resultsan incremental cost - effectiveness ratio of 6,982/quality - adjusted life years ( qaly ) was estimated for the baseline cohort of 50-year - old male patients . given the commonly accepted threshold of 40,000/qaly , hdf is cost - effective . the probabilistic sensitivity analysis showed that hdf is cost - effective with a probability of ~81% at a threshold of 40,000/qaly . it is fundamental to measure the outcome also in terms of quality of life . hdf is more cost - effective for younger patients.conclusionhdf can be considered cost - effective compared to hf - hd .

alzheimer disease ad frequent neurodegenerative disease ferri et al 2005 characterized progressive dementia occurs middle late life the neuropathological hallmark ad presence cortical intracellular neurofibrillary tangles nft extracellular -amyloid plaques braak braak 1991 leads synapse dysfunction neuronal cell loss subsequent brain atrophy ballard et al 2011 in past decades knowledge key pathogenic mechanisms disease improved still completely understood querfurth laferla 2010 natural history ad could divided three different phases pre clinical phase pathogenic mechanisms disease started symptoms identified prodromal phase mild cognitive symptoms appear severe enough meet dementia criteria dementia phase dubois et al 2007 hand mild cognitive impairment mci clinical construct created capture patients subtle cognitive symptoms risk ad petersen et al 2009 however subjects develop types dementias progress even revert normal cognition ganguli et al 2004 brooks loewenstein 2010 therefore difficult label mci patients prodromal ad currently frequently used clinical diagnostic criteria ad diagnostic statistical manual mental disorders dsm iv tr apa 2000 national institute neurological disorders stroke alzheimer disease related disorders nincds adrda mckhann et al 1984 working group first dementia syndrome established criteria based clinical features ad type dementia applied the dsm iv tr criteria require presence impairment multiple cognitive domains sufficient severity interfere individuals activities daily living diagnose dementia hence clinical diagnosis ad possible patient reach dementia phase disease hand these criteria shown good sensitivity specificity distinguish patients ad type dementia non demented subjects less accurate differentiate ad dementia primary dementias knopman et al in fact 20% patients clinically diagnosed ad ad pathology autopsy mayeux et al a accurate earlier diagnosis ad e.g. mci patients progress ad dementia phase ideally subjects pre clinical phase could enable administration potential disease modifying drugs would great impact patients life profound implications public health brookmeyer et al 1998 context need specific biological markers ad diagnosis earliest stages a biomarker defined measureable feature used diagnose physiologic pathologic condition the ideal biomarker ad would 1 directed fundamental pathophysiology disease 2 marker presence disease 3 efficacious prodromal even pre clinical stages ad 4 indicator disease severity 5 marker treatment effectiveness 6 inexpensive non invasive klunk 1998 case ad different biomarkers described using diverse approaches the development cerebrospinal fluid csf assays neuroimaging techniques provide information presence ad pathological changes major step forward field consequently biomarkers ad called play central role clinical characterization disease recently nincds adrda criteria revised national institute aging alzheimer association nia aa workgroup include experience research past 25 years mckhann et al 2011 the nia aa proposed series diagnostic classification pre clinical ad mci full blown ad introduced csf neuroimaging biomarkers supportive features disease albert et al 2011 mckhann et al 2011 sperling et al 2011 however authors cautioned use biomarkers stated use limited research drug trials nevertheless core clinical criteria ad remained relatively intact new criteria this review focus widely studied currently accepted sources biomarkers ad csf magnetic resonance imaging mri nuclear medicine techniques including amyloid imaging typically csf better reflects brain neurochemistry biochemical values plasma serum may affected many non neurological factors csf biomarker research focused mainly molecules related central neuropathological features ad main component senile neuritic plaques snp tau proteins part nfts a 3643 amino acid peptide originated proteolysis amyloid protein precursor querfurth laferla 2010 snps mainly composed protein 40 a40 42 a42 amino acids seems deposition begins a42 plaque maturation acquire a40 iwatsubo et al 1994 by contrast nfts formed tau proteins microtubule associated stabilizing protein essential axonal transport tau hyperphosphorylated ad tends aggregate leading synaptic neuronal dysfunction lee 1996 blennow et al 2010 ; this process nfts formation starts entorhinal cortex spreading hippocampus rest brain these neuropathological changes begin early even decades onset clinical symptoms dementia braak braak 1991 relative contributions altered tau amyloid metabolism neurodegeneration remain investigation nevertheless nfts robust associated measures synapse loss cognitive impairment snps gomez isla et al 1997 typically ad patients lower levels a42 csf compared normal controls a40 tends remain constant tau tau phosphorylated tau p tau increase arai et al 1995 blennow et al 1995 ; 1995 galasko et al 1998 kurz et al 1998 shoji et al 1998 hulstaert et al 1999 ; 2001 sunderland et al 2003 lewczuk et al 2004 ; the low csf a42 levels entity may represent retention peptide plaques resulting reduced availability diffuse csf this supported findings a42 csf inversely correlated amyloid plaque load autopsy strozyk et al 2003 tapiola et al 2009 measured vivo amyloid imaging fagan et al 2006 the increase tau p tau csf ad patients may stem release csf neurodegeneration blennow et al 1995 although exact mechanism known t tau p tau concentrations csf associated nft load autopsy buerger et al 2009 well cognitive decline brain atrophy buerger et al 2002 ; low csf a42 high tau p tau proteins shown high accuracy ad diagnosis galasko et al 1998 andreasen et al 2001 schoonenboom et al 2004 studies compared ad patients normal control subjects demonstrated low a42 78100% sensitivity 4781% specificity ad diagnosis high csf tau levels 70% sensitivity 92% specificity p tau showed 77% sensitivity 87% specificity however p tau appears better tau diagnosis ad shown positive predictive value 90% especially p tau phosphorylated threonine 181 p tau181 hampel et al figure 1 shows tau levels ad frontotemporal dementia ftd normal autopsied subjects figure 2 shows a42 levels population schoonenboom et al ad alzheimer disease ftd frontotemporal dementia studies found a42 tau levels alone sufficient differentiate ad normal controls dementias a42/tau ratio used improve diagnosis ad a meta analysis showed a42/tau ratio sensitivity 71% specificity 83% ad hampel et al 2004b recent study showed signature ad based csf a42/p tau181 ratio cutoffs present 90% ad patients compared 36% normal control group de meyer et al 2010 although csf biomarkers seem useful diagnose ad sensitive enough assess disease progression sunderland et al 1999 vemuri et al 2010b nevertheless higher csf tau lower a42 values associated rapid cognitive decline response cholinesterase inhibitor treatment higher mortality patients ad dementia wallin et al 2010 cerebrospinal fluid biomarkers might role differential diagnosis ad dementias patients dementia lewy bodies dlb like ad patients lower levels a42 csf compared normal controls reflecting subjects also present ad pathology nevertheless shorter amyloid peptides a37 a38 a40 appear slightly elevated ad dementia bibl et al 2006 consequently a42/a37 a42/a38 ratios seemed better discriminate ad dlb bibl et al t tau p tau levels normal high dlb studies shown markers levels dlb definitely lower found ad other proposed biomarker differentiation ad dlb monomeric soluble -synuclein available studies dlb patients showed conflicting results elevated unchanged decreased total -synuclein levels reported patients compared ad control subjects mukaetova ladinska et al 2010 in addition limited data oligomeric -synuclein csf although could potential biomarker synucleinopathies tokuda et al 2010 possibly combination markers help distinguish ad dlb future ftd csf a42 levels lower control subjects higher ad tau p tau levels higher controls lower ad patients hulstaert et al hence tau a42 p tau a42 ratios could useful tool distinguish ad ftd bian et al creutzfeldt jakob disease cjd characterized decreased csf a42 concentrations high tau levels reflecting intense neuronal damage p tau levels normal kapaki et al 2001 ; current data suggest p tau levels could relevant marker differentiate ad types dementias hampel et al cerebrospinal fluid biomarkers may also useful predicting progression ad subjects mci andreasen et al 1999 hampel et al 2004c ewers et al 2007 low csf a42 levels predicted conversion 6080% sensitivity 65100% specificity tau levels 8386% sensitivity 5690% specificity p tau 7384% sensitivity 4788% specificity hampel et al 2004b however conversion ad dementia mci subjects shown significant variability among different csf studies ( 2006 found high sensitivity 95% specificity 87% conversion mci ad dementia using combination tau a42/p tau181 ratio cutoff values by contrast multicenter csf study found lower sensitivity 83% specificity 72% conversion mci ad single site studies considerable inter site assay variability highlights need standardization analytical techniques subject selection mattsson et al 2009 other large multicenter studies confirmed high predictive value csf biomarkers identification cases prodromal ad context mci syndrome shaw et al 2009 visser et al 2009 the csf biomarkers used identify pre clinical stage ad cross sectional studies normal control individuals carrying apoe4 allele showed lower csf a42 higher p tau181 levels peskind et al interestingly csf levels a42 correlated brain volume group subjects mci ad dementia patients whole brain volume correlated csf tau p tau181 fagan et al 2009 a longitudinal csf study normal controls found increased tau levels change a42 1-year follow vemuri a longitudinal studies assessed csf biomarker combination biomarkers better predict ad dementia cognitive decline fagan et al 2007 demonstrated csf tau a42 ratios may predict future dementia cognitively normal older subjects two studies showed low levels csf a42 predicted cognitive decline measured mini mental status examination mmse gustafson et al although studies showed csf a42 could predict prodromal ad wide sd subjects made difficult determine individually progress ad further studies needed assess changes csf markers time determine best csf markers change structural neuroimaging techniques normally performed clinical assessment patients dementia mainly rule structural lesions brain tumors normal pressure hydrocephalus vascular lesions the mri shown high sensitivity detect brain atrophy caused neurodegenerative process bobinski et al 2000 several methods used analyze mri data including visual inspection volumetry region interest roi analysis applying manual tracing automated semi automated techniques voxel based morphometry vbm vemuri jack 2010 the visual rating images roi analysis depend priori choice structures vbm whole brain operator independent analysis ashburner friston 2000 however vbm analysis allows comparing groups patients useful classify individual cases visual assessment mri scans fast method assess brain atrophy precise raji et al 2010 volumetry medial temporal lobe structures common quantitative method used ad the traditional manual tracing time consuming consequently automated semi automated methods require significant manual intervention developed other methods analyze sequential mris boundary shift integral bsi tensor based morphometry tbm bsi developed quantify global percentage change brain surfaces two scans tbm provides three dimension profile brain atrophy vemuri jack 2010 brain mri studies shown ad patients widespread cortical volume loss including frontal lobes temporoparietal regions precuneus mesial temporal lobes relative sparing sensorimotor visual cerebellar regions du et al 2001 good et al 2001 ; thompson et al 2001 karas et al 2003 testa et al 2004 ishii et al 2005 dickerson et al 2008 burton et al 2009 ) longitudinal studies suggested atrophy stars medial temporal lobes fusiform gyrus prodromal phase disease spreads posterior temporal parietal lobes time dementia phase involves medial temporal lobes temporoparietal cortex frontal lobe whitwell et al 2007 this pattern atrophy progression mri appears follow distribution nft different braak braak 1991 pathological stages whitwell et al 2008 in ad also greater rate brain atrophy time compared healthy elderly subjects 1.92.2% per year versus 0.50.7% per year obrien et al 2001 sluimer et al hippocampal whole brain atrophy ad correlated well cognitive performance measures disease severity jack et al 2008a ridha et al 2008 mri studies show similar pattern brain atrophy observed ad jack et al 2000 ; chetelat et al 2002 karas et al 2004 bell mcginty et al 2005 whitwell et al 2008 rates whole brain atrophy around 1% per year ad control subjects jack et al 2004 ) whole brain atrophy rate mci associated impaired cognitive performance evans et al 2010 predicts progression ad dementia spulber et al 2008 whole brain techniques also shown higher rates atrophy specific areas hippocampus posterior cingulated gyrus superior parietal cortex associated incident ad mci cases chetelat et al 2005 hippocampal basal forebrain volumes normal subjects predicted future development ad hall et al 2008 interestingly recent study showed normal subjects ad csf profile increased rates brain atrophy time suggesting may pre clinical phase neurodegenerative process schott et al 2010 nevertheless observation requires longer follow period demonstrate particular group subjects higher risk develop ad dementia important clinical manifestation ad memory loss the majority researchers focused study hippocampal atrophy marker disease different neuroimaging studies assessing mesial temporal lobe structures consistently shown smaller hippocampal volumes ad patients compared controls pennanen et al 2004 measures hippocampal atrophy correlate well degree cognitive impairment jack et al 2000 mortimer et al 2004 ad pathology hippocampal sclerosis mri post mortem studies jagust et al 2008 both normal aging ad gradual volume loss overtime whole brain volume greater ad annual rate hippocampal atrophy 1.41% healthy controls and several studies shown hippocampal atrophy measured mri predicts conversion mci ad jack et al 1999 visser et al 1999 2002 geroldi et al 2006 devanand et al 2007 teipel et al 2007 a recent meta analysis showed measure could identify mci converters sensitivity 73% specificity 81% yuan et al 2009 although decreased entorhinal cortical thickness proposed predictor conversion mci ad measures decrease hippocampal volume appeared robust predictor there several limitations use whole brain hippocampal volume sole biomarker ad one important small brain volumes also seen normal aging consequently difficult separate cases early ad normal controls hand rate change whole brain entorhinal hippocampal volumes seemed good markers neurodegeneration useful determining effects ad therapies techniques labor intensive limited research studies brain functional neuroimaging techniques positron emission tomography pet single photon emission computed tomography spect allow broad range cerebral functions assessed patients currently living dementia actually brain metabolism brain perfusion different neurotransmitter systems measured great variety pet spect tracers however brain metabolism glucose analog 2-[f]-fluoro-2-deoxy glucose f fdg brain perfusion technetium-99 hexamethylpropylamine oxime 99mtc hmpao two widely employed techniques evaluation patients dementia clinical practice research brain spect 99mtc hmpao studies provide information regional cerebral blood flow f fdg pet estimates regional brain rate glucose consumption therefore providing information pattern neuronal loss synapse dysfunction patients dementia the pattern spect hypoperfusion pet hypometabolism usually seen ad involves anterior medial temporal lobes posterior cingulate posterior temporoparietal cortex kogure et al 2000 ; nevertheless spect seems less accurate pet diagnosis ad one autopsy study found technique alone sensitivity 63% specificity 93% diagnose ad jagust et al 2001 hand the sensitivity procedure discriminating ad ftd 71.5% specificity 78.2% sensitivity specificity differentiate ad vascular dementia 71.3 75.9% respectively dougall et al spect shown predict conversion ad test accuracy 82% huang et al 2007 positron emission tomography studies early ad shown pattern reduced metabolism posterior cingulate minoshima et al 1997 mesial temporal cortices as disease progresses greater involvement parietotemporal frontal cortices ishii et al 2001 ; this progression areas hypometabolism correlated cognitive deterioration time measured neuropsychological tests engler et al 1984 minoshima et al 1995 silverman et al 2001 mosconi et al 2005 a meta analysis showed pooled f fdg pet sensitivity specificity differentiate ad patients normal subjects 86% cases variability important studies sensitivity 61 100% specificity 54 100% patwardhan et al 2004 pet also seems good tool differentiate ad forms dementia autopsy confirmed study presence occipital hypometabolism able distinguish dlb ad 90% sensitivity 80% specificity minoshima et al this technique also high specificity sensibility differentiate ad ftd koeppe et al 2005 ; f fdg pet appears promising instrument discriminate patients mci progress ad dementia these patients show medial temporal posterior cingulate cortices hypometabolism compared healthy controls chetelat et al has also shown f fdg pet performs slightly better spect structural mri identifying prodromal phase ad patients clinical diagnosis mci pooled sensitivity 89% specificity 85% yuan et al 2009 interestingly similar metabolic reductions regions found mci ad patients seen asymptomatic subjects risk ad i.e. persons subjective memory complains caselli et al 2008 carriers apolipoprotein e 4 apoe4 allele reiman et al 1996 2001 2004 2005 abnormalities csf markers petrie et al 2009 maternal history ad mosconi et al 2007 2009 furthermore hippocampal hypometabolism normal aging predicted cognitive decline years advance clinical diagnosis de leon et al 2001 jagust et al these data indicate f fdg pet high sensitivity distinguish ad controls dementing entities also identify subjects high ad risk consequently pet ad pattern incorporated new clinical criteria prodromal ad dubois et al 2007 pre clinical ad sperling et al 2011 mci due ad albert et al the development pet radiotracers bind brain amyloid revolutionized use neuroimaging techniques patients dementia these molecules allow localization ad pathology vivo helped understanding underlying biology ad the actively tested tracers n methyl-[c]2-(4-methylaminophenyl)-6-hydroxybenzothiazole c pib klunk et al 2003 e)-4-(2-(6-(2-(2-(2-(18)f fluoroethoxy)ethoxy)ethoxy)pyridin-3-yl)vinyl)-n methyl benzenamine f av-45 choi et al 2009 2-(1 96-(2-f fluoroethyl)(methyl)amino)-2-naphthyl)ethyldene malono nitrile f fddnp small et al 2006 shin et al 2008 multiple c f compounds investigation trans-4-(n methyl amino)-4-{2-[2-(2-[18f]fluoro ethoxy)-ethoxy]-ethoxy}-stilbene f bay94 9172 rowe et al 2008 4-n-[c methyl]amino-4-hydroxystilbene c sb-13 verhoeff et al 2004 2-(2-[2-dimethylaminothiazol-5-yl]ethenyl)-6-(2-[fluoro]ethoxy)benzoxazole c bf-227 kudo et al 2007 while c better affinity fibrillar amyloid f compounds former short life limits use centers available cyclotron produce among tracers this thioflavin derived binds fibrillar high affinity brain retention correlates well levels amyloid ad brain tissue mathis et al 2002 2003 ; positron emission tomography studies c pib demonstrated increased retention tracer frontal parietotemporal cortices well striatum almost ad patients compared controls klunk et al 2004 edison et al 2006 rowe et al 2007 however important note positive c pib pet scan also seen entities often misdiagnosed ad dlb cerebral amyloid angiopathy edison et al 2008 gomperts et al 2008 dierksen et al c pib retention ad correlates rates whole brain atrophy archer et al 2006 parietotemporal hypometabolism f fdg pet klunk et al 2004 edison et al 2006 engler et al 2006 decreased csf a42 levels fagan et al 2006 grimmer et al there longitudinal studies using amyloid ligands observation time relatively short techniques available research since 2004 recent longitudinal studies ad patients assessing amyloid load progression neurodegeneration using structural mri f fdg pet found c pib retention relatively stable time neurodegenerative markers worsen parallel cognitive decline engler et al 2006 jack et al 2009 scheinin et al 2009 kadir et al these findings consistent hypothesis dissociation amyloid deposition neurodegenerative process amyloid accumulation precedes clinical symptoms reaches maximum detectable level cognitive deficits evident jack et al 2010a however another recent study shown increases c pib retention mild moderate ad dementia questioning hypothesis suggesting deposition might slow later stages disease still present villemagne et al 2011 mci patients c pib pet shows bimodal distribution subjects pattern retention similar ad patients others similar control subjects pike et al 2007 rowe et al in patients burden related episodic memory impairment cognitive tasks pike et al 2007 the pib positive mci subjects likely carry apoe4 allele progress ad pike et al 2007 ; factors seem influence shorter time progression pib positive subjects presence hippocampal atrophy higher c pib retention values apoe4 allele okello et al 2009 ; ( 2009 rescanned mci subjects single year minimal change seen group persons 2011 shown small significant increases c pib cortical retention mci patients baseline studies scans performed 20 months 3 years later studies reported similar results kadir et al 2010 koivunen et al approximately 2030% elderly cognitively normal subjects show degree c pib retention mainly prefrontal cortex posterior cingulate precuneus regions mintun et al 2006 pike et al 2007 rowe et al 2007 aizenstein et al 2008 in fact proportion c pib positive healthy control subjects increases age morris et al 2010 subjects some studies reported strong relationship impaired episodic memory performance c pib binding could modified cognitive reserve pike et al 2010 others found association mintun et al 2006 jack et al 2008b stornadt et al 2009 studies directly relating structural mri data burden control subjects yield contradictory results 2009 cortical thinning pattern consistent early ad dickerson et al 2008 ; becker et al 2010 increased rate brain atrophy scheinin et al 2009 others found brain atrophy c pib positive subjects normal individuals subjective cognitive impairment chetelat et al there two longitudinal studies reporting progression cognitively normal subjects scanned c pib pet symptomatic ad one study 2009 reported relative risk conversion cognitive normal ad increased almost five fold c pib positive pet scan ( 2011 performed serial c pib pet scans group normal subjects found 25% individuals positive scan developed mci ad dementia 3 years in addition authors also reported increase c pib brain retention control subjects positive baseline scan suggesting slow process accumulation brain time villemagne et al these data consistent hypothesis amyloid imaging detect accumulation advance onset dementia jack et al 2010a although longitudinal studies required determinate sequence pathological events process normalcy ad figure 3 shows spectrum amyloid deposition normal controls mci ad cases measured c pib pet mathis et al 2007 transaxial sagittal planes c pib pet scans normal control nc c pib positive nc nc+ c pib negative mci subject mci c pib positive mci subject mci+ highly c pib positive mci subject mci++ c pib positive ad patient ad reprinted mathis et al although amyloid imaging relatively new technique state accurately detects accumulation brain however reported case patient clinically diagnosed mild ad negative c pib pet scan low levels amyloid pathology autopsy found cairns et al 2009 on hand reported cases subjects positive amyloid pet without presence autopsy these data suggests c pib pet technique high specificity positive predictive value early ad diagnosis nevertheless c pib positive scan mean pre dementia stage ad prognostic value increased load pet established extensive longitudinal studies in scenario adding techniques assess functional brain changes suggesting early ad could special interest definitely information obtained current amyloid tracers bring light important issues biology amyloid deposition transition normal ad current data suggest diagnosis ad enhanced use promising biomarkers increase accuracy identify early stages disease each biomarkers seems indicate specific process ad amyloid imaging decreased csf a42 indicators brain amyloid burden csf tau brain atrophy brain metabolism biomarkers neurodegenerative process some authors proposed model disease timing use different biomarkers jack et al 2010a the ad pathologic cascade appears sequential two stage process initiated amyloid accumulation followed neuronal pathology validate hypothesis right use biomarkers clinical practice it necessary determine dynamics amyloid process biomarkers different stages disease an early ad diagnosis could enable administer treatments potential disease modifying effect neurodegeneration becomes severe the authors declare research conducted absence commercial financial relationships could construed potential conflict interest

alzheimer s disease ( ad ) is the most common form of dementia in the elderly , and it is characterized by progressive impairment in multiple cognitive domains of sufficient severity to interfere with individuals daily living activities . historically , the diagnosis of ad has been based on the identification of a clinical syndrome , and accuracy studies of the current clinical criteria conducted in referral clinics have shown high sensitivity for ad . however , the identification of the disease is still not perfect , and there is growing evidence that the use of biomarkers will increase our ability to better indentify the underlying biology of ad , especially in its early stages . these biomarkers will improve the detection of the patients suitable for research studies and drug trials , and they will contribute to a better management of the disease in the clinical practice . in this review , we discuss the most studied biomarkers in ad : cerebrospinal fluid proteins , structural magnetic resonance imaging , functional neuroimaging techniques , and amyloid imaging .

cardinal clinical characteristics sca3 include gait stance unsteadiness limb ataxia dysarthria oculomotor dysfunction sensory disorder pyramidal extrapyramidal dysfunction sca3 slowly progressive unremitting disease patients generally become wheelchair bound bedridden end stage median survival time disease onset approximately 21 years the resulting loss working ability reduced survival confer significant disease burden patients families society thus vital importance explore effective therapeutic options order alleviate symptoms retard disease progression sca3 nerve growth factor ngf founding member neurotrophin family essential proper development patterning maintenance mammalian nervous system previous studies revealed ngf specifically targets sensory sympathetic neurons peripheral nervous system well basal forebrain cholinergic neurons central nervous system also growing evidence support role ngf development differentiation maintenance human cerebellar connectivity context ngf high affinity receptor tachykinin receptor antagonist tkra ) these data imply ngf may neuroprotective effects cerebellar neurons hence might serve therapeutic candidate sca3 therefore clinical pilot study set forth examine efficacy ngf patients sca3 this study open label clinical trial assessing efficacy ngf patients sca3 conducted first affiliated hospital zhengzhou university november 2011 november 2012 this study approved ethics committee first affiliated hospital zhengzhou university registered chinese clinical trial registry www.chictr.org chictr onc-11001954 all study procedures accordance declaration helsinki recruited subjects provided written informed consents ataxia patients family history screened department neurology first affiliated hospital zhengzhou university referred genetic testing department genetic diagnosis patients fulfilled following inclusion criteria 1 ataxia patients family history checked diagnosed two independent doctors genotype sca3 confirmed genetic test 2 older 18 years 3 willing give informed consent recruited the exclusion criteria follows 1 allergy neurotrophin 2 concomitant severe systematic diseases psychiatric disorders 3 unable finish scale assessment rating ataxia sara score 4 refuse attend study 5 ataxias attributed secondary causes alcohol drug abuse toxic exposure all enrolled patients underwent standard neurological electrophysiological neuroimaging examinations sca3 subtype classified according clinical findings china used study extracted purified submandibular gland male mouse high homology amino acid sequence human ngf the mngf safely used series clinical studies approved china food drug administration mngf administered peripherally intramuscular injection dose 18 g daily 4 weeks consecutively it proven good reliability validity among chinese patients degenerative cerebellar the sara evaluates axial gait stance sitting speech appendicular finger chase nose finger test fast alternating hand movements fahms heel shin slide functions the sara sum score ranges 0 40 0 indicating ataxia 40 severe ataxia thus deterioration improvement disease severity respectively represented increase decrease sara score patient chinese version sara performed baseline 2 weeks midpoint 4 weeks endpoint treatment these sara evaluations videotaped reviewed independently two investigators attend original assessment random order the average score rated two evaluators denoted final sara score the primary outcome measure change sara score treatment compared baseline continuous variables expressed mean standard deviation sd categorical variables expressed frequencies proportions appropriate the observed changes sara score treatment baseline analyzed nonparametric wilcoxon signed rank test continuous variables expressed mean standard deviation sd categorical variables expressed frequencies proportions appropriate the observed changes sara score treatment baseline analyzed nonparametric wilcoxon signed rank test the mean age 39.14 7.81 years mean age onset 35.00 6.53 years mean disease duration 4.14 1.90 years mean cag repeats number 77.57 2.27 the mean sara score dropped 8.48 2.40 6.94 2.34 p 0.001 6.30 1.87 p 0.001 2 4 weeks treatment respectively table 2 significant decrease subsections sara scores also observed stance p 0.008 0.003 speech p 0.046 0.023 finger chase p 0.026 0.015 fahms 0.015 0.009 heel shin slide p 0.006 0.001 2 4 weeks therapy respectively the mean improvement total sara score 2.18 1.30 ranging 0 5.75 study scores sara subsections baseline 2 4 weeks ngf therapy comparisons midpoint therapy baseline comparisons endpoint therapy baseline sara scale assessment rating ataxia fahm fast alternating hand movements ngf nerve growth factor one previous study shown insulin like growth factor-1 one neurotrophic factors may effective reducing disease progression sca3 previous studies reported ngf improve cognitive decline patients alzheimer disease may also potential therapeutic roles neurodegenerative diseases postmortem histopathological study patients sca3 revealed considerable neuronal loss cerebellar purkinje cell layer four deep cerebellar nuclei ngf prevent neuronal death age related atrophy adult brain inhibiting apoptosis cholinergic neurons basal forebrain would reasonable postulate may also inhibit apoptosis cerebellum neurons expressing tyrosine kinase trka serve potential therapy sca3 such treatment effect observed early 2 weeks therapy sustained 4 weeks knowledge study first investigate efficacy ngf sca3 postmortem study shown ngf high affinity receptor tkra distributed neurons human cerebellum cortex deep nuclei throughout life these findings support involvement ngf development differentiation maintenance cerebellar connectivity although blood brain barrier bbb low permeability large proteins autoradiography studies suggested ed blood borne ngf subunit -ngf cross bbb mice arrive brain parenchyma direct permeation second therapeutic effect ngf might also mediated via proprioceptive sensation system it reported 87% sca3 patients somatosensory evoked potential abnormalities especially lower limbs due degenerative lesions dorsal column spinal cord hence ngf therapy may improve stance heel knee shin slide due improved proprioception these two proposed mechanisms may explain observed improvement therapy substantiate use ngf treat patient sca3 first open label study observed therapeutic efficacy might contributed placebo effects however one randomized double blind placebo controlled study evaluate efficacy varenicline sca3 patients mean improvements sara score therapeutic group placebo group 1.97 0.86 respectively the sara score improvement 2.18 current study unlikely accounted placebo effect alone furthermore sara reliable valid scale linearly assess ataxia symptoms changes sara scores exceeding 1.1 points considered clinically relevant nevertheless pilot data suggest ngf may promising treatment patients sca3 large scale randomized double blind placebo controlled trial would worthwhile evaluate efficacy tolerability ngf sca3 patients

background : spinocerebellar ataxia type 3 ( sca3 ) is the most common subtype of sca worldwide , and runs a slowly progressive and unremitting disease course . there is currently no curable treatment available . growing evidence has suggested that nerve growth factor ( ngf ) may have therapeutic effects in neurodegenerative diseases , and possibly also in sca3 . the objective of this study was to test the efficacy of ngf in sca3 patients.methods:we performed an open - label prospective study in genetically confirmed adult ( > 18 years old ) sca3 patients . ngf was administered by intramuscular injection ( 18 g once daily ) for 28 days consecutively . all the patients were evaluated at baseline and 2 and 4 weeks after treatment using the chinese version of the scale for assessment and rating of ataxia ( sara).results : twenty - one sca3 patients ( 10 men and 11 women , mean age 39.14 7.81 years , mean disease duration 4.14 1.90 years , mean cag repeats number 77.57 2.27 ) were enrolled . after 28 days of ngf treatment , the mean total sara score decreased significantly from a baseline of 8.48 2.40 to 6.30 1.87 ( p < 0.001 ) . subsections sara scores also showed significant improvements in stance ( p = 0.003 ) , speech ( p = 0.023 ) , finger chase ( p = 0.015 ) , fast alternating hand movements ( p = 0.009 ) , and heel - shin slide ( p = 0.001).conclusions : our preliminary data suggest that ngf may be effective in treating patients with sca3 .

nanopores powerful research tools investigate structural dynamic properties single biomolecules nanopores inner diameters nanometers following size exclusion principle individual dna protein molecules fit pass pore importantly passing molecule blocks pore leading transient change current parameters current blockade duration amplitude give information length size molecule one widely published scientifically attractive subjects sensing dna nanopores ultimate goal studies develop inexpensive method sequence dna dekker 2007 branton et al 2010 large array experiments also shed light biophysics dna rna translocation biological pores kasianowicz et al ; healy 2007 vercoutere akeson 2002 deamer branton 2002 marziali akeson 2001 well inorganic pores reviewed e.g. howorka siwy 2009 healy et al 2007 dekker 2007 examined aspects include frequency strands thread pore henrickson et al 2000 meller branton 2002 nakane et al 2008 ii orientation strands butler et al 2006 wang et al 2004 mathe et al 2005 wanunu et al 2008 wiggin et al 2008 li et al 2003 chen et al 2004 storm et al 2005 fologea et al 2007 steinbock et al 2010 iii speed dna transport meller et al 2001 wanunu et al 2008 iv influence transmembrane potential aksimentiev schulten 2005 mathe et al 2005 kathawalla et al 1989 heng et al 2005 keyser et al 2006 v pore geometry howorka bayley 2002 well vi interaction pore walls wiggin et al 2008 wanunu et al 2008 covalently attaching dna pore wall attractive variation single molecule research previous studies aimed dna sensing one multiple single stranded dna ssdna molecules end tethered pore wall protein howorka et al complementary mismatched dna molecules showed dna duplexes free tethered strands form inside nanopore as perfect mismatched duplexes respectively different lifetimes dna modified nanopores could used biosensor elements distinguish nucleic acids single point mutations howorka et al the covalent attachment dna strands also exploited control electronic properties pore general artificial pores exhibit engineered properties voltage gating ion selectivity attractive model systems biological voltage gated ion channels hille 2001 ( 2004 first demonstrate dna modified nanopores function ionic switches a single gold nanotube carrying thiol terminated dna strands shown preferentially transport cations one direction hindering transport the ion current rectification explained assuming dna molecules deflect external electric field cause voltage dependent pore opening the attached dna strands localized single subnanoscale position covered entire pore additionally length fully extended dna molecules smaller pore diameter hence possible completely block current e.g. 59-nm diameter gold tube modified 30-mer dna aim achieving greater pore blockade ion current modulations study attempts tune ionic pore properties restricting dna immobilization small nanoscale region single conically shaped nanopores opening 8 12 nm selectively modified narrow section ssdna oligonucleotides fig 1 length 30 nucleotides compared current voltage curves single nanopores dna modification different kcl concentrations 1 10 mm the experimental results indicate attached dna either reduced blocked ionic current flow pore dependent ionic strength also influenced conformation dna strands odijk 1977 skolnick fixman 1977 kaiser rant 2010 1scheme surface charge pattern obtained process one sided modification single conically shaped nanopore point tethered dna molecules we estimate position junction dna modified zone zone carboxyl groups several tens nanometers small opening pore vlassiouk siwy 2007 carboxyl groups formed process pore preparation scheme surface charge pattern obtained process one sided modification single conically shaped nanopore point tethered dna molecules we estimate position junction dna modified zone zone carboxyl groups several tens nanometers small opening pore vlassiouk siwy 2007 carboxyl groups formed process pore preparation addition dna modified conical nanopores presented function rectifiers rectifying systems preferential direction mass flow cases entirely stop flow opposite direction forming diode rectifiers diodes based surface charge patterns pore walls previously created previous embodiments able switch transport ions charged species bassignana reiss 1983 mafe ramirez 1997 daiguji et al 2005 vlassiouk siwy 2007 nguyen et al 2010 cheng guo 2009 example ionic bipolar diode built based nanopores contained junction positive negative charges pore walls vlassiouk siwy 2007 cheng guo 2009 a unipolar diode formed junction positive negative surface charges neutral zone karnik et al by contrast dna modified nanopores presented feature opening diameter tuned operation conditions e.g. kcl concentration voltage the rationally engineered pores thus applied tune switch transport ionic neutral species single conically shaped nanopores prepared track etching technique according published procedure fleischer et al briefly 12-m thick films polyethylene terephthalate pet irradiated single heavy ions e.g. au u kinetic energy ~2 gev irradiation performed linear accelerator unilac institute heavy ions research gsi darmstadt germany spohr 1983 tracking foils etched one side 9 naoh form conically shaped nanopores apel et al the side membrane protected etching acidic stopping solution the preparation performed conductivity cell pore opening could observed electrochemically once etching current ~200 pa recorded membrane washed stopping medium kcl solution the transport properties single pet nanopores investigated kcl solutions buffered ph 8 tris buffer an approximate diameter small opening conical pore determined using conductance measurements described previously apel et al sides single nanopore membrane placed contact 1 kcl ag agcl electrodes immersed solution current voltage curve recorded voltages 100 100 mv voltage regime the nanopores produce linear current voltage curve whose slope provides ionic resistance rp nanopore the tip diameter related rp via \documentclass[12pt]{minimal \usepackage{amsmath \usepackage{wasysym \usepackage{amsfonts \usepackage{amssymb \usepackage{amsbsy \usepackage{mathrsfs \usepackage{upgreek \setlength{\oddsidemargin}{-69pt \begin{document}$$ r_{p \frac{4\rho l}{\pi dd \end{document l membrane thickness specific ionic resistance electrolyte base big opening cone diameter the diameter determined called bulk etch rate polymer material pet etching conditions 20c 9 naoh bulk etch rate equal 2.13 nm min apel et al 2001 the value determined formula 2 2.13 nm time etching minutes validity relation confirmed scanning electron microscopy studies wharton et al pore walls surface track etched pet films contain carboxyl groups density ~1 group nm wolf reber 2002 the carboxyl groups attachment points covalently couple amino modified dna using procedure previously applied surface modification purposes procedure carboxyl groups activated 1-ethyl-3-(3-dimethylaminopropyl carbodiimide hydrochloride edc coupled amine forming amide vlassiouk siwy 2007 a dna oligonucleotide 5-terminal amino c12 spacer sequence 5-cgc gag aagtta cat gac ctg tag acg atc-3 purchased integrated dna technologies coralville ia the received amount 1,267 nm dissolved 500 l 0.1 mes buffer ph 5.5 amidation protocol dna edc solution placed contact small opening pore large opening pore contact 0.1 2-(n morpholino)ethanesulfonic acid mes solution ph 5.5 24-h incubation placing modifying agent one side membrane is known create nonhomogeneous distribution chemicals along pore axis vlassiouk siwy 2007 due conical pore shape the concentrations edc dna high first tens nanometers narrow tip decayed hyperbolically along pore axis consequently dna attachment expected occur region close small opening reagents concentration sufficiently high it expected nanopore system surface chemistry schematically shown fig 1 four dna modified nanopores opening diameters 4 12 nm studied the ionic properties nanopores studied conductivity cell etching performed two ag agcl electrodes used apply transmembrane potential measure ion current we used two electrode setup electrode placed narrow end conical nanopore grounded electrode next big pore opening used apply given potential difference respect ground electrode the voltage swept 3 3 v 1 kcl 4 4 v lower concentrations voltage step 100 mv the presented current voltage curves averages typically three sweeps unless otherwise stated figs the current variations averaged signals typically exceed 8% current variations strongest negative voltages 2 v single conically shaped nanopores prepared track etching technique according published procedure fleischer et al briefly 12-m thick films polyethylene terephthalate pet irradiated single heavy ions e.g. au u kinetic energy ~2 gev irradiation performed linear accelerator unilac institute heavy ions research gsi darmstadt germany spohr 1983 tracking foils etched one side 9 naoh form conically shaped nanopores apel et al the side membrane protected etching acidic stopping solution the preparation performed conductivity cell pore opening could observed electrochemically once etching current ~200 pa recorded membrane washed stopping medium kcl solution the transport properties single pet nanopores investigated kcl solutions buffered ph 8 tris buffer an approximate diameter small opening conical pore determined using conductance measurements described previously apel et al sides single nanopore membrane placed contact 1 kcl ag agcl electrodes immersed solution current voltage curve recorded voltages 100 100 mv voltage regime the nanopores produce linear current voltage curve whose slope provides ionic resistance rp nanopore the tip diameter related rp via \documentclass[12pt]{minimal \usepackage{amsmath \usepackage{wasysym \usepackage{amsfonts \usepackage{amssymb \usepackage{amsbsy \usepackage{mathrsfs \usepackage{upgreek \setlength{\oddsidemargin}{-69pt \begin{document}$$ r_{p \frac{4\rho l}{\pi dd \end{document l membrane thickness specific ionic resistance electrolyte base big opening cone diameter the diameter determined called bulk etch rate polymer material pet etching conditions 20c 9 naoh bulk etch rate equal 2.13 nm min apel et al 2001 the value determined formula 2 2.13 nm time etching minutes validity relation confirmed scanning electron microscopy studies wharton et al pore walls surface track etched pet films contain carboxyl groups density ~1 group nm wolf reber 2002 the carboxyl groups attachment points covalently couple amino modified dna using procedure previously applied surface modification purposes procedure carboxyl groups activated 1-ethyl-3-(3-dimethylaminopropyl carbodiimide hydrochloride edc coupled amine forming amide vlassiouk siwy 2007 a dna oligonucleotide 5-terminal amino c12 spacer sequence 5-cgc gag aagtta cat gac ctg tag acg atc-3 purchased integrated dna technologies coralville ia the received amount 1,267 nm dissolved 500 l 0.1 mes buffer ph 5.5 amidation protocol dna edc solution placed contact small opening pore large opening pore contact 0.1 2-(n morpholino)ethanesulfonic acid mes solution ph 5.5 24-h incubation placing modifying agent one side membrane is known create nonhomogeneous distribution chemicals along pore axis vlassiouk siwy 2007 due conical pore shape the concentrations edc dna high first tens nanometers narrow tip decayed hyperbolically along pore axis consequently dna attachment expected occur region close small opening reagents concentration sufficiently high it expected nanopore system surface chemistry schematically shown fig 1 four dna modified nanopores opening diameters 4 12 nm studied the ionic properties nanopores studied conductivity cell etching performed two ag agcl electrodes used apply transmembrane potential measure ion current we used two electrode setup electrode placed narrow end conical nanopore grounded electrode next big pore opening used apply given potential difference respect ground electrode the voltage swept 3 3 v 1 kcl 4 4 v lower concentrations voltage step 100 mv the presented current voltage curves averages typically three sweeps unless otherwise stated figs the current variations averaged signals typically exceed 8% current variations strongest negative voltages 2 v figure 2a presents set current voltage curves single conically shaped nanopore small big openings 8 400 nm respectively a positive voltage electrode configuration corresponds positively biased electrode placed big opening conical nanopore vice versa negative voltage similar previous reports conical nanopore rectified ion current due breaking symmetry electric potential inside pore siwy fulinski 2002 hanggi marchesoni 2009 cervera et al 2005 in particular broken symmetry affected interactions k ions negatively charged carboxyl groups pore walls the rectification quantified calculating called rectification degree ratio currents voltages absolute value opposite polarity e.g. i(3 v)/i(+3 v negatively charged nanopores transference numbers found reversal potential hille 2001 least 0.8 indicates ~80% current carried potassium ions cervera et al the larger currents negative voltages correspond cations moving small opening large opening pore the experimentally found ion current rectification selectivity properties conically shaped nanopores modeled using poisson nernst planck pnp equations cervera et al 2005 2006 2007 white bund 2008 well monte carlo et al figure 2b presents average ionic concentrations along pore axis 1 1 v 0.1 kcl obtained bynumerically solving pnp equations described vlassiouk et al 2009 negative voltages ionic concentrations ions potassium chloride are significantly higher corresponding positive voltage thereby validating negative voltage corresponds high conductance state given concentration cations higher anions negative positive potentials plot fig the modeling furthermore revealed case conically shaped nanopores quantitative estimation cation selectivity based reversal potential measurements holds true low voltages high negative potentials 2b indicated increase concentration potassium chloride ions within pore cervera et al 2006 2a current voltage curves single conically shaped nanopore openings ~8 400 nm recorded ph 8 three kcl concentrations indicated insets show position electrical ground external voltage relative two pore openings b numerical solutions pnp equation conically shaped nanopore surface charge density 0.5 e nm showing average ionic concentrations along pore axis opening diameters modeled nanopore set 5 500 nm current voltage curves single conically shaped nanopore openings ~8 400 nm recorded ph 8 three kcl concentrations indicated insets show position electrical ground external voltage relative two pore openings b numerical solutions pnp equation conically shaped nanopore surface charge density 0.5 e nm showing average ionic concentrations along pore axis 2009 opening diameters modeled nanopore set 5 500 nm well known pores rectify walls excess surface charge pore diameter comparable thickness electrical double layer siwy fulinski 2002 cervera et al 2006 consequence rectification degree conically shaped nanopores 1 m kcl lower 0.1 10 mm kcl 2a 2.7 |15.4| na 5.68 na 1 4.7 |8.32| na 1.75 na 0.1 kcl 4.9 |4.08| na 0.828 na 10 mm kcl the small difference rectification degrees 0.1 10 mm kcl agreement predictions based pnp equations point nonlinear dependence rectification degree kcl concentration cervera et al the modeling also showed optimal ratio pore diameter screening length produces highest rectification degree 6-nm diameter conical pore concentration range pore predicted rectify ~0.12 0.40 kcl cervera et al the optimal concentration range shifted diluted solutions 8-nm pore studied 10 mm 0.1 kcl pore rectified almost equally well a different behavior observed modifying tip nanopore 30-nucleotide long ssdna figure 3 compares current voltage curves pore dna modification 1 0.1 10 mm kcl for concentrations dna modification caused significant decrease current suggests long dna molecules physically occluded pore diminished effective opening diameter.fig 2 modification 30-nucleotide ssdna 1 kcl b 0.1 kcl c 10 mm kcl two sweeps 4 4 v shown dna modified nanopore red curves present three consecutive reverse sweeps voltages 4 4 v showing big variation sweeps hysteresis compared forward sweeps 4 4 v shown gray 1 0.1 m kcl differences currents recorded forward reverse voltage sweeps observed current voltage curves nanopore studied fig 2 modification 30-nucleotide ssdna 1 kcl b 0.1 kcl c 10 mm kcl two sweeps 4 4 v shown dna modified nanopore red curves present three consecutive reverse sweeps voltages 4 4 v showing big variation sweeps hysteresis compared forward sweeps 4 4 v shown gray 1 0.1 m kcl differences currents recorded forward reverse voltage sweeps observed experimental results suggest occlusion pore dna becomes pronounced lowering ionic strength 1 m kcl attachment dna caused decrease ~2.5-fold ion current compared non modified pore fig 3a 10 mm kcl dna modified nanopore exhibited least five times lower current fig any explanation observed current change consider two effects initially used describe experiments performed non attached pore translocating dna strands smeets et al firstly dna molecule excludes certain volume pore fewer ions available transport secondly dna molecules heavily charged thus presence brings counter ions pore lumen insertion dna molecules pore therefore cause decrease increase current depending whether number excluded ions larger smaller number additionally introduced dna counterions both effects observed systematic experiments threading single dna molecules unmodified nanopores smeets et al 2006 dependence concentration background electrolyte pore threaded dna molecules caused positive negative spike current reported cases lowering the kcl concentration background electrolyte caused dna counter ions become dominant our observation decrease kcl concentration leads increase pore blockage suggests simple explanation based volume exclusion electrostatic effects insufficient it likely transmembrane current additionally influenced changes conformation dna molecules this effect recently observed dna molecules attached gold surfaces kaiser rant 2010 experiments the background electrolyte concentration caused dna molecules extended concentrations ~1 mm molecules became entirely stretched the length dna strand determined applying negative potential gold surface determining quenching fluorophore attached dna end these results explained considering dependence mechanical rigidity ssdna ionic strength environment theories odijk 1977 well skolnick fixman 1977 distinguish bare l0 electrostatic le contributions total persistence length charged molecule lp \documentclass[12pt]{minimal \usepackage{amsmath \usepackage{wasysym \usepackage{amsfonts \usepackage{amssymb \usepackage{amsbsy \usepackage{mathrsfs \usepackage{upgreek \setlength{\oddsidemargin}{-69pt \begin{document}$$ l_{p l_{0 l_{e \end{document}. bare persistence length ssdna known 23 nm tinland et al 1997 value le ionic strength dependent calculated \documentclass[12pt]{minimal \usepackage{amsmath \usepackage{wasysym \usepackage{amsfonts \usepackage{amssymb \usepackage{amsbsy \usepackage{mathrsfs \usepackage{upgreek \setlength{\oddsidemargin}{-69pt \begin{document}$$ l_{e \frac{{l_{b 4}}\lambda^{2 l_{d}^{2 \end{document \documentclass[12pt]{minimal \usepackage{amsmath \usepackage{wasysym \usepackage{amsfonts \usepackage{amssymb \usepackage{amsbsy \usepackage{mathrsfs \usepackage{upgreek \setlength{\oddsidemargin}{-69pt \begin{document}$$ l_{b \frac{{e^{2 4\pi \varepsilon \varepsilon_{0 k_{b \end{document bjerum length linear density polymer ld stands screening length given debye hueckel theory the parameters kb usual meaning boltzmann constant temperature respectively length le becomes important lower concentrations e.g. 10 mm kcl equals ~10 nm applied dna modified pore high length obviously increases volume dna strands could thereby help explain high level occlusion observed nanopore measures 8 nm diameter fig it also important mention lowest studied kcl concentration 10 mm current voltage curves exhibited strong hysteresis especially negative voltages fig when voltage sweep started negative voltages currents typically significantly lower case sweep started positive voltages we think hysteresis caused voltage induced movement attached dna strands well voltage dependent ionic concentrations discussed figure 4 summarizes model accounts observed hysteresis high currents sweep positive negative transmembrane potentials when positively biased electrode placed wide opening pore dna molecules expected deflected away narrow opening toward wide opening present significant steric hindrance strands fig 4 region due conical shape pore voltage polarity constitutes lower conductance state device reducing positive voltage switching negative values forces dna strands deflect toward small opening pore fig 4 region b it recalled negative voltages concentrations potassium chloride ions region close small pore opening increase least several times compared bulk concentration fig this region elevated ionic concentration rather wide reaches several hundreds nanometers away narrow opening consequently dna strands modified pore contact high concentrations potassium chloride ions turn likely causes dna assume compact form fig 4 region b)in line results kaiser rant 2010 at moderate negative voltages currents pore large leading high rectification degrees negative voltages threshold value ~2 3d 4 dna molecules might become extended due electric field heng et al 2005 randall et al 2006 nanopore blocked fig 4 region c).fig 3d 8-nm diameter nanopore modified dna recorded voltage changed 4 4 v. insets schematically indicate possible configurations attached dna molecules function applied voltage two current voltage curves shown fig 3d 8-nm diameter nanopore modified dna recorded voltage changed 4 4 v. insets schematically indicate possible configurations attached dna molecules function applied voltage contrast voltage swept negative positive values dna molecules deflect small opening toward large opening pore we think process occurs significantly severe steric hindrance lowering negative applied voltage decreases ionic concentrations pore causing dna maintain elongated form induced high voltage thereby leading greater pore blockade order support general hypothesis conformation dependent occlusion nanopores dna molecules we expected case dna would lead less extensive pore blockade based geometric grounds figure 5 shows current voltage curves 12-nm pore 0.1 kcl ph 8 dna attachment 3 wider structure characterized less extensive blockade also higher negative currents dna modification larger current dna modification suggests according smeets et al ( 2006 number dna counter ions exceeds number ions excluded nanopore most likely dna attachment increases local surface charge density consequently enhances ionic concentrations narrow opening.fig 5a current voltage curves modification 30-mer ssdna single conically shaped nanopore narrow opening 12 nm b current voltage curves 12-nm dna modified nanopore 10 mm kcl ph 8 forward bias gray squares reverse bias red squares current voltage curves modification 30-mer ssdna single conically shaped nanopore narrow opening 12 nm b current voltage curves 12-nm dna modified nanopore 10 mm kcl ph 8 forward bias gray squares reverse bias red squares wider nanopore also exhibited ion current rectification degree 0.1 kcl 4 v equal almost 17 three times higher rectification degree 8-nm dna modified pore fig this observation first puzzling us one would expect higher rectification degree narrower pore one possible explanation dna conformational change makes pore structure less asymmetric less rectifying another explanation takes account dependence rectification degree conical nanopore ionic strength already discussed there optimal ratio pore diameter screening length maximal rectification degree cervera et al 2006 concentrations lower optimum given pore diameter with dna nanopore effectively smaller pore might outside optimal rectifying regime as expected wider conical nanopore 10 mm kcl hysteresis ion currents recorded forward reverse voltage sweeps much less pronounced compared 8-nm nanopore see figs we prepared ionic rectifying system based single conically shaped nanopores whose small opening modified ssdna in contrast previously designed bipolar unipolar diodes vlassiouk siwy 2007 karnik et al 2007 containing stationary surface charges attached dna molecules flexible assume different configurations depending operation conditions kcl concentration voltage our experimental results provide evidence level nanopore blockage likely persistence length tethered dna increase lower kcl concentration due salt dependent dna volume nanopore changes effective diameter thus applicable building systems control transport charged neutral species future research systematically study transport properties dna modified pores function dna length we expect able tune rectification properties pores well effective pore diameter function external voltage we also try identify particular ratio dna length pore diameter dna modified nanopores exhibit spontaneous openings closings pore similar biological voltage gated channels

single nanopores attract a great deal of scientific interest as a basis for biosensors and as a system to study the interactions and behavior of molecules in a confined volume . tuning the geometry and surface chemistry of nanopores helps create devices that control transport of ions and molecules in solution . here , we present single conically shaped nanopores whose narrow opening of 8 or 12 nm is modified with single - stranded dna molecules . we find that the dna occludes the narrow opening of nanopores and that the blockade extent decreases with the ionic strength of the background electrolyte . the results are explained by the ionic strength dependence of the persistence length of dna . at low kcl concentrations ( 10 mm ) the molecules assume an extended and rigid conformation , thereby blocking the pore lumen and reducing the flow of ionic current to a greater extent than compacted dna at high salt concentrations . attaching flexible polymers to the pore walls hence creates a system with tunable opening diameters in order to regulate transport of both neutral and charged species .

skin largest sensory organ body densely equipped highly specialized sensory nerve endings capable sensing wide range sensory stimuli light touch mechanical pressure temperature itch pain these cutaneous sensory afferents distinguished many morphological anatomical electrophysiological molecular criteria liu 2011 owens lumpkin 2014 for example based cell body size degree myelination axonal conduction velocity cutaneous fibers split c fibers abraira ginty 2013 bessou perl 1969 li et al 2011 smith lewin 2009 zimmermann et al 2009 they split free nerve endings found glabrous hairy skin embedded keratinocytes epidermis particular population c unmyelinated fibers found exclusively hairy skin form longitudinal lanceolate endings around hair follicles delfini et al 2013 li et al 2011 terminate large arborizations similar c fiber tactile afferent receptive fields liu et al 2007 cutaneous free nerve endings tuned respond itch inducing compounds pain evoking thermal mechanical chemical stimuli zimmermann et al 2009 mrgprd free nerve endings belong subset non peptidergic nociceptors convey noxious mechanical -alanin induced itch stimuli cavanaugh et al 2009 liu et al 2012 whereas c low threshold mechanoreceptors c ltmrs proposed contribute light touch normal conditions bessou et al 1971 douglas ritchie 1957 johansson et al 1988 lken et al 2009 maruhashi et al 1952 olausson et al 2002 seal et al 2009 zotterman 1939 touch hypersensitivity injury delfini et al 2013 liljencrantz et al 2013 seal et al 2009 our knowledge molecular contents contribute functional specialization diametrically different subpopulations cutaneous afferents still infancy recent study gaillard et al 2014 ) identified novel g inhibitory interacting protein ginip marks two distinct subsets non peptidergic neurons cutaneous free nerve endings mrgprd neurons dong et al 2001 v glut3- th- tafa4-expressing c ltmrs delfini et al 2013 li et al 2011 seal et al 2009 took advantage ginip mouse model combination isolectin b4 ib4 cell surface staining fluorescence activated cell sorting facs we succeeded purify three distinct populations drg neurons subjected respective total rna contents rna deep sequencing technology rna seq as expected ginip ib4 neurons showed remarkable enrichment mrgprd transcripts ginip ib4 neurons revealed striking enrichment three known markers c ltmrs tafa4 th vglut3 deeper comparison rna seq data revealed distinct transcriptional signatures mrgprd expressing neurons c ltmrs confirmed situ hybridization analysis 100 genes most importantly addition providing transcriptional signatures two categories primary sensory neurons study expands molecular characterization c ltmrs suggests particular subset primary sensory neurons shares many molecular features ltmrs functional readout used electrophysiological recording unravel specific exclusive functional expression low voltage gated ca channel cav3.3 c ltmrs likely plays key role shaping functional specialization in recent study generated mouse model expresses fluorescent protein cherry ginip locus gaillard et al 2014 double labeling experiments using anti ginip antibody combination ib4 staining showed drg neurons split four distinct categories ginip ib4 ginip ib4 ginip ib4 ginip ib4 neurons the ginip ib4 double positive dp population corresponds cutaneous free nerve endings mrgprd neurons figures 1a 1b ginip ib4 population corresponds tafa4-expressing c ltmrs figures 1a 1b ginip ib4 population contains 20% remaining ib4 neurons figure 1a ginip ib4 double negative dn neurons represents heterogeneous population neurons composed peptidergic nociceptors subset ret neurons trkb trkc neurons figure 1a as ginip mouse model allows high fidelity expression cherry ginip neurons sought combine live cherry fluorescence ib4 cell surface staining facs purify four categories drg cells figures 1c 1d neurons cell body size 70 eliminated filtering suspension axonal debris removed using percoll gradient negative gating used exclude dead dying cells incorporated sytox blue dye cells autofluorescent v500 channel figures 1c s1 next gated cherry positive cherry negative cells subdivided ib4 ib4 subsets figures 1c s1 facs sorted ib4 ib4 cherry positive neurons gave rise expected proportions cells two thirds ib4 corresponding ginip ib4 remaining one third ib4 corresponding ginip ib4 figure 1d the cherry ib4 subset excluded transcriptional analysis highly contaminated cd31 pecam)-expressing endothelial cells consistent ib4 binding cell type data shown remaining three sorted samples hereafter called dp ginip ib4 c ltmrs ginip ib4 dn ginip ib4 first confirmed rt pcr accurate expression ginip cherry transcripts figure s2b extended purity evaluation analyzing expression mrgprd tafa4 trka trpv1 sorted sample figure s2c expected mrgprd transcripts highly detected dp sample low two others whereas tafa4 expression high c ltmrs trka trpv1 transcripts enriched dn sample consistent enrichment peptidergic nociceptors dn fraction figure s2c high quality rna figure s2a extracted three purified populations neurons respective transcriptional profiles generated using rna seq after one round rna amplification libraries built biological duplicate sequenced 100-bp paired end cycles using illumina hiseq2000 system sbs technology image analysis base calling performed using hiseq control software rta component illumina this approach yielded 40 50 millions reads submitted sequence mapping mouse reference genome mm10 casava 1.8.2 software after removal contaminants 28s 18s 5s rrna mitochondrial chromosomes phix illumina controls illumina adaptors reads mapping multiple splicing positions reads match obtained 32 42 millions reads used generation raw data file table s1 among first ranked genes raw data file found genes known related structural organization neurons tuba1a prph sncg mtap1b table s2 all genes exhibited strong expression three sorted samples generated least 50,000 reads table s2 accordingly highly broadly expressed drg neurons allen brain atlas database interestingly genes associated cellular stress hsp90ab1 fos also highly expressed samples likely due cell dissociation facs process table s2 most importantly quick analysis reads per kilobase per million reads rpkm generated genes expected enriched sorted subpopulation revealed high enrichment mrgprd p2rx3 dp subset th tafa4 c ltmrs cgrp sp dn neurons figure 1e providing first proof concept successful outcome experimental paradigm identify genes preferentially enriched one sample carried pairwise comparative analysis 2-fold enrichment p 0.01 libraries normalization trimmed mean values method tmm provided edger statistical package then subset extracted list genes upregulated one subset compared two others we found 486 genes enriched dp subset 549 c ltmrs 2,916 dn subset tables s3 s4 s5 these genes encode variety molecules including ion channels receptors signaling molecules previously uncharacterized genes neuronal subsets included facs strategy and because facs acutely dissociated neurons may induce cellular stress impacts gene expression follow strategy could employed validate specificity data sets situ hybridization ish approach fixed adult drg sections knowing might miss genes expressed subsets sorted neuronal subpopulations assumed 1,000 number reads transcripts detection ish would difficult monitor therefore carried second pairwise comparative analysis considering genes values 1,000 reads figure 2a result obtained 156 transcripts preferentially expressed dp subset 184 c ltmrs 784 dn population figure 2b tables s6 s7 s8 we monitored expression 48 dp- 68 c ltmrs- 13 dn enriched genes using ish followed ginip immunostaining ib4 labeling dp c ltmrs candidate genes ginip trka immunolabeling dn candidate genes these triple labeling experiments provided unambiguous accurate expression pattern tested gene the 13 dn enriched tested genes revealed two different expression patterns genes mainly expressed neurons genes exclusively expressed non neuronal cells harbor satellite and/or glial cell morphology figure s3 agreement rna seq data genes expressed neurons excluded dp neurons c ltmrs furthermore found 6/13 genes expressed trka peptidergic neurons others 2/13 excluded neuronal population revealing heterogeneity dn subset figure s3 aim study consisted deciphering transcriptional signatures confer functional specializations mrgprd expressing cutaneous free nerve ending c ltmrs analyzed expression patterns 48 dp enriched genes 68 c ltmtrs enriched genes in line rna seq data sets 48 dp enriched probes highly expressed dp neurons virtually excluded c ltmrs http://www.ibdm.univ-mrs.fr/equipes/reynders/dp-ishlibrary.pptx however vast majority dp enriched genes displayed additional expression pattern outside dp neurons 12 genes showed expression ginip ib4 neurons 17 genes showed lower expression discrete subsets small 3/17 medium large size neurons 14/17 19 genes expressed broader neuronal subsets addition intense expression dp neurons figures 2c2e dp ish library the strategy applied c ltmrs enriched genes showed 68 tested genes expressed c ltmrs massively excluded dp neurons http://www.ibdm.univ-mrs.fr/equipes/reynders/cltmrs-ishlibrary.pptx one striking feature c ltmrs enriched genes vast majority also expressed medium large size drg neurons whereas two expressed small neurons indeed 68 tested genes six specifically expressed c ltmrs marked subset ginip neurons bind ib4 figures 3a3f quantitative analyses showed ceacam10 expressed 71.2% 6.3% c ltmrs wfdc2 73.4% 1.7% cacna1i 89.2% 5.9% c1ql4 70% 1.8% fbp2 19.9% 4.7% bok 62.7% 6.4% c ltmrs figure 3 g highlighting previously suggested molecular heterogeneity particular class neurons delfini et al the remaining 60 genes could split three categories 18 genes exhibited high expression c ltmrs also marked discrete subset large diameter neurons 23 genes showed equal levels expression c ltmrs large diameter neurons 19 genes exhibited high enrichment larger diameter neurons discrete expression c ltmrs figures 4a4c cltmrs ishlibrary because 88% c ltmrs enriched genes displayed additional expression preferentially large diameter neurons sought unravel molecular identity neurons to performed double ish using nf200 antisense probe combination selected c ltmrs enriched probes followed trka immunolabeling this triple staining experiment showed selected probes massively excluded trka peptidergic neurons expressed subsets nf200 neurons figure s4 knowing nf200 hallmark proprioceptors ltmrs bourane et al 2009 li et al 2011 luo et al 2009 wende et al 2012 data suggest c ltmrs share molecular features ltmrs mrgprd expressing neurons c ltmrs exhibit fundamental differences anatomical functional levels however little known molecular contents instruct respective functional specializations in line pooled dp c ltmrs enriched genes according biological functions searching statistically represented associations gene ontology go terms using gorillla database figures 5 6 s5 this analysis yielded respectively 20 33 functional annotations dp cltmrs dp neurons exhibited high enrichment g protein coupled receptors well transcripts encoding signaling molecules gna14 gna1 gnaq plcb3 prkca prkcd prkcq figures 5a s5a table s9 whereas c ltmrs exhibited striking enrichment transcripts associated voltage gated ion channel activity figures 6a s5b table s10 detailed analysis genes associated g protein receptor binding signal transducer activity led identification two transcripts encoding g protein coupled receptors namely angiotensin receptor type 1a agtr1a prostaglandin receptor d2 ptgdr figure 5a our ish experiments showed agtr1a expressed dp neurons c ltmrs subsets drg neurons figure 5b whereas ptgdr found higher intensity dp neurons compared c ltmrs large size neurons figure 5c in addition gpcrs go analysis revealed striking enrichment transcripts encoding signaling molecules dp neurons including gna14 gna1 gnaq plcb3 prkca prkcd prkcq table s9 our situ screen showed transcripts highly expressed dp neurons virtually absent c ltmrs figures 5d5 g the restricted enrichment number signaling molecules likely endows dp neurons molecular machinery required sense transduce injury induced pain hypersensitivity regarding c ltmrs one striking observations respect go analysis high enrichment transcripts associated voltage gated ion channel activity other physiologically pertinent functional annotations encompass substrate specific channel ion transmembrane transporter g protein coupled receptor activities figures 6a s5b voltage gated ion channels included potassium channels kcnq2 kcnj12 kcnv1 kcna2 kcna4 sodium channels associated beta subunits scn1a scn1b scn3b scn4b scn7a scn8a low voltage activated calcium channels cacna1h cacna1i figures 6a s5b table s10 ish screen showed cacna1i specifically expressed c ltmrs figure 3c kcnj12 cacna1h expressed c ltmrs well large diameter neurons excluded dp neurons figures 6b 6c scn4b scn8a exhibited low expression c ltmrs high expression myelinated large neurons detectable signal dp neurons c ltmrs ish library slides 67 68 the combinatorial enrichment channels c ltmrs likely explain tonic repetitive firing properties particular class neurons delfini et al interestingly substrate specific channels ion transmembrane transporters encompassed anoctamin 6 ano6 solute carrier 17a2 slc17a2 ano6 part outwardly rectifying chloride channels martins et al 2011 slc12a7 encodes k cl co transporter kcc4 boettger et al 2002 suggesting channels could involved membrane excitability in situ analysis ano6 slc12a7 showed transcripts expressed c ltmrs dp neurons exhibited expression large drg neurons figures 6d 6e gpcr activity related transcripts also represented go analysis c ltmrs data set including cholecystokinin receptor cckar purinergic receptor y1 p2ry1 our ish data showed cckar p2ry1 transcripts highly enriched c ltmrs totally excluded dp neurons displayed expression large diameter neurons figures 6f 6 g s4b together data provide functional description sets genes differentially expressed dp versus c ltmrs the differential expression clusters genes likely contributes differences signal transduction intrinsic membrane excitability firing properties c ltmrs mrgprd neurons our data also provide predictive basis deciphering mechanisms specific external stimuli downstream associated signaling pathways fine tune activity two neuronal subsets our rna seq data revealed high enrichment cacna1h cav3.2 cacna1i cav3.3 c ltmrs our ish showed transcripts encoding channels expressed c ltmrs cav3.3 exclusively expressed neuronal subset figures 3e 7a explore whether channels functional c ltmrs used whole cell patch clamp recordings primary culture heterozygous cherry expressing neurons presence alexa 488 ib4 live staining distinguish c ltmrs dp neurons ionic conditions designed isolate calcium currents current voltage relationships showed inward low voltage activated lva high voltage activated hva currents recorded c ltmrs soma confirming systematic presence type currents neurons figures 7b 7c in sharp contrast dp neurons express lva current agreement absence cav3 transcripts population figures 7b 7c test whether cav3.2 cav3.3 functional c lmtrs took advantage cav3.2 high sensitivity nickel ions ni lee et al 1999 blockade type calcium currents c ltmrs low concentration ni 30 revealed ni insensitive current slow kinetics activation inactivation typical cav3.3 channel figure 7d chemin et al 2002 in contrast ni sensitive fraction obtained subtraction revealed faster kinetics explicit criss cross pattern typical cav3.2 figure 7d date zinc zn available pharmacological tool interferes gating kinetics cav3.3 cataldi et al 2007 traboulsie et al 2007 after blockade cav3.2-like fraction ni subsequent application zn 100 slowed remaining native cav3.3-like current prominent slowing deactivation figure 7e analysis several c ltmr soma revealed cav3.3-like currents account third total amplitude c ltmrs type calcium currents figure 7f the dramatic slowdown deactivation kinetics zn shows cav3.3 unique feature portfolio ion channels specific c ltmrs figure 7 g test whether two type calcium channel isoforms impact c ltmr excitability performed current clamp experiments neurons t type calcium channels well known generate rebound low threshold spikes lts following transient hyperpolarization c ltmrs membrane depolarization triggered small lts membrane held negative values figure 7h inhibition cav3.2 ni 50 significantly alter lts action potential ap threshold whereas potentiation cav3.3 zn 100 increased lts amplitude kinetics figure 7h inset this resulted increased excitability lowering ap activation threshold crowning potentiated lts figures 7h 7i two cells ten sole application zn resting potential c ltmrs elicited depolarization sufficient trigger spontaneous ap trains figure 7j application ni conditions produce change membrane potential shown exclude trpa1 channel contribution also activated zn experiments performed presence trpa1 blocker hc030031 10 together electrophysiological data nicely corroborate rna seq ish expression data demonstrating cav3.2 cav3.3 channels concomitantly expressed c ltmrs channels absent free nerve ending mrgprd expressing neurons in study used recently generated mouse model cutaneous mrgprd expressing neurons c ltmrs genetically marked cherry protein combination ib4 live staining followed cell sorting rna deep sequencing gene profile three categories primary sensory neurons free nerve ending mrgpd expressing neurons c ltmrs heterogeneous population drg cells neither bind ib4 labeled cherry protein we provide digital measure presence prevalence transcripts known previously unknown genes three neuronal subsets because dn population composed variety neuronal subsets glial cells dn data set used identify genes specifically enriched free nerve ending mrgprd expressing neurons specifically enriched c ltmrs this analysis yielded 156 genes specifically enriched dp neurons 184 c ltmrs these genes encode variety molecules including ion channels receptors signaling molecules previously uncharacterized genes major concern gene profiling experiment follow procedure which genes one pursue done lists genes ? the answer question motivated objectives expand molecular characterization c ltmrs identify combinatorial expression key genes shape functional specialization free nerve endings mrgprd neurons c ltmrs mrgprd neurons required acute injury induced mechanical pain well sensation itch cavanaugh et al 2012 shields et al 2010 whereas c ltmrs postulated convey light touch physiological conditions mechanical chemical pain injury abraira ginty 2013 delfini et al 2013 our rna seq data combination ish gene ontology analysis revealed great number genes expressed dp neurons c ltmrs vice versa reflecting anatomical functional differences indeed dp neurons showed high enrichment genes encoding gpcrs signaling molecules addition selective expression three isoforms protein kinase c family the enrichment receptors signaling molecules agreement involvement dp neurons acute injury induced mechanical pain itch cavanaugh et al we show dp neurons express high levels agtr1a ptgdr suggesting pro nociceptive effect respective agonists angiotensin ii prostaglandin d2 likely occurs dp neurons activation pgd2 shown enhance neuronal excitability increasing amplitude ttx sensitive currents ebersberger et al 2011 nakae et al 2005 continuous perfusion angiotensin ii enhanced tactile thermal hypersensitivity setting nerve injury via activation agtr1a pavel et al 2013 furthermore dp neurons exclusively express mrgprd gpcr activated -alanine histamine liu et al 2012 suggesting dp neurons part histamine independent itch neural circuit neurons targeted treating clinical itch resistant antihistamines c ltmrs hand expressed different gpcrs expressed dp neurons cckar metabotropic gq p2y1 receptor showed striking enrichment voltage gated channels including potassium channels kcnq2 kcnj12 kcnv1 kcna2 kcna4 sodium channels associated beta subunits scn1a scn1b scn3b scn4b scn7a scn8a low voltage gated calcium channels cacna1h cacna1i it shown cckar restricted 10% neurons overlap peptidergic cgrp nociceptors broberger et al 2001 our rna seq ish data demonstrate cckar expressed c ltmrs excluded dp neurons cholecystokinin octapeptide sulfated form binds gq/11-coupled cckar involved regulation large variety physiological functions among opioid induced anti nociception suh et al 1995 electrophysiological studies rat drg neurons shown activation cckar evokes membrane depolarization indicating cckar mediates excitatory responses et al 2006 psychophysiological studies human using cholecystokinin octapeptide could lend new insights role c ltmrs touch sensation normal pathological conditions c ltmrs also exhibited high expression metabotropic gq p2y1 receptor whereas dp neurons mainly expressed ionotropic purinergic receptor p2x3 demonstrating dp neurons atp adp compounds sensed p2x3 whereas c ltmrs signaling intermediates likely detected though metabotropic gq p2y1 receptor our data also highlighted specific functional enrichment lva type channels c ltmrs among three type channels found cav3.2 cav3.3 highly present c ltmrs cav3.3 exclusively expressed particular class neurons t type currents slow kinetics resistance nickel block described subset small sized rat drg trigeminal neurons expressing slowly adapting mechano activated currents coste et al our rna seq data combination ish electrophysiological recordings provided clear demonstration cav3.3 specific marker c ltmrs offers new possible selective pharmacological target control c ltmrs excitability mice although zinc plethoric effects use investigate cav3.3 contribution c ltmr excitability shows specificity particularly slowing lts kinetics highly correlated massive changes cav3.3 currents line depolarizing effects observed result least part persistent depolarizing zinc modulated cav3.3 current whereas contribution zinc targets remains possible trpa1 effect eliminated use specific blocker possible inhibition two pore potassium task3 channel kcnk9 unlikely transcript present c ltmrs study usoskin et al 2015 mechanistically findings suggest substantial contribution cav3.3 rebound firing reminiscent observed nuclear reticularis thalamic neurons like c ltmr express cav3.2 cav3.3 prominent cav3.3 effect burst firing astori et al 2011 finally recent elegant study using unbiased large scale single cell rna sequencing revealed 11 molecularly distinct subsets primary sensory neurons including th expressing c ltmrs population usoskin et al 2015 very interestingly genes enriched c ltmrs study similar upregulated th population described usoskin colleagues study however ish experiments showed vast majority c ltmrs enriched genes also expressed neurons preferentially nf200 neurons supporting strategy validate specific expression identified enriched genes using ish approach given majority c ltmrs enriched genes also expressed nf200 neurons nf200 hallmark proprioceptive ltmrs abraira ginty 2013 postulate c ltmrs share many molecular features ltmrs line hypothesis studies last years demonstrated c ltmrs share many functional anatomical features ltmrs abraira ginty 2013 c ltmrs described convey low threshold mechanical stimuli bessou et al 1971 douglas ritchie 1957 johansson et al 1988 1952 seal et al 2009 zotterman 1939 they sensitive skin indentation respond sustained mechanical stimuli slow intermediate adaptation rates form longitudinal lanceolate endings around hair follicles abraira ginty 2013 delfini et al 2013 li et al 2011 lou et al 2013 cross comparison rna seq data sets five nf subsets recently published usoskin colleagues usoskin et al 2015 provide comprehensive view molecular signatures c ltmrs ltmrs conclusion data presented study provide wealth information presence prevalence transcripts known previously unknown genes three categories primary sensory neurons highlight differential expression great number genes two diametrically opposite classes cutaneous afferents ish screen followed immunostaining ib4 binding led generation two open access libraries allow monitoring cellular distribution 115 genes provide first electrophysiological recording low voltage gated ca channel exclusively present c ltmrs suggest c ltmrs share many molecular features ltmrs the expression patterns dn enriched genes co expression c ltmrs enriched genes nf200 neurons supplemental tables enclosing dp- c ltmrs- dn enriched genes well go analysis provided supplemental information mice maintained standard housing conditions 23c 40% humidity 12 hr light cycles free access food water c57bl/6 mice charles river laboratories heterozygous ginip knockin mice previously generated laboratory gaillard et al 2014 special effort made minimize number well stress suffering mice used study all protocols agreement european union recommendations animal experimentation ib4ginip ib4ginip ib4ginip ib4ginip cells sorted facs aria cell sorter bd bioscience gating sytox blue cherry ginip cells sytox blue cherry ginip cells drgs cell suspensions c57bl/6 mouse used negative control cherry fluorescence cells sorted directly ice cold rlt lysis buffer rneasy micro kit qiagen supplemented 10% v v -mercaptoethanol sigma snap frozen 80c rna extraction rna seq libraries constructed truseq rna sample preparation low throughput protocol kit illumina were performed using hiseq control software real time analysis component provided illumina the quality data assessed fastqc babraham institute illumina software sav sequence analysis viewer demultiplexing performed using illumina sequencing analysis software casava 1.8.2 eland_rna module casava was used align rna seq reads mus musculus genome ucsc mm10 set gene model annotations refflat.txt file downloaded ucsc october 10 2012 order generate automatically splice junctions reads also aligned set contaminants including rrnas mitochondrial chromosome phix genome illumina control illumina adapters then exon counts summed gene divided length reads i.e. 100 obtain gene counts downstream statistical analysis made 1 genes generated least ten reads 2 genes generated least 1,000 reads differentially expressed genes identified using bioconductor robinson et al 2010 package edger alexa et al 2006 version 2.6.2 data normalized using tmm robinson oshlack 2010 normalization factors genes adjusted p value less 1% according fdr method benjamini hochberg declared differentially expressed genes classified using custom rules 1 genes declared differentially expressed condition relative condition b condition c fold change least 2 absolute value cases considered specific condition a. 2 gene specific two conditions b simultaneously differentially expressed versus c b versus c absolute fold change 2 absolute fold change 2 b. 3 filtered genes considered specific none condition ( 4 genes belonging none previous categories considered specific three conditions simultaneously free web access gorilla database used perform go functional annotation resulting list genes described eden et al lumbar drgs prepared cultured described previously francois et al we incubated cultures alexa-488-conjugated ib4 1 g ml 5 min 37c invitrogen diluted extracellular solution washed recordings conditions recordings voltage gated calcium currents membrane potential described delfini et al patterns expression 100 genes enclosed dp c ltmrs ish libraries dp and a. mantilleri performed situ analyses p.m. performed pcrs probe synthesis s.l

summarycutaneous c - unmyelinated mrgprd+ free nerve endings and c - ltmrs innervating hair follicles convey two opposite aspects of touch sensation : a sensation of pain and a sensation of pleasant touch . the molecular mechanisms underlying these diametrically opposite functions are unknown . here , we used a mouse model that genetically marks c - ltmrs and mrgprd+ neurons in combination with fluorescent cell surface labeling , flow cytometry , and rna deep - sequencing technology ( rna - seq ) . cluster analysis of rna - seq profiles of the purified neuronal subsets revealed 486 and 549 genes differentially expressed in mrgprd - expressing neurons and c - ltmrs , respectively . we validated 48 mrgpd- and 68 c - ltmrs - enriched genes using a triple - staining approach , and the cav3.3 channel , found to be exclusively expressed in c - ltmrs , was validated using electrophysiology . our study greatly expands the molecular characterization of c - ltmrs and suggests that this particular population of neurons shares some molecular features with a and a low - threshold mechanoreceptors .

endoplasmic reticulum er organelle crucial biosynthetic signaling functions eukaryotic cells the er major intracellular calcium ca storage organelle critically involved ca homeostasis ca mediated signaling pathways also provides environment synthesis folding modification proteins destined secreted embedded plasma membrane reviewed 1 2 moreover er major site biosynthesis steroids cholesterol lipids proper folding maturation stabilization nascent protein er require highly oxidizing ca rich er environment essential diverse posttranslational cotranslational modifications including glycosylation disulfide bridge formation proteins subjected entering er processes assisted monitored several resident chaperones ca binding proteins including glucose regulated proteins grp78 bip immunoglobulin heavy chain binding protein calreticulin calnexin several folding enzymes thioredoxin like protein disulfide isomerase pdi pdi oxidizes cysteine residues nascent proteins i.e. oxidative folding resulting formation intra- intermolecular disulphide bonds reduced pdi turn oxidized thiol oxidoreductase ero1 ero1 transfers reducing equivalents molecular oxygen generating stoichiometric amounts h2o2 per newly formed disulphide coupled depletion reduced gluthatione pool proteins fail adopt correctly folded native conformation proper oligomeric assembly case multisubunit proteins retrotranslocated cytosol process known er associated protein degradation erad degraded 26s proteasome various physiological pathological conditions including hypoxia er ca depletion oxidative injury high fat diet hypoglycemia viral infections may cause imbalance er protein folding load capacity leading accumulation unfolded proteins er lumen condition referred er stress er stress sets motion evolutionary conserved integrated signal transduction pathway known unfolded protein response upr the upr primarily aims ameliorating protein load er coordinating temporal shut protein translation along complex program gene transcription increase er folding capacity if transcriptional program fails reestablish proper er homeostasis persistence er stress induces cell death severe er stress cause cell death usually activating intrinsic apoptosis moreover order clear er accumulation terminally misfolded protein aggregates degraded proteasome upr may upregulate autophagy machinery 5 6 macroautophagy hereafter referred autophagy major lysosomal pathway bulk degradation cytoplasmic materials including proteins damaged organelles characterized sequestration entire portions cytoplasm double membrane bounded vacuole called autophagosome 7 8 spite role self digestion mechanism autophagy mainly activated protect cell death however like case upr stimulation autophagy certain circumstances required activate cell death machinery although upr autophagy function independently recent reports show may interlinked share functional duality exerting cytoprotective basal metabolic stress conditions cytocidial activity acute cellular damage tumor cells bathed hostile microenvironment confronted chronic metabolic stress conditions favor activation adaptive mechanisms upr autophagy 10 11].moreover certain promising anticancer regimens shown activate concomitantly er stress autophagy cancer cells see section 4 molecular link upr autophagic response er stress stress pathways influence therapeutic outcome remain largely undefined making topic important area future research cancer therapy here review molecular mechanisms underlying emerging connections upr autophagy pathways discuss potential implications context anticancer therapy the unfolded protein response mammalian cells governed three transmembrane er stress sensors namely perk pkr like er kinase ire1 inositol requiring enzyme 1 atf6 activating transcription factor 6 kept inactive state binding er chaperone bip preventing oligomerization induced activation er homeostasis perturbed accumulating misfolded proteins become progressively bound bip titrating away bip interaction transmembrane signaling proteins upon deinhibition homodimerization these er sensors activate complex er nucleus signaling pathway transmits information across er membrane extensive gene expression program mediated activation downstream transcription factors genetic program activated upr results upregulation folding machinery along expansion er lumen enhanced degradation terminally misfolded proteins erad additional mechanisms include general translational shutdown well degradation select group secretory mrnas proteins delayed translocon process also known pre emptive quality control the mechanisms upr induction coupled failure reestablishing er folding capacity leads cell death requirement upr signaling autophagy stimulation still unsettled questions furthermore recent studies revealed er serves subcellular platform formation signaling complexes comprising molecular elements upr bcl-2 family members pro- antiapoptotic reviewed perhaps regulators autophagy following sections discuss current knowledge main signaling pathways emanating branch upr along downstream targets figure 1 ire expressed cell types tissues whereas expression ire1 primarily restricted epithelial cells gastrointestinal tract 14 15 ire1 type transmembrane protein n terminal luminal sensor domain c terminal cytosolic effector region contains kinase endoribonuclease rnase domains cells undergoing er stress oligomerization ire1 results trans autophosphorylation activation rnase domains excise 26 nt sequence xbp1u unspliced xbp1 producing mature xbp1s mrna spliced xbp1 xbp1s encodes active leucine zipper bzip transcription factor xbp1s regulates transcription several genes involved er quality control mechanisms er golgi biogenesis well erad components 1822 recently revealed also genes involved redox homeostasis oxidative stress responses consistent xbp1 deficient cells found susceptible exogenous agents causing oxidative stress h2o2 parthenolide concomitant reduced expression several antioxidant enzymes including catalase thioredoxin trx1 overexpression xbp1 restored catalase expression reduced ros generation h2o2 thus implicating protective role xbp1 oxidative stress intriguingly antioxidant effect mediated xbp1u i.e. protein encoded unspliced xbp1 mrna whereas xbp1s i.e. product ire1 activation failed induce changes catalase expression response ros following er stress thus underscoring ire1 activity dispensable although molecular mechanism underlying differential function unspliced xbp1u spliced xbp1s products xbp1 still elusive- may involve binding regulation selected targets dependent relative abundance different cell types study highlights new role xbp1 ros signaling independent ire1 rnase activity although ire1 displays intrinsic kinase activity substrates known thus far ire1 however prolonged activation ire1 capable transmit map kinase activation cascade it shown ire1 serve molecular platform recruitment adaptor protein tnf receptor associated receptor 2 traf2 e3 ubiquitin ligase leads activation apoptosis signal regulating kinase 1 ask1 map3k jnk p38 mapk pathway 25 26 depending cellular context activation jnk either allow cells adapt er stress initiating autophagy discussed promote apoptosis response persistent irrecoverable er stress like ire1 perk type transmembrane protein luminal sensing domain cytosolic kinase domain becomes activated following dimerization the resulting transautophosphorylation induces conformational change enhances affinity perk eif2 eukaryotic initiation factor 2 alpha phosphorylation eif2 ser51 perk results rapid shut general translation relieving protein burden stressed er concomitant loss cyclin d1 arrests er stressed cells g1 addition recent study shown eif2 phosphorylation also regulates translation via inhibition rrna synthesis coordinately regulating translation ribosome biogenesis cellular stress paradoxically translational shutdown leads selective translation transcription factor atf4 member bzip family transcription factors the perk eif2-atf4 axis regulates expression genes involved amino acid biosynthesis transport functions antioxidant stress responses apoptosis in addition eif2 perk also phosphorylates nuclear factor e2-related factor 2 nrf2 nrf2 maintained inactive cytoplasmic complex microtubule associated protein keap1 kelch like ech associated protein 1 nrf2 phosphorylation promotes dissociation keap1 leading nuclear accumulation nrf2 binding antioxidant response element promotor genes encoding detoxifying enzymes heme oxygenase 1 ho-1 line results shown nrf2 cells prone er stress induced apoptosis likewise perk cells along impaired attenuation protein synthesis found mount high amount endogenous peroxides preceding apoptotic induction response agents causing perturbation er functions interfering ero1 blocked increased ros production thus providing link protein oxidation er ros production er stress this perk function linked ability atf4 regulate expression genes involved glutathione biosynthesis antioxidant response these studies suggest perk branch upr bifurcates two parallel integrated signaling pathways perk eif2-atf4 perk nrf2 key role adaptation oxidative stress metabolic consequence biosynthesis posttranslational oxidative processing er isoforms atf6 atf6 atf6 present cell lines type ii transmembrane er proteins release bip cause atf6 oligomerization instead reveals golgi localization sequence once translocated golgi atf6 cleaved juxtamembrane site site 1 site 2 proteases s1p s2p also involved cleavage er membrane transcription factor srebp sterol response element binding protein 35 36 processed atf6 moves nucleus forms active homodimers dimerizes bzip transcription factors like nf caat binding factor well xbp1s regulate transcription atf camp response elements cres erses 38;39 interestingly yoshida et al found xbp1u interacts directly active form atf6 atf4 targeting proteasomal degradation may provide negative feedback loop decrease xbp1 expression other transcriptional targets include proteins increasing er chaperone activity degrading er client proteins 37 39 although atf6 neither essential basal expression er chaperones embryonic postnatal development plays important role recovery acute er stress adapting cells chronic er stress additionally recent study shows atf6 also contributes xpb1s independent manner lipid biogenesis er expansion er stress response thought predominantly mediated irei pathway bcl-2 family proteins consist proapoptotic multidomain proteins e.g. bax bak antiapoptotic multidomain proteins e.g. bcl-2 bh3-only proteins e.g. bid bim bad key regulators mitochondrial apoptosis 41 42 they function gatekeepers antiapoptotic bcl-2 gatecrashers proapoptotic bax bak outer mitochondrial membrane while molecular mechanism underlying mitochondrial action still matter debate becoming clear bcl-2 family proteins exert tight control apoptosis different subcellular sites a constellation er localized bcl-2 family members including bax bak bik nbk bcl-2 shown engaged control er ca homeostasis 4446 extensive review see 13 47 moreover recent reports identified bcl-2 members vital regulators upr sensor mechanisms cellular fate following er stress for instance atf6 negatively regulates bad proapoptotic activity upregulating regulator calcineurin 1 rcan1 endogenous inhibitor calcineurin protein phosphatase b bad dephosphorylation allows dimerization antiapoptotic bcl-2 protein family members like bcl xl thus inhibiting activity this mechanism underscores prosurvival role genetic program activated atf6 suppression bad proapoptotic activity the proapoptotic multidomain proteins bax bak form protein complex cytosolic domain ire1 interaction shown essential ire1 signaling genetic ablation bax bak mice caused abnormal response tunicamycin induced er stress liver along extensive tissue damage decreased expression xbp1 reduced jnk activation furthermore requirement bax bak proteins proper ire1 signaling confirmed mefs doubly deficient dko proapoptotic proteins recent report klee et al showed reconstituting bak expression er membranes dko cells sufficient reestablish ire1-traf2 activation mitochondrial apoptosis discussed section 2.3 instigated reticular forms bh3-only proteins bim puma interestingly ire1 pathway activated reticular bh3-only effectors atypical lead xbp1 splicing likely arms upr required upregulation xbp1 mrna levels atf6 sufficiently activated however alternative intriguing possibility could involve differential regulation ire1 rnase activity required xbp1 mrna splicing ire1-traf2 complex formation required activate proapoptotic jnk signaling different subset proapoptotic proteins er membrane clearly studies required shed light mechanisms regulating ire1 signal transduction recently er associated bax inhibitor-1 bi-1 evolutionary conserved antiapoptotic protein identified new player regulation ire1 bcl-2 family members modulators bi-1 block bax mediated apoptosis following er stress intrinsic stress signals directly interacting antiapoptotic bcl-2 family members enhancing antiapoptotic function bi-1 er found capable interact c terminus domain ire1 inhibit ire1 signaling vitro well mice flies conferring increased resistance conditions mild er stress er stressed bi-1 deficient cells displayed ire1 hyperactivation along increased xbp1 mrna splicing expression xbp1s dependent genes thus unraveling paradoxical role bi-1 inhibitor cytoprotective ire1 branch upr mildly er stressed cells interestingly another study using human fibrosarcoma cells overexpression bi-1 inhibited ros production downstream er stress upregulation ho-1 effector perk pathway described previously bi-1 deficiency mefs affect expression ho-1 raises intriguing possibility bi-1 may affect ire1 possibly perk pathway cell type specific manner whereas mechanistic studies required solve discrepancies findings reveal subtle cross talk molecular sensors upr cell death regulators might affect amplitude function upr moreover abundant evidence claims critical role proapoptotic bcl-2 family proteins induction apoptosis following er stress initial cellular responses fail restore er homeostasis sustained er stress causes upr switch adaptive cell death pathway however molecular elements switch still elusive exception components upr dominant prosurvival i.e. bip proapoptotic i.e. chop 56 57 role assigned genetic studies each apical upr sensor holds dualistic role propagating adaptive well toxic signals example genetic deletion perk interference eif2 phosphorylation impairs cell survival 58 59 tumor growth hypoxia artificially increasing perk activity increases cell survival however lin et al shown sustained perk signaling lethal whereas equivalent duration ire1 signaling suggesting transition protective proapoptotic upr function involves switch ire1 signaling along enduring perk activity the main effector perk mediated apoptosis proapoptotic transcription factor chop c ebp homologous protein gadd153 induced atf4 atf6 well xbp1s however perk eif2 branch appeared essential chop upregulation perk atf4 eif2 ser51ala knock cells failed induce chop er stress 32 58 59 chop activity also regulated translationally limited chop mrna lifetime posttranslationally p38mapk phosphorylation enhances proapoptotic activity 57 64 the latter mechanism may provide point convergence perk ire1 signaling pathways since p38mapk downstream target ire1-traf2-ask1 signaling complex 25 26 genetic studies shown chop loss function results cytoprotection whereas chop gain function enhances sensitivity variety stresses perturbing er function 56 65 chop mediated cell death entails induction variety genes may potentiate apoptosis including gadd34 ero1 bim trb3 tribbles homologue 3 gadd34 regulatory subunit protein phosphatase 1 pp1 targets pp1 dephoshorylate eif2 promotes resumption protein synthesis if protein folding capacity er reestablished premature deinhibition translation increase client protein load er may favor improper disulphide bond formation unfolded proteins thus amplifying damage addition elevated expression ero1 chop thought instigate hyperoxidizing conditions er 67 68 thus perk axis involved maintaining redox state er stress discussed also ability turn prooxidant signal transcriptional program chop efficiently set motion suggested recent study wherein stability prosurvival prodeath mrnas proteins was studied conditions mild severe er stress atf4-dependent prosurvival gene expression likely sustained perk activated transiently limited extent in contrast consequence intrinsic instability proapoptotic mrnas proteins apoptotic program mediated atf4 target chop would activated protective mechanisms fail require sustained perk activation chop also regulate expression number bcl-2 family proteins yet unidentified mechanism suppresses expression antiapoptotic bcl-2 directly promoting transcription proapoptotic bh3-only protein bim although clear chop fulfills important role er stress induced apoptosis fact perk eif2 ser51ala knock cells unable induce chop yet susceptible er stress 58 59 unravels dual role perk axis triggering adaptive proapoptotic processes increased sensitivity perk deficient cells could explained least part impaired activation prosurvival pi3k phosphatidylinositol 3 kinase)-akt signaling pathway shown promote expression inhibitor apoptosis proteins iaps thus conferring cellular resistance er stress 70 71 an interesting molecular switch prosurvival prodeath functions perk pathway could involve human orthologue drosophila tribble protein trb3 downstream transcriptional target chop showed trb3 knock sensitized cells cell death tunicamycin treatment remarkably trb3 could downregulate induction repressing chop atf4 functions 72 73 a mechanism proposed wherein trb3 exerts negative feedback chop mild er stress allowing cell adapt er stress 72 73 in contrast severe persistent er stress induction trb3 would robust leading apoptosis mechanism involving trb3-mediated inhibition dephosphorylation akt 74 75 this feedback mechanism could facilitate er stress mediated apoptosis severely er stressed cells successfully mounted proapoptotic threshold levels chop similar perk ire1 signaling also implicated promoting impairing cell survival for instance unfolded proteins accumulate artificially extending ire1 rnase function led enhanced survival 62 76 knock xbp1 impaired cell survival 77 78 pointing general protective role ire1-xbp1 signaling er stress however another report ire1 overexpression hek293 cells led activation absence er stress subsequent cell death discussed ire1 apparently gained signaling properties independent xbp1 splicing strongly dependent interaction bcl-2 proapoptotics bcl2 modulators er membrane thus ire1 promote cell death recruiting traf2-ask1 complex leading activation jnk p38 mapk cascades 25 26 jnk turn exert proapoptotic effect activating certain bh3-only proteins bim 79 80 suppressing antiapoptotic activity bcl-2 the apoptotic pathway evoked upr still unclear mounting observations indicate mitochondrial pathway heavily involved since cells lacking bax bak apaf-1 resistant apoptosis induction different er stressors 44 82 83 moreover mentioned several bcl-2 family members localize er regulate calcium levels well signal transduction upr in addition bim bh3-only proteins noxa puma transcriptionally activated p53-dependent independent mechanisms depending type er stressor thus bridging er stress bax bak mediated mitochondrial membrane permeabilization recently klee coworkers using bax bak cells showed bak targeted er membrane sufficient engage mitochondrial apoptosis activated bh3-only molecules puma bim er thus bypassing need localized mitochondria reticular bak engaged atypical ire-traf2 activation pathway wherein mobilization ca facilitated persistent jnk activation intriguingly er ca release per se able incite mitochondrial apoptosis unless bak expressed reticulum whereas favored nonapoptotic cell death shown also previous study whether pathway role normal cells expressing mitochondrial and er bax bak still needs proven however already argued jnk functions master regulator apoptosis perhaps autophagy pathways er stress thus together the emerging consensus amplitude temporal activation specific arms upr along repertoire signaling platforms formed er membrane upr interactome crucial elements determining cellular fate following er stress proteasomal degradation autophagy two main mechanisms charge protein clearance cell unlike proteasomal degradation digests soluble ubiquitin conjugated proteins specific way autophagy degrade soluble aggregated proteins 8 86 thus autophagic process entire cytoplasmic portions including organelles cytoplasmic components engulfed within double membrane vesicle designated autophagosome the maturation vesicles involves fusion lysosomes leads turn degradation autophagosome components lysosomal degradative enzymes 8 86 as discussed variety stress signals nutrient starvation treatment different anticancer agents including induce er stress stimulate autophagy process nowadays considered essential cellular process participating number physiological functions within cell the molecular mechanisms responsible regulation autophagy completely elucidated yet although genetic biochemical analyses performed last years identified several autophagy genes atg participate regulation cellular process researchers working autophagy field formally divided autophagic process several steps initiation autophagy relies formation isolation membrane called preautophagosomal site the autophagy process ends fusion autophagosome lysosome digestion autophagosome content release digested components back cytosol 8 86 sections briefly summarize mechanisms different stages autophagy regulated normal rate autophagy cell low therefore cellular process becomes activated response certain situations thus exposure cell autophagic stimulus triggers series modifications autophagic machinery allow formation elongation the precise origin mammalian cells still unknown although proposed could either derived de novo synthesized lipids generated vesicle budding er golgi apparatus endosomes transmembrane proteins atg9 vmp-1 88 89 required autophagosome formation suggested could play role transport lipids well recruitment additional proteins involved initiation autophagy thus movement atg9 trans golgi location preautophagosomal site seems crucial event initiation autophagy 87 90 the relocation transmembrane protein atg9 autophagosome thought require activation complex formed proteins atg1 atg13 atg17/fip200 .. activity atg1 complex modulated mammalian target rapamycin complex 1 mtorc1 mtorc1 protein complex formed mtor raptor regulatory associated protein mtor mlst8 pras40 proline rich akt substrate 40 kda plays central role control protein synthesis cell growth cell proliferation regulation several downstream targets in addition mtorc1 proposed regulate autophagy repressing activity atg1-atg13-atg17/fip200 complex 9295 regulation mtorc1 relies small g protein rheb ras homologue enriched brain still completely elucidated mechanism activates mtorc1 the tuberous sclerosis proteins tsc1 tsc2 gtpase activating protein gap activity rheb therefore promote inhibition hence inactivation tsc1/2 stimulates rheb mtorc1 inhibits autophagy result central position control cellular homeostasis one important upstream regulators mtorc1 prosurvival kinase akt phosphorylates inactivates tsc2 well pras40 another important regulator tsc2 amp activated protein kinase ampk phosphorylates tsc2 different residue akt leading activation tsc1/2 inactivation rheb inhibition mtorc1 discussed following sections modulation mtorc1 activity one mechanisms er stress autophagy become connected another important step initiation autophagy generation specific pool phosphatidylinositol-3-p pip3 autophagosome mammals this event catalyzed class iii phosphatidylinositol 3 kinase pi3k complex consists vps34 vacuolar protein sorting 34 regulatory protein p150 homolog yeast vps15 protein accumulation pip3 seems crucial recruitment autophagy proteins atg18/wipi-1 important atg9 trafficking therefore initiation autophagic process in addition proteins matg2 dfcp1 double fyve domain containing protein 1 may also regulated pip3 play role regulation formation elongation autophagosome underlining importance pip3 early stage autophagy specific phosphoinositide 3-phosphatase jumpy ) importantly vps34-interacting proteins required autophagy including vps30/atg6/beclin1 atg14 autophagy beclin-1 regulator 1 ambra-1 uvrag among different partners vps34 beclin-1 bh3-only domain permits interaction protein antiapoptotic proteins bcl-2 bcl xl different stimuli including er stress modulate interaction beclin-1 bcl-2 family members see also following sections considered important mechanism autophagy regulation atg14 uvrag also interactors vps34 although presence class iii pi3k complex seems mutually exclusive 87 103 104 recent findings support atg14 plays important role early stages autophagy activation response starvation case further research still necessary understand complex lipid protein protein protein interactions regulate formation the elongation initial autophagic membrane requires participation two ubiquitin like protein conjugation systems modify autophagy proteins atg5 atg8/lc3 thus upon autophagy stimulation atg5 conjugated atg12 process atg12 is activated e1 activating enzyme atg7 transferred e2-like protein atg10 finally atg12 attached internal lysine atg5 process seem require e3 ligase protein is cleaved protease atg4 generates glycine ct residue atg8/lc3 then e1 enzyme atg7 activates atg8 transferred e2-like protein atg3 the last step atg8/lc3 modification involves conjugation protein phosphatidylethanolamine pe process facilitated e3-like activity atg12-atg5 conjugate 105 106 upon autophagy induction atg8/lc3 becomes lipidated associates autophagosome widely used monitor activation autophagy immunofluorescence 8 86 107 the atg16l atg8/lc3 complexes play crucial role modification autophagosomal membrane therefore elongation closure autophagosome the last step autophagic process fusion autophagosome lysosomes the canonical machinery vacuole membrane fusion seems participate regulation process 87 90 thus lysosomal protein lamp2 small gtpase rab7 implicated autophagosome lysosome fusion mammalian cells nevertheless many additional proteins including belonging rab soluble n ethylmaleimide sensitive factor attachment protein receptors family snare believed play important role autophagosome lysosome fusion process 8 86 the lysosomal degradation autophagosomal content relies several lysosomal hydrolases including cathepsins b l. final outcome activation autophagy program highly dependent cellular context strength duration stress inducing signals thus besides role cellular homeostasis autophagy form programmed cell death play cytoprotective role example situations nutrient starvation 108 109 accordingly autophagy plays dual role cancer one hand cellular process may help overcome stress evoked lack nutrients oxygen initial steps tumorigenesis hand autophagy proposed play tumor suppressor function providing minimal supply atp required dna repair preventing oxidative stress reducing intratumoral necrosis local inflammation 110113 moreover different anticancer treatments activate autophagy tumor cells proposed either enhance cancer cell death act mechanism resistance chemotherapy 110115 different situations induce er stress also lead induction autophagy discussed er stress response is activated protect cells different alterations affecting organelle however intensity duration er damage restored response er stress also lead cell death likewise autophagy help cells cope er stress instance contributing elimination unfolded aggregated proteins participate mechanism er stress induced cell death 115 117119 section we try delineate proposed mechanisms er stress autophagy become connected certain cellular situations figure 2 as described accumulation unfolded proteins triggers upr thus promoting inhibition general protein synthesis well increased translation several transcription factors enhance expression er stress genes see previous sections details evidence link upr autophagy obtained ectopic expression polyglutamine polyq proteins experiments dominant negative form perk genetic substitution serine 51 eif2 ala prevents phosphorylation protein prevented polyq protein induced autophagy strongly suggesting perk dependent eif2 phosphorylation plays important role activation autophagy response accumulation unfolded proteins hand eif2 phosphorylation seems also important autophagy induced er stress related unrelated stimuli 75 120 121 it important bear mind perk protein kinase regulating eif2 phosphorylation see reference review double stranded rna activated protein kinase pkr activated viral responses general control nonderepressible 2 gcn2 activated upon aminoacid starvation heme regulated inhibitor hri activated heme depletion also phosphorylate eif2. accordingly pkr dependent eif2 phosphorylation modulates autophagy response viral infection likewise small heat shock 22 kda protein 8 hspb8 cochaperone bcl-2-associated athanogene 3 bag3 proposed mediate mutated hungtingtin induced eif2 phosphorylation autophagy via gcn2 activation regarding signalling pathways eif2 phosphorylation modulate autophagy kouroku et al showed perk eif2-dependent atg12 upregulation required induction autophagy response polyq protein accumulation suggests controlling expression autophagy related genes eif2 downstream targets could one mechanisms connecting events hand we recently found treatment cancer cells -tetrahydrocannabinol thc active component marihuana activates autophagy via er stress eif2 phosphorylation effect mediated perk pkr gcn2 salazar m. velasco g. unpublished observations our data indicate induction autophagy response thc treatment relies eif2 phosphorylation dependent upregulation transcription factors p8 atf-4 chop well pseudokinase trb3 four genes previously identified essential mediators thc action cancer cells 122 123 we also showed important step induction autophagy inhibition akt mtorc1 axis pseudokinase trb3 see additional details figure 2 case further research still necessary clarify precise mechanisms eif2 phosphorylation regulates autophagy response different er stress signals activation ire1 arm er stress response also shown regulate autophagy thus treatment tunicamycin thapsigargin treatment proteasome inhibitors induced autophagy ire1-dependent manner the proautophagic actions ire1 seem rely ability protein interact cytosolic adaptor traf-2 activate jnk interest jnk proposed regulate autophagy bcl-2 phosphorylation prevents protein interacting inhibiting essential autophagy regulator beclin-1 99 124 125 in addition jnk shown control beclin-1 expression regulate ceramide induced autophagy as discussed beclin-1 associated vps34 plays important role regulation autophagy see figure 2 it therefore conceivable activation ire1/traf2/jnk arm er stress may regulate autophagy modulation beclin-1 function expression intriguingly recently shown xbp-1 ablation increases autophagy protects toxicity induced aggregates enzyme superoxide dismutase 1 model amyotrophic lateral sclerosis these observations suggest xbp-1 may play different role traf2/jnk regulation autophagy ire1 arm upr er stress activation frequently accompanied calcium release cytosol leads activation several ca regulated signalling pathways different agents including er stress inducers shown produce increase cytosolic calcium concentration activate autophagy one mechanisms connecting ca release er autophagy stimulation ampk explained several kinases regulate mtorc1 including ampk inhibits mtorc1 activating tsc2 ampk considered important energy sensor becomes activated upon atp cellular depletion phosphorylation different kinases three ampk upstream kinases identified date lkb1 ca calmodulin dependent kinase kinase cacmkk transforming growth factor beta activating kinase 1 tak1 jttel coworkers showed increases cytosolic ca concentration upon treatment different er stress inducers stimulate camkk leading turn ampk activation inhibition mtorc1 autophagy stimulation the group recently shown trail induced autophagy also mediated ampk case mechanism involves phosphorylation ampk tak1 lkb1 cam kk these observations suggest ampk may play important role regulation autophagy response different ca dependent independent stress signals another ca activated kinase regulates autophagy response er stress death associated protein kinase 1 dapk dapk ser thr kinase plays important role tumor suppressor due ability promote apoptosis autophagy thus dapk deficient mefs less sensitive er stress induced autophagy wild type counterparts activation dapk upon er stress relies dephosphorylation inhibitory autophosphorylation site kinase pp2a phosphatase suggests additional er stress activated signals apart ca release required stimulate proautophagic activity kinase regarding mechanisms dapk regulates autophagy recently shown dapk phosphorylates beclin-1 bh3-only domain preventing thus interaction protein bcl-2 133 134 as p53 modulates autophagy different mechanisms 112 136138 could another way dapk could regulate autophagy response certain er stress stimuli the protein kinase c theta pkc also implicated regulating autophagy response er stress calcium dependent manner thus knock pkc inactivation upr signalling routes prevented autophagy induced acute er stress study inactivation mtorc1 used concentrations thapsigargin thapsigargin observed suggests different signalling routes may converge regulation autophagy er stress situations involving calcium mobilization another link ca er stress autophagy relies modulation inositol 1,4,5-trisphosphate receptor ip3r this receptor releases ca er stores response different cellular signals although could also play additional functions derived ability interact different proteins including members bcl-2 family inhibition ip3r xestospongin b lithium induced decrease myo inositol-1,4,5-triphosphate ip3 levels promotes autophagy intriguingly effects seem independent ca mobilization function ip3r thus recently shown use pharmacological inhibitors ip3r disrupts interaction protein beclin-1 could additional way regulating pro autophagic function protein further investigation nevertheless necessary clarify whether mechanism participates activation autophagy response er stress discussed case er stress autophagy is currently considered cell survival mechanism certain cellular settings also promote cell death consequently depending whether pharmacological genetic inhibition autophagy enhances prevents cell death activation autophagy er stress assigned respectively cytotoxic 75 115 119 132 145147 protective 5 6 115 119 128 role it worth noting depending intensity stimulus cell type normal versus cancer cells cellular context hypoxia starvation treatment antitumoral agents presence mutations final outcome autophagy activation could different an important problem time predicting whether induction er stress activate autophagy protective cytotoxic way relative lack understanding molecular mechanisms autophagy regulates cell death thus autophagy proposed protect apoptosis operate alternative cell death mechanism e.g. cells defective apoptosis act upstream apoptosis activate cellular process reviewed 133 148 discussed previous section key regulatory steps activation autophagy upon stimulation er stress mtorc1 inhibition interaction beclin-1 bcl-2 ) can also receive signals derived different inputs including directly related er stress moreover regulatory proteins transmitting signals akt ampk dapk jnk play also major role modulation cell survival independently autophagy it therefore essential consider cellular context order understand different er stress signals integrated yield protective cytotoxic autophagic response from discussion clear er stress autophagy activate prosurvival mechanisms well lethal programs especially conditions enduring er stress organellar damage thus activation upr autophagy may either impede facilitate drug mediated cell killing plausible depend type cancer cytotoxic agents used while growing number reports started identify molecular elements cross talk er stress autophagy see section 3.2 thus unraveling potential druggable targets knowledge functional outcome activation pathways cancer cells responding chemotherapeutics still limited terms therapeutic outcome drugs combination thereof capable activating proapoptotic branch upr simultaneously inhibiting prosurvival function provide highest therapeutic benefit moreover autophagy activated following er stress survival response restoring er homeostasis e.g. removal protein aggregates pharmacological blockage could protract upr activation critical threshold reached may precipitate proapoptotic function hand autophagy may endorse proapoptotic functions certain er stress pathways see also section 4.1 become lethal backup pathway cancer cells defect apoptotic signaling 133 148 a wide array conventional experimental chemotherapeutic agents shown stimulate er stress activation upr along autophagy cancer cells for example tunicamycin thapsigargin brefeldin activate autophagy colon prostate cancer cells thus mitigating er stress protecting cell death however autophagy induced chemicals confer protection normal human colon cell line nontransformed murine embryonic fibroblasts rather contributes cell death the combined administration vorinostat histone deacetylase inhibitor sorafenib tyrosine kinase inhibitor carcinoma cells promotes cell death although activates time protective er stress driven autophagic response similarly resistance imatinib mesylate bcr abl tyrosine kinase inhibitor used treatment chronic myeloid leukaemia might also rely least part secondary activation er stress induced autophagy by contrast cannabinoid treatment activates er stress autophagy leading apoptotic cell death glioma pancreatic cancer cells nontransformed embryonic fribroblasts primary astrocytes neither er stress autophagy activated response treatment compounds likewise agents nelfinavir hiv protease inhibitor anticancer activity 151 152 melanoma differentiation associated gene-7/interleukin 24 mda-7/il-24 145 153 activate er stress response promotes autophagy apoptosis cancer cells increased expression tetraspanins family proteins facilitate spatial organisation localisation multiprotein complexes distinct membranal microdomains also shown activate er stress autophagic cell death understanding precise molecular mechanisms regulate extent autophagy activation response different triggering signals well ones control interplay cellular process apoptosis therefore crucial design new antitumoral therapies based modulation er stress autophagy response here we discuss selected group clinically used promising cytotoxic drugs demonstrated ability inducing upr autophagy paradigms discuss potential targeting pathways cancer therapy cannabinoids active components marijuana thc important owing high abundance potency exert wide variety biological effects mimicking endogenous substances endocannabinoids bind activate specific cannabinoid receptors thus treatment agents shown curb tumor growth various animal models cancer 157159 the antitumoral action cannabinoids based ability agents inhibit tumor angiogenesis activate apoptosis cancer cells 157 158 our recent findings unravelled cannabinoids induce autophagy different types tumor cells including glioma astrocytoma pancreatic cancer cells whereas activate cellular process nontransformed cells resistant cell death promoting activity cannabinoids interest pharmacological genetic inhibition autophagy prevented cannabinoid induced cell death well apoptosis whereas abrogation apoptosis prevented cell death autophagy induced agents these observations led us conclude induction autophagy part mechanism cannabinoids promote apoptotic death cancer cells the vivo relevance findings demonstrated observation cannabinoid treatment reduced tumor growth activated autophagy apoptosis subcutaneous tumor xenografts derived human u87 mg astrocytoma cells transformed mouse embryonic fibroblasts mefs likewise similar results obtained orthotopic model pancreatic cancer previously shown proapoptotic antitumoral action cannabinoids 122 123 salazar m. velasco g. unpublished observations furthermore autophagy deficient tumors generated subcutaneous injection transformed atg5 mefs resistant thc antitumoral action strongly supporting autophagy essential antineoplastic activity cannabinoids in addition analysis samples obtained two glioblastoma multiforme patients indicated thc administration might also trigger autophagy mediated cell death human tumors as discussed previous section mechanism responsible activation autophagy upon thc administration relies cannabinoid receptors induced early accumulation de novo synthesized ceramide event takes place er leads turn er dilation increased eif2 phosphorylation activation er stress response induces regulation several genes including stress regulated protein p8 downstream targets atf-4 chop pseudokinase trb3 required stimulation autophagy response cannabinoid action trb3 plays crucial role induction autophagy upon thc administration inhibitory interaction akt leads turn mtorc1 inhibition agreement observations treatment mice thc decreased mtorc1 activity stimulated autophagy apoptosis reduced tumor growth xenografts generated p8 cells generated p8 cells trb3 regulated response thc confirming p8/trb3 pathway plays essential role activation autophagy cell death cannabinoids also vivo cannabinoids activate therefore cell death promoting signalling route involves stimulation er stress autophagy apoptosis cancer cells thus cannabinoids constitute interesting tool investigate differential molecular mechanisms responsible stimulation autophagy mediated cell death hand selectivity cannabinoids stimulate described cell death promoting pathway cancer cells together low toxicity good safety profile makes agents promising antineoplastic tools photodynamic therapy pdt anticancer therapy involving selective photosensitization malignant cell types usually involving porphyrins porphyrin analogs agents suitable photophysical properties the initial step photodynamic process involves localization photosensitizing agent subcellular loci followed irradiation visible light appropriate wavelength 161 162 this results formation singlet oxygen ros cause photodamage sites photosensitizing agent localized since singlet oxygen migrate fraction micron site formation result photodamage quite specific thus distinguished property pdt ros formation mainly targeted particular subcellular site affects rather specific subset molecular targets pdt various agents shown induce apoptosis along autophagy cases autophagy activated mean protect cells killing agents found clinically useful reported show affinity er mitochondria lysosomes combinations sites a well studied paradigm er localizing dye hypericin naturally occurring phototoxin promising applications bladder cancer consistent predominant reticular localization cultured cells light activation hypericin coupled massive er expansion preceding ultrastructural features apoptosis vitro bladder cancer bearing rats verfaillie t. agostinis p. unpublished observations stimulation upr upr activation likely result immediate ros damage serca pump depleting er ca store followed concomitant activation autophagy mitochondrial apoptosis this ros paradigm er stress linked persistent activation perk eif2-chop axis proapoptotic function verfaillie t. agostinis p. unpublished observations induction autophagy er stressed cells unable mount apoptotic response bax bak deficiency results increased photokilling suggesting activation conversely apoptosis competent cells blocking autophagy stimulation following er stress sirnas target essential modulators autophagic machinery sensitizes cell death thus revealing cytoprotective role pathway dewaele m. agostinis p. unpublished results hence tempting speculate autophagy inhibition may potentiate proapoptotic perk pathway resulting better therapeutic opportunity cancer cell apoptotic machinery fully disabled further studies required establish whether suppression autophagic pathway along upr stimulation may represent valuable therapeutic strategy hypericin based pdt targeting proteasomal degradation proven valuable approach various cancer treatments proteasome inhibitors emerged new class er stress agents moreover recent evidence suggests used combination certain cytotoxic drugs pdt proteasomal inhibitors capable enhancing anticancer efficacy making agents promising class pharmacological agents combinatorial therapy bortezomib ps-341 velcade distinguishes proteasome inhibitors specifically inhibits 26s proteasome selectively blocking chymotryptic activity velcade clinically approved treatment multiple myeloma mantle cell lymphoma 166 167 shown successfully induce apoptosis various human cancer cell lines including myeloma prostate breast cancers well squamous cell carcinoma 168170 moreover preclinical studies indicate bortezomib displays anticancer activity pancreatic cancers one aggressive human diseases one potential mechanisms underlying apoptotic effects bortezomib cancer cells relies ability inhibit nf-b pathway blocking degradation cytoplasmic inhibitor ib however inhibition nf-b alone could fully account antitumor effect bortezomib suggesting additional pathways involved since proteasomal degradation misfolded proteins retrotranslocated er cytosol represents final step erad proteasomal inhibition causes additional burden unfolded proteins er this explains high efficacy bortezomib treatment types cancer cells er already predisposed considerable load for instance hypoxic cancer cells otherwise show increased resistance genotoxic agents well myeloma cells producing high amounts immunoglobulins hypersensitive treatment proteasome inhibitors 174 175 therefore therapies target er response combination bortezomib ought successful indeed shown bortezomib sensitized pancreatic cancer cells er stress mediated apoptosis additionally recently found significant retardation tumor growth vivo two different murine tumor models photofrin based pdt pdt approach stimulating upr combined bortezomib clinically used proteasome inhibitors this suggests blocking proteasome might offer new therapeutic avenue potentiate antitumor effect pdt interestingly schewe aguirre ghiso found myeloma cells surviving bortezomib treatment attenuated eif2 phosphorylation induction chop combined treatment gadd34-pp1 complex inhibitor salubrinal restored eif2 phosphorylation chop induction maximizing bortezomib induced apoptosis thus suggesting strategies capable sustaining chop expression might required successfully eradicate tumors furthermore processes functionally coupled proteasomal inhibition shown stimulate autophagy likely compensatory mechanism surprisingly whether autophagy enhances apoptosis induced proteasomal inhibitors seems depend whether treated cells transformed these findings suggest combined inhibition cellular degradation systems would enhance antitumoral efficacy indeed autophagy shown activated mcf-7 cells treated bortezomib mechanism involved proteasomal stabilization atf4 atf4 dependent upregulation lc3b this mechanism suggested contribute resistance breast cancer cells towards bortezomib however recent study wherein myeloma cells treated bortezomib combination autophagy inhibitor 3-methyl adenine 3-ma resulted antagonistic response instead expected synergizing effect research last decade contributed highlight important role er stress autophagy maintenance cellular homeostasis the last years also evidenced processes closely related signalling routes activated er stress response involved stimulating autophagy intriguingly for example accumulation unfolded proteins neurodegenerative diseases may activate protective autophagy response by contrast induction er stress cancer cells may promote stimulation autophagy mediated cell death activation protective autophagy may contribute resistance certain antitumoral therapies thus different factors intensity er stress signal simultaneous activation additional pathways cell type forth must integrated yield specific autophagic response considering escape drug mediated cell killing one major obstacles current cancer therapy better understanding role played processes cancer cells response chemotherapy would help us devise new efficient therapeutic opportunities utilizing inhibitors activators er stress pathways

different physiological and pathological conditions can perturb protein folding in the endoplasmic reticulum , leading to a condition known as er stress . er stress activates a complex intracellular signal transduction pathway , called unfolded protein response ( upr ) . the upr is tailored essentially to reestablish er homeostasis also through adaptive mechanisms involving the stimulation of autophagy . however , when persistent , er stress can switch the cytoprotective functions of upr and autophagy into cell death promoting mechanisms . recently , a variety of anticancer therapies have been linked to the induction of er stress in cancer cells , suggesting that strategies devised to stimulate its prodeath function or block its prosurvival function , could be envisaged to improve their tumoricidial action . a better understanding of the molecular mechanisms that determine the final outcome of upr and autophagy activation by chemotherapeutic agents , will offer new opportunities to improve existing cancer therapies as well as unravel novel targets for cancer treatment .

bevacizumab became standard care first line treatment metastatic colorectal cancer mcrc association chemotherapy patients benefit bevacizumab treatment cumulative data gathered retrospective studies show bevacizumab induced hypertension htn appears associated statistically significant improvement response rates progression free survival pfs overall survival os bevacizumab related htn may serve predictor biomarker response survival patients mcrc treated bevacizumb first line setting in subset patients htn g2 3 demonstrated predictive response treatment bevacizumab pfs trend towards favourable os observed these results strengthen previous evidence bevacizumab related htn may potential predictive biomarker bevacizumab efficacy in context exponential increase economic treatment costs contributed targeted therapy identification reliable non invasive feasible costly predictive factor would allow selection patients tumours likely benefit treatment spare would additional toxicity great value clinical practice colorectal cancer crc incidence mortality rates vary markedly around world globally crc third commonly diagnosed cancer males second females.1 approximately 25% patients present initial stage iv disease almost half patients crc develop metastases crc related 5-year survival rate approaching 60%.2 within past 10 years increase number chemotherapeutic agents available treat disease seen novel biologically targeted therapies interfere specific molecular pathways cancer proliferation metastasis developed.3 vascular endothelial growth factor vegf key mediator angiogenesis plays pivotal role pathogenesis wide range human cancers potential target new anticancer therapies bevacizumab humanised anti vegf monoclonal antibody became standard care first line treatment mcrc showing effectivity treatment mcrc improvement response rates survival outcomes.46 although patients benefit bevacizumab selection profit therapy still difficult biomarker yet identified allows us predict benefit association bevacizumab chemotherapy mcrc different biomarkers b raf k ras mutation microvessel density vegf receptor vegfr expression widely studied none proven reliable predictive factor.7 8 hypertension htn common complication treatment bevacizumab easily manageable antihypertensive agents the pathogenesis vegf signal inhibition induced htn still unclear reports suggesting impaired angiogenesis endothelial dysfunction associated bevacizumab treatment may responsible.5 9 fact vegf signal antagonism may lead inhibition nitric synthase consequent decrease nitric oxide level turn leads vasoconstriction decrease sodium renal excretion ultimately resulting htn.9 cumulative data recent studies suggested htn may biomarker efficacy vegf signal inhibition namely bevacizumab due underlying mechanism action retrospective studies shown patients develop bevacizumab induced htn showed increased response rates progression free survival pfs comparison these findings build hypothesis bevacizumab induced htn may represent predictive prognostic biomarker patients advanced colorectal cancer receiving first line bevacizumab.5 10 11 aim study retrospectively evaluate htn grades 2 3 correlated response treatment bevacizumab first line well improved pfs overall survival os series patients mcrc eligible patients 18 years age older histologically proven mcrc measurable disease receiving bevacizumab first line therapy dose 5 mg kg every 2 weeks association irinotecan 5-fluorouracil folinic acid according folfiri regimen oxaliplatin 5-fluorouracil folinic acid according folfox regimen tumour response evaluation performed use ct mri depending imaging methods used baseline blood pressure measurements recorded infusion bevacizumab nurses daily record blood pressure values carried patient the highest value arterial blood pressure recorded taken account define grade bevacizumab induced arterial htn according grading national cancer institute grade 23 arterial htn cut level htn definition accordance previous reports indicating level biologically clinically relevant.3 9 patients stratified two groups present htn grade 01 presented bevacizumab related htn grade 23 tumour response progression assessed radiologists home institution according response evaluation criteria solid tumors v.1.1 every 8 weeks response evaluated independent radiologists assessment performed clinical trial setting daily clinical practice context the best tumour response taken account define responders patients showing complete partial response non responders progressive disease disease control dc defined patients achieved complete response partial response stable disease the disease staged time diagnosis according guidelines american joint committee cancer v.7 the primary end point objective response rate defined number patients complete partial response divided number patients evaluated secondary efficacy end points duration pfs defined interval start bevacizumab therapy clinical progression death cause last follow visit progressed os defined interval start bevacizumab therapy death last follow visit odds ratios used compare relative odds response disease control given development bevacizumab related htn data patients lost follow censored time last evaluation a significant level 0.05 chosen assess statistical significance determine htn independent predictive factor response treatment bevacizumab cox regression model used adjusted age eastern cooperative oncology group ecog ps performance status kras status eligible patients 18 years age older histologically proven mcrc measurable disease receiving bevacizumab first line therapy dose 5 mg kg every 2 weeks association irinotecan 5-fluorouracil folinic acid according folfiri regimen oxaliplatin 5-fluorouracil folinic acid according folfox regimen tumour response evaluation performed use ct mri depending imaging methods used baseline blood pressure measurements recorded infusion bevacizumab nurses daily record blood pressure values carried patient the highest value arterial blood pressure recorded taken account define grade bevacizumab induced arterial htn according grading national cancer institute grade 23 arterial htn cut level htn definition accordance previous reports indicating level biologically clinically relevant.3 9 patients stratified two groups present htn grade 01 presented bevacizumab related htn grade 23 tumour response progression assessed radiologists home institution according response evaluation criteria solid tumors v.1.1 every 8 weeks response evaluated independent radiologists assessment performed clinical trial setting daily clinical practice context the best tumour response taken account define responders patients showing complete partial response non responders progressive disease disease control dc defined patients achieved complete response partial response stable disease the disease staged time diagnosis according guidelines american joint committee cancer v.7 the primary end point objective response rate defined number patients complete partial response divided number patients evaluated secondary efficacy end points duration pfs defined interval start bevacizumab therapy clinical progression death cause last follow visit progressed os defined interval start bevacizumab therapy death last follow visit odds ratios used compare relative odds response disease control given development bevacizumab related htn data patients lost follow censored time last evaluation a significant level 0.05 chosen assess statistical significance determine htn independent predictive factor response treatment bevacizumab cox regression model used adjusted age eastern cooperative oncology group ecog ps performance status kras status since january 2008 december 2013 79 patients records evaluated median follow time 19 months seventeen patients excluded present consistent record blood pressure monitoring patients characteristics according development bevacizumab related hypertension presented table 1 patients characteristics arb angiotensin receptor blockers htn hypertension n applicable os overall survival the median age diagnosis similar two groups 60 years predominance male gender observed arms 68.2% vs 65.8% all patients evaluated terms ps presenting ecog ps 0 1 the majority patients 85.4% group developed bevacizumab related htn ecog ps 1 group almost 66% presented ecog ps 0 the majority patients primary tumour colon 63% groups hepatic lung metastases frequently seen arms similar number patients presenting liver lung metastases peritoneal lymph node bone ovarian and/or penile metastases observed although lesser extent group patients developed bevacizumab related htn more patients seem previously submitted surgery primary 56.1% vs 36.8% colon 19.5% vs 10.5% rectum a similar proportion patients received chemotherapy stage ii iii crc groups the chemotherapy regimen often used association bevacizumab first line treatment mcrc undoubtedly folfiri 90% patients arms received treatment the majority patients arms evaluated kras status 14 group developed bevacizumab related htn 15 group presenting kras mutation nras status evaluated minority patients patients nras wild type about 40% patients groups presented previous diagnosis htn managed medical treatment forty one patients 51.9% developed htn g2-g3 chemotherapy bevacizumab treatment 35 patients 85.4% grade 2 6 patients 14.6% grade 3 thirty eight patients 92.7% beginning treatment adequate antihypertensive therapy maintained bevacizumab progression 3 suspended 2 developed thrombotic events 1 fistula all patients evaluated terms response 5 patients 6.3% presented complete response cr 32 patients 40.5% partial response pr 28 patients 35.4% stable disease group patients developed bevacizumab related htn 30 73.2% presented response treatment complete response cr)+partial response pr 40 97.6% achieved dc cr pr stable disease compared 7 patients 18.4% response 24 63.2% dc group develop htn 12.08 95% ci 4.13 35.29 p<0.001 responders vs non responders 20.8 95% ci 2.56 168.91 p 0.005 controlled vs non controlled disease figures 1 2 htn g2 3 independent predictor progression free survival adjusted factors os see online supplementary data number patients response groups grade 23 bevacizumab induced htn without bevacizumab related htn number patients disease control groups grade 23 bevacizumab induced htn without bevacizumab related htn a significant statistical difference obtained pfs two groups p<0.001 figure 3 the median os 28 months 22.733.3 group developed bevacizumab related htn the median os 33 months 25.740.3 group 21 months 16.525.5 significant statistical difference 2 groups p 0.114 figure 4 pfs patients colorectal cancer grade 23 bevacizumab induced htn without bevacizumab related htn p<0.001 os patients colorectal cancer grade 23 bevacizumab induced htn without bevacizumab related htn p 0.114 target therapies like bevacizumab affect tumour growth targeting specifically tumour activated biological pathways new toxicity profiles new agents emerged acneiform skin rash anti egfr therapy vascular abnormalities associated anti vegfr agents.12 htn common adverse event treatment antiangiogenic therapy bevacizumab correlated biological inhibition vegf related pathway may represent possible clinical marker treatment efficacy analogously demonstrated skin rash cetuximab.3 13 cumulative data gathered retrospective studies show bevacizumab induced htn appears associated statistically significant improvement response rates pfs os raising hypothesis bevacizumab related htn may serve predictor biomarker response survival patients mcrc treated bevacizumb first line setting scartozzi et al12 conducted retrospective study included 39 patients mcrc treated bevacizumab part front line therapy twenty per cent patients developed grades 23 htn significant improvement response rate 73.2% vs 18.4% p<0.001 pfs 14.5 months vs 3.1 months p<0.001 observed group patients developed bevacizumab related htn statistically significant difference median os sterlund et al14 study 56% patients presented htn results showing htn predicted bevacizumab treatment efficacy regardless analysed end point os pfs rr early htn defined htn within 3 months treatment initiation shown predictive os study similar data metastatic renal cell carcinoma breast cancer lung cancer also published.1517 dahlberg et al16 showed htn within 1 month bevacizumab therapy lung cancer predictive survival in study 51.9% patients developed htn g2-g3 chemotherapy bevacizumab treatment superior reported retrospective trials.6 12 16 according recently published large meta analysis incidence htn bevacizumab treated patients cancer 23.6%.18 possible explanation may study population presents high predominance male gender frequently affected primary htn suggesting bevacizumab related htn may present biological pathway increase may possibly due selection bias since retrospective trial our results seem line published data patients developed bevacizumab related htn showing improvement clinical outcomes namely response rates 73.2% vs 18.4% p<0.001 responders vs non responders disease control 97.6% vs 63.2% p 0.005 controlled disease vs non controlled pfs p<0.001 group no statistical difference median os observed 33 months vs 21 months p 0.114 although trend towards favourable outcome seen patients bevacizumab induced htn htn g2 3 independent predictor progression free survival adjusted factors age ecog ps kras status os this apparently conflicting result might explained fact htn important risk factor cardiovascular disease associated increase cardiovascular mortality possibly counteracting long term potential beneficial impact os the fact patients submitted surgery primary tumour group patients developed bevacizumab related htn may reflect population less advanced disease diagnosis only minority patients evaluated nras status explained fact mutation analysis became available clinical use institution recently the fact retrospective analysis majority previous studies subject presents limitations lack randomisation presenting possible selection bias accuracy data carried retrospective fashion the small sample size may also limitation although sample larger previous ones reported scartozzi sterlund would interesting perform analysis potential effect antihypertensive medication treatment efficacy bevacizumab treated mcrc owing sample size authors adequate statistical power evaluate hypothesis it known inflammation might affect blood pressure however owing fact c reactive protein level baseline systematically evaluated patients since normally requested infectious setting effect could assessed the accumulating evidence seems imply identification reliable clinical factor bevacizumab induced grades 23 arterial htn may constitute early indicator efficacy whereas lack side effect could represent warning lack activity maybe justify early change treatment strategy example egfr inhibitors.9 10 context exponential increase economic treatment costs contributed targeted therapy identification reliable non invasive feasible costly predictive factor would allow selection patients tumours likely benefit treatment spare would additional toxicity urgent.3 19 conclusion subset patients htn g2 3 demonstrated predictive response treatment bevacizumab pfs bevacizumab treatment related htn interesting subject moment prospective studies disclose whether bevacizumab associated htn predicts outcome exact mechanism development anti vegf related htn needed

backgroundbevacizumab has become standard of care as first - line treatment of metastatic colorectal cancer ( mcrc ) , after proving increased response rates and improvement in survival outcomes . hypertension ( htn ) is a common complication of the treatment with bevacizumab and , owing to its close relation with antiangiogenic mechanism , may represent a clinical biomarker to predict the efficacy of the treatment . the aim of this study was to retrospectively evaluate if htn grades 2 to 3 were correlated with response to treatment with bevacizumab in first line , as well as with improved progression - free survival ( pfs ) and overall survival ( os ) , in a series of patients with mcrc.methodsretrospective evaluation of clinical records of patients with histologically proven mcrc , treated with bevacizumab as first - line treatment , between january 2008 and december 2013.results79 patients were evaluated . 51.9% of patients developed htn g2-g3 during chemotherapy - bevacizumab treatment . in the group of patients who developed bevacizumab - related htn , 73.2% showed response to treatment ( complete response ( cr)+ partial response ( pr ) ) and 97.6% achieved disease control ( cr , pr or stable disease ) compared to 18.4% of patients with response and 63.2% with disease control in the group that did not ( or 12.08 ; 95% ci 4.13 to 35.29 ; p<0.001 responders vs non - responders ; or 20.8 ; 95% ci 2.56 to 168.91 ; p 0.005 controlled vs non controlled disease ) . the median os was 28 months ( 22.733.3 ) . significant statistical difference was obtained in pfs between the two groups ( p<0.001 ) . in the group that developed bevacizumab - related htn , the median os was 33 months ( 25.740.3 ) , and in the group that did not , it was 21 months ( 16.525.5 ) with no significant statistical difference between the two groups ( p 0.114).conclusionsin this subset of patients , htn g2 - 3 was predictive of response to treatment with bevacizumab and of pfs but not of os .

need advance saliva research strongly recognized strategic plan national institute dental craniofacial research the ability monitor health status disease onset progression recurrence treatment outcome non invasive means highly important advancing health care management saliva oral fluid perfect medium explored offering potential non invasive easy obtain means detecting monitoring disease the adoption saliva testing would allow patient collect specimens home yielding savings health costs convenience patient facilitating multiple sampling specimen collection less objectionable patients case bodily fluids easier children older individuals the analysis saliva thus provide cost effective approach screening large populations due significant advantages developing biomarkers saliva detection serious illnesses oral systemic cancers national healthcare agenda several years government performance results act 2008 one mandate formulated government performance results act report year 2013 proof principle obtained ability saliva monitor health diagnose one systemic disease a vast amount saliva omics data generated recent studies using high throughput technologies however still barriers researchers must overcome data exploited lack computationally accessible salivary data information inability cross reference salivaomics data could potentially made available different proteomics transcriptomics genomics metabolomics studies reasons urgent need create salivaomics knowledge base skb data management system web resource constructed support saliva diagnostics research present informatics advances brought skb associated tools resources ontologies controlled structured vocabularies designed provide consensus based means ensure consistent description data scientists working disparate domains applied biomedical domain ontology plays key role providing consensus based controlled vocabularies serving consistent annotation biological medical data information conspicuously within framework gene ontology sister ontologies within open biomedical ontologies foundry http://obofoundry.org the basic formal ontology bfo formal ontological framework developed barry smith pierre grenon others serves starting point 100 ontology projects primarily biomedical domain http://www.ifomis.uni-saarland.de/bfo/ the bfo framework readily extended treatment families ontologies types treatment relations ontologies different levels granularity genes species single patient epidemics geographical scale combining applications bfo medical geographical domain the framework may also used tool dealing relations distinct perspectives biomedical domain including culturally generated perspectives sort studied linguists anthropologists two bfo based ontologies special significance work ontology biomedical investigations obi ontology general medical sciences the obi ontology designed serve coordinated representation designs protocols instrumentation materials processes data types analysis areas biological biomedical investigation ontology general medical science ontology entities involved clinical encounter thus includes general terms used across medical disciplines including disease disorder disease course diagnosis patient healthcare provider to advance consistency data dental research community smith et al propose approach building consensus based ontology support dental research odr in analogy efforts fields consortium research groups specializing different areas study would undertake effort building different components ontology support dental research initial efforts direction scientists dental research biomedical ontology university buffalo university california include work ontology oral pathology oral maxillofacial anatomy dental disease dental procedures discuss saliva ontology integral work plan allow seamless connection use ontology support dental research dental domain use existing ontology resources developed areas biology medicine reusing elements strategies the work dental diseases carried conjunction development ontology general medical science the saliva ontology salo figure 1 detailed ontology bodily fluid optimized meet needs clinical diagnostic community cross disciplinary community omics researchers the salo created cross disciplinary interaction saliva experts protein experts diagnosticians ontologists aid development testing salo develop corpus saliva relevant literature skb assist characterizing core terms synonyms within ontology provide links salo content relevant items pubmed skb also incorporate results experiments data text mining using ontology salo incorporate links existing ontologies terminology resources involving treatment saliva relevant phenomena we also identify represent within salo relationships saliva relevant types represented ontologies gene ontology protein ontology chemical entities biological interest ontology also provide links corresponding snomed ct terms available each term ontology url points webpage providing definitions pubmed sources references annotations skb external databases it cross platform supports many popular relational database managements systems including mysql oracle postgresql sql server db2 the software data agnostic therefore easily adapted existing data sets it expandable customizable plug system open source community participate deeper development furthermore biomart seamlessly connect geographically disparate databases facilitating collaboration different groups these features catalyzed creation biomart central portal first kind community supported effort create single access point integrating many different independently administered biological databases anybody contribute independently maintained resource central portal allowing exposed shared research community linking resources portal users take advantage common interface quickly utilize different sources without learning new system the system also simplifies cross database searches might otherwise require several complicated steps several integrated tools streamline common tasks converting formats retrieving sequences the combination wide variety databases easy use interface robust programmatic access array tools make central portal one stop shop biological data querying sdxmart biomart data portal hosts salivary proteomic transcriptomic metabolomic microrna data offers access data using biomart interface querying environment the sdxmart designed provide variety queries facilitate saliva biomarker discovery including complex queries integrate genomic clinical functional information the sdxmart holds data projects oral diseases systemic diseases including oral cancer sjgren syndrome pancreatic cancer breast cancer the types datasets proteomics ii transcriptomics iii microrna iv metabolomics in addition sdxmart imported several public databases including ensembl genome database ensembl release 37 number resources continuously growing it built tandem salo sdxmart allow skb interoperate omics databases part general strategy facilitate integration heterogeneous disparate data sources enable system biology approaches either salo sdxmart first resource kind field dentistry

there is a need recognized by the national institute of dental & craniofacial research and the national cancer institute to advance basic , translational and clinical saliva research . the goal of the salivaomics knowledge base ( skb ) is to create a data management system and web resource constructed to support human salivaomics research . to maximize the utility of the skb for retrieval , integration and analysis of data , we have developed the saliva ontology and sdxmart . this article reviews the informatics advances in saliva diagnostics made possible by the saliva ontology and sdxmart .

emergence new concepts management oncologic malignancies fast changing trends modern medicine necessitate development strategies customized treatment suited patient variable requirements response chemotherapy predicted certainty individual presently moreover histopathological changes chemotherapy vary patients studies systematically described various histopathological changes seen neoadjuvant chemotherapy variety tumors including breast carcinoma rectal carcinoma ovarian carcinoma head neck carcinomas esophageal carcinoma wilms tumor non small cell lung carcinoma however emphasis studies varied study described pathologic changes following chemotherapy comprehensively this study describes various histopathologic changes seen neoadjuvant chemotherapy breast malignancies squamous cell carcinomas adenocarcinomas wilms tumor the present prospective study carried 60 patients including 40 patients carcinoma breast 20 patients malignancies received neoadjuvant chemotherapy study period one half years total 355 patients treated breast carcinoma neoadjuvant chemotherapy these 40 patients fulfilling inclusion criteria per protocol thesis project included study inclusion criteria patients prechemotherapy clinicoimaging assessment tumor size established histological diagnosis carcinoma biopsy received least two cycles neoadjuvant chemotherapy the study group divided three subgroups group n 30 group b n 10 included breast carcinoma patients initial pathologic material submitted trucut wedge biopsy group lumpectomy specimens group b. group c n 20 included cases malignancies besides breast carcinoma received neoadjuvant chemotherapy initial tissue diagnosis patients clinically examined detail investigated including radiological imaging laboratory tests decide stage cancer the initial biopsies subjected routine formalin fixation paraffin processing microscopic analysis hematoxylin- eosin stained sections supplemented special stains including immunohistochemistry decide histological type grade tumor according world helath organization classification detailed histopathological examination carried especially looking chemotherapy induced histopathologic changes like necrosis fibrosis inflammatory reactions retrogressive changes carcinoma breast neoadjuvant chemotherapy regimens either included cyclophosphamide 50 60 mg m2 iv iv intravenous doxorubicin 40 50 mg m2 iv 5-fluorouracil 500 800 mg m2 iv caf cyclophosphamide epirubicin 5-fluorouracil cef 21 days apart number chemotherapy cycles varied two six depending initial size tumor make operable the drugs doses neoadjuvant chemotherapy given patient recorded pro forma the tumor cells evaluated dissociation dyscohesion loss organization tumor cells necrobiotic changes necrosis vacuolation nucleus cytoplasm karyorrhexis pyknosis karyolysis change pattern type carcinoma noted the stroma examined host response including fibrosis elastosis collagenization infiltration lymphocytes plasma cells fibroblasts histiocytes giant cell formation observed epithelial stromal ratio calculated mean readings sections viable nonviable tumor cell ratios calculated pretreatment biopsies postchemotherapy specimens viability defined distinct nuclear chromatin intact nuclear cytoplasmic membrane absence criteria necrosis karyorrhexis karyolysis pyknosis a note made tumor type change chemotherapy tumor grading done based elston ellis modification bloom richardson mrb grading system nottingham prognostic index npi calculated lymphocytic response graded grade 1 scattered lymphocytes tumor cells grade 2 formation microaggregates lymphocytes grade 3 dense infiltration lymphocytes destroying tumor cells forming masses the presence lymphovascular embolization situ disease cancerization ducts separately noted twenty cases malignancies group c besides breast included 12 cases squamous cell carcinoma seven head neck four cervix one esophagus five cases adenocarcinoma three wilms tumor neoadjuvant chemotherapy squamous cell carcinoma head neck included chemotherapy based docetaxel cisplatin 5-fluorouracil tpf regimen patients carcinoma cervix received paclitaxel carboplatin regimen esophageal carcinoma cisplatin 5-fluorouracil given chemotherapy given ovarian carcinoma paclitaxel carboplatin patients anorectal carcinoma received 5-fluorouracil- oxaliplatin based regimen folfox children wilms tumor received vincristine dactinomycin cycles varying three four cycles considered appropriate reducing bulk tumor histologic sections malignancies observed change differentiation changes architecture necrobiotic changes host tissue response pearson coefficient correlation applied correlate three types response patterns parameters observed breast tumors showed complete response neoadjuvant chemotherapy included five ductal carcinoma otherwise specified nos type one schirrhous apocrine tubular types apocrine change tubular papillary pattern disappeared postchemotherapy specimens mucinous change metaplastic change appeared two ductal nos types ductal nos metaplastic change differentiated completely metaplastic mastectomy specimens table 1 carcinoma breast histological types tumor chemotherapy number cases metastasis 22 number lymph nodes metastatic deposits 88 the common pathologic changes noted necrosis elastosis collagenization lymphocytic response relation lymphocytic response clinical pathologic response irrespective presence pre postchemotherapy specimens giant cell response significantly correlated types response grades p 0.05 collagenization significantly correlated pathologic tumor regression grade p 0.05 tables 2 3 carcinoma breast inflammatory stromal response seen neoadjuvant chemotherapy carcinoma breast inflammatory stromal response seen neoadjuvant chemotherapy axillary lymph nodes present study total 20 cases group c 12 cases squamous cell carcinoma seven head neck four cervix one esophagus five cases adenocarcinoma three cases wilms tumor of 12 cases squamous cell carcinoma present study improvement histological differentiation moderate well differentiated carcinoma two cases head neck cancers whereas complete pathologic disappearance tumor another two cases comprising one case carcinoma tongue cervical carcinoma other histological changes observed increase keratinization formation keratin pearls acellular keratin islands nonviable tumor cells histiocytic giant cells increase lymphocytes surrounding residual tumor cells of five cases adenocarcinoma change histological differentiation seen case anorectal carcinoma revealed poor differentiation solid sheets small amount mucin initial biopsy moderately differentiated mucin secreting adenocarcinoma large mucin pools chemotherapy serous papillary adenocarcinoma ovary degenerative changes observed karyorrhexis pyknosis smudging dyscohesion loss papillary architecture areas single case esophageal adenocarcinoma increase signet ring cells adenocarcinoma histological differentiation chemotherapy three wilms tumors one biphasic two triphasic in two cases revealing triphasic wilms tumor pretreatment biopsy blastemal component decreased marked necrobiotic changes totally disappeared one biphasic wilms tumor mesenchymal component revealed rhabdoid differentiation one chondroid differentiation presence smooth skeletal muscles in present prospective study spectrum histopathologic changes observed use chemotherapy the effects divided two pathologic changes tumor cells changes stroma tumor cells nuclear enlargement nuclear shrinkage necrosis vacuolation nucleus cytoplasm pyknotic nuclei degenerative changes described literature.[159 present study loss architecture dyscohesion shrinkage tumor cells retrogressive changes like karyorrhexis karyolysis pyknosis observed addition mentioned changes view presence large variety retrogressive changes the emphasis trying recognize viable tumor cells morphologically identified cells distinct nuclear chromatin intact nuclear cytoplasmic membrane absence criteria necrosis karyorrhexis karyolysis pyknosis necrosis common event observed stroma fibrosis elastosis collagenization hyalinization microcalcification neovascularization observed described.[56813 present study addition prominent findings elastosis/ collagenization stroma hyalinization walls blood vessels atrophy adjacent breast parenchyma cancerization ducts even atrophic lobules collagenization found significantly correlated pathologic response tumor regression grade p 0.05 lymphocytic reaction presence plasma cells macrophages formation histiocytic giant cells observed many studies may indicative host tissue response necrobiotic tumor.[5101214 common inflammatory host response observed present study lymphocytic others included mixed inflammation plasmacytic prominent histiocytic giant cell types giant cell reaction significantly correlated types tumor responses p 0.05 pathologically similar changes observed response chemotherapy draining lymph nodes pronounced primary site though changes observed many workers positive correlation presence changes effect chemotherapy significant many studies according the presence fibrosis elastosis collagenization hyalinization necrosis inflammatory infiltrate lymphocytic response giant cell reaction plasma cells foamy macrophages microcalcification neovascularization significantly related response chemotherapy honkoop et al concluded none pretreatment pathologic biologic characteristics predictive good pathologic response present study the common type ductal carcinoma nos mucinous change appeared three patients chemotherapy diagnosed ductal carcinoma nos type initial biopsy transformation ductal metaplastic carcinoma papillary pattern ductal nos seen one case a change either higher lower grade noted rasbridge et al complete response seen one case apocrine carcinoma disappearance apocrine component mixed ductal apocrine differentiation another the response case ductal carcinoma nos significantly different response invasive lobular carcinoma present study the clinical pathologic response found significant correlation prechemotherapy grade however significant correlation observed postchemotherapy grade pathologic tumor regression grades clinical response so better response chemotherapy observed poorly differentiated malignancies higher mrb npi grades poorer response well differentiated ones p 0.05 figure 1 in contrast sanchez et al observed poor response poorly differentiated carcinomas carcinoma breast occasional nests viable tumor cells left post chemotherapy h e 100 post neoadjuvant chemotherapy hyalinization blood vessel wall common finding the adjoining breast showed atrophy parenchyma even terminal duct lobular units tdlus revealed ductal carcinoma situ dcis)/ cancerization ducts surviving tumor cells cases no correlation size response grades observed size reduction chemotherapy seen small large tumors restricted one group in squamous cell carcinoma changes observed improvement histologic differentiation complete pathologic disappearance tumor increase keratinization formation keratin pearls acellular keratin islands nonviable tumor cells histiocytic giant cells increase lymphocytes surrounding residual tumor cells cases figure 2 in adenocarcinoma improvement histologic differentiation seen large mucin pools chemotherapy degenerative changes observed serous papillary adenocarcinoma ovary increase signet ring cells noticed esophageal carcinoma figure 3 wilms tumor almost complete disappearance blastemal component retrogressive changes epithelial component squamous cell carcinoma chemotherapy necrotic masses keratin areas calcification left h e 100 adenocarcinoma abundant pools mucin occasional scattered tumor cells chemotherapy h e 40 wilms tumor bizarre tumor cells persisting along mesenchymal component complete disappearance blastemal component triphasic tumor chemotherapy h e 200 results study reveal response chemotherapy may markedly variable patients desired response some may achieved fewer number cycles whereas patients tumor may resist even maximum number neoadjuvant chemotherapy cycles presently employed thus defeating purpose even potential risk toxicity it also concluded tumor grade decreases differentiation improves addition retrogressive changes increase stromal component result chemotherapy carcinoma breast well malignancies

background : various histopathological changes have been observed following neoadjuvant chemotherapy in individual tumors in the literature.aims and objectives : to observe histopathologic changes seen after neoadjuvant chemotherapy in breast malignancies , squamous cell carcinomas , adenocarcinomas , and wilms tumor using breast cancer predominantly as the model.materials and methods : the present prospective study was carried out on 60 patients including 40 patients with carcinoma breast and 20 patients with other malignancies who received neoadjuvant chemotherapy.results:post neoadjuvant chemotherapy , mastectomy specimens revealed nuclear enlargement , nuclear shrinkage , necrosis , vacuolation of nucleus , vacuolation of cytoplasm , dyscohesion , and shrinkage of tumor cells with nuclear changes of nonviability like karyorrhexis , karyolysis , and pyknosis . stromal reactions manifested as fibrosis , elastosis , collagenization , hyalinization , microcalcification , and neovascularization . areas of necrosis included both vascular and avascular pattern . the stroma also revealed fibrinoid necrosis and mucinous change . hyalinization of the blood vessel wall was a common finding . the most common inflammatory host response observed in the present study was lymphocytic ; others included mixed inflammation , plasmacytic , prominent histiocytic , and giant cell types . giant cell reaction was significantly correlated to all types of tumor responses ( p < 0.05 ) . similar changes were also observed in other malignancies . a detailed review of the literature has also been done and presented.conclusion:the tumor grade decreases and differentiation improves , in addition to the retrogressive changes and increase in stromal component , as a result of chemotherapy in carcinoma breast as well as in other malignancies .

acute myeloid leukemia aml clinically morphologically genetically heterogeneous disease characterized clonal expansion myeloid blasts peripheral bone marrow bm blood tissues the incidence aml increases age particularly 65 years old median age diagnosis around 70 years age one powerful prognostic factors survival aml elderly patients present comorbidities worse performance status also adverse molecular features compared younger patients due presence comorbidities poor performance status intensive chemotherapy ic or allogeneic stem cell transplant often unsuitable large proportion elderly patients aml resulting early mortality prolonged hospitalization however feasible treatment associated better survival compared best supportive care median overall survival achieved ic elderly 513 months therefore increasing need new treatment options elderly patients aml the aza-001 phase iii trial demonstrated effectiveness azacitidine aza dose 75 mg m2 daily 7 consecutive days patients high risk myelodysplastic syndromes subset analysis aza-001 trial restricted patients 2030% bm blasts demonstrated superior efficacy aza compared conventional care regimens leading aza international approval treatment low bm blast count 2030% defined aml these results raise question potential use aza aml irrespectively bm blast count this supported identification somatic mutations genes involved epigenetic regulation approximately 30% aml patients consequently several groups assessed reported efficacy aza patients aml recently randomized phase iii trial comparing first line aza conventional therapies including intensive chemotherapy elderly patients aml revealed aza achieved clinically meaningful survival benefit compared conventional treatments we present herein multi centre retrospective analysis 77 aml patients treated aza first line due relapsed refractory disease the main aim characterization efficacy safety aza treatment aml patients portugal we also conducted exploratory comparison overall survival patients receiving 1st line treatment aza elderly aml patients treated ic alone patients aml received least 1 cycle aza selected inclusion the comparator group comprised 50 matched patients historical cohort aml patients single institute patient data collected retrospectively patient files 4 portuguese institutions following written informed consent the study approved ethical committee instituto portugus de oncologia de lisboa francisco gentil epe data treated according declaration helsinki overall survival os defined time start treatment aza either 1st 2nd line ict 1st line death cause last follow progression free survival pfs defined time start treatment disease relapse progression death cause survival curves different treatment groups compared using log rank test univariate analysis all tests two tailed p values less 0.05 considered statistically significant seventy seven patients treated aza 50 elderly aml patients treated ict included 1line n=51ict n n=50p value aza1st line vs ictage diagnosis69 years 2789]73 years 4689)74.5 years 6686)0.015median min max]33 65% 70yrs41 82% 70yrsgendermale54 70%)36 72%)28 56%)0.13female23 30%)15 29%)22 44%)percentage blasts start treatmentmedian min max]32 093]35 2092]50 20100]0.06mean standard deviation)37 23)40 20)52 30)haemoglobin10 g dl58 75%)38 74.5%)38 76%)0.39wbc x109/l)0.72 0.01 30.60]0.71 0.03 17.05]0.81 0.01 23.91]0.56neutrophils0.5 x10/l26 34%)20 39%)13 26%)0.05platelets50 x10/l34 44%)23 45%)24 48%)0.59aml subtypeprimary44 57%)33 64%)30 60%)0.21secondary33 43%)18 36%)20 40%)cytogenetics risk groupnormal36 47%)17 33%)16 32%)intermediate*22 29%)23 45%)17 34%)0.45poor**17 22%)11 21%)17 34%)unknown2 3%)*all others.**chromosome 7 3 abnormalities other higher age ict control group significant difference patients treated aza ict first line table 2treatment details.table 2aza n n=77aza 1line n n=51ict n n=50p valueaza1st line vs icttime diagnosis start aza months1.4 089.3]0.6 048.3]1.3 09.2]0.195median min max]first line treatmentaza n 51 100%)ict 3 7)24 31%)n adauno 90 arac 10010 20%)dauno 60 arac 10021 42%)ida 12 arac 1009 18%)total cyclesmedian min max]6 128]1.7 13)n aaza azacitidine ict intensive chemotherapy dauno 90+arac 100=daunorubicin 90 mg d1 3 cytarabine 100 mg d1 7 dauno 60+arac 100=daunorubicin 60 mg d1 3 cytarabine 100 mg d1 7 ida 12 arac 100=idarubicin 12 mg d1 3 cytarabine 100 mg d1 7 aza azacitidine ict intensive chemotherapy dauno 90+arac 100=daunorubicin 90 mg d1 3 cytarabine 100 mg d1 7 dauno 60+arac 100=daunorubicin 60 mg d1 3 cytarabine 100 mg d1 7 ida 12 arac 100=idarubicin 12 mg d1 3 cytarabine 100 mg d1 7 start aza and table 3response therapy.table 3n aza 1line n=51ict n n=50p value aza1st line vs ictresponse ratesoverall response pr+cr)34 44%)26 51%)38 76%)partial response10 31%)7 14%)11 22%)complete response24 13%)19 37%)27 54%)0.02stable disease22 29%)13 25%)progression refractory21 27%)12 24%)12 24%)transfusion independenceno44 57%)27 53%)yes30 39%)24 47%)n aunknown3 4% four 3.3% patients treated aza evaluable survival median follow time patients 12.4 months patients receiving aza first line treatment 6.9 months patients treated second subsequent lines period 1 survival estimates patients treated aza according line treatment fig median age diagnosis comparator group 74.5 years old range 6686 44% 22 patients female table 1 the median time diagnosis start ic comparator group 0.13 months range 09.2 months table 2 all patients treated oct alone received supportive care prior induction chemotherapy fig 4survival aml patients treated aza first line compared treated ict alone.fig 4 survival aml patients treated aza first line compared treated ict alone table 3response therapy.table 3n aza 1line n=51ict n n=50p value aza1st line vs ictresponse ratesoverall response pr+cr)34 44%)26 51%)38 76%)partial response10 31%)7 14%)11 22%)complete response24 13%)19 37%)27 54%)0.02stable disease22 29%)13 25%)progression refractory21 27%)12 24%)12 24%)transfusion independenceno44 57%)27 53%)yes30 39%)24 47%)n aunknown3 4% four 3.3% patients treated aza evaluable survival median follow time patients 12.4 months patients receiving aza first line treatment 6.9 months patients treated second subsequent lines period 1 survival estimates patients treated aza according line treatment fig median age diagnosis comparator group 74.5 years old range 6686 44% 22 patients female table 1 the median time diagnosis start ic comparator group 0.13 months range 09.2 months table 2 4survival aml patients treated aza first line compared treated ict alone.fig 4 survival aml patients treated aza first line compared treated ict alone their frailty imposes limitations upon aggressiveness treatment administered in addition poor prognostic disease features often seen population mean refractoriness relapse frequent treatments population require optimal balance tolerability efficacy the main aim cases prolong survival reduce impact cytopenias without undue toxicity aza demonstrated efficacy high risk mds reducing transfusion requirements prolonging survival this setting proven well tolerated including elderly based experience several groups tested efficacy safety aza elderly patients aml promising results our results line previous reports aza efficacious treatment aml regardless blast count the response rates overall survival observed study consonance reported groups it noteworthy rates cr obtained aza equivalent obtained published series elderly patients treated ic improved survival cohort significantly associated quality response including maintenance stable disease this suggests aza capable delaying progression thus prolong survival even hematological marrow improvement obtained this supports use population frank progression detected patient receives another definitive treatment allogeneic bone marrow transplant another significant finding analysis efficacy aza refractory relapsed following ic the fact survival start aza similar first second line patients indicates relapsed refractory patients benefit equally the comparison cohort treated aza first line comparator group treated ic highlights advantages brought former treatment modality it important note finding confirmed randomized trial however 41% trial patients treated aza following failure ict whereas comparator group never received aza as aza demonstrated efficacy second line therapy could explain difference findings the significantly lower survival ic cohort indicates population benefits less intensive treatment options target pathophysiological processes retrospective nature limited numbers patients in the collaborative nature study helps reduce bias may introduced single centre data however study adds new evidence use aza elderly patients aml indicates least inferior conventional ic the response rates survival data presented support use first line candidates ic second line refractory relapsed following ic this future collaborative studies add trial data better identify patients benefit new treatment modalities thus improving management elderly patients aml

retrospective data was collected from 77 elderly acute myeloid leukemia ( aml ) patients treated with azacitidine ( aza ) and 50 elderly aml patients treated with intensive chemotherapy ( ic ) from 4 portuguese hospitals.median os was 10.6 months in those receiving aza as 1st line . response ( overall response rate 44% ) had a significant impact on overall survival ( p=<0.0001 ) . median os of the comparator ic cohort was significantly inferior to that observed in the cohort treated with aza in first line ( p=0.0104).these results support the efficacy of aza in aml in the elderly in any line of treatment .

one important organ systems cardiovascular disorders specially ischemic heart diseases cause disability death some people specially workers work special industries risk disorders there many studies work workers health study cardiovascular system disorders one etiology one modifiable risk factors etiology work exposure according realities cardiovascular disorders risk factors assessed measured triglyceride total cholesterol low density lipoprotein ldl high density lipoprotein hdl blood pressure obesity smoking genetic also shift work stress carboxi hemoglobin cohb solvents noise vibration temperature environment air pollution can find high risk personnel non occupational occupational risk factors cardiovascular disorders exposure fumes solvents additives drugs carbon monoxide carbon disulfide pesticides heavy metals cold warm environment air pollution vibration noise cause occupational cardiovascular disorders had demonstrated work farmer heavy manual work exposure noise physical chemical cardiovascular disorders risk factors ghiasvand et al study shown work rail road industry risk factor cause hyper low density lipoprotein specially workers shift work study national heart lung blood institute shown work tunnel cause exposure carbon monoxide study foundry workers cause exposure kim et al study demonstrated work refrigerated industry risk factor workers work cold place environment diastolic blood pressure amount comparison exposure cold non exposure had shown work engine driver cardiovascular risk factors hypertension hyperlipidemia control group kang et al study found work companies related job mental stress specially background decision making related amount cholesterol triglyceride background demands work related systolic blood pressure chin ming lin et al demonstrated medical technicians hypertensive disorders healthcare workers also kim shown relationship cold exposure increasing blood pressure hypertension the objective study comparison cardiovascular risk factors workers different industries iran this study performed khorasan razavi tehran industries 2007 2011 workers divided three groups automobile industry work production saloon food industry light works office worker administrative healthcare sanitary workers cross sectional study simple randomized sampling method workers selected 0.05 0.80 p 25% population least 150 person group 10 factories automobile food industries light work this study done completing checklist medical issues workers validity reliability research tool checklist written improved educational department professors opinions pilot study correlation coefficient 85% used data gathered medical issues workers periodic physical paraclinic examinations blood pressure blood sampling triglyceride total cholesterol low density lipoprotein high density lipoprotein fasting blood sugar age body mass index work duration smoking shift work inclusion criteria least three year work duration industries exclusion criteria previous cardiovascular disorders dyslipidemias three groups observed age work duration body mass index shift work data gathered spss 11.5 analyzed calculation frequency mean anova quantitative variables chi-2 qualitative variables multi nominal logistic regression p 0.05 regression cardiovascular risk factors hypertension hyper triglyceridemia hyper cholesterolemia hyper low density lipoproteinemia ldl lower high density lipoproteinemia hdl diabetes mellitus smoking analyzed this study approved university board research ethics oral satisfaction told cumulative data used result without name industries this study performed khorasan razavi tehran industries 2007 2011 workers divided three groups automobile industry work production saloon food industry light works office worker administrative healthcare sanitary workers in cross sectional study simple randomized sampling method workers selected 0.05 0.80 p 25% population least 150 person group 10 factories automobile food industries light work this study done completing checklist medical issues workers validity reliability research tool checklist written improved educational department professors opinions pilot study correlation coefficient 85% used data gathered medical issues workers periodic physical paraclinic examinations blood pressure blood sampling triglyceride total cholesterol low density lipoprotein high density lipoprotein fasting blood sugar age body mass index work duration smoking shift work inclusion criteria least three year work duration industries exclusion criteria previous cardiovascular disorders dyslipidemias three groups observed age work duration body mass index shift work data gathered spss 11.5 analyzed calculation frequency mean anova quantitative variables chi-2 qualitative variables multi nominal logistic regression p 0.05 regression cardiovascular risk factors hypertension hyper triglyceridemia hyper cholesterolemia hyper low density lipoproteinemia ldl lower high density lipoproteinemia hdl diabetes mellitus smoking analyzed this study approved university board research ethics oral satisfaction told cumulative data used result without name industries results three sections general information specific information cardiovascular risk factors 875 workers industries 403 46.0% automobile industry 166 19% food industry 304 35% light works participated study in general mean age work duration body mass index smoking calculated the mean age automobile industry 34.87 6.19 years old food industry 34.13 8.64 years old light works 31.40 5.70 years old f 25.146 p 0.001 the mean work duration automobile industry 10.37 4.86 years food industry 5.94 2.46 years light works 5.16 1.98 years f 117.286 p 0.001 76 18.8% automobile industry workers smoker none food industry worker 11 3.6% light works smoker specific information mean systolic blood pressure diastolic blood pressure triglyceride total cholesterol low density lipoprotein ldl high density lipoprotein hdl fasting blood sugar measured table 1 these information demonstrated comparison related variables cardiovascular disorders industries workers comparison risk factors cardiovascular disorders industries workers number percent cardiovascular risk factors obesity hypertension hyper triglyceridemia hyper cholestrolemia hyper low density lipoprotein lower high density lipoprotein diabetes mellitus determined compared table 2 although person time automobile industry food industry light works 4179.11 986.04 1568.64 according results workers food industry healthy industries cardiovascular disorders mean high density lipoprotein hdl 63 mg dl preventive factor also according results automobile industry workers obesity hypertension hyper triglyceridemia hyper low density lipoprotein lower high density lipoprotein diabetes mellitus regression analysis hypertension hyper triglyceridemia lower high density lipoprotein significant difference automobile industries there results previous studies total cholesterol systolic diastolic blood pressure food industry workers normal range sancini et al demonstrated exposure noise physical chemical cardiovascular disorders risk factors had demonstrated work engine driver cardiovascular risk factors hypertension hyperlipidemia control group ghiasvand et al study shown work railroad industry risk factor cardiovascular diseases cause hyper low density lipoprotein total cholesterol blood specially workers shift work cardiovascular disorders hypertension cause stroke heart diseases relation long time disability work forbidden smoking control temperature popular advices managers industries healthy workers there fumes solvents stress heavy metals automobile industry affect cardiovascular system although controlled in study national heart lung blood institute shown work tunnel cause exposure carbon monoxide study foundry workers cause exposure but food industry temperature environment important risk factor controlled food industries iran the workers automobile industries work duration older body mass index also light works office worker administrative healthcare sanitary workers risk factors food industry workers may better health follow screening results screening cardiovascular disorders risk factors works industries helpful benefit according studies industries controls shift work stress noise vibration chemical exposures helpful study multi nominal logistic regression hypertension hyper triglyceridemia lower high density lipoprotein significant workers automobile industry automobile industry lot cardiovascular risk factors fumes chemicals physicals control reduce risk occupational cardiovascular disorders one ways control engineering method administrative end personal protective devices medical surveillance periodic examination for screening non occupational occupational disorders specially risk factors cardiovascular disorders helpful screening cardiovascular disorders risk factors important helpful industries specially automobile industry might preventive method disorders future

background : disorders of cardiovascular system can cause disability or death , screening is necessary specially in workers who maybe had risk factors . hypertension , hyperlipidemia , obesity , smoking , genetic , exposure to chemicals , fumes , solvents , coldness are non occupational and occupational risk factors . objective was comparison of cardiovascular disorders risk factors between workers in different industries of iran.methods:in a cross - sectional study , workers of automobile , food industries and light works had been selected and cardiovascular disorders risk factors had been gathered then data analyzed in spss with one - way anova , chi-2 and multi nominal logistic regression with p < 0.05.results:875 workers had been participated in the study , all of the cardiovascular disorders risk factors were in the normal range . mean of high density lipoprotein ( hdl ) in food industry workers was 63.83 17.42 mg / dl and it was protective , but in workers who work in automobile industry was 38.97 11.08 mg / dl and the lowest , also hypertension and hypertriglyceridemia were more prominent in this industry and after regression with p < 0.05 , the differences were significant.conclusions:screening of cardiovascular disorders risk factors were important and helpful in industries specially automobile industry , that might be preventive method for these disorders in the future .

vesicoureteral reflux vur occurs commonly children resulting potentially serious complications this could result renal scarring hypertension even end stage renal failure treatment options children vur include continuous antibiotic prophylaxis endoscopic injections laparoscopic open ureteroneocystostomy since hutch introduced open ureteral reimplantation correct vur 1952 many techniques developed 1984 endoscopic injections tissueaugmenting material correction vur introduced since many urologists prefer procedure endoscopic injection frequently used firstline treatment involves less pain shorter hospitalization period leaves scar contrast open reimplantation however injection treatment fails open ureteral reimplantation considered treating persistent vur we aimed test usefulness open reimplantations performed primarily failed endoscopic injection procedures we analyzed records 81 children underwent open reimplantation surgery 10 years among 64 underwent open reimplantation primary procedure group 17 underwent failed endoscopic injection group b we retrospectively analyzed clinical data groups including age sex preoperative reflux grade operation time laterality surgery postoperative complications hospitalization period all patients classed terms preoperative reflux grade using voiding cystourethrography vcug reflux grade classed grades v according international classification system international reflux study committee we classified patients reflux grades iii low grade reflux group patients grades iv v high grade reflux group success surgery defined resolution vur determined vcug 6 months surgery in addition conducted telephone based survey parents group b preference endoscopic injection open reimplantation reasons preference asked parents surgery choose primarily endoscopic injection ureteral reimplantation child undergo surgery vesicoureteral reflux reason statistical analysis performed using ibm spss statistics ver 20.0 ibm co. armonk student tests chi square tests applied compare parameters group p values 0.05 considered statistically significant the mean ages groups b 49.637.1 months 56.522.5 months respectively the male female ratios groups b 43:21 12:5 respectively there statistically significant differences age sex distribution 2 groups p=0.236 p=0.312 respectively the low grade reflux rates groups b 12 18.8% grade ii 2 3.2% grade iii 10 15.6% 8 47.1% grade ii 3 17.7% grade iii 5 29.4% respectively high grade reflux rates 52 81.2% grade iv 34 53.1% grade v 18 28.1% 9 52.9% grade iv 7 41.3% grade v 2 22.2% respectively patients high grade reflux significantly common group b p=0.022 6 months endoscopic surgery group b the numbers patients low- high grade reflux 10 58.8% grade ii 5 29.4% grade iii 5 29.4% seven 41.2% grade iv 6 35.3% grade v 1 5.9% respectively there significant change numbers primary endoscopic surgery group b p=0.738 however group still patients high grade reflux endoscopic surgery group b p=0.020 the operation times unilateral cases groups b 16736 minutes 15518 minutes respectively those bilateral cases groups b 21533 minutes 21623 minutes respectively the mean hospitalization periods groups b 10.45 days 8.82 days respectively there serious postoperative complications except bleeding occurred groups groups a b 7 11.0% 2 patients 11.9% respectively needed postoperative blood transfusions anemia however difference statistically significant p=0.107 6 months surgery resolution vur found patients underwent vcug survey parents children group b eleven parents 64.7% answered would choose endoscopic injection first situation the reasons included convenience endoscopic treatment need hospitalization involved less pain open surgery avoided discomfort urethral catheterization open surgery the main reason want children multiple procedures anesthesia among eleven parents chose primary endoscopic surgery 7 children low grade reflux 4 high grade reflux among 6 parents chose primary open surgery one child low grade reflux 5 child high grade reflux parents children low grade reflux preferred endoscopic surgery however statistically significant difference p=0.064 table 2 selecting treatment option different grades vur reflux depends clinical presentation renal function however minimally invasive treatments endoscopic injection advantages open surgery including less postoperative pain fewer bladder spasms infections absence surgical scarring furthermore procedure performed short operation time outpatient setting minimal use postoperative analgesics therefore endoscopic treatments preferred first line treatment children vur meta analysis elder et al showed one injection reflux resolution rate 78.5% grades ii reflux 73% grade iii 63% grade iv 51% grade v the american urological association aua vesicoureteral reflux guideline update committee analyzed data 17,972 patients reported overall success rate single endoscopic treatment 83.0% seen endoscopic treatment children vur many advantages high success rate also many failures especially children severe reflux first endoscopic treatment fails endoscopic treatment needs repeated reimplantation surgery indicated study analyzed characteristics patients underwent open reimplantation surgery patients underwent reimplantation primary treatment differ underwent endoscopic injection terms age sex operation time hospitalization period postoperative complications it generally assumed secondary operation failed primary procedure difficult complications open reimplantation vur after failed endoscopic surgery also supposed may show less success rate operation time complications hospitalization periods ureteral adhesion inflammation due primary injection patient severe vur however findings several studies reported previous endoscopic treatment adverse effect success subsequent open reimplantation many studies open reimplantation vur showed much higher success rates endoscopic treatment grades reflux aua guidelines success rate open surgical procedures 98.1% chertin et al reported success rate open reimplantation following failed deflux injections 100% similar study moreira pinto et al sencan et al showed previous endoscopic injections could cause difficulties dissection ureter fibrosis however alter success rate complications following open surgery parental preference major role selection endoscopic treatment compared long term antibiotic treatment survey 80% parents preferred endoscopic treatment rather antibiotic prophylaxis open surgery given option three treatment modalities this due failed cases endoscopic injection small high success rate endoscopic injection these patients received endoscopic injection ureteral reimplantation treat vur failed endoscopic injection according patients conditions parents demand therefore number patients underwent ureteral reimplantation little study analyzed patient matched anlaysis in addition follow duration surgery long several patients undergo follow vcug accordingly studies patients longer follow duration needed however meaningful study showed open reimplantation applied effectively failed endoscopic surgery in addition even parent whose child experienced failed endoscopic injection prefer endoscopic injection primary treatment vur patients high grade vur tended open ureteral reimplantation primary surgery rather endoscopic injections however significant differences operation time postoperative complications hospitalization period surgical success rates open ureteral reimplantation surgery failed endoscopic injection vur primary open reimplantation we conclude open reimplantation conducted safely effectively failed endoscopic treatment in addition parents prefer endoscopic injection first line treatment option children vur convenience reduced postoperative discomfort

purposeas endoscopic treatment for vesicoureteral reflux ( vur ) has increased , secondary ureteral reimplantation ( ur ) after failure of endoscopic treatment has increased . we studied the clinical feature and efficacy of secondary ur after failure of endoscopic treatment compared with primary ur.materials and methodseighty - one children who had ur for vur were enrolled . charts were reviewed retrospectively for age , sex , grade of vur before surgery , operative time , hospitalization period , postoperative complication , and success rate . primary ur ( group a , n=64 ) was compared with secondary ur after failed endoscopic treatment ( group b , n=17 ) . in group b , telephone survey for the satisfaction of endoscopic treatment and surgery was done.resultsmean age of each group was 49.637.1 and 56.622.5 months ( p=0.236 ) . there was no significant difference between each group in sex , mean operative time , postoperative transfusion , complication rate , and success rate . as telephone survey in group b , eleven responders preferred endoscopic treatment as primary treatment of vur because it was a simple method and no hospitalization.conclusionssecondary ur after failure of endoscopic treatment was similar to primary ur . parents preferred endoscopic treatment as first line treatment for vur in spite of the need for secondary ur after failure of endoscopic treatment .

well developed societies tend barriers different organizations different professions even professions want co operate help individuals satisfy needs health social services today face groups patients composite problems often unstable they include obviously elderly persons multiple problems chronically ill children persons suffering mental ill health they continuing need care search care move primary care hospital care municipality care provided elderly persons their situation demands form integration health social services 35 benefits identified including reduced hospital use strong focus prevention keeping patients healthy provision care closer home perspective person seeking care medical social needs connected individuals see multi ill needing support needs know it must said 1970s onwards number integrative approaches tried least education although exceptions learn generally speaking european health social services fragmented poorly equipped take care patients composite needs so far great extent task integrating different delivery systems managing transitions one provider another fallen shoulders patients relatives much evidence indicates problem face result prevailing mindset how increase understanding health social services located different organizations ? in well developed societies tend barriers different organizations different professions even professions want co operate help individuals satisfy needs health social services today face groups patients composite problems often unstable they include obviously elderly persons multiple problems chronically ill children persons suffering mental ill health they continuing need care search care move primary care hospital care municipality care provided elderly persons their situation demands form integration health social services 35 benefits identified including reduced hospital use strong focus prevention keeping patients healthy provision care closer home perspective person seeking care medical social needs connected individuals see multi ill needing support needs know it must said 1970s onwards number integrative approaches tried least education although exceptions learn generally speaking european health social services fragmented poorly equipped take care patients composite needs so far great extent task integrating different delivery systems managing transitions one provider another fallen shoulders patients relatives much evidence indicates problem face result prevailing mindset how increase understanding health social services located different organizations ? lindberg observed examples meaningful co operation local level colleagues different organizations meeting pooling knowledge local conditions patient user focal point the phenomenon variously called chain care integrated care seamless care shared care this co operation aims creating continuing relationship patient user regardless given moment responsible provider edgren stenberg found practical attributes co operation cass common task shared among co working agents.collaboration people minds instinctive behaviour.each actor capabilities known respected.a combination monetary non monetary incentives exists order create lasting mutually acceptable solutions collaboration people minds instinctive behaviour actor capabilities known respected a combination monetary non monetary incentives exists order create lasting mutually acceptable solutions according brommels presentation ehma conference 2006 cas approach means among things identifying supporting constructive relations agents within system understanding tensions conflicts.avoiding strict definitions roles concentrating agreed actions.giving agents freedom organize current activities identifying supporting constructive relations agents within system understanding tensions conflicts avoiding strict definitions roles concentrating agreed actions giving agents freedom organize current activities if improve understanding health social service provider functions integrated part locally driven health social service system need innovative terms changing practice model guide thinking traditional models view systems machines it helps us understand happens dynamic living systems many agents interconnected order make clear significance let us first recapitulate machine model long time effective organizations the machine constructor top manager describes integral parts supposed co operate hospitals health services organizations usually designed function according principles scientific management 15 16 rationality objectivity stability predictability terms associate approach rationality example requires integral parts act perfect information background similar values work towards goals system designer top manager outside system change seen linier predictable process controlled top managers carried works managers plans made followed intended change takes place direct consequence problems arise implementation change either wrong expectations inability refusal take prescribed action the change takes time energy outcome difficult take political decisions setting precise goals centralized detailed rules laid lines action specified constant top monitoring assessment real risk destroying capacity motivation called and unforeseen happens whole system breaks readiness capability adapt solutions pre programmed beforehand this we learn motivation renew system inability innovate survival system threatened if health social service providers meet changing demands expectations patients users must able move quickly find mutually acceptable locally developed forms integration points intersection separate services coming together instead theories assuming cause effect linkages separate details need theories deal patterns principles a years ago concepts knowledge society learning organizations associated mechanisms technologies internet e mail mobile phones digital imaging unknown davis coined expression time place describes time space restrictions this given us enormous possibilities communicate become connected network as advance knowledge society basic assumptions behind much taught practiced name management hopelessly date indeed assumptions organizations least fifty years old so turn complexity science complex adaptive systems cas according zimmerman et al they provide alternative traditional management principles offer patterns principles whereby better act increasingly complex world attempt harness health social care services meet particular needs individual way complexity science serve sense making tool it also enables us develop locally adapted solutions order manage complex tasks find advanced home health care complexity expressed amount information needed describe understand something and the term complex emphasizes necessary competence perform task owned one part comes result co operation within system a cas consists several subsystems called agents act dependence one another may either compete co operate according sense interests bring advantage self organization creating order increasing regularity system without help outside good examples would ant hill human immune defence financial market surgical operating theatre team important work carried new england complex system institute santa fe institute elsewhere researchers chemistry physics biology medicine anthropology economics sociology asking fundamental questions living systems living systems fixed change grow heal adjust renew develop organically prominent figures field include nobel laureates ilya prigogine chemistry 26 27 murray gell mann theoretical physics within medicine we find stuart kauffman 30 31 physics russ marion mary uhl bien according augustinsson one way explain phenomenon complexity reference possibility apply routines carry particular task the task characterized regularities think terms applying routines when everyday work characterized regularities irregularities mix predictable unpredictable highest degree complexity although research needed achieve greater understanding complex adaptive systems strengthen knowledge base action growing number examples show cas concept gaining ground within health social services the agents see point create order many local interactions complexity science offers new ways understand complementary knowledge organizes co operation we regard integrated care partners partners common system regard logically agents system emergency treatment clear example independent agents interacting locally independent agents another example comes elderly care cas concept applied secure agreement politicians civil servants the objective identify local politicians managers understand problems goals regarding structures processes involved care elderly common vision easy understand communicate there external constructor superior centrally located source govern design system when study cas focus interaction communication agents rewriting old clich whole greater sum parts whole relations parts whether two persons human machine machine machine intensity relations determines complexity system constant change adaptation development system unforeseeable non linear way today digital imaging diagnostic technology brings together specialist radiologist primary practitioner treating patient way virtually impossible previously and thereby generate new possibilities professional interaction patient involvement well new forms inter organizational collaboration complexity science emphasizes inherent power development self organizing nature system attempts reduce complexity system order gain control often counterproductive in practice assume provider top attempt specifying tasks risk complexity patient such top attempts usually made order make simpler provider person seeking care when address given community health social problems process local intelligence gathering using multiple local sources build knowledge base action connecting locally based agencies institutions individuals competence something one important aspect complexity science shown complementary knowledge organizes co operation later studies cass emphasized interplay environment system results sort co development whereby influences so example hospital inpatient time part hospital system another system workplace positive feedback loops enhance amplify changes tend move system away equilibrium stage for example general terms positive feedback loop income consumption economy bigger income individuals negative feedbacks tend dampen buffer changes hold system equilibrium state like thermostat fridge loops carriers information material energy agents system facilitate adaptability entire system complexity science positive negative feedback there much sense agents spending time separately detailed planning since functioning system result interactions instead holden talks direction without directives found purposes fostering connectivity among diverse agents effective coupling structures ideas innovations ensuring neither loose tightly interdependent complex systems better led indirect direct leadership behaviours the cas approach helps agents see co workers part innovative team great potential local knowledge needs they much better placed act centrally located management ever could they sense control work situation perhaps important change needed create good workplace way we see cas approach satisfies fundamental human need participate feeling solidarity part greater whole general criticisms cas approach coming practitioners concern lack recommendations behave part cas others may argue one several possible approaches promote integrated care communication- co ordination conflicts among participating agents rather free riding agents noted a certain level system inertia may also develop time learn use different approach organizing may mean insecurity risk similar expected accept new technology seen staff perspective cas approach may mean increased insecurity greater responsibility decision making elements risk management the cas approach raises ethical concerns refer decision making neither supported science objective criteria first due nature system determined sum choices made secondly appears simple final objective calculable ground decisions shift responsibility decision onto something somebody else nt blame genetic algorithm said sell know choices extent represent step dark therefore responsible but cas approach lays considerations taken account clearly alternatives for long time effective organizations looked upon like well oiled machines the machine constructor top manager describes integral parts supposed co operate hospitals health services organizations usually designed function according principles scientific management 15 16 rationality objectivity stability predictability terms associate approach rationality example requires integral parts act perfect information background similar values work towards goals system designer top manager outside system change seen linier predictable process controlled top managers carried works managers plans made followed intended change takes place direct consequence problems arise implementation change either wrong expectations inability refusal take prescribed action the change takes time energy outcome difficult take political decisions setting precise goals centralized detailed rules laid lines action specified constant top monitoring assessment real risk destroying capacity motivation called and unforeseen happens whole system breaks readiness capability adapt solutions pre programmed beforehand this we learn motivation renew system inability innovate survival system threatened if health social service providers meet changing demands expectations patients users must able move quickly find mutually acceptable locally developed forms integration points intersection separate services coming together instead theories assuming cause effect linkages separate details need theories deal patterns principles a years ago concepts knowledge society learning organizations associated mechanisms technologies internet e mail mobile phones digital imaging unknown davis coined expression time place describes time space restrictions this given us enormous possibilities communicate become connected network as advance knowledge society basic assumptions behind much taught practiced name management hopelessly date indeed assumptions organizations least fifty years old so turn complexity science complex adaptive systems cas according zimmerman et al they provide alternative traditional management principles offer patterns principles whereby better act increasingly complex world attempt harness health social care services meet particular needs individual way complexity science serve sense making tool it also enables us develop locally adapted solutions order manage complex tasks find advanced home health care complexity expressed amount information needed describe understand something important part complexity science complex adaptive system cas the term complex emphasizes necessary competence perform task owned one part comes result co operation within system a cas consists several subsystems called agents act dependence one another may either compete co operate according sense interests bring advantage self organization creating order increasing regularity system without help outside good examples would ant hill human immune defence financial market surgical operating theatre team important work carried new england complex system institute santa fe institute elsewhere researchers chemistry physics biology medicine anthropology economics sociology asking fundamental questions living systems living systems fixed change grow heal adjust renew develop organically prominent figures field include nobel laureates ilya prigogine chemistry 26 27 murray gell mann theoretical physics within medicine we find stuart kauffman 30 31 physics russ marion mary uhl bien according augustinsson one way explain phenomenon complexity reference possibility apply routines carry particular task the task characterized regularities think terms applying routines everyday work characterized regularities irregularities mix predictable unpredictable highest degree complexity although research needed achieve greater understanding complex adaptive systems strengthen knowledge base action growing number examples show cas concept gaining ground within health social services the agents see point create order many local interactions complexity science offers new ways understand complementary knowledge organizes co operation we regard integrated care partners partners common system regard logically agents system emergency treatment clear example independent agents interacting locally independent agents another example comes elderly care cas concept applied secure agreement politicians civil servants the objective identify local politicians managers understand problems goals regarding structures processes involved care elderly a common vision easy understand communicate created connect two groups there external constructor superior centrally located source govern design system when study cas focus interaction communication agents rewriting old clich whole greater sum parts whole relations parts whether two persons human machine machine machine intensity relations determines complexity system constant change adaptation development system unforeseeable non linear way today digital imaging diagnostic technology brings together specialist radiologist primary practitioner treating patient way virtually impossible previously thereby generate new possibilities professional interaction patient involvement well new forms inter organizational collaboration complexity science emphasizes inherent power development self organizing nature system attempts reduce complexity system order gain control often counterproductive practice we assume provider top attempt specifying tasks risk complexity patient such top attempts usually made order make simpler provider person seeking care working isolation burden coordination passes patient when address given community health social problems process local intelligence gathering using multiple local sources build knowledge base action connecting locally based agencies institutions individuals competence something one important aspect complexity science shown complementary knowledge organizes co operation later studies cass emphasized interplay environment system results sort co development whereby influences so example hospital inpatient time part hospital system another system workplace positive feedback loops enhance amplify changes tend move system away equilibrium stage for example general terms positive feedback loop income consumption economy the bigger income individuals whole population consume increasing income individuals negative feedbacks tend dampen buffer changes hold system equilibrium state like thermostat fridge loops carriers information material energy agents system facilitate adaptability entire system complexity science there much sense agents spending time separately detailed planning since functioning system result interactions instead holden talks direction without directives it found purposes fostering connectivity among diverse agents effective coupling structures ideas innovations ensuring neither loose tightly interdependent complex systems better led indirect direct leadership behaviours the cas approach helps agents see co workers part innovative team great potential local knowledge needs they much better placed act centrally located management ever could they sense control work situation perhaps important change needed create good workplace way we see cas approach satisfies fundamental human need participate feeling solidarity part greater whole general criticisms cas approach coming practitioners concern lack recommendations behave part cas theorists would claim cas nothing emperor new clothes others may argue one several possible approaches promote integrated care communication- co ordination conflicts among participating agents rather free riding agents noted a certain level system inertia may also develop time learn use different approach organizing may mean insecurity risk similar expected accept new technology seen staff perspective cas approach may mean increased insecurity greater responsibility decision making elements risk management the cas approach raises ethical concerns refer decision making neither supported science objective criteria first due nature system determined sum choices made secondly appears simple final objective calculable ground decisions shift responsibility decision onto something somebody else nt blame genetic algorithm said sell know choices extent represent step dark therefore responsible but cas approach lays considerations taken account clearly alternatives when competence necessary carrying given task lie within one individual provider organization co operation agents within system comes play discharge task we longer talking individual organization agent shaping overall workable solutions taking patient user perspective this applies matching care needs different patient groups individual patients the cas approach helps management understand traditional top way managing may meet problems organizations complex tasks an important discussion top management fact executes steering function leaders may consider accepting complexity instead trying reduce formulate simple concrete goals communicate give feedback measure performance when perceive health social service organizations cass gain insight processes go within organizations are willing face interdependence health social services dependence collaboration deliver appropriate integrated care ? if complexity science could important step towards fresh thinking order fulfil patients users presently unfulfilled needs edgar borgenhammar phd economics mba berkeley dsi lund professor emeritus nordic school public health gteborg former hospital chief executive sweden peter carswell dr centre health services research policy faculty medical health sciences university auckland auckland new zealand alene hokenstad project director united hospital fund new york usa

introductionorganizations can be regarded as systems . the traditional model of systems views them as machines . this seems to be insufficient when it comes to understanding and organizing complex tasks . to better understand integrated care we should approach organizations as constantly changing living organisms , where many agents are interconnected in so - called complex adaptive systems ( cas).theory and discussionthe term complex emphasizes that the necessary competence to perform a task is not owned by any one part , but comes as a result of co - operation within the system . adaptive means that system change occurs through successive adaptations . a cas consists of several subsystems called agents , which act in dependence of one another . examples would be the ant - hill , the human immune defence , the financial market and the surgical operating theatre team . studying a cas , the focus is on the interaction and communication between agents . although these thoughts are not new , the cas - approach has not yet been widely applied to the management of integrated care . this helps the management to understand why the traditional top down way of managing , following the machine model thinking , may meet with problems in interdependent organizations with complex tasks.conclusionwhen we perceive health and social services as cass we should gain more insight into the processes that go on within and between organizations and how top management , for example within a hospital , in fact executes its steering function .

introduce recent advances study post translational processing modification targeting cathepsins cystatins almost intracellular proteins passed principally similar processes synthesis degradation general therefore would like introduce general fate intracellular proteins post translational processing modification targeting ordered particles as figure 1 shows intracellular proteins synthesized pre promature complex polysomes prepart removed cotranslationally promature parts translocated golgi apparatus glycosylated mannose rich sugar the glycosylated mature part translocated target organelles degradation started splitting ordered nicked bonds make hydrophobic peptides these hydrophobic peptides secreted cytoplasm incorporated phagosomes proteosomes ubiquitination biological merit post translational processing modifications proteins possible considerations the capability take variable forms way biosynthesis important keep adaptability changing biological requirements intracellular translocation maturation must regulated pro parts bound sugar targeting signals cases cases carbamoyl phosphate synthetase cps ornithine transcarbamylase otc pro - parts play role signals recognized receptors located target organella membrane lysosomes shown figure 4 the glycosylated cathepsins targeted lysosomes mediated mannose-6 phosphate receptors located lysosomal as figures 4(a 4(b show lysosomes cathepsin h b located attached cell membrane contrary cathepsin l located lysosomes distributed diffusely liver cells figure 4(a shows using immunodouble gold particle staining cathepsin b small gold particle cathepsin h large gold particle located clearly different lysosomes cathepsin l translated 17 amino acids prepart 96 amino acids pro part 221 amino acids mature part 9 10 the prepart removed cotranslationally formed procathepsin translocated golgi apparatus 108-asn 155-asn mature parts glycosylated high mannose type sugar shown figure 2(a initiation degradation started nicking bond 178 bond cleavage cysteine protease cathepsin b translated 17 amino acids prepart 62 amino acids pro part 252 amino acids mature part 12 13 the prepart also removed cotranslationally formed procathepsins translocated golgi apparatus 38th asn pro part 111th asn mature part glycosylated high mannose type sugar mannose-6-phosphate moities play role targeting marker lysosomes cathepsin h translated 21 amino acids prepart 114 amino acids pro part 217 amino acids mature part the pro part two carbohydrate chains 70th asn 90th asn mature part consisted 217 amino acids one carbohydrate chain bound 99th asn 8 15 initiation degradation started 177th nicking bond cysteine protease intracellular protein degradation (= autophagosome formation regulated nutritional hormonal conditions fasting insulin enrichment caused stimulation phagosome formation cathepsin l amount increased contrary refeeding glucagon enrichment resulted suppression phagosome formation decreasing cathepsin l amount as see figure 2 cathepsins b l h individual ordered nicking bond initiation degradations bonds cleaved cysteine protease therefore table 1 shows half lives t1/2 cathepsins contents lysosomes clearly increased treatment e-64 inhibitor cysteine proteases the initiations degradation cathepsins started ordered limited proteolysis cysteine protease lysosomes figure 2 shows the formed hydrophobic products translocated autophagosome proteasome ubiquitinated degrade amino acids procathepsins mature cathepsins secreted various cells play individual physiological pathological roles figure 5 shows bone metabolism consisted functional balances osteoblastic cell function osteoclastic cell function bone collagen degraded mainly cathepsin l k therefore called collagenolytic cathepsins the secretion cathepsins osteoclasts stimulated pht parathyroid hormone suppressed vitamin d3 e-64 ca-148 eta therefore bone metastasis cancers inhibited cathepsin inhibitors e-64 clik-148 as pit formation test table 4 shows bone matrix degradation catalyzed cathepsin l cathepsin b. endogenous low molecular weight cathepsin inhibitors consisted two big groups one located skin ubiquitously located organs cystatin located epidermis cystatin b located ubiquitously cells organs the secretary formed cystatins cystatin c eggwhite cystatin saliva another secretary group high molecular weight repeated low molecular cystatins kininogen family serum each cystatin shows different inhibitory specificity individual cathepsins example cystatin inhibit cathepsin b activity strongly inhibit cathepsin l activity cathepsin h activities 7 19 the amino acid sequences low molecular weight cystatin family strong homology however post translational processing quite different localizations biological functions quite different skin stained immunohistochemistry using anticystatin antibody cornified envelope skin stained the sphingosine treatment new born skin resulted strong suppression targeting cystatin cornified envelope the sphingosine powerful inhibitor protein kinase c. shown figures 7(a 7(b threonine residue near c terminus 92th threonine figure 9 shows cystatin phosphorylated protein kinase c phosphorglated cystatin incorporated cornified envelope the participation protein kinase c phosphorylation cystatin also confirmed using specific inhibitor protein kinase c hidaka h-7 inhibited incorporation p cystatin phosphorylated cystatin incorporated skin cornified envelop envelop the phosphorylated cystatin conjugated filaggrin linker sediment peptide rich glutamine residues mediated epidermal transglutaminase presence calcium yield high molecular weight protein skin fiber shown figure 10 the phosphorylated cystatin showed strong inhibition cathepsin l cathepsin b h also showed strong inhibition bacterial cysteine proteases virus cathepsins therefore cystatin phosphorylated cystatin showed strong protection infections bacteria viruses skin post translational covalent modification cystatin changing inhibitory activities the third position n termius cysteine residue cystatin reacts glutathione form mixed disulfate complex also able make dimmer disulfate bond as figure 8 shows n terminus cystatins inserted substrate binding pocket cathepsins glutathionated dimmer form unable insert binding pocket cathepsins the coefficient oxidized reduced forms glutathiones regulates inhibitory activity cystatin therefore activities cathepsins regulated intracellular redox potentials spite changes inhibitory activities cystatin figure 7(b shows using -cell pancreas cystatin located insulin secretion particles therefore cystatin secreted insulin pancreas

cathepsins are an essential protease family in all living cells . the cathepsins play an essential roles such as protein catabolism and protein synthesis . to targeting to various organella and to regulate their activity , the post translational - processing and modification play an important role cathepsins are translated in polysome as the pre - pro - mature forms . the pre - peptide is removed cotranslationally and then translocated to golgi - apparatus and the pro - part is removed and the mature - part is glycosylated , and the mature - part is targeted into the lysosome mediated by mannose-6-phosphate signal and the mature - part is bound with their coenzymes . the degradation of the mature - part is started by the limited proteolysis of the ordered nicked bonds to make hydrophobic peptides . the peptides are incorporated into phagosome or proteasome after ubiquitinated and are degrade into amino - acids . cystatins are endogenous inhibitors of cathepsins . cystatin which is only located in skin is phosphorylated at the near c - terminus by protein kinase - c , and the phosphorylate - cystatin is incorporated into cornified envelope and conjugated with filaggrin - fiber by transglutaminase to form the linker - fiber of skin . the cystatin is modified by glutathione or make their dimmer , and they are inactive . those modifications are regulated by the redox - potential by the glutathione .

endocrine disorders may complicate cause mimic otolaryngologic disorders may anatomical due enlargement thyroid gland others physiological resulting increased decreased glandular activity.1 hypothyroidism characterized slowing mental motor activity depression constipation cold intolerance menorrhagia stiff muscles carpal tunnel syndrome sleep apnea dry hair skin weight gain snoring hoarse voice.1 less common symptoms involve heart muscles joints blood.2 dysarthria presenting symptom hypothyroidism reported before.3 present unusual case hypo thyroidism presenting dysarthria a 39-year old female presented clinic dysarthria six months duration revealed episodes dysphonea snoring sleep apnea dysphagia choking eating drinking her past medical history normal apart one occasion delayed recovery anaesthesia surgery fractured femur year clinical examination including central nervous system normal apart slightly puffy face there abnormality movement tongue pharygeal palate muscles pmol l normal 9.2 23.9 pmol l thyroid stimulating hormone 82.7 miu l normal 0.32 5.00 miu l antithyroglobulin antibodies 1:320 u ml normal 1:40 u ml antiperoxidase autoantibodies 1:1600 u ml normal 1:40 u ml a complete blood count film consistent iron deficiency haemoglobin 7grams normal 11 16 grams low serum iron 2 umol l normal 10 28 umol l increased red cell distribution width rdw 17.2 normal 11.6 13.7% other biochemical abnormalities high serum cholesterol 6.9 mmol l normal 3.6 6.8 hypo thyroidism diagnosed light patient diagnosis a second history taken showed suffered symptoms hypothyroidism dry skin generalised weakness excessive sleeping hoarse voice menorrhagia two months initiation therapy patient dysarthria associated symptoms dysarthria disturbance articulation may caused neuromuscular lesion abnormality vocal cords the first may result damage central peripheral nervous system head trauma brain stem infarction bulbar palsy motor neuron disease peripheral neuropathy huntington chorea parkinson disease multiple sclerosis myasthenia gravis muscle disease.4 second may attributable congenital traumatic inflammatory tumors post operative lesions vocal cords these causes unlikely patient showed associated clinical features diseases besides normal neurological examination investigations other causes congenital aquired storage disorders amyloidosis endocrine disorders acromegaly hypo thyroidism,3 presented case may lead enlargement one components vocal cords.56 likely cause dysarthria patient hypothyroidism this supported abnormal thyroid functions response dysarthria thyroxin dysarthria due hypothyroidism reported previous case.3 pathophysiology dysarthria hypothyroidism explained edematous swelling laryngeal hypopharyngeal structures combination macroglossia.3 shown macroglossia hypothyroidism caused thickening epithelial tissue.6 changes also explain choking dysphagia patient experienced there reports hypothyroidism responsible secondary dysphagia.1012 sleep apnea may also manifestation hypothyroidism likely caused edema myopathy.7 sleep apnea attributable hypothyroidism reversible thyroxin replacement therapy.8 episodic hoarseness voice also explained hypothyroidism,1 well delayed recovery anaethesia year probably result undiagnosed hypothyroidism.91314 unfortunately thyroid function tests performed patient time iron deficiency anemia patient due menorrhagia one characteristic features disease.1 hyperlipidaemia may also due hypothyroidism known association.1 clinical implication presented patient dysarthria may presenting symptom hypothyroidism even symptoms present long time hypothyroidism cause dysarthria confirmed discovery additional symptoms patient history otolaryngeal symptoms therefore considered possible symptoms hypothyroidism.15 prompt recovery dysarthria expected hormone replacement therapy

hypothyroidism is a common endocrine disorder with characteristic clinical symptoms and signs . typical symptoms of hypothyroidism are lethargy , cold intolerance , slowing of intellectual and motor activity , decreased appetite , weight gain , and dry skin . a 39-year - old female presented to the clinic with dysarthria as the chief symptom . subsequent questions revealed that other symptoms were confined to the otolaryngeal region , which were episodes of mild dysphonia , dysphagia , sleep apnea , and snoring . laboratory data revealed marked hypothyroidism and positive tests for antithyroglobulin and antimicrosomal antibodies . after administration of thyroxin , the dysarthria and the other symptoms rapidly disappeared . dysarthria may be the presenting symptom of hypothyroidism and can be resolved after hormone replacement therapy .

bicipital groove bg indentation anterior aspect proximal part humerus this groove allows tendon long head biceps brachi muscle enveloped synovial sheath ascending branch anterior circumflex humeral artery pass it bounded medially lesser tubercle laterally greater tubercle superiorly bridging transverse humeral ligament /muscle fibers subscapularis supraspinatus pectoralis major muscles 2 3 groove transverse humeral ligament muscle fibers bridging provides stability smooth functioning tendon long head biceps brachi muscle prevents subluxation multidirectional biomechanical movements arms apart greater function biceps brachi muscle whose tendon enshrined bicipital groove suppination flexion screwing biomechanical movements motion humerus the proximal humerus moves relation fixed biceps tendon firmly held place level intertubercular sulcus tuberosities humeral transverse ligament elevation arm humerus moves 3.8 cm fixed tendon dynamics external rotation internal rotation tendon forced medially lesser tubercle superiorly transverse humeral ligament coracohumeral ligament directly overlies transverse humeral ligament muscle fibres continuous rotator cuff morphometry bg may influence functions surrounding structures leading various pathological conditions 8 9 supratubercular ridge originally described meyer 1928 later hitchcock bechtol 1948 consists bony protuberance continuous superior aspect lesser tuberosity it allows tendon gradual change direction enters bicipital groove elevating forcing laterally therefore incidence spurs supratubercular ridge indian population study also observed apart bicipital groove important landmark replacement prosthesis shoulder thus knowledge bg highly useful prosthetic sizing positioning designing bicipital groove also acts important landmark placement lateral fin prosthesis shoulder arthroplasty humeral head replacement fractures upper end humerus series classic reports various authors papers 4 1215 have discussed primary versus secondary biceps tendinitis different treatment regimens entities 16 17 the association shoulder pain pathology lhb currently attributed inflammation synovitis impingement prerupture instability tendon entry bicipital groove subluxation dislocation 16 1824 lesions affecting tendon lhb brachii postulated among frequent causes pain disability shoulder this pain may caused rotator cuff supraspinatus biceps tendon diseases pathologies biceps tendon broadly divided two classes namely follows primary tendonitis berlemann bayley reported long term results 14 patients 15 shoulders following keyhole biceps tenodesis fifty three percent patients previously undergone subacromial decompression symptoms persisted biceps tenodesis carried this would suggest biceps tendinitis primary event.secondary tendonitis 14 15 17 27 may subdivided three main types inflammatory instability traumatic clearly huge overlap categories fact biceps pathology rarely single entity apart recent biomechanical data come youm et al found loading long head biceps tendon significantly affects glenohumeral joint range movement translations kinematics therefore varied anatomical knowledge bg important abnormalities bicipital tendon synovial sheath implicated variety causes shoulder pain disability 6 7 a radiological study recommended entire length bg examined determine osseous anatomy groove few authors studied morphology upper end humerus geographically diversified regions 10 3133 primary tendonitis berlemann bayley reported long term results 14 patients 15 shoulders following keyhole biceps tenodesis fifty three percent patients previously undergone subacromial decompression symptoms persisted biceps tenodesis carried secondary tendonitis 14 15 17 27 may subdivided three main types inflammatory instability traumatic clearly huge overlap categories fact biceps pathology rarely single entity apart the recent biomechanical data come youm et al found loading long head biceps tendon significantly affects glenohumeral joint range movement translations kinematics therefore varied anatomical knowledge bg important abnormalities bicipital tendon synovial sheath implicated variety causes shoulder pain disability 6 7 a radiological study recommended entire length bg examined determine osseous anatomy groove few authors studied morphology upper end humerus geographically diversified regions 10 3133 hence attempt made examine length width depth opening angle bicipital groove statistically correlate clinical implications north indian population along review literature the study carried using hundred one humeri assorted sex pair the subjects consisted 45 left 56 right humeri obtained osteology laboratory kg medical university lucknow india the lengths medial lateral walls depth lengths width bicipital groove figure 1 measured digital vernier callipers the statistical analyses consisting incidences supratuberecular ridge meyer presence bony spurs also mean standard deviation range median mode length medial lateral walls width depth opening medial wall angles bg carried the opening medial wall angles bg figure 2 computed the narrowness shallowness bg redefined objectively relation dimensions bicipital groove biceps tendon adequate prediction pathologies biceps tendon though precise definition narrowness shallowness bg difficult yet systematic relative constrained definition depending dimensions bicep tendon natural abode bicipital groove is formulated follows.if width bg less width biceps tendon narrow bg may produce attritional changes causing impingement inflammation degeneration similarly depth groove less height bicep tendon shallow causing subluxation dislocation long run may cause degenerative changes rupture width bg less width biceps tendon narrow bg may produce attritional changes causing impingement inflammation degeneration similarly depth groove less height bicep tendon shallow causing subluxation dislocation long run may cause degenerative changes rupture new definition narrowness shallowness requires width height biceps tendon width height 4 biceps tendons 2 cadavers have also measured provide realistic definition narrow shallow bicipital groove the means standard deviation lengths medial lateral walls width depth opening medial wall angles bg computed shown table 1 mean length medial wall bg right side 22 4 left side 23 5 mm table 1 mean length lateral wall bg right side 31 6 mm left side 31 5 mm mean width superior part bg right side 8 2 mm left side 8 2 mm the depth bg right side 5 1 mm left side 6 1 mm the incidence supratubercular ridge 37% total 17% right side 20% left side the average length bg 26.7% total length humerus average width bg 52% average width humerus the width height biceps tendon figure 3 displayed table 2 the morphometric study carried various authors 25 3437 compared present study depicted table 3 lengths medial wall lateral walls bg reported far except present study data comparison length bg present study comparable murlimanju slightly higher observed wafae et al the width bg present study close cone et al but slightly higher murlimanju lower wafae et al median width bg right left sides 8 10 mm respectively mode 8 mm side it indicates width bg north indian population 8 mm present study depth bg 3 mm depth ranging 46 mm 98% 96% humeri respectively 90% 86% study cone et al median mode depth right left side 5/6 mm medial wall angle bg slightly higher joseph lower cone et al medial wall angle mwa study lower cone higher joseph the median mode morphometric parameters bg useful prosthetic sizing positioning designing supratubercular ridge present study is found 17% right 20% left totalling 37% humerii per hitchcock bechtol cone et al radiographic interpretations observed ridge 50% cases reported pathologically significant observed ridge 88% right side 57% left side emphasised important right side left prevent medial displacement long head biceps bg in present study mean width height biceps tendon figure 3 10.5 1.6 mm respectively higher observed lam mok similarly dimensions tendon exit bg 7 1.8 mm respectively the width tendon exit height less observed lam mok if tendon encased median lateral walls bg due shallow depth may dislocated either partially fully biomechanical movements arms this dislocation biceps tendon associated impingement may cause degeneration leading partial full rupture passage time apart if movements tendon free narrow presence bony spurs bg biomechanical movements shoulder joint constant fraying might give rise pathologies biceps tendon reported wide grooves i.e. 17 mm often shallow but groove 3 mm deep less viewed suspicion managing pathologic conditions shoulder patient radiographs per cone view pfahler et al found flat groove angle associated radiologic depth less 2 mm they found significant accumulation pathologically changed biceps tendons flat groove angle present according several authors subluxation dislocation biceps tendon common presence shallow bicipital groove 1 4 5 7 39 it also reported shallow bicipital groove tendon susceptible chronic trauma due impingement overlying acromion rotator cuff coracoacromial arch shoulder movement a shallow intertubercular groove vulnerable impingement damage subluxation 37 40 rupture biceps tendon commonly occurs proximally near glenoid labrum distally bicipital groove smith designated bicipital groove types narrow normal shallow depending mean opening angle less 66 94 118. seen description objective realistic definition given author therefore present author attempted reliable realistic objective definition shallowness bg relation biceps tendon given section 2 paper this definition shallowness realised situ live patients expected elucidate pathologies associated shallowness groove per morphometric data table 3 bg biceps tendon observed two cadavers present study groove shallow according new definition shallowness figure 3 shows biceps tendon bg dissected cadaver wherein biceps tendon positioned perfectly well protected bg the bg case shallow seen mentioned figure height biceps tendon less depth bg continual mechanical stress anatomically narrow sites i.e. distal bicipital groove beneath acromion coracoacromial ligament impingement biceps tendon coracoacromial arch flexion may cause well known degenerative changes 19 41 present study data bg biceps tendon observed two cadavers do indicate narrow bg per new definition narrowness shallowness given section 2 did find conspicuous anatomic findings bicipital groove shoulders effected rotator cuff diseases mri narrow groove flat groove small medial groove predictive biceps pathology time arthroscopy 37 38 this may following limitations josheph study subjective qualitative definition shallowness narrowness limitations josheph study subjective qualitative definition shallowness narrowness the limitations josheph study follows clearly selection bias surgical patients suffering primary rotator cuff disease patients mri done classification biceps tendon pathology arbitrary based purely visual inspection histopathologic changes mri often considered less accurate x ray computed tomography scan evaluating bony dimensions clearly selection bias surgical patients suffering primary rotator cuff disease patients mri done surgical patients suffering primary rotator cuff disease patients mri done classification biceps tendon pathology arbitrary based purely visual inspection histopathologic changes mri often considered less accurate x ray computed tomography scan evaluating bony dimensions lesions due pathology biceps tendon postulated among frequent causes pain disability shoulder biceps tendon pathology visualized three main categories namely instability inflammatory traumatic abboud et al divided biceps tendon pathology normal inflamed partially torn ruptured tendon acute inflammatory chronic degenerative alterations causing partial complete rupture subluxation dislocation found long head biceps tendon instability biceps tendon besides factors may attributed length medial lateral walls opening medial wall angles depending width depth constituting shallowness bg presence supratubercular ridge 37 40 41 the implication longer walls expected ensure greater stability biceps tendon lying bicipital groove shorter walls multidirectional biomechanical movements but rider fact may also cause attritional friction longer length biceps tendon surrounded longer walls creating inflammation narrow conditions bg as lengths medial lateral walls decrease instability increases tendon likely damaged inference drawn based reconstruction anatomical model part human body advancing knowledge experience anatomy clinical studies recorded literature supported logical force study dry bones range provides idea length walls north indian population whereas mean sd reveals average size bg median may useful planning surgical procedures part body mode representative frequent incidence lengths walls subject population if instability biceps tendon studied relation lengths bg frequent value length walls may play vital role diagnostics tendon instability attritional damage felt groove 3 mm deep less 17 mm wide may predispose tendon subluxation dislocation patient radiographs the flat groove pfahler et al found depict significant accumulation pathologic changes biceps tendon 62% cases sonography the supratubercular ridge meyer prematurely shallow bicipital intertubercular sulcus postulated result variety lesions repetitive use acute trauma 37 40 41 include acute chronic peritendonitis varying degrees attrition damage tendon subluxation complete dislocation biceps tendon is enshrined bg width may influence pathology occurring tendon wider groove tendon morphometry bicipital groove terms length media wall lateral wall length bg width depth medial wall angle opening angle elucidated reference north indian population.the data morphometry bg utmost use anatomist radiologists orthopaedic surgeons physicians.the new definition narrow shallow bg given the morphometry bicipital groove terms length media wall lateral wall length bg width depth medial wall angle opening angle elucidated reference north indian population the data morphometry bg utmost use anatomist radiologists orthopaedic surgeons physicians new definition narrow shallow bg given

the variant morphometry of bicipital groove is reported to be associated with pathologies of biceps tendon and is useful in surgical procedures in this region . the pathologies of biceps tendon are frequent causes of shoulder pain . therefore , under the condition of paucity of data pertaining to north indians , not only morphometric analysis of bicipital groove and a new definition of narrow / shallow groove to provide logical explanation for dependence of pathologies of biceps tendon on groove morphology is done but also a review of the literature has been carried out . various dimensions such as lengths of medial and lateral walls , width , depth , medial wall , and opening angles including incidence of supratubercular ridge of bicipital groove from 101 humerii are 23 5 , 32 5 , 8 2 , 6 1 , 48.91 10.31 , 82.20 22.62 , and 37% , respectively . the average height along with average width of biceps tendon and average width along with average depth of bicipital groove from two cadavers are 1.8 , 10.5 , 11.3 , and 5.5 mm , respectively . the knowledge of bicipital groove will be of paramount importance to anatomists for new data , for orthopaedic surgeons in carrying out surgical procedures in this region , and for physicians in the management of anterior shoulder pain in north indian population .

five world fatal work safety accidents past decade 20012010 occurred china 1 work safety accidents become first cause deaths chinese citizens 44 yr old 2 work safety accidents pose great threat people life especially major accidents defined ones result least 10 deaths the chinese government established surveillance system monitoring work safety accident since 2001 state saws 3 authority system work safety accidents inquiry china injuries related transportation mining fire explosion majority occurrence 46 accidents occurred construction certain geographic regions china 7 8) previous researches focused characteristics work safety accidents research analyzing major accidents mass casualties there clear answer yet whether accidents mass casualties simple enlargement general accidents inherent mechanism accident cause special risk factors the depth research focused cause mechanism work accidents least 10 fatalities important theoretical value practical meaning improve relevance effectiveness accident prevention examine national profile extent type major accidents work safety causes 10 fatalities per accident analyzed data accident inquiry system saws website period 2003 2012 provide scientific basis prevention measures strategies reduce major work safety accidents deaths china data state saws corresponding period 20032012 used analysis saws competent agency state council charge comprehensive supervision administration work safety coalmines whole country china made mandatory provisions focusing implementation safety standards risk forecasting accident reporting accident accountability aspects development relevant laws regulations departmental regulations safety standards the saws law based administration strengthen comprehensive supervision administration work safety coal mines whole country the saws collects information provinces cities local data sources including details regarding fatal accidents three deaths non fatal injuries associated economic losses the information work safety accidents coalmine incidents required report enterprise involved regional agency provincial department finally submitted saws conducting investigation the saws announces findings determines penalty study data covered industries except military forces private enterprises additionally checked statistical year book road traffic accidents public literature supplement information details the analysis conducted using registered data 10 deaths per incident major accidents transportation involved victim operator passenger pedestrian stuck side road extraction details including types event year region number deaths injuries causes accidents transformed spss v13.0 chicago il usa we performed analysis frequency accidents deaths trend geographic distribution type major accidents we analyzed geographic distribution using hierarchical cluster analysis focused frequency accidents deaths incidence major accident shows occurrence probability person major accident mortality shows dying probability the incidence mortality major accident defined follows incidence number major accident/ total national population106)mortality number death/ total national population 106 data state saws corresponding period 20032012 used analysis saws competent agency state council charge comprehensive supervision administration work safety coalmines whole country china made mandatory provisions focusing implementation safety standards risk forecasting accident reporting accident accountability aspects development relevant laws regulations departmental regulations safety standards the saws law based administration strengthen comprehensive supervision administration work safety coal mines whole country the saws collects information provinces cities local data sources including details regarding fatal accidents three deaths non fatal injuries associated economic losses the information work safety accidents coalmine incidents required report enterprise involved regional agency provincial department finally submitted saws conducting investigation the saws announces findings determines penalty study data covered industries except military forces private enterprises additionally checked statistical year book road traffic accidents public literature supplement information details the analysis conducted using registered data 10 deaths per incident major accidents transportation involved victim operator passenger pedestrian stuck side road after extraction details including types event year region number deaths injuries causes accidents transformed spss v13.0 chicago il usa we performed analysis frequency accidents deaths trend geographic distribution type major accidents we analyzed geographic distribution using hierarchical cluster analysis focused frequency accidents deaths incidence major accident shows occurrence probability person major accident mortality shows dying probability the incidence mortality major accident defined follows incidence number major accident/ total national population106)mortality number death/ total national population 106 we found dramatic decline numbers accidents deaths since 2006 number accidents deaths dropped 52.0% 66.6% peak year 2005 2012 respectively 10-yr period 2003 2012 866 major accidents work safety china resulting 16795 deaths 9183 injuries the incidence major accident mean mortality rate 1.93 1.34 per one million populations annual recent years major accident mortality rate incidence major accident declined fig 1 mortality rate incidence major accidents work safety china year 20032012 fig the deeper color region higher number accidents deaths occurred using hierarchical cluster analysis found regions reporting highest number injuries deaths hunan 62/936 guizhou 69/1101 henan 54/1302 shanxi 79/2045 total 264 incidents 5384 deaths accounting 30% nation wide geographic distribution major work safety accidents china 20032012 number incidents number deaths transportation related accident responsible highest frequency deaths 2003 2012 n=437 table 1 shows transportation mining caused injuries deaths dates evaluated number injuries declined last years number injuries caused types industries however noticed tendency manufacturing construction service furthermore mining declined percentage injuries percentage transportation related accident increased type industry related accident china year 20032012 mining contributed highest number deaths n=7646 2003 2012 309 mining related accidents caused 24.7 fatalities as indicated table 2 80% major work safety accident related deaths occurred mining transportation declined recent years number deaths industry china year 20032012 table 3 displays number deaths per accident industry year the average number death per accident service mining shows decline recent years found similar trend manufacturing construction transportation coal mine related road traffic related accidents dominant nearly accounted four fifths types major accidents the evaluation types accidents revealed tailing dam related accident severe terms number fatalities per incident causing 105 deaths per incident number deaths per accident industry china year 20032012 aviation related railway related accidents caused 49.5 43.3 deaths per incident respectively table 4 the lethal type major work safety accident highest death injury ratio tailing dam accident machinery collapse electric shock accident coalmine accident specifically higher death injury ratio aviation accident railway accident specifically higher number deaths per incident fig 3 number deaths frequency type accidents china 20032012 death per incident death injury ratio different types accident we found dramatic decline numbers accidents deaths since 2006 number accidents deaths dropped 52.0% 66.6% peak year 2005 2012 respectively 10-yr period 2003 2012 866 major accidents work safety china resulting 16795 deaths 9183 injuries the incidence major accident mean mortality rate 1.93 1.34 per one million populations annual recent years major accident mortality rate incidence major accident declined fig 1 mortality rate incidence major accidents work safety china year 20032012 fig 2 shows major work safety accidents deaths relation geographic distribution deeper color region higher number accidents deaths occurred using hierarchical cluster analysis found regions reporting highest number injuries deaths hunan 62/936 guizhou 69/1101 henan 54/1302 shanxi 79/2045 total 264 incidents 5384 deaths accounting 30% nation wide geographic distribution major work safety accidents china 20032012 number incidents number deaths ) transportation related accident responsible highest frequency deaths 2003 2012 n=437 table 1 shows transportation mining caused injuries deaths dates evaluated number injuries declined last years number injuries caused types industries however noticed tendency manufacturing construction service furthermore mining declined percentage injuries percentage transportation related accident increased type industry related accident china year 20032012 mining contributed highest number deaths n=7646 2003 2012 309 mining related accidents caused 24.7 fatalities as indicated table 2 80% major work safety accident related deaths occurred mining transportation declined recent years the average number death per accident service mining shows decline recent years found similar trend manufacturing construction transportation coal mine related road traffic related accidents dominant nearly accounted four fifths types major accidents the evaluation types accidents revealed tailing dam related accident severe terms number fatalities per incident causing 105 deaths per incident number deaths per accident industry china year 20032012 aviation related railway related accidents caused 49.5 43.3 deaths per incident respectively table 4 the lethal type major work safety accident highest death injury ratio tailing dam accident machinery collapse electric shock accident coalmine accident specifically higher death injury ratio aviation accident railway accident specifically higher number deaths per incident fig 3 number deaths frequency type accidents china 20032012 death per incident death injury ratio different types accident economic development related incidence accident developing countries china increases growth rate economic may caused work safety accident rate rise 9 china major work safety accidents deaths appear decreased since 2006 growth rate gdp shows increase steadily varying range 7.8% 14.2% 10 efforts regulations surveillance safety initiatives innovations safety controls chinese government attribute decline improve whole country work safety chinese government issued series supervision regulations especially fatal serious work safety accidents fire regulation transportation regulation coal mine non coal mine regulations 11 five year plan national economic social development people republic china 11 part move chinese government also strengthened penalties fatal major work safety formulation implementation regulations provinces made great improvements preventing serious major accidents the number accidents deaths show sharp drop ten years broadly consistent published studies 1217 while decline frequency accidents deaths recent years mortality rate still relatively high according zhang et al ( 12 risk death 6.1 per million populations work safety whole country higher countries 18 using hierarchical cluster analysis recognized provinces hunan guizhou henan shanxi reported highest number major accidents deaths provinces shanghai qinghai hainan ningxia beijing tianjin reported least consistent regional distribution work safety accidents whole country 12 after detailed analyzing found hunan guizhou henan shanxi 55 major accidents resulting 900 deaths among accidents mining transportation contributed majority accounting 89% these statistics may relate differences natural environment working conditions level economic development economic patterns this percentage comparable published studies work safety accidents china 19 20 based counts proportions accidents transportation mining mostly produce casualties still high risk industrial safety accident types china transportation section data analysis revealed one major road traffic accident occurred every 10 days average china average number deaths aviation 49.5 this may important reason fewer aviation accidents caused widespread concern international community road transportation regulations people republic china issued end 2004 21 efforts made better disseminate regulation safety procedures tips operators increase drivers awareness risk improve emergency response capabilities reduce traffic accidents prevention strategies including driver training working conditions weather conditions hours service regulations safety culture better considered 2225 mining section coalmine accidents contributed majority literature reported coal mine accidents china occurred ten times frequently india 1 death rate per million tons 6.66 past 24 yr ninety four times larger america 0.07 26 the distribution exploitation mineral resources important factor significant difference china america fortunately provisions prevention coal mine accidents end 2005 27 closure half operating mines 2006 death rate per million tons showed rapid decline since 2006 26 number mining accidents deaths including major mining accidents declined however several safety concerns still persist mines ignoring safety professionals lack emergency rescue measures employee professional safety training some effective interventions needed mitigate worst health safety performance china this study used death per incident death injury ratio surrogate measure describe severity major accidents urgency emergency rescue the declines number death per service mining accident indicated improvement special intervention however obstacles exist transportation construction manufacturing intervention show fig 4 occurrence tailing dam accident associated major casualties economic losses cause high case fatality occurred like tailing dam incident collapse therefore full risk assessment prevention measures primary intervention prevent accidents aviation railway accidents recognized result relatively higher case fatality higher number occupational injuries we discussed major work safety accidents nationwide severity emergency rescue urgency accident using scatter plot death per accident death injury ratio improvement compared similar studies 7 12 additionally searched published literature statistical yearbook supplement detailed information the information registered saws system comprehensive cause per incident thus could analyze ten years work safety data indicate frequency incidents number deaths major work safety accidents declined ethical issues including plagiarism informed consent misconduct data fabrication and/or falsification double publication /or submission redundancy etc completely observed authors

background : this study provides a national profile of major work safety accidents in china , which cause more than 10 fatalities per accident , intended to provide scientific basis for prevention measures and strategies to reduce major work safety accidents and deaths.methods:data from 20032012 census of major work safety accidents were collected from state administration of work safety system ( saws ) . published literature and statistical yearbook were also included to implement information . we analyzed the frequency of accidents and deaths , trend , geographic distribution and injury types . additionally , we discussed the severity and urgency of emergency rescue by types of accidents.results:a total of 877 major work safety accidents were reported , resulting in 16,795 deaths and 9,183 injuries . the numbers of accidents and deaths , mortality rate and incidence of major accidents have declined in recent years . the mortality rate and incidence was 0.71 and 1.20 per 106 populations in 2012 , respectively . transportation and mining contributed to the highest number of major accidents and deaths . major aviation and railway accidents caused more casualties per incident , while collapse , machinery , electrical shock accidents and tailing dam accidents were the most severe situation that resulted in bigger proportion of death.conclusion:ten years major work safety accident data indicate that the frequency of accidents and number of eaths was declined and several safety concerns persist in some segments .

previously described growth conditions including inducing development used a. nidulans 17 18 expression anea via niia promoter induced 0.6% sodium nitrate repressed 0.2% ammonium tartrate an anea disruption cassette dc constructed according methods described yu et al using genomic dna prepared a. nidulans fgsc a4 dna fragments dc amplified pcr using appropriate primer sets anea a1/-a2 anea b1/-b2 argb for/-rev the complete dc amplified nested pcr primer set anea c1/-c2 purified used transform tj1 strain nucleic acid preparation northern blot analysis performed described previously southern blotting genomic dna treated appropriate restriction enzymes separated 1% agarose gels the gels washed distilled water dw soaked twice depurination solution 250 mm hcl 15 min reaction gel washed dw soaked denaturation solution 1.0 nacl 0.4 naoh 1 hr genomic dna contained processed gel transferred onto hybond n membrane amersham biosciences pittsburg pa usa gene specific probes prepared pcr generated fragments labeled using ecl direct nucleic acid labeling system amersham biosciences previously described growth conditions including inducing development used a. nidulans 17 18 expression anea via niia promoter induced 0.6% sodium nitrate repressed 0.2% ammonium tartrate an anea disruption cassette dc constructed according methods described yu et al using genomic dna prepared a. nidulans fgsc a4 dna fragments dc amplified pcr using appropriate primer sets anea a1/-a2 anea b1/-b2 argb for/-rev the complete dc amplified nested pcr primer set anea c1/-c2 purified used transform tj1 strain nucleic acid preparation northern blot analysis performed described previously southern blotting genomic dna treated appropriate restriction enzymes separated 1% agarose gels the gels washed distilled water dw soaked twice depurination solution 250 mm hcl 15 min reaction gel washed dw soaked denaturation solution 1.0 nacl 0.4 naoh 1 hr genomic dna contained processed gel transferred onto hybond n membrane amersham biosciences pittsburg pa usa gene specific probes prepared pcr generated fragments labeled using ecl direct nucleic acid labeling system amersham biosciences the dc deletion anea gene constructed used transform recipient strain southern blot analysis pst digested genomic dna candidate strains pcr amplified probe specific 5'-flanking region anea confirmed deletion chromosomal anea gene revealing expected band 5.7 kb fig 1 investigate function anea development a. nidulans pattern hyphal growth including radial growth septation asexual sporulation examined one deletion mutant strains however observed differences wild type deletion mutant data shown these results indicated -cop essential viability a. nidulans results similar obtained yeasts recently copi proteins reported play roles responses er stress thermal stress yeast 11 13 here tested effect -cop deletion sensitivity environmental stresses caused treatment fungi several drugs known exert adverse effects cellular processes related stress response no detectable changes observed -cop defective strain treatment drugs including inhibitors cell wall biosynthesis calcofluor white congo red caspofungin n glycosylation tunicamycin ergosterol biosynthesis terbinafine glycerol biosynthesis fludioxonil inducer apoptosis farnesol ca chelator egta thermal stress makes -cop defective yeast strain unviable showed effect viability vegetative growth -cop deletion strains data shown taken together these results show -cop a. nidulans neither essential viability involved stress responses unlike yeast a. nidulans mutation sodc gene copi depletion also results failure cytokinesis reduction number overlapping central spindle microtubules meiotic divisions spermatogenesis drosophila -cop depleted anea a. nidulans strain observed defects asexual development data shown significant defects sexual development fig even culturing conditions favorable cleistothecium development hypoxic treatment 1% glucose dark anea strain showed 50% decrease fruiting body formation table 3 in addition 1% glucose without hypoxic treatment fruiting body formation almost completely abolished -cop depletion 2% lactose without hypoxic treatment predominantly induces formation fruiting bodies -cop depletion showed deleterious effect fruiting body formation table 3 because effect c sources hypoxic treatment tested presence 0.1% sodium nitrate nitrogen source tested 0.2% yeast extract preferentially induces sexual development found defect fruiting body formation 1% glucose without hypoxic treatment recovered anea deletion strain data shown although experiments required determine underlying mechanisms results suggested -cop plays role sexual development a. nidulans certain environmental conditions possibly affecting cytokinesis and/or construction er based spindle envelopes observed fruit fly spermatogenesis our previous study showed c terminal domains -cop -cop essential interaction n terminal wd40 motif -cop tpr region -cop involved controlling interaction two cops a. nidulans we also reported a. nidulans -cop substitute s. cerevisiae -cop functions vivo yeast -cop known stabilize thermo sensitive -cop mutation thus expression -cop confers viability -cop mutant elevated temperatures therefore investigated whether phenotype sodc1 mutant suppressed expression anea a. nidulans prg3-ama1 plasmid containing anea gene encoding -cop introduced sodc1 mutant expression anea however expression -cop could rescue thermo sensitive phenotype sodc1 mutant non permissive temperature 42 fig these results indicate -cop able interact -cop stabilize -cop elevated temperatures unlike yeast summary results indicate -cop a. nidulans essential related formation sexual reproductive organ response particular environmental factor(s it worth highlighting recent reports indicate function copi complex subunits confined intracellular vesicular trafficking the copi complex also involved cellular events chromosome disjunction a. nidulans male development chicken embryos er stress responses yeast association calcineurin heat stress meiotic divisions spermatogenesis drosophila induction productive autophagy cellular survival intercompartmental trafficking specific rnas neuronal cells neurite outgrowth although studies necessary confirm actual role -cop and/or copi sexual development fungi results open interesting avenues studies function copi complex the dc deletion anea gene constructed used transform recipient strain southern blot analysis pst digested genomic dna candidate strains pcr amplified probe specific 5'-flanking region anea confirmed deletion chromosomal anea gene revealing expected band 5.7 kb fig 1 investigate function anea development a. nidulans pattern hyphal growth including radial growth septation asexual sporulation examined one deletion mutant strains however observed differences wild type deletion mutant data shown these results indicated -cop essential viability a. nidulans results similar obtained yeasts recently copi proteins reported play roles responses er stress thermal stress yeast 11 13 here tested effect -cop deletion sensitivity environmental stresses caused treatment fungi several drugs known exert adverse effects cellular processes related stress response no detectable changes observed -cop defective strain treatment drugs including inhibitors cell wall biosynthesis calcofluor white congo red caspofungin n glycosylation tunicamycin ergosterol biosynthesis terbinafine glycerol biosynthesis fludioxonil inducer apoptosis farnesol ca chelator egta thermal stress makes -cop defective yeast strain unviable showed effect viability vegetative growth -cop deletion strains data shown taken together these results show -cop a. nidulans neither essential viability involved stress responses unlike yeast in a. nidulans mutation sodc gene encoding -cop responsible non disjunction chromosome cell division copi depletion also results failure cytokinesis reduction number overlapping central spindle microtubules meiotic divisions spermatogenesis drosophila -cop depleted anea a. nidulans strain observed defects asexual development data shown significant defects sexual development fig even culturing conditions favorable cleistothecium development hypoxic treatment 1% glucose dark anea strain showed 50% decrease fruiting body formation table 3 in addition 1% glucose without hypoxic treatment fruiting body formation almost completely abolished -cop depletion 2% lactose without hypoxic treatment predominantly induces formation fruiting bodies -cop depletion showed deleterious effect fruiting body formation table 3 because effect c sources hypoxic treatment tested presence 0.1% sodium nitrate nitrogen source tested 0.2% yeast extract preferentially induces sexual development found defect fruiting body formation 1% glucose without hypoxic treatment recovered anea deletion strain data shown although experiments required determine underlying mechanisms results suggested -cop plays role sexual development a. nidulans certain environmental conditions possibly affecting cytokinesis and/or construction er based spindle envelopes observed fruit fly spermatogenesis our previous study showed c terminal domains -cop -cop essential interaction n terminal wd40 motif -cop tpr region -cop involved controlling interaction two cops a. nidulans we also reported a. nidulans -cop substitute s. cerevisiae -cop functions vivo yeast -cop known stabilize thermo sensitive -cop mutation thus expression -cop confers viability -cop mutant elevated temperatures therefore investigated whether phenotype sodc1 mutant suppressed expression anea a. nidulans prg3-ama1 plasmid containing anea gene encoding -cop introduced sodc1 mutant expression anea however expression -cop could rescue thermo sensitive phenotype sodc1 mutant non permissive temperature 42 fig these results indicate -cop able interact -cop stabilize -cop elevated temperatures unlike yeast summary results indicate -cop a. nidulans essential related formation sexual reproductive organ response particular environmental factor(s it worth highlighting recent reports indicate function copi complex subunits confined intracellular vesicular trafficking the copi complex also involved cellular events chromosome disjunction a. nidulans male development chicken embryos er stress responses yeast association calcineurin heat stress meiotic divisions spermatogenesis drosophila induction productive autophagy cellular survival intercompartmental trafficking specific rnas neuronal cells neurite outgrowth although studies necessary confirm actual role -cop and/or copi sexual development fungi results open interesting avenues studies function copi complex

we have previously isolated -cop , the -cop interactor in copi of aspergillus nidulans , by yeast two - hybrid screening . to understand the function of -cop , the anea+ gene for -cop / anea was deleted by homologous recombination using a gene - specific disruption cassette . deletion of the -cop gene showed no detectable changes in vegetative growth or asexual development , but resulted in decrease in the production of the fruiting body , cleistothecium , under conditions favorable for sexual development . unlike in the budding yeast saccharomyces cerevisiae , in a. nidulans , over - expression of -cop did not rescue the thermo - sensitive growth defect of the -cop mutant at 42. together , these data show that -cop is not essential for viability , but it plays a role in fruiting body formation in a. nidulans .

diagnosis extent resection management hd depend sensitive specific identification ganglion cells.[13 however documenting aganglionosis often difficult tedious routine hematoxylin eosin h e stained sections acetylcholinesterase ache evolved gold standard diagnosing hd however histochemical analysis technically challenging date gained worldwide utilization applicability the aim study evaluate efficacy calretinin immunostaining ganglionic aganglionic hd colon biopsy specimens correlate h e thereby exploring utility suspicious cases hd our standard protocol care child presenting hd barium enema study presentation followed laparotomy multiple biopsies taken spastic aganglionic segment transition zone normal colon a colostomy sited level junction normal colon transition zone the definitive surgery choice duhamel pull 6 months age thirty six full thickness rectal biopsies suspected hd 24 bowel segments resected definitive pull surgery calretinin monoclonal mouse antihuman antibody dako clone dak calret 1 code ir627 immunohistochemistry ihc staining done paraffin embedded blocks routine h e examination the age patients ranged 1 day 14 years mean 8.2 months 46 boys 14 girls f 3.2 3.3:1 normal ratio 3:1 4:1 twenty three patients 63.8% presented less 1 month age five 8.3% long segment disease 6 10% total colonic aganglionosis analysis 36 initial full thickness colon biopsy specimens h e staining revealed absence ganglion cells negative 19 cases 52.7% presence ganglion cells positive 2 cases 0.05% suspected presence ganglion cells 15 cases 41.6% of 19 cases reported negative h e staining 17 47.22% reported negative 2 0.05% positive calretinin histochemistry ganglion cells nerve fibers table 1 hematoxylin eosin staining versus calretinin rectal biopsies 15 patients h e sections suspicious presence ganglion cell calretinin ihc showed immunopositivity 3 slides whereas 5 slides showed immunopositivity nerve fibers among 5 slides positive nerve fibers 2 cases 2/60 calretinin gave slight positive staining nerve fibers staining areas the slight calretinin positivity 2 specimens observed large bundles staining areas thus indicating beginning transition zone 24 patients resected specimens definitive surgery sent serial biopsies taken aganglionic segment transition zone ganglionic segment table 2 calretinin expressed ganglion cells nerve fibers submucosal myenteric plexus 24 aganglionic spastic segments transition zone calretinin staining positive ganglion cells 20 cases 83.3% focally positive nerve fibers 22 cases 91.6% submucosal myenteric plexus in ganglionic bowel segments hd calretinin showed immunopositivity 90% ganglion cells nerve fibers submucosal myenteric plexus the histological diagnosis hd challenging requiring expertise senior pathologist access specialized techniques handling frozen specimens ache staining this makes diagnosis hd difficult centers cases infrequent causing delay treatment child calretinin proven highly sensitive presence ganglion cells nerve fibers this protein involved calcium transport absence allows accumulation cytoplasmic calcium causing excess neuroexcitability ultimate neurodegeneration barshack et al procured colons 10 patients proven hd calretinin ihc performed sections aganglionic zone ganglionic zone transition zone colons they identified calretinin staining interstitial nerve fibres inf ganglion cells normal colon figures 1 2 focal inf staining 92% transition zones in contrast inf ganglion cell staining aganglionic zone it interpreted positive negative immune reactivity reducing ambiguity diagnosis study rectal biopsy specimens diagnosis ambiguous 15 patients h e examination using calretinin immune positivity demonstrated 3 patients 3 ganglion cells 5 nerve fibers thereby ruling hd patients hematoxylin eosin section ganglion cell arrow h e 10 calretinin staining ganglion cells h e 40 guinard samuel et al studied largest number suction biopsies using calretinin they concluded calretinin accurate proving absence ganglion cells easy interpret replace ache diagnose hd they however recommend used aid along h e examination especially ultrashort segment disease transition zone specimens study found calretinin correlated h e examination rectal biopsies resected bowel specimens rectal biopsy specimens calretinin also aided diagnosis 15 patients ambiguous findings calretinin ihc accurate detecting presence absence ganglion cells holds several advantages follows 1 carried paraffin embedded tissue sections 2 staining pattern simple 3 binary pattern interpretation negative positive 4 cost effective present study it serve valuable cost effective diagnostic aid centers ache enzyme histochemistry available

aim : to evaluate the efficacy of calretinin immunostaining in diagnosing hirschsprung 's disease ( hd).materials and methods : sixty cases were studied over a period of 1 year ( july 2010-june 2011 ) . there were 36 full - thickness biopsies and 24 resected specimens . calretinin processing was done on the paraffin - embedded blocks after routine histopathological examination.results:of the 36 biopsy specimens , in 19 cases hd was diagnosed by hematoxylin and eosin ( h and e ) staining earlier . in 2 patients , ganglion cells were seen and hd was ruled out . in 15 cases , there was a diagnostic dilemma and calretinin was used . ganglion cells were found in 3 specimens and nerve fibers in 5 . in all 24 resected specimens , calretinin correlated with the findings on h and e staining.conclusions:calretinin was extremely useful in solving the suspicious and indeterminate cases of hd . it can serve as a valuable cost - effective diagnostic aid in the centers where acetylcholinesterase enzyme histochemistry is not available .

drug abuse considered critical health related social economic problem countries last three decades world experienced shocking figures expressing drug abuse prevalence societies esp among teenagers youth united nation office crime drug abuse prevention recently reported 185 million drug consumers world wide increasing treatment demand world iranian drug abuse prevention headquarter estimated 4.5 million opium consumers iran end 2004 study conducted serajzadeh feyzi 21 iranian state universities 5.8% interviewed drug consumers claimed drug dependent 3.1% claimed high drug dependency making total 8.9% drug dependent university students regarding harmful pharmaceutical social legal health economic effects drug abuse need serious preventive action one divides drug abuse prevention three comprehensive selective obligatory categories other preventive strategies include life skills training lst information improvement affective education social influence drug abuse resistance education life skills abilities needed provide groundwork effective stress management presentation positive behaviors these skills enable individual accept social role responsibilities face others demands expectations daily interpersonal problems effectively without hurting others the term life skills involves big class socio psychological interpersonal skills help individual take conscious decisions communicate effectively improve interactive self management skills adopt active healthy life style life skills organize personal interpersonal environmental actions way lead better health turn leads physical psychological social comfort lst program aimed drug abuse prevention new preventive strategy primarily focuses socio psychological factors leading drug abuse mainly emphasizes promotion personal social skills this program first planned gilbert botvin kernel university prevent smoking abuse among teenagers 1997 used alcohol drug abuse prevention researchers confirmed positive influence lst drug abuse reduction effective use intelligence capacity furthering self confidence ego improvement prevention aggressive behaviors suicide aids prevention lots investigations also conducted individual social problems solution lst studied impact lst middle schoolers knowledge insight ability adopt healthy life style findings reported lower rate smoking among attended program compared refused attend eisen et al showed significant difference pre- post test scores 6239 students study drug abuse rate among intervention group significantly lower norm compared control group another study botvin griffin claimed positive effect lst addiction prevention the survey tested 4466 students new york schools 3 years investigate effectiveness cognitive behavioral program drug abuse prevention those received least 60% intervention program showed significant drug abuse reduction furthermore qaderi indicated training courses could reduce drug abuse tendency affect addicts attitude one survey carried nazarpoor et al 243 students tabriz medical university investigate lst affects individual opinion toward drug abuse prevention besides change rise knowledge insight assertiveness skills rate attending lst workshops a significant relationship also observed participants change attitude toward drug abuse rise social skills due participation workshops furthermore significant relationship detected major gender social skills promotion rate greater change observed among boys they concluded due impact cognitive skills development attending lst workshops necessary university students results survey feredrick university germany demonstrated preventive effect lst non smokers non drinkers also great impact smokers drinkers moradi et al investigated effect drug abuse resistance prevention skills training 181 workers asalooyeh petrochemical company two intervention control groups results indicated positive impact training intervention group knowledge insight drug abuse resistance skills self efficacy decision making preventive actions study barati et al showed positive role lst reduction abstract norms encouraging drug abuse among university students this prove positive impact assertiveness training program modification beliefs abstract norms youth also great influential role planning performance drug abuse prevention training programs especially universities the present study aimed investigating effect lst promotion drug abuse preventive behaviors among university students stability results 4-year follow this field trial study conducted using pre- post experimental design control group samples 60 university students 50% male 50% female entering university different majors selected quota random sampling assigned randomly two equal intervention control groups matching the selection criterion included factors student gonabad medical universityentering university second semester 2007giving consent able attend workshops student gonabad medical university entering university second semester 2007 giving consent able attend workshops the intervention group took part two one day lst workshops held weekly first workshop four years intervention 2011 examinees except 9 tested using instrument 4 intervention group 5 control group ceased continue study due attrition control confounding variables participants inspection period researchers data collected use researcher made questionnaire including demographic information age gender major father mother educational level father job habitation ( b drug abuse preventive behaviors including self awareness interactive skills decisiveness ability say problem solving ability resist others illogical demands stress management familiarity drug abuse side effects negative attitude toward drug abuse the questionnaire included following items regarding self awareness items familiarity components self awareness self evaluation skills knowledge self strengths weaknesses positive thinking abilities positive ego development skills relationship self esteem drug abuse self esteem endurance features tolerant people factors affecting features investigatedthe domain interactive skills investigated issues knowledge definition objectives communication components communication communication barriers ineffective interaction effective listening interpersonal relations importance ways improve themregarding decisiveness saying others resistance others illogical demands items familiarity basic concepts assertiveness interactive styles types assertiveness cognitive barriers assertiveness crucial steps behavior change steps assertive behavior advice say special techniques assertive behavior discussedproblem solving domain paid issues familiarity basic concepts problem solving problem centered coping emotion centered coping steps problem centered copingdecision making skill involved familiarity decision making process factors affecting decision different styles decision making steps logical decision makingcritical thinking domain studied items knowledge basic concepts critical thinking components critical thinking thinking instrumentsin stress management domain factors knowledge concept stress related factors model application coping strategies stress symptoms stress management strategies descriptions self cooling adaptive introspections investigateddrug abuse side effects focused types drug mechanism side effects risk factors preventive factorsattitude toward drug abuse included items general attitude toward addiction drug abuse social opinion addiction drug abuse solution problems addiction avoidance addicts regarding self awareness items familiarity components self awareness self evaluation skills knowledge self strengths weaknesses positive thinking abilities positive ego development skills relationship self esteem drug abuse self esteem endurance features tolerant people factors affecting features investigated domain interactive skills investigated issues knowledge definition objectives communication components communication communication barriers ineffective interaction effective listening interpersonal relations importance ways improve regarding decisiveness saying others resistance others illogical demands items familiarity basic concepts assertiveness interactive styles types assertiveness cognitive barriers assertiveness crucial steps behavior change steps assertive behavior advice say special techniques assertive behavior discussed problem solving domain paid issues familiarity basic concepts problem solving problem centered coping emotion centered coping steps problem centered coping decision making skill involved familiarity decision making process factors affecting decision different styles decision making steps logical decision making critical thinking domain studied items knowledge basic concepts critical thinking components critical thinking thinking instruments stress management domain factors knowledge concept stress related factors model application coping strategies stress symptoms stress management strategies descriptions self cooling adaptive introspections investigated drug abuse side effects focused types drug mechanism side effects risk factors preventive factors attitude toward drug abuse included items general attitude toward addiction drug abuse social opinion addiction drug abuse solution problems addiction avoidance addicts the questionnaire made use related references texts expert panel eight experts field commented face qualitative content validity questionnaire having taken required permits validation aims study emphasizing confidentiality results attracting trust consent participants data collected distribution questionnaires among target group lst workshops study pre- post test design questionnaires encoded examinees could tested workshops 4 years educational intervention furthermore ensure one way blindness intervention pre test post test administered experienced test taker unaware type intervention workshops held four experts including two psychiatrists two clinical psychology masters passed lst courses training techniques included lecturing discussion question answer role play modeling handing educational booklets chicago il usa descriptive analytical analyses frequency mean standard deviation tests chi square test p 0.0.5 permission data collection gained research ethics committee gonabad university medical sciences their responses questionnaire anonymous respondents participated study voluntarily permission data collection gained research ethics committee gonabad university medical sciences their responses questionnaire anonymous respondents participated study voluntarily 51.6% samples 17 19 years 38.3% 20 22 years 10.2% 23 24 50% participants studying public health 26.7% nursing 23.3% operation room anesthesia regarding father educational level 10% primary school education 18.3% middle school education 35% diploma 20% aa 10% ba 6.7% higher as mother educational level 13.3% primary school education 38.3% middle school education 30% diploma 10% aa 8.3% ba higher considering father job 8.3% jobless 33.3% workers farmers 26.7% employees 31.7% self employed nearly 66.7% participants resided towns urban 33.3% lived villages rural table 1 descriptive data demographic variables independent test results indicated significant difference pre test post test drug abuse preventive behaviors control intervention groups intervention p 0.36 significant difference observed immediately intervention p 0.001 words lst could successfully promote drug abuse preventive behaviors reduce risk factors leading drug abuse table 2 comparison drug abuse preventive behaviors based independent test intervention follow test administered also paired test showed significant difference control group pre- post intervention insight toward drug abuse prevention p 0.05 regarding factor intervention group showed significant change intervention p 0.001 remained stable follow test administered 4 years p 0.01 table 3 comparison drug abuse preventive behaviors based paired test intervention follow test administered chi square test results detected significant relationship factors gender age major drug abuse preventive behaviors p 0.05 demonstrated significant relationship father educational level promotion drug abuse preventive behaviors p 0.05 way father higher educational level led participant awareness behaviors also participants whose fathers clerks employees thought tobacco harmful whose fathers workers jobless though difference statistically significant p 0.05 those inhabiting towns knowledge decisive behaviors compared villagers difference n't significant either p 0.05 results showed lst could either promote participant knowledge drug abuse preventive behaviors decrease risk factors leading drug abuse significant post test increase intervention group drug abuse preventive behaviors mean scores observed the findings study line studies e.g. zollinger et al botvin griffin botvin et al moradi et al confirmed positive impact lst promotion intervention group insight drug abuse resistance skills self efficacy decision making balance preventive activities also barati et al showed effectiveness lst reduction abstract norms encouraging drug abuse among university students affirm positive impact training modification beliefs abstract norms youth also mentioned factors study results show directly related psychological capacity improved lst the findings study also line bohler feredrick university demonstrated preventive role lst among non smokers non drinkers also great impact smokers drinkers smith et al also claimed significant impact lst leadership management skills youth findings also confirm results studies samari laalyphase asserted lst promote one interactive skills social acceptability turn affect drug abuse preventive behaviors valyany et al emphasized importance lst workshops reduce drug abuse tendency the study results indicated intervention group observed pre- post intervention difference remained stable even follow test conducted 4 years main treatment this line findings botvin griffin suggested 6-year long educational program confirms effectiveness lst drug abuse prevention nazarpoor et al claimed promotion participants knowledge social skills also meaningful increase change outlook toward drug abuse attending workshops it seems change outlook long run reduce drug abuse tendency our study proved significant positive relationship father educational level children knowledge drug abuse preventive behaviors this confirms findings babayi showed students whose parents higher academic level better information drug abuse probably families higher academic levels potential skills use various techniques promote children insight toward drug abuse this proves crucial role parents play forming proper insight toward drug abuse turn formation drug abuse preventive behaviors children our study included limitations samples ceased go study available follow test lst promote interactive decision making problem solving critical thinking stress management skills lead social acceptability turn reduce drug abuse tendency it recommended plan perform constant lst workshops effective tools drug abuse prevention universities

background : drug abuse is now - a - days one of the gravest social harms . recent years have experienced a drastic rise in drug abuse among school and university students . thus , the need for special attention to the issue is deemed important . the present study was conducted with the aim of assessing the impact of life skills training on promotion of drug abuse preventive behaviors.methods:this field trial experimental study was conducted on 60 students of gonabad medical university selected through quota random sampling and assigned randomly into two intervention and control groups . data were collected through a questionnaire , including two sections of demographic information and drug abuse preventive behaviors . the questionnaire was first assessed as to its validity and reliability and then administered both before and after educational intervention and also as a follow - up 4 years after intervention data were then analyzed using t - tests and chi-square.results:comparison of post - test mean scores of drug abuse preventive behaviors of both groups showed a significant difference ( p < 0.01 ) which remained stable 4 years after intervention . there was a significant relationship between father 's educational level and drug abuse preventive behaviors ( p < 0.01).conclusions : life skills training is effective in the promotion of drug abuse preventive behaviors of university students .

q fever zoonotic disease caused coxiella burnetii microorganism frequently infects domestic ungulates well wild mammals many genera 1 animals the disease transmitted humans incidentally inhalation aerosols infected cattle sheep 1 2 humans c. burnetii infection may asymptomatic acute chronic chronic q fever rare endocarditis presenting common complication 1 3 q fever reported almost every country except new zealand 4 canada burnetii antibodies 5 japan 60 84% cattle reproductive disorders seropositive 6 a study conducted southern france showed 5 8% endocarditis cases humans due c. burnetii prevalence acute q fever 50 cases per 100,000 inhabitants 7 researchers suggested incidence q fever chronically underestimated clinical manifestations disease often nonspecific even absent therefore concerns disease focus importance detection 1 3 8) korea little information concerning epidemiology c. burnetii infection either animals humans a cases acute q fever humans reported 9 10 we examined prevalence antibodies c. burnetii dairy cattle nationwide people health screening rural area korea used data evaluate impact q fever animals humans korea upon agreement dairy owners serum samples 414 dairy cattle collected 31 farms march june 2001 serum collected people visited kangwon national university hospital health examinations april december 2002 the subjects interviewed confirm absence symptoms respiratory tract infection preceding two weeks informed consent obtained people health screening animals treated ethical guidelines kangwon national univesity coxiella burnetii phase ii antigen nine mile whole cell antigen prepared previously described national institute infectious diseases niid tokyo japan 12 dotted onto teflon printed glass slides each serum sample diluted 1:16 phosphate buffered saline pbs overlaid antigen dots incubated 45 min 37 moist chamber the slides subsequently washed twice 5 min pbs plus 0.05% tween-20 incubated 1:1,400 dilution fluorescein isothiocyanate fitc)-conjugated rabbit anti bovine igg sigma aldrich st louis mo u.s.a fitc conjugated rabbit anti human igg dakocytomation glostrup denmark 45 min 37 moist chamber the slides washed twice using method examined using fluorescence microscopy axioskop 2 zeiss germany 200 magnification all sera produced positive equivocal reactions 1:32 analyzed using 2-fold serial dilutions 1:4,096 approximately 10% sera divided tested concurrently kangwon national university niid quality control test reproducibility greater 95% concordance results two laboratories upon agreement dairy owners serum samples 414 dairy cattle collected 31 farms march june 2001 serum collected people visited kangwon national university hospital health examinations april december 2002 the subjects interviewed confirm absence symptoms respiratory tract infection preceding two weeks informed consent obtained people health screening animals treated ethical guidelines kangwon national univesity coxiella burnetii phase ii antigen nine mile whole cell antigen prepared previously described national institute infectious diseases niid tokyo japan 12 dotted onto teflon printed glass slides each serum sample diluted 1:16 phosphate buffered saline pbs overlaid antigen dots incubated 45 min 37 moist chamber the slides subsequently washed twice 5 min pbs plus 0.05% tween-20 incubated 1:1,400 dilution fluorescein isothiocyanate fitc)-conjugated rabbit anti bovine igg sigma aldrich st louis mo u.s.a fitc conjugated rabbit anti human igg dakocytomation glostrup denmark 45 min 37 moist chamber the slides washed twice using method examined using fluorescence microscopy axioskop 2 zeiss germany 200 magnification all sera produced positive equivocal reactions 1:32 analyzed using 2-fold serial dilutions 1:4,096 approximately 10% sera divided tested concurrently kangwon national university niid quality control test reproducibility greater 95% concordance results two laboratories each province relatively high prevalence ranging 8.9 59.3% overall national prevalence 25.6% of positive sera n=106 80 75.5% high antibody titers 1:256 the mean age subjects 43.7 yr standard deviation 15.9 range 19 82 male female ratio 1:0.86 n=110:95 of one serum sample high antibody titer 1:512 two samples low antibody titers 1:64 table 2 this study examined seroprevalence c. burnetii infection cattle people health screening korea the sera cattle showed high prevalence 25.6% anti c burnetii antibodies 75.5% positive sera high titers 1:256 by contrast sera healthy people showed relatively low prevalence 1.5% anti c the sera dairy cattle collected regardless disease status animal limitation study since could find association history reproductive failure seropositivity cows absence data disease status prediction concerning seropositive status reproductive problems made however c. burnetii infection prevalent areas korea every region seroprevalence 8% national prevalence 25.6% moreover high prevalence high titer sera provides evidence disease might active country neighboring japan seroprevalence c. burnetii healthy cattle ranges 2 46% cattle reproductive disorders range 60 84% 6 reported bovine placentitis highly associated presence c. burnetti 13 in addition 9% abortions goats reported caused microorganism 14 this first report provide data suggest c. burnetii infection might one important causes reproductive problems cattle korea further studies based isolation c. burnetii needed elucidate etiologic role microorganism reproductive problems cattle country furthermore high seroprevalence c. burnetii among cattle suggests possibility contamination environment around farms since c. burnetii widely distributed wild animals ticks causes q fever humans 1 3 necessary evaluate environmental hazards associated c. burnetii infection may threaten public health near future in addition survey high risk group farmers butchers strongly needed the finding people rural area demonstrated relatively low seroprevalence c. burnetii well lower titers suggests infection microorganism seems relatively low rural area korea previously published report ( 11 less 1% healthy people patients fever unknown origin tested positive anti c another report showed none sera 70 healthy people reactive c. burnetii antigen 15 recent data showed 11 448 healthy people reactive 16 on hand group previously reported one 88 patients community acquired pneumonia diagnosed q fever ifa assay using phase ii antigens 18 despite low seroprevalence c. burnetii humans exclude risk q fever individuals high risk occupations farmers veterinarians meat processing workers considering high rate infection observed cattle study previous korean report showed two 46 stock breeders antibody titer 1:20 15 5 sera 202 abattoir workers reacted phase ii antigen 16 conclusion c. burnetii appears highly prevalent pathogen cattle korea accordingly studies high risk groups needed evaluate seroprevalence organism korea

we report results on the seroprevalence of antibodies to coxiella burnetii in cattle and healthy people in korea . upon agreement with dairy owners , serum samples from 414 dairy cattle were collected between march and june 2001 and samples from 205 people for health screening were collected between april and december 2002 . the sera were analyzed for the presence of anti - c . burnetii phase ii antibodies using an indirect microimmunofluorescence test ; strong fluorescence at a 1:32 dilution was regarded as positive . the overall seroprevalence of c. burnetii in cattle in korea was 25.6% , with regional variation from 8.9 to 59.3% . of the positive serum samples , 75.5% had antibody titers 1:256 . by contrast , only 1.5% of people in a rural area were seropositive , and most of the positive samples had low antibody titers . in conclusion , this study showed that relatively high seropositivity of c. burnetii in dairy cattle , accordingly , the studies on the high - risk groups are needed to evaluate the seroprevalence for this organism in korea .