Datasets:

ronitHF

/

pubmed-10k

like 0

brown - squard syndrome ( bss ) , which occurs due to dysfunction of the spinothalamic tract , typically reflects the hemisection of the spinal cord at the cervical or thoracic level . the syndrome mainly occurs as a result of penetrating trauma , syringomyelia , hematomyelia , tumor , severe discs , or blunt trauma . among the multiple etiologies , the most common cause is penetrating trauma , such as a gunshot7,8 ) . therefore , most management guidelines focus on penetrating cervical injuries and/or vertebral artery ( va ) injury12 ) . non - missile penetrating spinal cord and va injuries are rare because of the bony structures that protect the spinal cord and va14 ) . thus , the treatment approach for wounds caused in non - missile penetrating spinal injuries such as a knife , a power drill bit , or even a pen could be different from common missile penetrating injuries5,6,13,17).to our knowledge , there are few reports in the literature of complete obstruction of the va due to penetration of a foreign body through the neural foramen into the spinal canal . herein , the authors report on va dissection and bss caused by penetration of an electric screw driver bit . a 25-year - old machine operator was involved in a violent episode and was stabbed in his right neck with an electric screw driver bit that was thrown by the opponent . on arrival at the emergency department , the electric screw driver bit was placed in the right lateral aspect of the neck at zone i ( fig . the tip of the electric screw driver bit was located at the center of the vertebral canal of c3 ( fig . he was given high - dose methylprednisolone ( bolus dose of 30mg / kg followed by 5.4mg / kg / hour for 23 hours ) according to the protocol for spinal cord injury . an immediate interventional angiography was undertaken without general anesthesia due to the nature of the emergency . the angiography revealed a total occlusion with dissection of the right va at the level of c3 . immediate coil embolization at both proximal and distal ends of the injury site was performed ( fig . an attempt at manual extraction of the electric screw driver bit failed with great resistance . after the patient was moved to the operating room , the electric screw driver bit was removed manually with muscle dissection under general anesthesia . venous blood spilled out and was controlled easily by application of several pieces of gelatin sponge . no postoperative complications such as wound dehiscence , cerebrospinal fluid ( csf ) leakage , or infection were observed . the neurological motor function of the right upper and lower extremities recovered to 3/5 and 4/5 , respectively , with persistent decreased sensory function after one year . fortunately , the patient experienced no neck swelling , auscultation of a neck bruit , or delayed ischemic complications . penetrating injury is the third most frequent cause of spinal cord injury in adults , surpassed only by traffic accidents and falls3,18 ) . stab wounds are associated with lesser surrounding tissue injury than gunshot wounds because the former delivers less energy than missile injuries9 ) . although vascular injury is the most common sequel of penetrating neck trauma , va injury is rare because it is well protected by the transverse foramen4,10 ) . therefore , penetrating injury of the va is mostly caused by gunshot wounds which deliver large kinetic energy , depending upon the bullet 's mass and speed12 ) . in this article , we report a rare case of va penetration by an electric screw driver bit with spinal cord insult , consequently presenting as bss . moreover , surgical exploration of the va can cause additional damage to the spine and surrounding tissues . therefore , it may be reasonable to embolize an occluded artery , because the unilateral ligation of the va rarely results in brainstem ischemia11,16 ) . there are a few reports regarding the treatment of traumatic va injury such as the arteriovenous fistulas and pseudoaneurysms2 ) . emergent surgical exploration is necessary for patients with hard signs of vascular injury , such as hemodynamic instability , hemorrhage exsanguinations , or expanding hematoma15 ) . patients that are hemodynamically stable and who are without respiratory compromise should undergo further diagnostic imaging evaluation15 ) . as presented in this case , endovascular techniques were a safe and effective method of treatment and were not associated with significant morbidity or mortality1 ) . airway management , intubation methods , and surgical positions can be points of debate between anesthesiologists and surgeons9 ) . if a lacerated va can be successfully obliterated , a penetrating electric screw driver bit may be extracted without general anesthesia . nevertheless , the authors recommend that surgeons should be prepared for conversion to open surgery and extraction should be performed with the support of a surgical team . we initially tried to extract the electric screw driver bit manually without general anesthesia in the intervention theater after va embolization . however , the electric screw driver bit was positioned firmly in the neural foramen , and the patient complained of severe pain when the electric screw driver bit was being pulled out . in addition , there was more important rationale that justified surgical exploration for extraction of the electric screw driver bit . on extraction of the electric screw driver bit , the authors describe a rare case of penetrating cervical injury caused by an electric screw driver bit with accompanying va penetration and 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 .

INTRODUCTION CASE REPORT DISCUSSION

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

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

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

Introduction Materials and Methods Results Discussion

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

Introduction Materials and Methods Results Discussion

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

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION Limitations of the study Recommendations CONCLUSION Summary

primary intracystic squamous cell carcinoma ( scc ) in the breast is an extremely rare neoplasm . primary sccs in the breast are quite rare , although scc mixed with ductal carcinoma is more common . it can arise from metaplastic epithelium associated with other primary malignancy of the breast , metastasis from primary elsewhere in the body or an extension of malignancy from the skin covering . a 45-year - old female presented with a lump in the right breast , which she had noticed since three weeks . her menstrual cycles were regular and she had two children . clinical examination revealed a firm cystic lump of 12 10 cm in the upper inner and central quadrant of the right breast . the skin over the right breast appeared stretched , with a slight retraction of the nipple . pre - operatively , the patient was relegated to clinical stage iii ( t3n0m0 ) . ultrasonographic examination of the right breast showed an irregular - shaped hypoechoic lesion measuring 12 6 cm with an internal anechoic area measuring 6 3 cm , suggesting a cystic malignant tumor . fine needle aspiration from the lump in the right breast yielded 10 ml of pale yellow serous fluid . wet - fixed and air - dried smears were prepared from the centrifuged aspirated fluid and were stained with papanicolaou ( pap ) stain and may - grnwald - giemsa ( mgg ) stain , respectively . the smears studied were cellular and showed malignant squamous cells predominantly in singles and occasional syncytial groups , and some cells were spindle shaped . the malignant squamous cells had rounded borders with hyperchromatic enlarged nucleus and coarse irregular granular chromatin . cytoplasm of the squamous cells showed a variable degree of keratinisation [ figure 2 ] . cytology smears showing malignant squamous cells in singles with numerous cyst macrophages ( mgg , 400 ) cytology smear showing malignant squamous cells in singles , demonstrating cytoplasmic keratinisation ( pap , 400 ) to exclude metastatic scc , a search for remote primary scc included chest radiograph , cystoscopy , colposcopy , oesophagogastroscopy , laryngoscopy and cervical pap smear , which did not reveal any extramammary cancer . the criteria include : ( 1 ) no other neoplastic element , such as ductal or mesenchymal ones , are present in the tumor , ( 2 ) the tumor is independent of adjacent cutaneous structures and ( 3 ) no other distant epidermoid tumor exists in the patient . later , simple mastectomy with axillary clearance was performed and the specimen was sent for histological examination . the cut - section of the mastectomy specimen showed a large cystic tumor measuring 8 3 cm , containing yellow serous fluid . the cystic cavity was lined by dysplastic squamous epithelium with an infiltrating tumor , showing malignant squamous cells in broad sheets , groups and whorls , with keratin - pearl formation and intercellular bridges . immunostaining for estrogen / progesterone receptor and her-2/neu oncoprotein were negative and that for cytokeratin was positive . the smears studied were cellular and showed malignant squamous cells predominantly in singles and occasional syncytial groups , and some cells were spindle shaped . the malignant squamous cells had rounded borders with hyperchromatic enlarged nucleus and coarse irregular granular chromatin . cytoplasm of the squamous cells showed a variable degree of keratinisation [ figure 2 ] . cytology smears showing malignant squamous cells in singles with numerous cyst macrophages ( mgg , 400 ) cytology smear showing malignant squamous cells in singles , demonstrating cytoplasmic keratinisation ( pap , 400 ) to exclude metastatic scc , a search for remote primary scc included chest radiograph , cystoscopy , colposcopy , oesophagogastroscopy , laryngoscopy and cervical pap smear , which did not reveal any extramammary cancer . the criteria include : ( 1 ) no other neoplastic element , such as ductal or mesenchymal ones , are present in the tumor , ( 2 ) the tumor is independent of adjacent cutaneous structures and ( 3 ) no other distant epidermoid tumor exists in the patient . later , simple mastectomy with axillary clearance was performed and the specimen was sent for histological examination . the cut - section of the mastectomy specimen showed a large cystic tumor measuring 8 3 cm , containing yellow serous fluid . multiple sections studied showed cystic breast tumor . the cystic cavity was lined by dysplastic squamous epithelium with an infiltrating tumor , showing malignant squamous cells in broad sheets , groups and whorls , with keratin - pearl formation and intercellular bridges . immunostaining for estrogen / progesterone receptor and her-2/neu oncoprotein were negative and that for cytokeratin was positive . pure scc and adenosquamous carcinoma have been listed under metaplastic breast carcinomas in the world health organization classification . the japanese breast cancer society has defined scc of the breast as a special type of cancer , in which malignant cells are arranged in broad sheets and whorls with keratin formation or intercellular bridge . the incidence of scc reported in the western countries is 0.13.6% and in japan , it is 0.17% . the age group affected is between 32 and 65 years , with an increased tendency of left - sided involvement . the characteristics of scc of the breast generally reported are a large - sized tumor , rapidly growing with a central cyst formed by necrosis . this is unlikely in our case because there was little necrosis elsewhere in the tumor . the possible mode of origin of scc is epidermoid cyst of the breast , chronic abscess and complete metaplasia of glandular breast tissue . lymph node involvement is reported to be less frequent than might be expected , given the larger tumor size . the tumor cells are negative for vimentin , estrogen and progesterone and are diffusely positive for high molecular weight cytokeratin and c - erbb-2 . squamous cells in fine needle aspiration cytology ( fnac ) of breast lesions can be found in various benign lesions , like epidermoid cyst , subareolar abscess , fibroadenoma , infracted papillomas , spindle cell metaplasia , cystic sarcoma phyllodes , pseudosarcoma and malignant breast tumors or metastatic malignancy . the benign breast conditions with abundant squamous cells may sometimes mimic malignant squamous lesion and vice versa . in general benign squamous cells are bland looking and are often associated with anucleated squames , and no tumor , cell cannibalism . the malignant squamous cells are more pleomorphic , mitotically active and dyskeratotic and , sometimes , bizarre - shaped cells can be seen . the differential diagnosis of malignant squamous cells in fnac of the breast includes primary scc and metastatic scc of the breast . careful assessment of cytological features of squamous cells and the background appearance appears to be critical for arriving at a correct diagnosis . however , in the case of intracystic scc , numerous foamy macrophages coexist with malignant squamous cells . hormonal therapy is not indicated as most cases of scc are negative for hormonal receptors . scc of the breast is reported to be resistant to both radiotherapy and standard chemotherapy performed for invasive ductal carcinoma . in our case , radical mastectomy with axillary clearance was performed with postoperative adjuvant therapy , as given for common types of breast cancers . there was no evidence of recurrence of the tumor after four years of treatment . in conclusion , the presence of malignant squamous cells in fine needle aspiration of the breast suggests primary scc or metastatic scc . the finding of a pure scc necessitates more accurate work - up to exclude skin lesion or metastasis . the primary scc should not be confused with a much more largely manifested metaplastic change in other usual breast cancers . although the presence of numerous cyst macrophages in the background of breast fnac smears suggests a benign lesion , when present with malignant squamous cells , they 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 .

Introduction Case Report Cytology Histology Discussion

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

1. Introduction 2. Method 3. Results 4. Discussion

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

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

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

Introduction Pathophysiology of type 2 diabetes, DPP-4 inhibitors, and vildagliptin Vildagliptin and Ramadan fasting Conclusion

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

INTRODUCTION MATERIALS AND METHODS Patients Treatment Assessment of response and statistical analysis RESULTS Patient characteristics Incidence of etPD, ltPD, and PsPD Survival outcomes Pseudoresponse DISCUSSION CONCLUSION

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

Introduction Case Report Discussion The authors state that they have no Conflict of Interest (COI).

this case describes a young non - pregnant cat that presented with uterine prolapse in association with an unusual diffuse , polypoid , fibrosing perimetritis and parametritis . no intra - abdominal complications were seen on ultrasound examination 3 months postsurgery . at the time of writing , uterine 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 . uterine prolapse is rare in the queen , usually occurring after delivery of kittens or abortion . we report uterine prolapse in a non - pregnant queen and in association with an unusual diffuse , polypoid , fibrosis of the perimetrium and parametrium . the perimetrium ( syn uterine tunica serosa ) is equivalent to the visceral peritoneal lining . it is composed of loose connective tissue containing nerves and smooth muscle cells covered by mesothelium . it blends in with the broad ligaments of the uterus , which is also covered by mesothelium and contains a core of connective tissue , the parametrium . while peritonitis in cats is frequently associated with feline infectious peritonitis , specific lesions unique to the perimetrium have not been documented . a stray 6- to 7-month - old domestic shorthair queen , weighing 2.15 kg , was presented to the university veterinary clinic with uterine prolapse after being observed for several days prior to capture . on examination , both uterine horns were prolapsed , everted and swollen , with regions of hyperaemia , particularly towards the tips of the uterine horns . no evidence of placental zoning or tissue necrosis , in particular gangrene , was noted ( figure 1 ) . prolapsed and everted uterus following lavage which revealed swollen , hyperaemic and viable tissue clinical assessment revealed normal appetite and demeanour . the vital parameters were 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 ) at 0.1 mg / kg iv q4h for pain management and to facilitate further examination . initial haematological and blood biochemical evaluations revealed a 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 ] ) and a neutrophilic leukocytosis with 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 a mild hyperkalaemia ( 6.0 mmol / l [ ri 3.75.8 mmol / l ] ) and mild hypoglycaemia ( 66 mg / dl [ ri 70150 mg / dl ] ) . oestrogen plasma concentrations were retrospectively evaluated at 25.7 pg / ml ( ri 1540 pg / ml ) . intravenous crystalloid fluid solution ( plasmalyte ; travenol laboratories ) was administered to correct an estimated 8% dehydration deficit and maintenance requirement over 12 h. antibiotic therapy consisting of intravenous cefazolin sodium ( cefazolin for injection ; west - ward pharmaceutical ) at a dose rate of 22 mg / kg iv q8h was also administered . attempts at reducing the uterus were again unsuccessful so an internal ovariectomy and external hysterectomy were performed . the ovarian pedicles were transfixed and ligated prior to transection and before manipulating the uterine body further . the everted uterus was circumferentially ligated distal to the cervix ( approximately 5 cm away from the vulva ) and transected . the remaining uterus was returned to the abdominal cavity . from within the abdominal cavity , the serosa of uterine body was noted to be diffusely thickened by multiple - to - coalescing white villous projections ( figure 2 ) . formalin - fixed perimetrial surface of uterine body revealing diffuse - to - coalescing polypoid lesions there was a small amount of mildly flocculent abdominal fluid present . the cat recovered uneventfully and follow - up examinations in a 6 month period , including ultrasound at 3 months postsurgery , detected no abdominal abnormalities . microscopic examination of the uterine body confirmed the presence of multiple nodular perimetrial proliferations of moderately loose connective tissue forming irregular polyps or plaques blending into the myometrium ( figures 3 and 4 ) . most polyps contained multifocal randomly distributed lymphocyte and plasma cell infiltrations , and regions of neovascularisation . polyps were covered by a combination of normal , attenuated and moderately swollen mesothelial cells . at sites of mesothelial cell loss , the stratum vascularis was moderately oedematous containing both congested medium - to - large size blood vessels and dilated lymphatics . endometrium and uterine epithelium appeared unremarkable with a low glandular content , intact mucosal epithelium and minimal - to - no inflammatory cell infiltrations . the lesion was characterised as a diffuse , chronic , polypoid , fibrotic perimetritis and parametritis . cross - section of uterine body ( u ) revealing multiple polypoid perimetrial projections ( p ) ( haematoxylin and eosin ) polypoid structures blending with myometrium and containing ( a ) foci of angiogenesis and ( b ) regions of active fibroplasia ( haematoxylin and eosin ) uterine prolapse has rarely been documented in cats , with individual case reports citing complete or unilateral uterine prolapse with or without uterine mucosal eversion . the majority have occurred in association with the periparturient period , one of which was proposed to be associated with oestrus . furthermore , a novel lesion inducing extensive perimetrial fibrosis , not previously reported in cats , was noted and could potentially be implicated as the cause . however , other factors have been suggested including uterine atony or excessive abdominal contractions , including tenesmus . for the uterus to prolapse there is a requirement for laxity , stretching or rupture of uterine ligaments , and for patency of the cervix . laxity of the uterine ligaments can occur due to repeated pregnancy , and changes in progesterone , oestrogen and relaxin concentrations near parturition . cervical patency occurs at two time points , oestrus ( with or without ovulation ) and at parturition . based on the absence of any newborn animals , no mammary development and no histopathological features to suggest recent pregnancy , such as placental zoning , recent uterine haemorrhage , inflammation and endometrial regeneration , it is unlikely this animal was in advanced pregnancy or had aborted . it was not possible , however , to establish if this animal had recently been in oestrus as oestrogen levels , which were within normal limits , can vary significantly over short periods of time within the oestrus cycle . while the uterus was large , this latter feature could potentially have contributed to uterine prolapse in at least two ways , namely by inducing ligament laxity and/or acting as a caudal abdominal irritant inducing abdominal contractions or tenesmus . while an active lesion , the extent of mature fibrous tissue would suggest the presence of this lesion in advance of the uterine prolapse . perimetrial inflammation can co - exist as part of peritonitis , and in cats peritonitis is frequently associated with feline infectious peritonitis or primary bacterial septic peritonitis where bacteroides species and fusobacterium species are mainly isolated . however , such lesions do not manifest as polypoid fibrous proliferations . similar proliferative fibrotic lesions focused on the perimetrium have been described in dogs associated with experimental stilboestrol ( synthetic oestrogen ) administration . perimetrial papillae consisting of proliferations of mesothelial cells and subjacent connective tissue were documented in ovariectomised dogs administered stilboestrol over 150350 days . similar to the present case , such lesions were confined to genital organ serosal surfaces , including the broad ligament . such perimetrial lesions were also noted in entire female dogs when administered synthetic oestrogens in association with other lesions , including oestrogen - induced renal fibrosis . proposed mechanisms of regional fibrous proliferations suggested involvement of stromal oestrogen receptors . while the cat develops oestrogen - associated pathology , similar perimetrial pathology has not been documented in association with oestrogen . the ovaries were not submitted for histopathological evaluation to eliminate ovarian pathology definitively ( cystic follicular disease , ovarian neoplasia ) as a potential source of hyperoestrogenism . cats in the tropics are likely to cycle continuously and it is recognised that some unmated cats may appear to be in persistent oestrous , owing to a relatively short interoestrous period and thus remain in a physiological hyperoestrogenic state . it was also unlikely this cat had access to exogenous oestrogenic compounds . in humans , more generalised intra - abdominal , fibrosing lesions of peritoneal surfaces ( intra - abdominal fibromatosis ) are described in mesenteric , retroperitoneal and pelvic locations . sclerosing peritonitis has been noted after surgery , following long - term continuous peritoneal dialysis and also as a side effect of utilising beta - blockers . the pathogenesis of these lesions is thought to be the result of a delayed healing response due to loss of mesothelial cells or continued serosal irritation . however , the appearance is not described as polypoid and ranges through a spectrum of gross changes in the appearance of the visceral peritoneum , from opacification of the serosa ( disorganised collagen fibres and stromal expansion ) through to tanned peritoneum ( hyalinised collagen , fibrin and vessel sclerosis ) , which can progress to a stage where the viscera are palpably stiffened ( mural fibrosis ) . the unique perimetritis and parametritis observed in this case highlights an additional potential cause of uterine prolapse and warrants that , in cases of uterine prolapse in non - pregnant animals , perimetrial and parametrial tissue should be evaluated for varying degrees of 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 .

Case summary Relevance and novel information Introduction Case description Discussion Conclusions

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

1. Background 2. Objectives 3. Patients and Methods 3.1. Analysis of Acinetobacter baumannii Species Isolated From Clinical Samples 3.2. Multilocus Sequence Typing 3.3. Analysis of Drug Resistance and Related Genes 3.4. Statistical Analysis 4. Results 4.1. Detection of the Drug-Resistance Ability of the Isolates 4.2. Results of Multilocus Sequence Typing 4.3. Detection of Drug-Resistance Genes Expression 5. Discussion

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

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

despite advancements in modern medical science and the health management industry , the incidence of cerebral palsy ( cp ) continues to rise1 . the most common features of cp are decreased muscle strength and abnormal muscle tone2 . cp sufferers lack theability to generate enough force to maintain antigravity postural control , which result in abnormal postures3 . the development of movement and posture may be altered by non - progressive damage to the brain and subsequent neurological impairments ( spasticity , muscle weakness , co - contractions and visual impairment)5 . studies indicate that children and adults with both mild and severe forms of cp have postural impairments6,7,8 . the emergence of sitting postural control in early infancy changes the way infants interact with the world . from the sitting position , looking , reaching , and interacting become functional and allow exploration that supports learning and further development of motor skills . therefore , independent sitting , defined as not needing support from a caregiver or pillow while sitting , is one of the first developmental goals for every child . individual differences are present between children , and characteristic signs of developmental disorders during infancy are relatively unspecific . therefore , why a specific child is not able to achieve sitting postural control is not always clear . one method of examining postural control in adults and children is to measure the center of pressure ( cop ) at the base of support using a force platform during the task of remaining upright . cop has frequently been used to investigate postural control during standing by young children who are healthy or have cp9 , 10 . the purpose of this study was to investigate the differences of the pressure distributions of the sitting postures of typical developmental ( td ) children and children with cp . twelve cp childrens were recruited from an outpatient rehabilitation clinic . they were hemiparetic , had an mmse - k score above 2411 , could maintain an independent sitting posture without support . two of the cp children were subsequently excluded because they refused to participate in this experiment . all parents of the enrolled participants provided their written informed consent to their children s participation prior to this experiment , in accordance with the ethical principles established in the declaration of helsinki . as a result of the exclusion , this study used two school chairs mounted on a force platform to assess the quiet - sitting pressure distribution of the subjects . fsa seating assessment ( canada ) the acquisition frequency was set at 5 hz . the stated working range of the device is 0200 mmhg , with a resolution of 1 mmhg . the system was also calibrated to assign absolute pressure values to the digital output from an a / d converter connected to the sensing pad . this was done by applying a pressure distribution as similar to actual conditions as possible . then thus subjects sat on one of two school chairs according to their height . the chairs were those generally used in school . for research purposes , this study used two basic school chair because students spend a long time of day - to - day sitting on them . one chair had a 40 cm floor to seat height , a 35 cm seat depth , and a 32 cm seat width and is designed for 122.4133.5 cm height of subjects . the another had a 35 cm floor to seat height , a 38 cm seat depth , and a 35 cm seat width , and is designed for 133.6152.7 cm height of subjects . it has been used for the posture symmetry in other study13 statistical analyses were performed using pasw 18.0 . descriptive statistics were calculated ( frequency , mean , standard deviation , range ) . the mann - whitney u - tests and wilcoxon s signed rank tests were used to analyze differences between the groups and differences in lesion side , respectively . the si of the age matched td group was employed as the normal criteria . table 1table 1.general characteristics of the 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 the general characteristics of the participants . the si of the td group was significantly closer to zero than that of the cp group of children . cp and right hemiparetic cp were respectively 4.96 ( 2.24 ) and 5.12 ( 0.83 ) with no significant difference between the hemiparetic sides . the purpose of the present study was to investigate the differences in sitting posture of children with cp while they sat on school chairs . it is known that dynamic postural control during sitting can be reliably assessed using cop data of infants who are developing typically or infants with or at risk of cp14 . the principal finding of this study is that cp children s sitting posture is asymmetrical , leaning to the less - paretic side . we thought that infants who are developing typically develop the ability to sit by exhibiting an optimal range of movement variability , whereas cp may present either too much or too little variability leading to a very rigid and narrow or unpredictable set of movement solutions to achieve independent sitting . the finding of this study is agreement with previous studies that have the dissimilarities of the cop patterns of infants with cp and td have been obviously demonstrated14 . the results can not be generalized to all cp children because the sample was limited to ten children and they were at the one stage of the developmental process . future studies should assess the relationship between symmetry of sitting posture and 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 .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

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

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

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

Introduction Methods RPE cell isolation and culture Central neuroretina explant preparation and culture Histological and immunochemical processing Neuroretina thickness quantification Statistical analysis Results RPE cells Neuroretina explants Neuroretina thickness quantification Discussion Conclusions

ectopic kidney is a relatively rare renal anomaly however , an endourologist does encounter stone disease in an ectopic kidney occasionally . factors such as anomalous blood vessels and tortuous ureter with high insertion can lead to poor drainage and predisposition to the formation of renal calculi in these patients . the common management option for such stones is laparoscopy or ultrasound guided percutaneous nephrolithotomy ( pcnl ) . we report our experience with the use the recently described micro - pcnl or microperc for two such cases . two male patients , aged 57 and 60 years respectively , presented to us with calculi in ectopic pelvic kidneys . the first patient had undergone a flexible ureterorenoscopy elsewhere during which the stone could not be reached due to difficult angulation and inflamed tissue leading to poor vision . computed tomography urogram revealed an ectopic malrotated left kidney lying over the sacrum . there was a 13 mm 11 mm sized calculus in the renal pelvis [ figure 1a and b ] . in the second patient , computed tomography urogram revealed a normal right kidney and an ectopic malrotated left kidney lying over the sacrum . there was an 18 mm 17 mm sized calculus ( 1232 hu ) in the pelvis and a 5 mm 4 mm sized calculus ( 423 hu ) in the lower calyx of the ectopic kidney [ figure 1c and d ] . ( a ) x - ray kub showing renal calculus in pelvis of pelvic ectopic kidney with double j stent in situ , ( b ) computed tomography urogram showing the pelvicalyceal system anatomy , ( c ) x - ray kub showing renal calculus ( yellow arrow ) in ectopic kidney , ( d ) computed tomography urogram showing the pelvicalyceal system anatomy ( location of pelvic stone - yellow arrow ; location of lower calyceal stone - red arrow ) both procedures were performed under general anesthesia . in lithotomy position , ureteric catheterization was carried out under cystoscopic guidance using 7 fr ureteric catheters over a 0.035 inch glidewire ( terumo , tokyo , japan ) . the position was changed to supine - oblique with a sandbag under the ipsilateral hemipelvis to move the overlying bowel away from the kidney . an ultrasound probe was pressed against the anterior abdominal wall to displace the bowel away from the line of access . further , colour doppler was used to rule out any significant blood vessel in the path of needle puncture . three - way connector was attached to the needle , allowing saline irrigation , passage of a 0.9 mm flexible microperc telescope , and a 272 m holmium : yttrium aluminum garnet ( ho : yag ) laser fiber [ figure 2 ] . the stones were completely fragmented to dust with the laser . an x - ray and ultrasound was obtained on the first post - operative day to document stone clearance and to rule out any fluid collection . the urethral catheter was removed on the first post - operative day and the patient was discharged . ( a ) surface view of patient position and ultrasound guided percutaneous renal access , ( b ) intraoperative ultrasonography picture showing entry of puncture needle ( yellow arrows ) into lower calyx containing calculus ( red arrow ) , ( c ) confirmation of access into lower calyx by antegrade contrast study to delineate pelvicalyceal system , ( d ) intraoperative surface view showing microperc instruments , ( e ) puncture site at the end of the procedure ( yellow arrow ) , ( f ) post - operative x - ray kub showing complete clearance operating times were 30 and 35 min respectively . in the first patient , ureteric catheter was left in situ for 1 day . in the second patient , ureteric catheter was replaced by a double - j stent at the end of the procedure . visual analog pain scores ( 1 - 10 scale ) on first post - operative day were two and three respectively . first post - operative day and 1 month follow - up x - ray and ultrasound kub revealed complete stone clearance and no evidence of fluid collection in the abdomen . options for managing small renal calculi in pelvic ectopic kidney are shock wave lithotripsy ( swl ) , retrograde intrarenal surgery ( rirs ) , ultrasound or laparoscopy guided pcnl . since these kidneys are surrounded by bowel and bone , moreover , the clearance of fragmented stones is also impaired due to high insertion of ureter and impaired pyeloureteral motility due to surrounding fibrous bands . rirs can be technically demanding due to abnormal and tortuous course of the ureter of a pelvic kidney . this was exemplified by our first patient in whom the stone could not be reached with flexible ureteroscope and the procedure had to be abandoned . the potential hazards in percutaneous access in an ectopic kidney are : ( 1 ) risk of injury to surrounding bowel , particularly if track dilatation is carried out through the bowel , ( 2 ) abnormal vasculature resulting in bleeding from tract dilatation and ( 3 ) spillage of fluid into peritoneal cavity . microperc is a minimally invasive form of pcnl in which percutaneous renal access and stone fragmentation are achieved in a single - step using a 16 g needle . since dilatation is not performed , potential hazards associated with it are avoided . fluid collection is less likely during puncture and at the end of the procedure since the needle puncture site closes quickly . both our patients had rapid post - operative recovery , probably due to lack of fluid spillage and hence no paralytic ileus . though laparoscopic guidance has been advocated for percutaneous access of an ectopic kidney , in experienced hands , ultrasound guidance can provide a safe entry into the appropriate calyx . appropriate patient positioning to move bowel away from the kidney , placing a sand bag to push the kidney towards the anterior abdominal wall and compression with the ultrasound transducer allow safe access into the kidney . further , the use of colour doppler rules out any significant blood vessel along the path of needle puncture . the hemoglobin drop in both of our patients was minimal , supporting the safety of this procedure . in laparoscopy guided pcnl , an abdominal drain may need to be left in situ for a prolonged period because of persistent urinary leakage . the average hospital stay in a series of 15 patients who underwent laparoscopy guided pcnl was 4.8 days ( 4 - 11 days ) . the patients in that series had sequential removal of nephrostomy tube , urethral catheter and abdominal drain , which lead to the prolonged post - operative stay . thus , ultrasound guided microperc is a safe and efficient technique in the management of small renal calculi in ectopic kidneys and it is a minimally invasive procedure with 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 .

INTRODUCTION CASE REPORT DISCUSSION

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

INTRODUCTION MATERIAL AND METHODS RESULTS DISCUSSION CONCLUSION

human metapneumovirus ( hmpv ) is an rna virus in the pneumovirinae subfamily of the paramyxoviridae family that was first isolated in the netherlands in 2001 and has subsequently been identified worldwide . it has been implicated as a significant cause of hospitalization for young children , second only to respiratory syncytial virus ( rsv ) in infants hospitalized with acute respiratory infections ( aris ) . hmpv has been detected in 1.543.0% of patients with aris . while it circulates predominantly in the winter , seroprevalence studies have shown that almost all children over five years of age have evidence of past infection . clinical syndromes associated with hmpv infection are similar to those of rsv infection , ranging from mild upper respiratory tract infections to wheezing and severe lower respiratory tract infections requiring mechanical ventilation . rare cases of fatalities have been associated with hmpv and it has been implicated in a handful of cases of encephalitis . although hmpv infections have been diagnosed in adults , their greatest impact occurs in children . a significant association with hmpv and wheezing is seen in young children , and hmpv has been linked to apparent life - threatening events in infants . hmpv has been associated with aris with super - infections as a result of staphylococcus aureus and streptococcus pneumoniae . to begin to understand the impact of hmpv on our institution , we analyzed children admitted to our tertiary care center in southeast michigan with respiratory symptoms during the respiratory season of 20062007 through an observational , retrospective study . the primary purposes of our study were to establish the utility of testing for hmpv in children who were admitted to our hospital during the respiratory virus season and to compare the impact of hmpv and rsv on the healthcare system . we identified a convenience sample of 256 nasopharyngeal ( np ) specimens from children younger than 18 years of age admitted with respiratory symptoms between november 1 , 2006 and may 31 , 2007 . the specimens were obtained by a np wash or swab based on the admitting physician s discretion . after routine testing by direct fluorescent antibody ( dfa ) and/or culture for rsv , parainfluenza viruses 13 , influenza viruses a and b , adenovirus , and rhinovirus , the np specimens were frozen at 70c and later subjected to nucleic acid extraction using the easymag system ( biomrieux , durham , nc , usa ) and following the manufacturer s protocol with slight modifications . two hundred microliters of each specimen were pre - treated with 20 units of dnase ( new england biolabs , uk ) at 37c for 45 min before extraction . extracts were used as the template for detection of hmpv using the nuclisense real - time analyte specific reagent ( asr ) assay performed on the easyq instrument ( biomrieux ) . a proprietary internal control containing the same primer binding sites as the hmpv target with unique internal sequences targeted by a separate molecular beacon probe were spiked into each specimen before extraction to monitor amplification integrity . laboratory personnel were blinded to the clinical data and the clinical investigator was blinded to the laboratory results . only the first specimen from which a virus was identified per admission was considered . the -test was used for analysis of categorical variables , comparing hmpv - positive patients to rsv - positive patients as well as patients who were negative for both hmpv and rsv . the student s t - test was used for an analysis that compared continu ous variables . of the 256 specimens , rsv was identified in 52 ( 20.3% ) , hmpv in 18 ( 7.0% ) , influenza in 9 ( 3.5% ) , rhinovirus in 5 ( 2.0% ) , parainfluenza in 8 ( 3.1% ) , and adenovirus in 4 ( 1.6% ) . three specimens had co - infections : hmpv and rsv in two and rsv and influenza a in one . hmpv was detected primarily in specimens collected between january and march ( 83.4% ) , while rsv was uniformly detected in those from november through february and then decreased into may . no hmpv was detected in specimens obtained in april and may ( figure 1 ) . because the primary goal of our study was to compare patients with hmpv to those with rsv , and because the number of patients with isolated viruses other than hmpv or rsv was low , cases with viruses other than hmpv or rsv and those with no specific virus isolated were combined as a separate group for the additional analyses detailed in the following section . while several studies have shown that hmpv occurs more in older children than does rsv , other reports showed no difference in age predilection , gender predominance , or presence of underlying medical disorders . in our study , the majority of hmpv - positive patients were aged 1324 months ( n=7 , 43.8% ) , whereas most rsv - positive patients ( n=35 ; 71.4% ) were younger than 12 months of age ( p<0.01 ) . underlying medical disorders considered were prematurity , chronic lung disease , bronchopulmonary dysplasia , asthma , congenital heart disease , congestive heart failure , immunosuppression , immunodeficiencies , hematological and solid organ malignancies , diabetes , and renal failure . we also found that children attending daycare or school were not at increased risk of being positive for hmpv or rsv or for having a respiratory illness not associated with either of these viruses ( table 1 ) . table 1demographic data.hmpv+(% ) n=16rsv+(% ) n=49negative for hmpv and 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 of underlying medical condition11 ( 68.8)32 ( 65.3)140 ( 74.5)p values not listed here were > 0.05 and thus deemed not statistically significant;*p<0.01 . p values not listed here were > 0.05 and thus deemed not statistically significant ; hmpv has previously been reported to be a rare cause of community - acquired pneumonia . we found that hmpv - positive patients were more likely to be diagnosed with pneumonia ( 37.5% ) than were the other two groups ( 14% , p=0.04 for rsv - positive , p=0.02 for negative for both hmpv and rsv ) . in addition , hmpv - positive patients were equally likely to be diagnosed with bronchiolitis as were rsv - positive children ( approximately 30% ) but less likely than children with respiratory symptoms who were negative for hmpv and rsv ( 11% , p=0.02 ) . in our study , the rate of abnormal chest radiographs was comparable in all three groups , but peribronchial cuffing was more likely to be present in hmpv - positive patients than in the other two groups ( table 2 ) . table 2clinical features.featurehmpv+(% ) n=16rsv+(% ) n=49negative for hmpv and 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 not listed here were > 0.05 and thus deemed not statistically significant;*p=0.04;**p=0.02;p=0.02;p<0.01;p=0.03;p=0.02 . pvalues not listed here were > 0.05 and thus deemed not statistically significant ; in order to compare severity of illness across the three patient populations , we examined their hospital course . mean duration of hospital stay was 6 days ( range 137 d ) for hmpv - positive patients , 6 days ( range 1112 d ) for rsv - positive patients , and 12 days ( range 1117 d ) for patients negative for hmpv and rsv ( p=0.83 for hmpv vs. rsv ; p=0.06 for hmpv vs. negative for hmpv and rsv ) . hmpv - positive patients were more likely to be treated with antibiotics than were rsv - positive patients ( 81.2% vs. 51.0% , p=0.03 ) and were more likely to be admitted to the intensive care unit ( icu ) ( 37.5% vs. 12.2% , p=0.02 ) . hmpv - positive patients were as likely to require oxygen supplementation , mechanical ventilation , and steroid use as were rsv - positive patients ( table 2 ) . infections with hmpv and rsv have been reported to be clinically indistinguishable , but subtle differences were identified in our study . the most common presenting symptom for hmpv - positive patients was fever , which occurred more often in hmpv - positive patients than in the other two patient populations . hmpv - positive patients were also more likely than patients negative for hmpv and rsv to have decreased urine output ( table 3 ; 43.8% vs. 14.4% , p<0.01 ) . on physical examination , hmpv - positive patients were more likely than rsv - positive patients to exhibit focal decreased breath sounds ( 18.8% vs. 2.0% , p=0.01 ) and were more likely than patients negative for hmpv and rsv to exhibit signs of otitis media ( 18.8% vs. 4.8% , p=0.02 ) . otherwise , there were no differences in the signs and symptoms noted in hmpv and rsv - positive patients ( table 4 ) . table 3clinical symptoms.hmpv+(% ) n=16rsv+(% ) n=49negative for hmpv and 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 not listed here were > 0.05 and thus deemed not statistically significant;*p=0.01;**p=<0.01;p<0.01 . pvalues not listed here were > 0.05 and thus deemed not statistically significant ; table 4clinical signs.hmpv+(% ) n=16rsv+(% ) n=49negative for hmpv and 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 of otitis media3 ( 18.8)8 ( 16.3)9 ( 4.8)lymphadenopathy of head / neck region1 ( 6.2)0 ( 0)11 ( 5.9)pvalues not listed here were > 0.05 and thus deemed not statistically significant;*p=0.01;p=0.02;p=0.02 . hmpv was the second most commonly identified respiratory virus during the respiratory season of 20062007 in our study . our results confirm the previous findings that children with symptomatic hmpv infection are older than those infected with rsv , possibly because of differences in the upper respiratory tract or lung anatomy of older children that allow for hmpv acquisition . in our study , although a previous report found that hmpv is a rare cause of community - acquired pneumonia among hospitalized patients ( 4.9% ) , 37.5% of our hmpv - positive patients were admitted with a diagnosis of pneumonia . this difference is likely because of a requirement of three independent radiologists interpretations of the radiographs in the earlier study compared to the diagnosis of one admitting physician in our study . hmpv - positive patients were more likely than the other two groups to have peribronchial cuffing on their chest radiographs , evidence suggesting that these patients have interstitial edema , likely a result of the disruption of the respiratory epithelial structure and inflammation that hmpv has been shown to cause in animal studies . hmpv may have a stronger predilection for the respiratory epithelial cells than rsv , corroborated by reports that hmpv - positive patients frequently exhibited signs of otitis media . further research is needed on the pathogenesis of hmpv in humans , specifically regarding the ability of hmpv to infect human respiratory epithelial cells compared to rsv and other viruses . the present study demonstrates , as previously reported , that hmpv - positive patients were as likely to be severely ill as were rsv - positive patients . in fact , in our study , hmpv - positive patients were more likely than rsv - positive patients to be admitted to the icu even though they were not more likely to have an underlying medical illness . in addition , hmpv - positive patients were more likely than were rsv - positive patients to receive antibiotics . during the study time period , our institution did not routinely test specimens for hmpv . physicians may have used antibiotics more often in patients only retrospectively shown to be hmpv - positive because they were not aware of a specific virus contributing to these patients illnesses . the finding of increased antibiotic use in children with other aris , most of whom had negative viral cultures , corroborates this suggestion . it is also possible that the increased diagnosis of pneumonia in the hmpv - positive patients contributed to the increased use of antibiotics . this study , however , shows that there may be subtle differences in the clinical features of hmpv and rsv infections . as previously reported , fever was the most common presenting symptom for hmpv - positive patients and was more common in hmpv - positive children than in the other two groups . hmpv - positive patients were also more likely to experience decreased urinary output than patients negative for hmpv and rsv . more information is needed to compare the level of inflammation produced by hmpv with that of other viruses . our study was limited by extraction of the data from a convenience sample and by the fact that only one respiratory viral season was studied . in addition , the signs and symptoms associated with the studied viruses may have been exaggerated as only hospitalized patients were included . lastly , we acknowledge the li mi tations associated with the use of different methods for detecting different viruses in our study . the real - time asr assay that we used to detect hmpv was likely more sensitive than the dfa- and culture - based techniques used to detect rsv and other viruses . as such , it is possible that children infected with rsv were assigned to the group of patients not infected with hmpv or rsv . even with this potential drawback , however , our data strongly suggest that hmpv was common in the patient population included in this study . furthermore , as molecular methods gain more widespread use for the detection of many respiratory pathogens , continued studies assessing the correlation between laboratory and clinical information are warranted . our study confirms that hmpv is a significant pathogen particularly in young children and is frequently associated with respiratory symptoms resulting in hospitalization . in general , the clinical manifestations of hmpv- and rsv - associated infections in children are similar , although our data suggest subtle differences in illness presentation but not in severity . our data also suggest that underidentification of children with hmpv may lead to inappropriate use of antibiotics . therefore , considering the high prevalence of hmpv , the severity of hmpv illness , and the ease and accuracy of detection , routine diagnostic testing for hmpv should be implemented . increasingly , hmpv is being recognized as a significant cause of disease in other populations such as elderly patients and immunocompromised cases , highlighting other groups that would likely benefit from routine testing for hmpv . future studies with expanded patient populations will help to determine how identifying cases with hmpv - associated disease will allow clinicians to anticipate the patient s clinical course , identify cohort patients appropriately , and decrease the use of 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 .

Introduction Materials and Methods Results Discussion

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

INTRODUCTION DATA COLLECTION AND PROCESSING STATISTICS OF NONCODE LNCRNAS AND DISEASES lNCRNA CONSERVATION QUALITY OF LNCRNAS DISCUSSION FUNDING

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

Introduction Results and Discussion Conclusion Experimental Section

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

INTRODUCTION CASE REPORT Diagnosis and etiology Treatment objectives and alternatives Treatment progress Treatment results DISCUSSION CONCLUSIONS

the laparoscopic removal of a cervical stump following a supra cervical ( subtotal ) hysterectomy was first described by nezhat et al , and they concluded that the cervical stump could be removed laparoscopically by an experienced surgeon . the advantages of the laparoscopic approach included possible stump adhesiolysis , providing adequate postoperative vault support , and assessment of the pelvic lymph nodes . the 43-year - old , presented with a history of persistent p v discharge and occasional post - coital bleeding . she had undergone subtotal hysterectomy in 1994 , due to postpartum hemorrhage following a normal delivery . a colposcopic biopsy done in january 2009 , reported severe dysplasia of the cervix , with a human papillomavirus ( hpv ) effect and crypt extension . there was a strong family history of cancer of the cervix , as her mother had succumbed to the disease . on general examination she was in fair general condition , well - built and well - nourished , with adequate hydration . the hemoglobin was 13.3 g / dl , blood sugar was 5.3 mmols / l , urea and electrolytes were normal . an initial diagnosis of abnormal pap smear was entertained and the patient opted for a laparoscopic trachelectomy , with the option of a laparotomy , after discussing all her options . there were dense adhesions in the pouch of douglas involving the bowel and the cervical stump . the pelvic lymph nodes were clearly visualized ( after intracervical methylene blue injection ) and did not appear to be enlarged . gentle adhesiolysis was undertaken using sharp dissection , bipolar cautery , and a harmonic scalpel . the vaginal vault was subsequently opened over the ceramic cup of a clermont ferrand elevator . a cystoscopy with retrograde ureteral catheterization , to confirm the integrity of the bladder and ureters , was undertaken . the cervical stump after laparoscopic trachelectomy at one week of follow - up the patient was well . a postoperative intravenous urogram ( ivu ) confirmed that both the ureters and bladder were intact . subtotal hysterectomy was developed as a procedure in the 1990s , and is regarded as a safe option to total abdominal hysterectomy in the management of benign uterine conditions and in obstetrics , due to severe postpartum hemorrhage . okaro et al , in an assessment of the long - term outcomes of laparoscopic supracervical hysterectomy analyzed the case records of 70 consecutive women undergoing the procedure . of these , 24.3% ( 17 cases ) reported symptoms related to the cervical stump , within 14 months of the original surgery . in his series 14 of these patients underwent laparoscopic trachelectomy , one had only laparoscopic adhesiolysis and two underwent a laparotomy with trachelectomy due to dense bowel adhesions on the cervical stump . histologically the stumps showed endometriosis ( 23.5% ) and mild dysplasia in 7.6% of the patients . in this case our patient presented with persistent p v discharge and occasional post - coital bleeding . the subsequent pap smears were abnormal . in a retrospective of 41 patients undergoing laparoscopic subtotal hysterectomy , van der stege et al , noted that 98% of the patients were satisfied with their procedure , with 10% of them having monthly spotting . they concluded that although laparoscopic hysterectomy for benign diseases was a satisfactory procedure , special attention should be paid to careful management of the cervical stump . hilger et al , reviewed the indications of 310 trachelectomies performed at the mayo clinic from 1974 to 2003 . they included stump prolapse ( 4% ) , fibroid mass ( 1% ) , cervical dysplasia ( 6% ) , carcinoma in situ ( 5% ) , irregular bleeding ( 2% ) , and cervicitis ( 53% ) . the complications following vaginal trachelectomies were encountered in 80% of the procedures against 37% in the abdominal procedure . in our report the cervical stump confirmed carcinoma in 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 .

INTRODUCTION CASE REPORT THE OPERATION DISCUSSION

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

1. Introduction 2. Organization of GSL-Enriched Lipid Microdomains 3. GSL Metabolism Diseases 4. GSL- and Ceramide-Enriched Membrane Microdomains Are Binding Targets for Pathogenic Microorganisms 5. Fatty Acid Chains of Ceramide Are Indispensable for GSL-Mediated Signaling 6. Role of Ceramide Metabolite Sphingosine-1-phosphate in Immunological Reactions of Human Keratinocytes 7. Conclusion

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

Introduction Results and Discussion Conclusion Experimental Section Supporting Information

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

Introduction Methods Subjects Measurement of the degree of stenosis, stroke severity, recurrent stroke, and functional outcome Measurement of plasma phospholipid FAs composition Risk factors Statistical analysis Results Demographic data and the relationship between clinical data and fatty acid composition Association of fatty acid composition with stroke severity on admission, recurrent stroke, and functional outcome Discussion Conclusions Supplementary Material

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

INTRODUCTION MATERIALS AND METHODS Statistical analysis RESULTS Evaluation of CVMS Evaluation of sagittal and vertical analysis Comparison between subgroups DISCUSSION CONCLUSION

established 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 . we 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 . 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 . we 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 .

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 .

Introduction Description Projects

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

I M Study question and design Participants Data collection and analysis Rigor Ethical considerations Findings Personal maturity and emotional growth Social development Closeness to God Alteration in value systems and beliefs Ethical and professional commitment D C

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

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

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

Introduction Results Discussion Experimental Procedures Author Contributions

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

Level of Evidence INTRODUCTION MATERIALS AND METHODS Objectives and Statistical Analyses RESULTS Demographics Primary Speech Perception Outcomes Subgroup Results Bilateral Outcomes Patient SelfAssessments Adverse Events DISCUSSION CONCLUSION

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

Introduction Materials and Methods Leishmania infantum Genomic DNA extraction PCR amplification and cloning in pTZ57R/T vector Ligation of LACK gene into pcDNA3.1 cloning vector Detection of pc-LACK recombinant plasmid SDS-PAGE and Western blot analysis Results Gene Cloning in pcDNA3.1 SDS PAGE and Western blot analysis Discussion Conclusion

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

Introduction Materials and Methods Subjects Questionnaire survey Blood and urine tests Measurement of baPWV Statistical analysis Results Discussion Conclusion Declarations Competing Interests Author Contributions Grant Support Abbreviations

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

What Can High-Reliability Organizations Teach Health Care? Assessing Hospitals Current Performance against the Principles of High Reliability Adapting High-Reliability Science to Hospitals The High-Reliability Health Care Maturity Model: A Practical Framework for Improvement Leadership Safety Culture Robust Process Improvement Future Research and Development Tasks Policy Implications Conclusion

acute generalized exanthematous pustulosis ( agep ) is a rare acute reaction that is drug - induced in 90% of the cases , characterized by a widespread , sterile pustular rash . cefepime is a fourth generation cephalosporin antibiotic used to treat febrile neutropenia , severe infections related to the urinary tract , skin , nosocomial pneumonia , brain abscess , and intra - abdominal and septic lateral / cavernous sinus thrombosis . a 67-year - old man with renal failure who had been on dialysis during the last 2 years and with an 8-year history of cardiac insufficiency was admitted to the hospital complaining of 6 days of diarrhea . the patient was taken to the semi - intensive care unit and treated with ciprofloxacin . as a consequence , his long - term medications had not been changed and consisted of acetylsalicylic acid , furosemide , captopril , carvedilol and clonazepam . on the seventh day , the patient became dyspneic and his chest radiograph showed a left lower lobe opacity . treatment for nosocomial pneumonia was promptly initiated with cefepime ( 1 g / day ) . five days later , he presented with a pruritic , erythematous , maculopapular eruption affecting the abdomen , neck and skin folds . one day later , he developed disseminated pustular lesions ( fig . 1 ) and his temperature was 37c . laboratory exams evidenced c - reactive protein 136 mg / l , white blood cells 14,700 cells/l ( normal 3,50010,500 cells/l ) with 11,995 cells/l neutrophils ( normal 1,7008,000 cells/l ) . histology showed a toxic pustuloderma with spongiform subcorneal pustules , edema in the papillary dermis and perivascular inflammatory infiltrate consisting of neutrophils ( fig . after withdrawal of cefepime and introduction of imipenem , the disseminated skin nonfollicular pustules cleared within 4 days following a desquamation . the patient denied previous adverse reaction to other drugs and no personal or family history of psoriasis was evident . agep is a disease characterized by the rapid onset of many sterile , nonfollicular pustules usually arising on an edematous erythema and frequently accompanied by leukocytosis and fever . skin symptoms usually arise rapidly after an insult and resolve spontaneously ( within a few days ) . agep often starts predominantly in intertriginous areas or on the face , spreading rapidly to the trunk and lower limbs . the mean duration of the pustules is 9.7 days , and an annular desquamation typically follows for a few days . complications are rare [ 1 , 3 ] . the agep validation score of the euroscar study group has been used to establish the diagnosis . a score between 8 and 12 for agep is a definitive diagnosis ( table 1 ) . the case score was 11 , according to the validation score of the euroscar study group ( table 2 ) . the main differential diagnosis of agep is pustular psoriasis . because the pustules clinically and histologically resemble the lesions of pustular psoriasis and because in a number of reports patients had a history of plaque psoriasis , some authors assume that agep is nothing more than an acute exacerbation of psoriasis caused by a variety of exogenous triggers however , many studies strongly suggest that agep is not associated with psoriasis [ 1 , 5 ] . up to now agep has been attributed to a variety of causes such as viral infections , chlamydia pneumoniae infection or hypersensitivity to mercury , but the skin reaction is primarily an adverse response to drugs . antibiotics , other than cefepime , have been implicated as the causative agents in 80% of individuals . in this group , the present case of agep has well defined criteria , and because correct diagnosis generally leads to spontaneous resolution once the causative drug is withdrawn , clinicians should keep the possibility of this cutaneous drug reaction in 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 .

Introduction Case Report Discussion

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

1. Introduction 2. Purpose 3. Methods 4. Results 5. Discussion

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

Introduction Patients and Methods Results Characteristics of the patients and dose of RUF Efficacy Response rate Retention rate Tolerability Discussion Conclusions

cluster headache is a form of primary neurovascular headache , which consists of unilateral head pain that occurs in association with cranial autonomic features and , in most patients , has a striking circannual and circadian periodicity . excruciating painful attacks are accompanied by restlessness or agitation , usually last less than 3 h , and occur in bouts for a few months during which the patient has one or more crisis per day . the international headache society defines cluster headache as attacks of severe , strictly unilateral pain which is orbital , supraorbital and temporal or in any combination of these sites , lasting for 15180 min and occurring from once every other day to 8 times a day . the attacks are associated with one or more of the following , all of which are ipsilateral : conjunctival injection , lacrimation , nasal congestion , rhinorrhoea , forehead and facial sweating , miosis , ptosis , eyelid oedema . epidemiological data for the general population are scarce ; only five studies have been carried out until now on cluster headache prevalence in the general population , with conflicting results ( prevalence rates vary between 56 and 326 cases in every 100,000 inhabitants ) . a recent study on a sample representative of the italian general population aged over 14 years , reported an estimated prevalence rate of 279/100,000 ( 95% ci : 173427 ) , 227/100,000 ( 95% ci : 104431 ) in women and 338/100,000 ( 95% ci : 175592 ) in men . cataract is clouding ( opacity ) of the lens of the eye that causes a progressive , painless loss of vision . the first symptom of cataract is usually blurred vision . other symptoms that may progressively appear are : glare , halos , double vision and perception of colours as more yellow and less vibrant . three different types of cataract can be identified on a histological basis : nuclear , cortical and posterior subcapsular . investigations [ 58 ] on possible risk factors for the development of cataract , showed positive correlation with myopia , diabetes , smoking [ 911 ] , use of systemic corticosteroids , exposition to uv - b , and other environmental factors , to which has to be added genetic predisposition . however , it should be underlined that different degrees of correlation between distinct risk factors and the three types of cataract have been reported [ 6 , 15 ] . prevalence studies of age - related cataracts are hampered by the absence of a uniform grading system for cataract opacities , by differing definitions of visual impairment , and by additional coexisting ocular pathologies causing loss of vision . nonetheless , the framingham eye study in 1977 reported that the proportion of people with age - related cataracts causing loss of vision of 20/30 ( 6/9 ) or worse was 15.5% for all ages and 45.9% for those older than 75 years . in the beaver dam eye study in 1992 , using a similar definition of loss of vision reported proportions were 38.8% for men and 45.9% for women older than 74 years . however , it is not clear if variations in frequency reflect methodological diversity or true differences between populations . interestingly , most of the cited studies analyzed the cataract prevalence in age groups which are substantially different from those of our two cases . one single study reported the prevalence of cataract at a younger age ( 40 years and older ) with identified risk factors for the three different histological types of cataract . this study showed age - specific rates of the different histological cataract type , by stratifying the population sample by age . prevalence of 1% ( cortical type ) , of 0.2% ( nuclear type ) and of 2.0% ( posterior subcapsular ) was reported in individuals between 40 and 49 years and a prevalence of 3.9% ( cortical type ) of 0.2% ( nuclear type ) and of 2.6% ( posterior subcapsular ) was reported in individuals between 50 and 59 years . here , we report two cases that both developed cataract before the age of 50 years on the side affected from the cluster headache and reviewed published similar cases and potential mechanisms for comorbidity . we present a 44-year - old man with a history of cluster headache , diagnosed in accordance with ihs guidelines , since he was 21 years old . daily pain attacks , lasting about 90 min , were localized to the left periorbital area with ipsilateral lacrimation and rinorrhea . the cluster headache that initially occurred sporadically , from the age of 43 years became chronic . at the age of 39 , the patient was diagnosed of cataract on the left eye that was in the same year treated by surgery . the sole risk factor for developing cataract at anamnesis was smoking habit ( 30 cigarettes / day ) . a detailed pharmacological anamnesis excluded any kind of exposure to prolonged treatment with steroid drugs . the second case was a 53-year - old man who experienced his first cluster headache episode when he was 44 years old . cluster periods were characterized by 34 attack / day of severe right - sided pain localized to the periorbital region , lasting about 45 min and associated to ipsilateral lacrimation . the reported risk factor for developing cataract was smoking habit ( 10 cigarettes / day ) . also in this case , there was no report of chronic treatment with steroid drugs . references were identified by searches of pubmed from 1966 until october 2007 with the terms cluster headache and cataract . our search in pubmed shows only one report of a patient with cluster - like headache , which began after surgical removal of the crystalline for cataract and intraocular lens implant . therefore , to our knowledge , this is the first report of cataract occurring in cluster headache patients and , interestingly , on the same eye affected by the pain attack . it is of interest that both patients developed cataract in a young age , after suffering for years from chronic cluster headache attacks and in the absence of main risk factors for the development of early onset cataract , including prolonged steroid drugs exposure . it is noteworthy that the two patients , although not relative , had the same , rather uncommon , last name that we found to be specifically represented in a defined area of north - eastern italy . however , because we could not investigate the pedigree of one patient , any hypothetical genetic link between the uncommon occurrence of early onset cataract and cluster headache can not be proposed . in addition , it was not possible to perform in either of the two patients any investigation on ocular inflammation . thus , no speculation can be made on a possible causal relationship between repeated episodes of pain , inflammation and autonomic abnormalities that are considered to contribute to the cluster headache attack , and the precocious occurrence of the cataract . however , this first evidence of the occurrence of cataract in relatively young adult men with a history of chronic cluster headache is of importance , because underlining this uncommon type of comorbidity may favour the report of 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 .

Introduction Case reports Search strategy and selection criteria Discussion

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

Introduction Materials and Methods 1. CT acquisition 2. Segmentation and planning 3. Fat measurement 4. Statistical analysis Results Discussion Conclusion

hepatitis b virus ( hbv ) is an important risk factor for developing hepatocellular carcinoma ( hcc ) ( 1 ) . hcc is the fifth most common cancer worldwide , and it causes significant public health problems , especially in association with chronic hepatitis b ( 2 ) . morphologic lesions during hepatocarcinogenesis include dysplastic lesions ( dysplastic foci ( df ) and dysplastic nodules ( dns ) ) with low- and high - grade dysplasia and small cancerous lesions ( 2 cm in diameter ; early hcc ) ( 4 - 7 ) . the most common cause of cirrhosis in shiraz transplant center , which is the largest liver transplant center in the country , is hbv related , and majority of patients with hcc in this center are also hbv related ( 1 ) . therefore , we attempted to determine the incidence of hcc and its precursors in this group of cirrhotic patients through a thorough examination of explanted hbv - related cirrhotic livers . a two - year cross - sectional study was performed on 103 explanted hbv - related cirrhotic livers from the pathology department of shiraz university of medical sciences . during the study period ( 2014 - 2015 ) , explanted cirrhotic hbv - related livers ( recipient cirrhotic liver after liver transplantation ) received in the department of pathology were fixed in formalin for 24 - 48 hours . histologic sections from any hepatic nodule larger than 1 cm in diameter or those with any kind of difference with background parenchyma , such as color or consistency , were studied ( figures 1 and 2 ) . sections were stained with hematoxylin and eosin method , and slides were examined under a double - headed microscope by two pathologists . df were classified into having either small cell changes ( scc ) , formerly called small cell dysplasia , or large cell changes ( lcc ) , formerly called large cell dysplasia . nodules were studied and classified as low - grade dysplastic nodules ( lgdn ) , high - grade dysplastic nodules ( hgdn ) , and hepatocellular carcinoma ( hcc ) ( 5 ) . the following criteria were used to classify the different nodules in cirrhotic livers : cellularity , thickness of hepatocellular plates , nuclear atypia ( either pleomorphism or irregular contour ) , nucleocytoplasmatic ratio , cytoplasmic staining ( eosinophilic , basophilic , amphophilic , and clear ) , pseudoacinar pattern , stromal invasion , portal tracts , biliary pigment , lipid vacuoles , and iron status of nodules ( 8) . in lgdn , hepatocytes are not completely abnormal . that is , they show normal or slightly increased nucleocytoplasmic ( n / c ) ratio , minimal nuclear atypia , and no mitotic activity , but portal tracts are still present . compared with lgdn , hgdn have all of their features except the n / c ratio is higher , nuclear atypia is more obvious , cytoplasmic basophilia is more significant , and liver plates ( more than two cells thick ) are thickened with mitotic figures . in hcc , cell size is usually decreased , nuclear density is at least twice that of normal , nuclear atypia is definite , mitotic figures are present , and pseudoacinar formation may be present ( 9 ) . the term early hcc mean hcc in an early stage that are small in size ( usually < 2 cm ) . histologically , hcc are well differentiated and lack prominent cellular and structural atypia ( 10 - 13 ) . for convenience , we used the term advanced hcc ( ahcc ) for hccs that are greater than 2 cm in size in this article . the chi - square test was used to evaluate the significance of the relation between the nodules and their categorical variables . a total of 103 patients were included in the study . among the patients , 14 ( 13.16% ) were women , 89 ( 86.4% ) were men , age ranged from 20 to 73 , and mean age was 51.4 10.5 . among all explanted cirrhotic livers , 92 ( 89.3% ) had df with lcc , 57 ( 55.3% ) of which showed scc as well . out of these 103 explanted livers , 39 ( 37.9% ) had lgdn , 38 ( 36.9% ) had hgdn , 19 ( 18.4% ) had early hepatocellular carcinoma ( ehcc ) , and 21 ( 20.4% ) had ahcc ( figures 3 - 6 ) . note that all the cases with ehcc and ahcc also had scc , lcc , hgdn , and lgdn . thirteen cases of ehcc were accompanied with ahcc , and 6 cases of ehcc did not show any ahcc ( size larger than 2 cm ) . all of the 21 ahcc cases were transplanted with the preoperative diagnosis of malignancy and hepatitis b - related cirrhosis . however , among livers with ehcc , 6 were not accompanied with ahcc , 2 of which were unsuspected before surgery and diagnosed on the explanted liver . the two ahcc cases were smaller than 2 cm in diameter ( table 1 ) . our results showed that the presence of scc , lgdn , and hgdn has a statistically significant association with ehcc ( p < 0.05 ) and hcc ( p < 0.05 ) . moreover , the presence of ehcc is associated with ahcc ( p < 0.05 ) . hcc is a poor prognostic cancer , and it is one of the most common causes of cancer death around the globe . hcc is also common in countries with high incidence of hbv - related cirrhosis ( 1 ) . despite the improvements in treatment modalities for hcc , the survival rate remains low even after liver transplantation because hcc can not be easily diagnosed before the advanced stage ( 14 ) . therefore , studies on the incidence and diagnosis of precancerous lesions of hcc are important , especially for patients who develop hcc secondary to hbv infection and cirrhosis . therefore , pathologists and clinicians should be able to diagnose ahccs and ehccs from their precursor lesions using various techniques preoperatively ( 10 ) . hepatic precancerous lesions are currently divided into two categories depending on cytological and histological changes : microscopic df and macroscopic dns ( 11 ) . df can be recognized only in microscopic examination as they are smaller than 1 mm . dns are recognizable both in gross examination of hepatic specimens and in microscopic examination as well - defined nodule lesions that are different from the background cirrhotic liver tissue in size , color , texture , or degree of bulging on the cut surface . hccs can be either ehcc ( less than 2 cm ) or ahcc ( larger than 2 cm ) ( 8 , 9 ) . globally , hbv is the most common cause of cirrhosis and hcc in iran ( 12 ) . in this study , we attempted to identify the true prevalence of hcc and its precursors in 103 explanted livers through thorough sectioning of at least 15 sections for each liver after precise inspection of the gross specimens . among all 103 explanted cirrhotic livers in our study , 92 ( 89.3% ) had df with lcc . according to this result , lcc is a common finding in cirrhotic livers of patients with chronic hepatitis b , but it does not seem to be associated with any malignant lesion , thus confirming the lack of premalignant potential in theses lesions . that is , some studies similar to our own found that lcc ( formerly called lcd ) had no histogenetic association with hcc ( 13 ) . others concluded that lcc in relation to hbv might not only be an innocent bystander but might be closely related to hepatocarcinogenesis ( 15 ) . it can be diagnosed by the presence of clusters of hepatocytes with a small size , minimal nuclear atypia , high n / c ratio , and high nuclear crowding . the report by plentz et al . indicated that severely decreased expression of p16 and p21 , telomere shortening , and accumulation of dna damage in scc and hcc compared with lcc and cirrhotic nodules were all suggestive of the malignant potential of scc compared with lcc ( 17 ) . in our study of 103 patients with hepatitis b - associated liver cirrhosis , 57 ( 55.3% ) had scc . out of these 57 livers with scc , 19 ( 70.4% ) had ehcc and 21 ( 36.8% ) had hcc . our results showed that the presence of scc is associated with ehcc ( p < 0.05 ) and hcc ( p < 0.05).therefore , scc may be associated with the presence of ehcc or hcc . according to our results , a thorough sampling is advised for every patient with scc to detect any malignant lesion . for example , in one study , scc was proved to be associated with aneuploidy and elevated dna index , and it was found to be a moderate - to - high risk for progression to hcc ( 18 ) . among the 103 explanted livers , 39 ( 37.9% ) had lgn , 38 ( 36.9% ) had hgdn , 19 ( 18.4% ) had ehcc , and 21 ( 20.4% ) had ahcc . thus , 70% and 53% of the cases with lgdn and 50% and 55% of the cases with hgdn showed ehcc and hcc , respectively . this association was statistically significant ( p value < 0.05 ) . according to previous studies , lgdn and hgdn are indicators of increased risk of hcc in cirrhotic livers . hcc nodules are commonly seen within dns ( i.e. , nodule - within - nodule pattern ) ( 14 ) . in conclusion , scc , lgdn , and hgdn seem to be common associated findings and precursors of hcc in livers infected with hepatitis b. a strict follow - up and a precise and thorough sampling and sectioning of livers with scc and any abnormal dns , especially those larger than 1 cm , are highly recommended because of their association with 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 .

1. Background 2. Objectives 3. Methods 4. Results 5. Discussion

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

Introduction Methods Results Tear osmolarity as a diagnostic tool Tear osmolarity compared to other diagnostic tests Factors affecting tear osmolarity External factors Environmental factors Contact lens wear Surgery Ocular and systemic conditions Pharmacological effects and treatment Discussion Conclusion

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

None Competing interests Abbreviations

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

Introduction Methods Collection of sperm Measurement of sperm forward motility Measurement of sperm intracellular pH Plasma membrane polarization of human and mouse sperm Statistical analysis Results Effect of curcumin on human and mouse sperm forward motility Effect of curcumin on human and mouse sperm intracellular pH (pHi) Effect of curcumin on human and mouse sperm plasma membrane polarization Discussion Conclusion Conflict of Interest

the incidence of splenic abscess is a clinically uncommon disease with current literature reporting a 0.140.7% occurrence rate [ 1 , 2 ] . although splenic abscesses are a clinical rarity , they have the potential to be fatal . the presentation of this disease is often vague and insidious including left upper quadrant abdominal pain , fever and chills . additionally , these patients may present with leukocytosis , left upper quadrant mass and pleural effusion on chest x - ray . splenic abscesses generally occur in patients with underlying comorbidities , which commonly include neoplasia , immunodeficiency , trauma , metastatic infection , splenic infarct or diabetes . the best management of splenic abscesses is still debatable with the various modalities including antibiotics , percutaneous drainage or splenectomy . the current literature supports a 67100% success rate with percutaneous drainage ; however , tung et al . states that the most optimal treatment for splenic abscess is splenectomy . of the 600 cases of splenic abscess documented in the current literature , most have been described as air confined to the left upper quadrant on chest x - ray . we report a young patient with a ruptured splenic abscess resulting in an acute abdomen and pneumoperitoneum . to our knowledge , there have only been four other reported cases of ruptured splenic abscess causing pneumoperitoneum [ 3 , 4 ] . a 48-year - old female with the past medical history of diabetes , coronary artery disease and psoriasis , presented with acute onset of diffuse abdominal pain 2 days prior to admission to the hospital . upon presentation , she was tachycardic with a heart rate of 130 and hypotensive with a blood pressure of 78/45 . she was resuscitated in the emergency department with intravenous fluids and her blood pressure and heart rate responded appropriately . her initial labs showed a normal wbc level of 8.3 thou / mcl ; however , the patient was acidotic with a ph of 7.29 and a lactic acid level of 3.5 mmol / l . an acute abdominal series was obtained demonstrating free air below the right hemidiaphragm ( fig . 1 ) . the patient was subsequently boarded for an exploratory laparotomy with repair of perforated viscus , as that is the usual cause of pneumoperitoneum , especially under the right hemidiaphragm . because the patient 's vital signs stabilized after being resuscitated , the decision was made to obtain a ct scan to better assess the location of the perforated viscus . much to our surprise , the patient did not have a perforated viscus , but a splenic abscess that had ruptured causing the pneumoperitoneum ( fig . 2 ) . figure 2:ct scan demonstrating gas - forming splenic abscess and free air in the peritoneal cavity . ct scan demonstrating gas - forming splenic abscess and free air in the peritoneal cavity . the patient then became increasingly confused and her vital signs again deteriorated demonstrating worsening sepsis . the patient was taken to the operating room where a laparoscopic splenectomy was attempted but was quickly converted to laparotomy with splenectomy due to the gross contamination of the abdomen . the patient was continued on antibiotics and taken to the intensive care unit for post - operative care . the splenic abscess grew prevotella intermedia , a bacterium commonly found in the oral flora . the patient underwent a full work - up looking for the source of the splenic abscess . a transesophageal echocardiogram was performed but was negative for any masses , thrombus or vegetation . furthermore , a panorex was performed and was negative as the patient had reported tooth pain 1 week prior to her admission . she returned to the clinic on post - operative day 14 to receive her splenectomy vaccinations . although a rare disease , splenic abscess should be included on the differential diagnosis of a patient presenting to the hospital with peritonitis or pneumoperitoneum . this is especially true for patients who are immunocompromised or have underlying comorbidities including neoplasia , diabetes , trauma or history of splenic infarct or embolization . in our case , ct scan provided important information about the cause of our patient 's pneumoperitoneum , as we had assumed it was due to a perforated ulcer or diverticulitis . this did change our operative management , as we attempted to perform a less invasive surgery on our patient by starting with a laparoscopic technique . however , we had to convert to an open splenectomy due to the gross contamination and adhesions from the splenic abscess . in retrospect , an open laparotomy was the best approach to this patient 's care as it allowed for better visualization and irrigation of the abdominal cavity . it is unknown if we had successfully performed the splenectomy laparoscopically if it would have decreased recovery time . the current literature supports the laparoscopic splenectomy as a safe and effective procedure in patients with splenic abscess demonstrating an average length of stay of 14 days . in our case , the patient remained in the hospital for only 10 days after converting to the open procedure . the current literature reports that the most common organisms found in splenic abscesses are aerobic microbes , particularly streptococci and escherichia coli . this microbe is commonly found in the oral flora and is associated with periodontal disease . although our transesophageal echocardiogram and panorex were both negative , we believe this splenic abscess developed as a result of translocation of the oral flora as the patient was complaining of a tooth ache 1 week prior to her presentation . in summary , we have presented a case of ruptured splenic abscess as a cause of pneumoperitoneum and peritonitis . although rare , we encourage splenic abscess be included on the differential diagnosis of patients presenting with peritonitis and pneumoperitoneum . although there is still controversy in the current literature when managing splenic abscesses , our recommendation is splenectomy , especially in the case of ruptured splenic abscess resulting in hemodynamic instability . whether it is better to perform the splenectomy laparoscopically or open is still debatable , the important thing is to obtain source control . furthermore , a full work - up is required for patients presenting with a splenic abscess as the underlying cause could cause more serious illnesses for the patient in the future . there are no conflicts of interests or financial funding to disclose for any of the 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 .

INTRODUCTION CASE REPORT DISCUSSION CONFLICT OF INTEREST

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

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

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

Age and gender effects on cognition and emotion processing Age effects on brain anatomy related to memory and emotion processing Age effects on brain physiologic activity related to memory and emotion processing The study of ovarian aging Conclusions

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

1. Introduction 2. Inflammatory Target Proteins and Respiratory Diseases 3. Inflammatory Signalings Involved in the Respiratory Diseases 4. Therapeutic Implications 5. Conclusions

polycystic liver disease is a condition characterized by the presence of multiple cysts in the liver , which may be inherited or sporadic , and most cases of adult polycystic liver disease ( apld ) are related to autosomal dominant polycystic kidney disease ( adpkd ) . in 1964 , there have been some reports , till date , that describe the association of malignant neoplasms with potter type iii cystic disease of the liver and kidney [ 2 , 3 , 4 , 5 , 6 , 7 , 8 ] ; however , most of these describe pancreatic and hepatobiliary neoplasms arising in patients with polycystic liver and kidney diseases including adpkd , and no report has described the association of gastric carcinoma with potter type iii cystic disease of the liver and kidney . this is the first case report of multiple gastric carcinomas associated with potter type iii cystic disease of the liver , mesenterium and kidney . although it is unclear whether the development of multiple gastric carcinomas is directly associated with the pathogenesis of multiple cysts in the liver , mesenterium and kidney , this case provides a perspective for etiology of gastric carcinoma with polycystic disease . he had been receiving hemodialysis treatment 3 times a week for 2 years because of chronic renal failure due to polycystic kidneys . he stated that his sister had suffered from polycystic kidney disease . physical examination on admission revealed distended abdomen due to polycystic disease . the patient had high serum levels of carcinoembryonic antigen ( 7.9 ng / ml ) and carbohydrate antigen 19 - 9 ( 202 mg / dl ) . an abdominal computed tomography scan showed multiple cysts in the liver and cystic lesions on both sides in the kidney and mesenterium ( fig . gastrointestinal fiberscopy showed the presence of two ulcerated lesions in the lesser curvature in the middle and lower portions of the stomach . histopathological analysis of the gastric tumor biopsy specimens revealed papillary adenocarcinoma in one tumor and poorly differentiated adenocarcinoma in another . distal gastrectomy with lymph node dissection was performed , and the gastric tumors were surgically diagnosed to be t2n1m0 , stage ii , according to the tnm classification for gastric carcinoma . macroscopic examination showed that two tumors of type ii were present in the middle and lower portions of the stomach ( fig . the distal lesion was diagnosed as a poorly differentiated adenocarcinoma ( 38 39 6 mm ) within the muscularis propria , with marked lymphatic invasion ( ly2 ) and vascular invasion ( v2 ) ( fig . 3b ) ; the lesion on the proximal side was diagnosed as a papillary adenocarcinoma ( 58 47 6 mm ) within the submucosal layer , with marked lymphatic invasion ( ly2 ) and vascular invasion ( v1 ) ( fig . adpkd is the most common of all inherited renal cystic diseases and has various external manifestations , including cysts in the pancreas , seminal vesicles , arachnoid membrane , and liver . the most accepted among them is the osathanondh and potter system that was established in 1964 , according to which polycystic kidneys are classified into three types . the cysts may be present in any portion of the nephron or tubule , but they are most commonly found in bowman 's space and the angle of the loop of henle . in the older classification , potter type iii cystic disease was considered to represent adult - type polycystic disease of the kidney . in 1972 , they also observed cysts in the liver and/or pancreas in 58 of these patients ; hence , they described this condition as potter type iii polycystic disease . in the present case , computed tomography and ultrasonography scans showed multiple renal and hepatic cysts in addition to multiple cysts in the mesenterium . therefore , according to hatfield and pfister 's criteria , we considered that this case of association of apld with multiple renal cysts could be classified as potter type iii cystic disease . the patient stated that his sister had suffered from polycystic kidney disease ; however , we could not confirm the presence of a family history of polycystic kidney disease because his sister had been out of contact for long . in the absence of a family history of adpkd , bilateral multiple renal cysts with hepatic cysts , together with the absence of other manifestations suggesting a different renal cystic disease , provide evidence for diagnosis . in these instances , a family history , however , may be absent in 2040% of new patients in whom the diagnosis of adpkd first is suspected from imaging studies , which can be due to a de novo mutation of pkd-1 or pkd-2 genes . although we could not confirm the mutation of pkd-1 or pkd-2 in this case , family history , clinical findings and imaging studies suggested that the patient suffered from adpkd . reports on the association of malignant neoplasm with apld and adpkd ( potter type iii cystic disease ) are extremely rare . to our knowledge , only 8 such cases have been reported in the english language literature ( table 1 ) [ 2 , 3 , 4 , 5 , 6 , 7 , 8 ] . till date , most cases of neoplasms associated with polycystic disease of the liver and kidney have been reported as either pancreatic or hepatobiliary neoplasms , namely 5 cases of pancreatic neoplasms and 3 cases of cholangiocarcinoma . since 5 of these cases were pancreatic neoplasms , we think that adpkd has some genetic influence on the development of pancreatic neoplasms . aziz et al . reported 2 patients from the same family ( a brother and a sister ) with adpkd who developed adenocarcinoma of the gastroesophageal junction ; however , these patients did not have cysts in the liver or other organs except for the kidney . in the present case , although a detailed family history could not be obtained , multiple cysts were observed in the liver , both kidneys , and mesenterium . these genes encode a member of the polycystin protein family , which is an integral membrane protein that functions as a regulator of calcium - permeable cation channels and intracellular calcium homoeostasis . some studies have described an association between the pkd genes and cancer cells ; polycystin-1 , encoded by pkd-1 , was reported to induce apoptosis and cell cycle arrest in the g0/g1 phase in cancer cells . moreover , pkd-1 inhibits cancer cell migration and invasion and may be considered a new member of the tumor suppressor family of genes . therefore , adpkd , which is caused by defective pkd-1 and pkd-2 , may be a carcinogenic condition . in addition , mutations in two genes , namely prksch ( which codes for hepatocystin ) and sec63 , have been found to cause apld . however , as mutations in these genes have not been found in all families , undiscovered genes responsible for causing apld probably still exist . although it is unclear whether the development of multiple gastric carcinomas is directly associated with the pathogenesis of cysts in the liver , mesenterium and kidney , the present case demonstrates that gastric carcinoma can be an external complication in patients with 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 .

Introduction Case Report Discussion Disclosure Statement

canaliculitis is a common encounter in ophthalmic practice but supernumerary puncta and canaliculi ( spc ) are rare congenital disorders . in a large series 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 ( figure 1a ) . eversion of the lower eyelid revealed two puncta , 0.5 mm apart ( figure 1b ) . the outer punctum was situated at the normal anatomical position ; whereas the inner punctum in the caruncle . gentle pressure did not result in any regurgitation from the both puncta . the patient was treated with oral cloxacillin 500 mg , 6 hourly for 5 days . the outer punctum had a soft stop with regurgitation of fluid from the same punctum . the outer punctum - canaliculus system was a cul - de - sac ( figure 1c ) . c ) dacryocystography showed pooling of dye in the cul - de - sac ( white arrow ) . c ) dacryocystography showed pooling of dye in the cul - de - sac ( white arrow ) . most spcs ( 78% ) present with epiphora . among the 23 patients reported by satchi et al . , none presented with canaliculitis . sequestration of tear and debris in the cul - de - sac served as nidus for infection . the resultant canaliculitis with its surrounding edema caused obstruction of the lacrimal drainage ; hence epiphora . epiphora however , may develop despite patent lacrimal drainage system . the 2-compartment model for lacrimal canalicular drainage of kakizaki et al . , suggested that the muscle of duverney - horner may deviate normal flow within the accessory canaliculus and thence transport tears back to the lacrimal tear lake , leading to epiphora . a solid epithelial cord forms in the region of the medial lower eyelid ( figure 2a ) and sends projections to form the canaliculi and the nasolacrimal duct ( figure 2b ) . spc is due to extra out - budding of the solid epithelial cord ( figure 2c ) . canalization begins at 4 months of gestation with disintegration of the central ectodermal core , forming lacrimal drainage outflow system . in this case , the extra inner canalicular epithelial bud ( nearer to the main epithelial cord ) underwent complete canalization and remained connected to the main epithelial cord . the outer canalicular epithelial bud , although its punctum is located at the normal anatomical position , was separated from the main epithelial cord ; forming a cul - de - sac ( figure 2d ) . c ) extra out - budding of the solid epithelial cord in supernumerary puncta and canaliculi . d ) the outer canalicular epithelial cord was separated from the main epithelial cord , forming a cul - de - sac . c ) extra out - budding of the solid epithelial cord in supernumerary puncta and canaliculi . d ) the outer canalicular epithelial cord was separated from the main epithelial cord , forming a 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 .

Introduction Case Report Discussion

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 .

Supplementary Material

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

INTRODUCTION MATERIALS AND METHODS RESULTS Population size with diabetes Healthcare expenditures attributed to diabetes Future economic burden of diabetes CONCLUSIONS

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

RESEARCH DESIGN AND METHODS Populations. Fasting glucose levels. History of diabetes. Cognitive function. Additional assessments. Statistical analyses. RESULTS DISCUSSION Supplementary Material

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

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

we are grateful for financial support from the industrial source technology development program of the ministry of knowledge economy ( 10044909 ) , the next - generation biogreen 21 program ( ssac grant # pj01111803 ) , rda , and the kribb initiative program of 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 .

Abbreviations Disclosure of potential conflicts of interest Funding

surgical 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 . a 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 . the 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 . cyanoacrylate 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 . corneal perforation caused by small corneal punctures can sometimes be managed with a bandage soft contact lens , patching , and/or tissue adhesive [ 2 , 3 , 4 ] . however , surgical intervention is usually indicated when the anterior chamber does not reform within a short period of time . herein , we report the successful experience of managing a small paracentral corneal perforation without good apposition , which could not be tamponaded by a soft bandage lens only , with iatrogenic iris incarceration followed by cyanoacrylate tissue adhesive . a 41-year - old man noted irritation in the right eye and was diagnosed to have a piece of metallic foreign body in his right cornea . iatrogenic corneal perforation was encountered , while the attending physician attempted to remove the residual rust ring with a corneal burr . slit - lamp examination revealed a paracentral corneal penetration with a diameter of approximately 0.5 mm ( fig . the anterior chamber was flat , and active aqueous leakage was confirmed by a positive seidel test with fluorescein . intravenous antibiotics with cefozopran hydrochloride were initiated , in conjunction with prophylactic topical antibiotic with 0.5% levofloxacin three times daily . after 48 h of treatment , there was no significant improvement in the right eye . concerns were raised about possible complications such as secondary angle closure glaucoma and prolonged hypotony . since the puncture was located paracentrally near the visual axis , simple primary closure of the wound or corneal patch graft was deemed undesirable . as the wound was completely punched out , injected viscoelastic materials in the anterior chamber came out from the wound , which made it quite difficult to dry the surface of the cornea for application of tissue adhesive . therefore , to provide a necessary support for the gluing , amniotic membrane plugging was tried , but it was too slippery to be fixed on the wound . a paracentesis port was created . a blunt 27-gauge needle ( with a 2-mm bent tip ) attached to a viscoelastic injecter was gently inserted into the anterior chamber through the paracentesis . using the bent tip , a small tug of iris care was taken not to touch or injure the corneal endothelium or anterior capsule of the lens . meticulous drying of the wound and incarcerated iris was performed and a tiny amount of ethyl-2-cyanoacrylate adhesive ( aron alpha a ; sankyo co. ltd . , attention was paid to ensure that the glue did not spread on to the surrounding cornea . the corneal puncture was thus successfully closed without any sutures , and the anterior chamber remained well formed . postoperatively , the patient was treated with topical 0.1% fluorometholone and 0.5% levofloxacin three times daily . pupillary distortion was observed when the iris was dilated ; however , the patient felt no visual disturbance . herein , we reported a case of iatrogenic paracentral corneal perforation without good apposition successfully treated with autologous iris incarceration and cyanoacrylate adhesive . conventionally , small corneal perforations were treated with stitches with or without corneal patch graft , tissue adhesives [ 2 , 3 , 4 ] , and recently with amniotic membrane and fibrin glue . the indications for the use of a bandage soft contact lens to treat a corneal laceration include small wounds with good apposition , good alignment of the wound edges , and no incarceration or prolapse of the uvea or lens . corneal puncture wounds of less than 2.0 mm in size can usually heal without being sutured . in this case , the wound was relatively small ( less than 1 mm ) , but the apposition of the wound was not optimal due to a sharply demarcated punch , which made restoring the anterior chamber extremely difficult using a bandage soft contact lens only . in cases with good apposition , a small amount of viscoelastic materials through the perforation site is often enough to allow safe application of tissue adhesive without gluing intraocular contents . the technique described in this case appears useful secondary to the minimal residual scarring and related astigmatism . a second advantage of this technique is that the iris is introduced to the perforation site from inside the eye in a controlled manner to prevent excessive iris prolapse and provide a stable support for the tissue adhesive . this is more adequate than the use of an amniotic membrane , which is very slippery and difficult to be fixed on the wound . furthermore , this technique requires no foreign tissue , thereby eliminating the risk of immunological tissue reactions . the tissue adhesive may also theoretically circumvent the potential risks of epithelial down - growth by preventing corneal epithelium from growing directly over the iris . this technique is safe and effective for paracentrally located corneal perforation ; however , significant synechiae involving the angle could prove to be problematic when the perforation is more peripheral . very small descemet 's membrane transplantation instead of iris incarceration may be the other option for this situation . in conclusion , application of cyanoacrylate adhesive following iatrogenic incarceration of the iris seems to be simple and effective in 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 .

Background Case Observations Conclusions Introduction Case Report Discussion Disclosure Statement

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

Background Copy number variant and rare allele studies Genome-wide association studies A biological process disrupted across traditional diagnostic boundaries? Conclusions Abbreviations Competing interests Authors' contributions

fever is a common reason for seeking medical attention and accounts for more than 20% of emergency department visits.1 many mothers and caregivers have perceived phobia associated with their child 's fever for reasons that include serious infection , seizure , brain damage and death.2 paediatricians have regularly advised parents to seek immediate medical attention if their children developed a fever because fever is of great diagnostic importance especially in developing countries where infectious diseases such as malaria and pneumonia are very prevalent.3 in developing countries , because of the relatively low level of literacy and financial constraint of procuring reliable thermometers , many parents rely on palpation to assess if their children have fever.4 the objective use of the thermometer to detect the presence of fever is often limited to health care settings in most resource - poor settings . an accurate determination of the absence of fever in a child assures parents and saves cost by preventing unnecessary investigations and medication . ownership of thermometer seems poor , although no study in our environment was found to support that . in a study in new york , usa , of the 78% of caregivers that owned thermometers , nearly half ( 48% ) still utilised tactile method to assess fever.4 in another study , in india in contrast , only 15% owned thermometer with 23.8% of them not knowing how to use it.5 this underscores the fact that the use of palpation as a means of assessing fever by mothers is common and universal . majority of mothers and care givers in our environment still utilise tactile assessment to determine if their children have fever . controversies over the reliability of tactile assessment of fever have been variously reported ; while a study found it accurate enough , at least in younger infants,4 others reported that it overestimates the presence of fever.67 the reliability of the use of palpation to detect fever still remains uncertain . this study was carried out to determine the reliability of tactile assessment of fever by nigerian mothers in under-5 children and also to determine if the surface of the hand used influence the accuracy . the study further ascertained if palpation of a single or multiple anatomical sites influence the accuracy of tactile assessment of fever . children under the age of 5 years presenting for care with their mothers were recruited for the study . ethical approval was obtained from the research and ethics committee of federal medical centre owerri , imo state nigeria before commencement of the study . the subjects were recruited from the children outpatient clinic and children emergency room of the hospital . information such as age and sex of the child , mother 's age and highest educational qualification ( heq ) were captured using a profoma . thereafter , each eligible child had a tactile assessment of fever by the mother . the response of the mother as regard presence or absence of fever was documented following which the child 's axillary temperature was measured and documented . the axillary temperature was measured using mercury in glass thermometer , which was left in the axilla for 5 minutes before reading . frequencies , percentages and mean were analysed where appropriate . the sensitivity , specificity , positive predictive values ( ppvs ) and negative predictive values ( npvs ) of tactile assessment was calculated using axillary temperature of 37.5c as cut - off for fever . the different hand surfaces used for palpation , as well single versus multiple sites of palpation were also compared . there were 60 males and 53 females giving a male : female ratio of 1.1:1 . seven ( 6.2% ) of the mothers had only primary school education , while 38 ( 33.6% ) and 68 ( 60.2% ) had secondary and tertiary education , respectively . fever detected by touch was reported in 81 ( 71.6% ) children by mothers while , only 65 ( 57.5% ) mothers were able to correctly predict the presence of fever in their children . there was no statistical significant difference in the ability of the mothers to correctly predict fever with respect to their heq ( = 2.66 , p = 0.265 ) . comparison of fever detection by palpation with the mothers highest educational qualification ( heq ) irrespective of the part of hand used for palpation , the sensitivity of detection of fever by palpation was 82.4% , while the specificity was 37.1% . the use of the palmer surface of the hand had a better sensitivity ( 95.2% ) than the dorsum of the hand ( 69.2% ) . table 2 shows the sensitivity , specificity , ppv and npv of the part of hand used for palpation . sensitivity , specificity , positive predictive value ( ppv ) and negative predictive value ( npv ) of the part of hand used for palpation a total of 71 ( 62.8% ) of the mothers palpated at more than one site while the remaining 42 ( 37.2% ) used only single site . table 3 shows the different sites used by the mothers in palpation , while table 4 compares the sensitivity , specificity , ppv and npv of use of single site versus multiple sites in palpation . the use of multiple sites had better sensitivity ( 86.7% ) than the use of single site ( 76.2% ) . sites used by the mothers to palpate for fever palpation of single site versus multiple sites the present study shows that mothers can be trusted to a good extent to assess for the presence of fever in children by palpation . a sensitivity of 82.4% obtained in this study is similar to those reported by okposio and abhulimhen - iyoha in benin , nigeria as well as graneto and soglin in chicago , usa , who documented sensitivity of 89.2% and 84% , respectively.89 some studies in nigeria have reported a higher sensitivity value of 96.3% and 94.6% , respectively.610 a specificity of 37.1% obtained in the present study is quite poor and is lower than the specificities reported by other workers ranging from 64.3% to 82%.489 it is , however , higher than 23% reported by akinbami et al.6 the heterogeneity in the methodology of these studies may have been responsible for the differences obtained between the present study and some of the earlier studies . for instance , akinbami et al.,6 used rectal temperature of 38c as the cut - off of fever , wammanda and onazi10 adopted axillary temperature of 37.2c as the cut - off for fever , while the present study and that of okposio and abhulimhen - iyoha8 used axillary temperature of 37.5c as the definition of fever . although the specificity in the present study was poor , the relatively high sensitivities obtained in all the studies still make maternal palpation for the presence of fever a considerably useful method of assessment of childhood fever . although , palpation overestimates fever , the elicitation of fever by mothers would encourage early presentation as it has been documented that the concern for fever is a strong motivational factor for seeking medical care.2 the educational qualification of a mother did not influence the accuracy of palpation for the presence of fever . it is therefore expected that any mother , irrespective of her educational background should be able to appreciate fever by palpation . with a npv of 71.9% , if a mother says her child does not have fever , her history of subjective assessment of fever without the use of a thermometer is about 72% reliable . the likelihood of a child adjudged to be febrile by a mother to be truly febrile measured by the ppv in the present study is 51.9% . this is higher than 39% reported by wheybrew and co - workers.7 this higher ppv reported in this study may have been influenced by the prevalence of fever in the present study . in the present study , 57.5% of the study subjects had fever , while in the study of wheybrew et al.,7 27% of children had fever . the palmer surface of the hand was the most commonly used hand surface by mothers to palpate for fever . the sensitivity , specificity , npv and ppv of palmer surface was higher than that of the dorsal surface or the use of both surfaces . this may be attributed to the fact that receptors for detection of heat and temperature are more concentrated on the palmer surface and finger tips.11 the use of both surfaces may confuse the mother as the sensation obtained from both are likely to vary , hence the reduced sensitivity obtained in this study when compared with palmer surface alone . the forehead was the most common site used for tactile assessment of fever in this study . this corroborates reports from previous studies , which also found the forehead to be the most common anatomical site used for tactile assessment of fever.4 this may possibly be explained by the fact that the head is easily accessible compared with the other parts of the body . it was observed that palpation of multiple sites was more sensitive than use of single site . this is in agreement with an earlier report by okposio and abhulimhen - iyoha as well as singhi and sood.812 this implies that if fever is to be assessed by palpation without the use of a thermometer , palpation of multiple body sites should be utilised . palpation with the palmer surface of the hand using multiple sites improves the reliability of tactile assessment of fever . clinicians should not discountenance mothers complaint of fever in their children detected by palpation without the use of a thermometer . mothers should be taught to palpate at multiple sites with the palmer surface when palpating for the presence of fever in their children to improve the 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 .

INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION CONCLUSION

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

Modelling human pancreatic cancer associated with None Pancreatic cancer histopathology and Checkpoint inactivation, Mouse models for PDAC associated with

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

Background Material and Methods Patients Data collection Grouping Statistical analysis Results Patients characteristics Risk factors for early CRBSI Type of CVC and position of CVC indwelling Pathogen identification Clinical manifestation Treatment and prognosis Discussion Conclusions

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

Introduction Prostate Cancer: A Global Concern Androgen Receptor: Structure and Function Androgen-Dependent and -Independent Prostate Cancer Targeting AR with Steroid Conjugates Toward Novel AR Antagonists by Molecular Design: Taking Inspiration from the Estrogen Receptor Targeting AR with Nonsteroidal Conjugates Conclusion

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

Introduction Objectives Methods Recruitment and procedure Exclusion criteria The ECG machine The V-Quick patch template Data management and ECG analyses Results Discussion Limitations and observations Conclusion

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

Introduction Materials and Methods Results Discussion

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

INTRODUCTION CASE REPORTS Case 1 Case 2 DISCUSSION

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

1. Introduction 2. Context 3. Alternative Models to a Weight-Centric Public Health 4. Obesity and Ethnography 5. Conclusions

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

INTRODUCTION MATERIALS AND METHODS Feature Extraction from Pathology Reports for Classification Building Celiac Disease Classifiers Classification model Variable configuration Feature selection Classifier Evaluation RESULTS DISCUSSION Limitations CONCLUSION Financial Support and Sponsorship Conflicts of Interest

the possible presence of infection in cervical smear tests is usually reported by the cytologists based on cytological criteria . therefore , non - specific cervicitis or inflammatory changes in a smear report are common . these findings are usually unclear for clinical approaches and there is not an appropriate guideline for management of such patients . it is not known if these women should be recalled for cultures and treatment or inflammatory change should be considered as minor . changes in balance of normal vaginal flora can cause an overgrowth of pathogens that lead to discharge . although it is a common complain among sexually active women , there are still gaps in our knowledge about it . bacterial vaginosis ( bv ) , vaginal candidiasis ( vc ) and trichomoniasis are the three infections most commonly associated with vaginal discharge ( 1 ) . a number of studies have indicated the association of bv with postoperative complications after gynecologic surgery as well as pregnancy complications ( 25 ) . trichomonas vaginalis ( tv ) is also another common cause of vaginal discharge , but some studies have not found its association with pregnancy complications ( 3 ) or some other indicated that tv during pregnancy was a predisposing factor for preterm delivery and delivering low - birth weight infants ( 6 ) . the pap smear test for detection of bv has showed a sensitivity of 88.2% , a specificity of 98.6% , and a positive predictive value ( ppv ) of 96.8% ( 7 ) . the sensitivity , specificity , and ppv of pap test for tv diagnosis has been reported 98% , 96% , and 88% , respectively ( 8) . also , some recent studies have demonstrated the co - infection of vaginal pathogenic organisms in pap tests ( 2 , 9 , 10 ) . this study aimed to investigate the prevalence of pathogenic vaginal microorganisms and the presence of inflammation in pap smear among an iranian women sample and to evaluate the possible co - infection of these organisms . this 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 . exclusion criteria were pregnancy , smoking , use of oral contraception and/or corticosteroids , regular use of vaginal douche , and women with chronic systemic diseases or systemic immunosuppression . prior to data collection , the objectives of the study were explained in community meetings . all women received their laboratory results in written form , and results were explained during a consultation . the study protocol was also approved by the institutional review boards of department of gynecology and obstetrics . data were obtained in privacy using an information form applied by one investigator , followed by gynecological examination and specimen collection all done by one gynecologist . cervical smear samples , based on the conventional method , were prepared with a spatula and fixed on glass slides and were stained with the papanicolaou technique . tv was defined as the presence of trophozoites in pap smears ; vc was diagnosed if fungal hyphae or budding yeasts were present in pap smears . the presence of inflammation in the smears was divided into mild , moderate , and severe categories . mild inflammation was defined as less than 30 inflammatory cells / high - power field ; moderate inflammation was defined as 30 to 100 inflammatory cells / high - power field ; and severe inflammation was defined as more than 100 inflammatory cells / high - power field . the frequency of pathogenic microorganism included bv , tv , and vc were determined in specimens . statistical significance was analyzed using statistical package for social sciences ( spss software version 17 ) . the comparison between data was performed with the pearson 's chi square or fisher 's exact test . mild inflammation was noted in 136 ( 26.1% ) , moderate in 155 ( 29.4% ) and severe in 117 ( 22.2% ) of them ( table 1 ) . the frequency of pathogenic vaginal microorganisms and inflammation among pap smear samples among all samples , bv ( the presence of clue cells ) was the most common pathogenic microorganism which was seen in 91 samples ( 17.2% ) , followed by vc ( n = 56 ; 10.6% ) . tv ( n = 2 ; 0.4% ) was shown to have a low prevalence among our patients ( table 1 ) . the overall prevalence of pathogenic microorganisms was greater among reproductive age participants , meanwhile the prevalence of bv as well as vc was significantly different ( p = 0.042 and p = 0.006 , respectively ) between non- and post- menopausal women . inflammation was detected in 76% ( n = 402 ) of pap smears , in which 80.1% ( n = 322 ) were reported among reproductive age women . however , the severity of inflammation was not significantly different between non- and post- menopausal women . overall , the presence of inflammation was significantly associated with vc ( p = 0.002 ) , but its severity was not correlated to the infection . although , there was no significant relation between the presence of inflammation and bv , the severity of inflammation in specimen was significantly increased in association with bv ( p < 0.001 ) . in our study , the prevalence of bv among pap smear specimens of women living in tehran was 17% which is similar to azargoon 's study ( 16% ) on a population in semnan ( another city in center of iran ) ( 3 ) , but it had a higher rate in hamadan ( a western city of iran ) and has been reported 28.5% ( 11 ) . in comparison with other asian countries , in india , but it was 38% in an african study in botswana on 703 pregnant women ( 13 ) . some predisposing factors for bv include having sexual relationship with more than one partner , early onset of sexual activity , orogenital sexual contacts , coitus during menstruation , use of iud , and smoking ( 2 , 14 ) . differences in sexual behavior and risk factors in different societies can justify the differences in bv prevalence of mentioned studies . reproductive age women indicated that the prevalence of bv in muslim women was lower than non - muslims . although the prevalence of tv in pap samples of our study was much less than several studies , similar reports demonstrated by depuydt et al in flanders detected by real - time pcr ( 0.37% ) ( 15 ) . however , this rate was 18% in the study in hamedan ( 11 ) , 8.5% among indian women ( 16 ) , and 19% in the african study ( 13 ) . a probable reason for these variations could be differences in pap sampling quality and cytologists skill . several studies show an increased vaginal ph ( 5 ) in both bv and tv infection ( 3 , 5 , 17 ) . demirezen et al in turkey on 600 pap tests detected tv in 6% of women and bv in 44% of tv cases . they also found a significant relationship between tv and bv infection , which might be due to this hypothesis that tv by phagocytosis the vaginal lactobacilli increases the vaginal ph and generates an anaerobic environment , providing an appropriate condition for the growth of anaerobic microorganisms ( 18 , 19 ) . also , it was confirmed by heller 's study on 400 pap samples which concluded that bv diagnosis should be considered at the presence of tv in pap smears ( 2 ) . in our study , we found no significant association between bv and tv in conventional pap smear sampling , which might be because of low prevalence of tv in our samples . the effect of habits and behaviors on vaginal flora among different population can be evaluated in further assessments . in our study , only the presence of candida in pap smear samples was associated with the report of inflammation . therefore , reporting inflammation in pap smear test might be considered for candidiasis evaluation and treatment . several studies investigated the association between infection and existence of inflammation in cervical smears ( 2023 ) . some of them found that inflammation on pap smear had a relatively low predictive value for the presence of infection ( 21 ) . they stated that the inflammation was not exactly a consequence of infection and other causes might be responsible for this appearance ( 20 ) . in contrast , others indicated a significant association between inflammatory smear and reproductive tract infections ( 22 , 24 ) . as burke et al ( 22 ) in their study on 256 women found an evidence of inflammation in 9.7% of smears and genital tract infection in 29.2% of their participants overall . they found infection in 48% of women having inflammatory changes on smear test vs. 27.3% of women whose smear tests showed no evidence of inflammation . they discovered that the prevalence of infection with chlamydia trachomatis , candida , bacteroides and gardnerella vaginalis was higher in the inflammatory smear group . they concluded that women with an inflammatory smear were more likely to mask the infection than women whose smear shows no evidence of inflammation ( 22 ) . prevalence of bv infection ( 17% ) in this study was similar to most other studies , which was not true for tv ( < 1% ) . the use of other diagnostic methods may be probably more valuable than conventional pap smear for evaluation of tv among iranian women and it needs further investigations . as the report of inflammation in our samples was very high , use the newer pap smear techniques such as thinprep ( liquid base preparation ) in the further studies is recommended . based on our results , inflammation reported in the pap smears demands the evaluation for vc and its 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 .

Introduction Materials and methods Results Discussion Conclusion

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

Introduction Materials and Methods Results Discussion Conclusions

behet 's disease ( bd ) is a chronic inflammatory disease that is characterized by recurrent oral and genital ulceration and uveitis1 ) , the other clinical manifestations include skin lesions , arthritis , thrombosis , neurologic symptoms and intestinal ulceration . polychondritis is known as a rare complication of bd and no case of bd combined with polychondritis had been reported on until firestein et al . reported on five patients with both bd and relapsing polychondritis ( rp ) in 19852 ) . we recently experienced a case of a 40-year - old male patient with both bd and polychondritis , and we report herein on this unusual case along with a review of the literature . a 40-year - old man was referred to our hospital for further evaluation of his fever , oral ulcers and skin rashes that had lasted for 10 days . he had previously suffered from intermittent oral ulceration and uveitis that had been treated with oral prednisolone and intraocular injections of triamcinolone at another hospital 1 year prior to this admission . on the physical examination , his blood pressure was 130/80 mmhg , the pulse rate was 80 per minute and the body temperature was 38.4. he had conjunctival injections in both eyes and several aphthous ulcers on the tongue ( figure 1a ) and buccal mucosa . a small round ulcer with tenderness was found on the penile root ( figure 1b ) . several round erythematous skin lesions were present on the chest and thigh ( figure 2a ) . his left knee joint was swollen ( figure 2b ) and tender , and needle aspiration revealed turbid inflammatory synovial fluid . the laboratory findings were as follows : hemoglobin 14.8 g / dl , white blood cell count 10,400/l , platelet count 291,000/l , erythrocyte sedimentation rate 104 mm / hr and c - reactive protein 205 mg / l ( normal range , 0~3.4 mg / l ) . the renal and liver function tests were normal and the tests for rheumatoid factor and antinuclear antibody were negative . the urinalysis was normal and no microorganisms were grown on both the blood and urine cultures . his chest radiography showed no abnormal findings and mild splenomegaly was observed on abdominal sonography . skin biopsy was performed and the histologic examination showed perivascular mononuclear cell infiltrations , extravasation of the red blood cells and fibrinoid necrosis of the blood vessel walls ; all of these findings were compatible with cutaneous vasculitis combined with bd ( figure 3 ) . on the 5th day of admission , both ear auricles became swollen , red and tender ( figure 4 ) . he remembered having a similar auricular inflammation 2 years ago that had improved after taking non - steroidal anti - inflammatory drugs . after he was administered with colchicine 1.2 mg / day and prednisolone 30 mg / day , his clinical symptoms improved and he was discharged on the tenth day of admission . polychondritis is an inflammation of the various catilagenous structures such as the auricle , nose and trachea , and it has also been known to involve the eye and the joints . when polychondritis is relapsing , it is called relapsing polychondritis ( rp)8 ) . in rp , various cutaneous manifestations are also seen , including palpable purura , erythema nodosum , panniculitis , livedo reticularis and urticaria . there have been case reports on polychondritis combined with other rheumatologic diseases such as systemic lupus erythematosus ( sle ) , rheumatoid arthritis ( ra ) and systemic vasculitis9 - 11 ) . however , there have been no reports of polychondritis combined with bd until firestein et al . first reported on five patients who had the features characteristic of both bd and rp , and those authors termed this as mouth and genital ulcers with inflamed cartilage ( magic ) syndrome2 ) . polychondritis is still known as a rare complication of bd and only five more cases of bd combined with polychondritis have been reported on since firestein 's report3 - 7 ) . in our patient , bd was diagnosed based upon the recurrent oral and genital ulcers , the skin vasculitis , the arthritis and the past history of uveitis . the chondritis developed at the bilateral auricles , eye and the joints during the acute exacerbation period of bd , and this resolved with the improvement of the symptoms of bd . this suggests that the development of polychondritis may have been associated with bd in our case . as for treatment for the combined cases of bd and rp , colchicine , steroid and dapsone skin involvement in bd includes various lesions such as erythema nodosum , pustules , papules , pseudofolliculitis , acneiform folliculits , sweet 's syndrome , or pyoderma gangrenosum12 ) . the histopathology of the papulopustular lesions usually shows vasculopathy that is characterized by the perivascular infiltration of mononuclear cells , and this infiltrate is mainly composed of lymphocytes . the erythema nodosum - like lesion is known to be characterized by extravascular neutrophilic infiltration and panniculitis , and this can be followed by lymphocyte infiltration during the evolution of the skin lesions14 ) . in our case , the main histopathylogic finding of the skin lesions was perivascular mononuclear cell infiltration and fibrinoid necrosis of the vessel walls , which are the characteristic findings of vasculopathy . although cases of bd combined with polychondritis have rarely been reported , bd and polychondritis do not seem to be separate entities . compared the frequency of the various clinical features in bd and rp ( table 1 ) , and they showed that some of the manifestations that are considered unique for one disease could be found in the other disease2 ) . these similarities between the clinical manifestations of bd and rp imply that there seems to be something in common for the pathogenesis of bd and polychondritis . the pathogenesis of both bd and rp are associated with autoimmunity . in patients with rp , the antibodies to collagen type ii , ix and xi are elevated in the sera15 , 16 ) and the frequency of human leukocyte antigen ( hla)-dr4 is increased17 ) . additionally , rp has been associated with other autoimmune diseases such as ra and sle9 , 10 ) . in patients with bd , autoantibodies such as antiendothelial cell antibody and antibody to -tropomyosin have been found18 - 20 ) and it has been postulated that cross - reactivity and molecular mimicry between the peptides from the streptococcal proteins or the viral heat shock proteins ( hsps ) , which are homologues of human hsps , and the mucosal antigens result in the selection of autoreactive t cells21 ) . have proposed that autoimmunity to components of the cartilage other than type ii collagen , such as proteoglycans or elastic tissue , could be the common mechanism of bd and rp2 ) . in particular , elastic tissue is ubiquitous in the human mucosa that is mainly involved in bd , and in the cartilage too , which is the major target of rp . however , because of the rarity of the combined cases , there have not yet been any investigations about the common pathogenesis of the two diseases , and so additional research is clearly needed . although there have been only a few reports about polychondritis combined with bd , we think this combination of diseases might have been overlooked so far and closer scrutiny would reveal more 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 .

INTRODUCTION CASE REPORT DISCUSSION

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

Subjects and Methods Software used in the study Preprocessing Postprocessing (simulating the movements and its analysis) Discussion

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

RESEARCH DESIGN AND METHODS Animals. Preparation of cells and Western blotting analysis. IPGTT and insulin tolerance test. Immunohistochemistry. Quantification of atherosclerotic lesions in the aortic sinus. Ex vivo treatment of macrophages. Isolation of tissue RNA and real-time quantitative RT-PCR. Flow cytometry. Data analysis. RESULTS GLP-1 receptor is abundantly expressed in monocytes/macrophages. Exendin-4 reduces monocyte adhesion in C57BL/6 mice without affecting glucose tolerance. Exendin-4 reduces monocyte adhesion and atherosclerotic lesions in apoE Exendin-4 reduces the inflammatory response through cAMP signaling pathway in macrophages. DISCUSSION

disability prevalence increases with advanced age , and disability in the elderly population is a major public health problem . this is especially crucial for chinese : there are 88 million persons aged 65 and above in china alone , and the number is projected to increase to 341 million in 2050 ; importantly , disability prevalence in chinese elderly population has been increasing [ 35 ] in contrast to a declining trend in the developed countries [ 6 , 7 ] . there is a big gap between the scale of the problem and the quantity and quality of available information . the first population - based report from china was published 16 years ago , and countable number of publications [ 35 , 812 ] exist in the literature . in this study , with large population - based data from shanghai ( the biggest city in china ) and singapore ( a city country in southeast asia , with majority of the residents being descendents of immigrants from south china ) , we aimed to examine the independent contributions of physical and cognitive health to disability in chinese older adults and explore the potential influences of environment . participants of the present study were identified from two big population - based studies : the shanghai survey of alzheimer 's disease and dementia ( ssadd ) and the singapore longitudinal ageing study ( slas ) . the ssadd was conducted in 1987 based on a probability sample of 5055 community - dwelling chinese older adults from jing'an district of shanghai city . the slas was conducted among a total population sample of 2808 older adults from south east region of singapore . baseline assessments of the slas were completed from september 2003 to december 2005 . details of the ssadd [ 8 , 13 ] and , we selected 4639 participants from the ssadd sample and 2397 participants from the slas cohort . all participants had complete data on demographic information , physical health , and cognitive function and obtained a mini - mental state examination ( mmse ) total score of at least 21 points . face - to - face interviews were conducted by psychiatrists / psychiatric nurses ( in shanghai ) or trained research nurses ( in singapore ) , and data were collected for a wide range of variables . for the present analysis , we extracted the following variables from the databases : age , sex , functional status , chronic diseases , self - rated health status , and mmse total score . functional status was assessed by the participant 's level of dependency in performing 8 activities of daily living ( adl ) : eating , grooming , dressing , transferring , walking , toileting , bathing , and climbing stairs . the participants were asked do you have or not have any of the following illnesses or conditions at the present time ? " in the slas , a list of 14 medical conditions was covered in the interview . the participants were asked do you have a history of this medical problem ? medical conditions that were not included in the list were recorded under any other problems . we selected ten chronic diseases on which data were available from both samples : hypertension , diabetes , heart diseases ( in singapore : defined as any of heart attack , heart failure , or atrial fibrillation ) , stroke ( in shanghai : effects of stroke ) , kidney disorder ( in singapore : kidney failure ) , chronic obstructive lung disease ( in shanghai : emphysema / bronchitis ) , asthma , arthritis ( in shanghai : arthritis or rheumatism ) , mental illness , and cancer ( in singapore , identified from any other problems ) . in statistical analysis , the number of chronic diseases was used as an objective measure of physical health . in singapore , self - rated health status was assessed using a single question : in general , would you say your health is : excellent , very good , good , fair , or poor ? in shanghai , the same question was asked but there were four choices : excellent , good , fair , poor . excellent and very good together and created a new variable self - rated health status : 1 = excellent or very good , 2 = good , 3 = fair , and 4 = poor . this was used as a continuous variable in multivariate analyses ( score ranged from 1 to 4 , with higher value representing poorer health status ) . the test has been validated in shanghai and singapore , respectively . summed scores of mmse range from 0 to 30 , higher values denoting better cognitive functioning . chi - square test ( for dichotomous variables ) or independent sample t test ( for continuous variables ) was used to compare the difference regarding various characteristics between the two study samples . multiple logistic regressions were used to examine the relationship between health factors and disability , and the influence from environment . in analysis based on the combined sample , age , sex , number of chronic diseases , self - rated health status , mmse total score , and environment ( singapore versus shanghai ) were included in the regression model . the same set of variables ( except environment ) was used in the stratified analysis . the prevalence of disability was much higher in shanghai sample ( 5.0% ) than that in the singapore sample ( 1.8% ) . however , the shanghai participants were also older , had more chronic diseases , had poorer self - rated health status , and obtained lower mmse total score as compared to their singapore counterparts ( table 1 ) . a scrutiny of the eight adl items revealed that the difference was only significant for bathing ( 3.5% versus 0.4% ) and climbing stairs ( 3.1% versus 1.2% ) . in multiple logistic model based on the combined sample , number of chronic diseases , self - rated health status , mmse total score , and environment the odds ratio was 1.35 ( 95% ci 1.221.50 ) for number of chronic diseases , 2.85 ( 95% ci 2.363.43 ) for self - rated health status ( continuous ) , 0.89 ( 95% ci 0.850.94 ) for mmse total score , and 0.68 ( 95% ci 0.480.96 ) for environment ( singapore versus shanghai ) , respectively . results from stratified analysis showed that the strength of associations between health variables and disability differed between the two samples ( table 3 ) . for example , in the shanghai sample , one point increase on the 4-point self - rated health status scale was associated with 3.13 times ( 95% ci 2.553.85 ) higher odds of having disability while the odds ratio was only 1.73 ( 95% ci 1.072.81 ) in the singapore sample . for mmse total score , the corresponding odds ratio was 0.91 ( 95% ci 0.860.96 ) in the shanghai sample and 0.83 ( 95% ci 0.730.94 ) in the singapore sample . based on data from two population - based studies , we found that the number of chronic diseases , self - rated health status , cognitive function , and unmeasured environment factors represented by study sample were significantly associated with functional disability among chinese older adults . the contributions of physical health [ 8 , 18 ] and cognitive function [ 19 , 20 ] to functional disability have been documented and were well replicated in our analysis . with large population - based data , we firstly quantified the strength of differential contributions of each of self - reported physical health , self - rated health status , and cognitive function to disability in chinese elderly population . the findings are relevant and important for policymakers , medical practitioners , and academics . participants in the singapore sample were 32% less likely to have disability compared with participants in the shanghai sample . participants from both studies were chinese older adults living in big city , and a number of important covariates were adjusted in multiple regression models . it is less likely that the observed difference was caused by unmeasured factors at individual level . however , a careful examination of the two most difficult adl tasks ( bathing and climbing stairs ) provided us with plausible explanations . in 1987 , most families in shanghai had no bathroom and shower , and bathing was considered as a complicated task . in contrast , the majority of slas participants lived in public houses that have standard bathroom and shower and easy - to - climb stairs . our study provided fresh evidence on the role of environmental factors in disablement process [ 2123 ] . as pointed out by verbrugge and jette : disability is not a personal characteristic , but is instead a gap between personal capability and environmental demand disability can be diminished swiftly and markedly if the physical or mental demands of a given task are reduced . our findings suggest that environment not only affects the prevalence of disability , but also modifies the strength of associations between disability and health variables . strengths of our study include concurrent measuring of various health factors and simultaneous inclusion of those factors in multivariate model . however , no causal relationship could be drawn given cross - sectional nature of the study design . in conclusion , physical health and cognitive function were significantly associated with disability among chinese older adults living in asian metropolises . all the authors contributed substantially to the design , analysis and interpretation of the data and participated in drafting or revising the 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 .

1. Introduction 2. Methods 3. Results 4. Discussion Conflict of Interests Authors' Contribution

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

Introduction Results Discussion

often , a new physics faculty member is faced with the duty of renovating the introductory physics labs . we 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 microcomputer - based laboratories ( mbls ) . our student body consists mostly of science majors that take the algebra - based course . but , the lab renovation described here could be used for the calculus - based group as well . we would suggest adjusting the lab manuals . nowadays , mbls are usually the choice when thinking of a renovation . they have effectively demonstrated an advantage to the learning process over the years.[14 ] moreover , for our science students , the labs are important in reinforcing the concepts learned in class . it is very common to hear , during the lab sections , students commenting on their grasp of the concept learned in class due to the experiment being performed . the overall 1-year lab experience follows the guidelines provided by the american association of physics teachers . it contains a brief theoretical description and the procedures to be followed on the day of the lab . on the lab day , the students should come prepared and ready to start without additional instructions . the instructor circles around the stations to guide and answer appropriate questions if needed . on the lab day , the students are handed the lab report . it contains a data analysis part , some discovery questions , and ends with a summary and conclusion part . the report is completed by the student during the session . we did not find an appropriate version for the other half to meet our overall goal . therefore , we kept a few traditional labs . the list of experiments is chosen based on the lecture material . one of the concerns was to always be able to cover the theory before the lab was performed by the student . one - year introductory microcomputer - based laboratory experiment list understanding motion , free fall , projectile motion , atwood 's machine , boyle 's law , electrical equivalent of heat , heat transfer , electrostatic charge , ohm 's law , rc circuit , and magnetic induction . a suggestion for the beginner is to try all the experiments before hand until you get really familiar with the sensors and software and how they work . most of the time , it is a lack of understanding of the use of the apparatus , assuming it is not defective of course . we will list here the total equipment needed per station to implement the 1-year lab described above . it is expected that the laboratory will have a printer that can be shared among all groups . each station consists of a laptop and the science workshop 750 interface ( ci-7650 ) with the datastudio software ( ci-6870 g ) . lab station : science workshop interface , laptop and motion sensor sensors to be used with the interface for data measurement : motion sensor ( ci-6742a ) , photogate and pulley system ( me-6838 ) , accessory photogate ( me-9204b ) , time - of - 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 a few sensors . from left to right : pressure sensor , charge sensor , and photogate head the datastudio software collects and analyzes the data . it has an easy - to - use interface , allowing the students to explore the data . for instance , the left screenshot in ure 3 displays a graph of voltage versus time . the data are collected using a voltage sensor when a magnet is dropped through a coil . the students can select a region on the graph and the software calculates the area under the curve . the screenshot on the right in figure 3 displays the curve - fitting feature of datastudio . left screenshot : induction lab using graph and area under the curve calculation . the pressure column will be filled as the measurements are taken using the pressure sensor . boyle 's law lab using a table display the additional equipment needed from pasco to perform the experiments are : 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 ) , and bar magnet ( em-8620 ) . general lab supplies needed include a pair of scissors and 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 and black ( 5 on set ) ( se-9750 ) or ( se-975 ) , masses and hanger set ( me-8979 ) , universal table clamp ( me-9376b ) , calorimetry cups ( td-8825a ) , and hot plates ( se-8830 ) . oftentimes , this task is hard to accomplish for a solo faculty in a small institution . we provided here a list of the experiments and equipments needed to upgrade about half the experiments to mbls on a 1-year introductory physics lab . we would like to add that although we used pasco , there are other comparable systems in the market . the intention of this paper is to help others with their own lab renovation . in order to better fulfill this 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 .

INTRODUCTION LAB SETUP EXPERIMENTS EQUIPMENT CONCLUSION

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 .

Images

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

Introduction Role of Sodium Glucose Transporter in Renal Glucose Excretion SGLT2 Inhibitors Clinical Expectations for SGLT-2 Inhibitors Efficacy Safety Positioning the SGLT2 Inhibitors Conclusion

the goal of defibrillation ( df ) testing is to verify the detection of ventricular tachyarrhythmias by implantable cardioverter - defibrillators ( icds ) , and to ascertain df efficacy . all clinical trials , which demonstrated the benefit of icd therapy , have included some kind of df testing . according to the food and drug administration , the instructions for use of icds include the recommendation with df testing at the time of implantation . the absence of df testing may have medico - legal implications whether a device fails to terminate ventricular fibrillation ( vf ) during follow - up . nevertheless , df testing has been deliberately omitted in the majority of patients enrolled in contemporary icd studies . despite the stochastic nature of df , ineffective shocks during vf induction result in system modifications in up to 5% of patients . system modifications , such as repositioning of the electrode , reversal of polarity , use of a high - energy device , use of a dual instead of a single - coil electrode , or the implantation of an additional subcutaneous electrode , may be unnecessary or even harmful since many patients have a successful retesting with the original system configuration . no single system modification in itself has ever been shown to improve outcome . with current devices and programming , an inadequate safety margin occurs infrequently , and the incidence of ventricular tachyarrhythmias requiring shocks is low at 46% per year . while df testing has never been shown to improve survival , it is associated with an increased , albeit small risk of major adverse events of 0.4% . by omitting routine df testing , the approach to icd implantation will likely change considerably , as the procedures will be shorter and performed without technical support and anaesthesia . similar to the simple trial , we hypothesized that , with current high - energy icd devices , the omission of df testing during implantation will not result in an inferior shock efficacy during follow - up when compared with standard df testing . as opposed to the simple trial , which used the composite outcome of arrhythmic death or failed appropriate shock , the primary end point of our study was the shock efficacy of all true ventricular tachycardia ( vt ) and vf episodes occurring in any patient during follow - up . the nordic icd trial ( nct01282918 ) was a prospective , randomized , parallel group , multi - centre non - inferiority trial conducted at 48 centres in five european countries , and designed to investigate the effect of df testing at the time of icd implantation on first shock efficacy ( fse ) during follow - up . briefly , patients with an indication for icd implantation according to the current european society of cardiology guidelines were included . patients with hypertrophic or arrhythmogenic right ventricular cardiomyopathy were excluded because of special considerations necessary during icd implantation . one thousand and seventy - seven patients were randomized ( 1 : 1 ) to first time icd implantation with or without df testing during the procedure . patients were followed for at least 12 months through regular on - site visits , and remotely via home monitoring ( hm ; biotronik se & co. kg , berlin , germany ) . all true ventricular tachyarrhythmias treated with an icd shock in any patient were included in the primary analysis . the study was conducted in accordance with the declaration of helsinki , and country - specific regulatory requirements . the protocol was approved by the institutional review board or ethics committee of each participating centre . a standard df testing protocol was used by all participating centres , as described previously . briefly , the initial shock for df was programmed to 15 j. if successful , df testing was terminated . if unsuccessful , a second shock programmed to 24 j was delivered , and had to be confirmed . if unsuccessful , a system revision was recommended , and the df testing procedure was repeated . the primary end point was the fse to terminate all true episodes of vt / vf during follow - up . a blinded two - member clinical event committee reviewed the diagnostic information of the vt / vf treated with icd shocks . safety end points included procedural serious adverse events , vt / vf conversion rate , and all - cause , cardiac , or arrhythmic mortality during follow - up . all adverse events were reviewed and adjudicated by a data and safety monitoring board ( dsmb ) , consisting of three members . all patient deaths were adjudicated by the dsmb according to a classification originally described by epstein et al . the goal of the nordic icd trial was to demonstrate the non - inferiority of no df testing when compared with df testing with respect to the primary endpoint of fse . this margin was chosen because the variation of fse in published trials was in the range of 8392% . after a pre - specified blinded re - assessment , the sample size was increased to 1080 patients recruited over a period of 28 months , with a follow - up of at least 12 months . the intention - to - treat ( itt ) population which included all randomized patients , the evaluated - for - safety population which included randomized patients who received an icd , and the per protocol ( pps ) population which included randomized patients who received an icd and did not have a major protocol violation . however , for time - dependent outcomes , these patients were included up to the date of the protocol violation . the primary analysis was performed in the per protocol episode data set ( p - pps ) , which included all sufficiently documented shocked episodes in pps patients . baseline characteristics are presented group wise for the itt population as mean ( sd ) or frequency ( % ) . to compensate for the dependence structure induced by recurrent episodes with similar outcome in some of the patients , a random effects logit model with patients as a random factor and group as a fixed factor was used for calculation of a two - sided 95% confidence interval ( ci ) of the difference fsenodftest fsedftest . to state non - inferiority , this ci had to lie completely above the pre - specified non - inferiority margin of 10% . without the use of a model that takes the correlation structure into account , the few patients with an extreme numbers of shocks would have dominated the results . procedural and safety outcomes were compared with a student 's t test , or test or fisher 's exact test , whichever was appropriate . hazard rate reduction was assessed using a cox proportional hazards model . a two - tailed p - value < 0.05 was considered to be statistically significant . data were analysed using sas ( version 9.4 ) and stata ( version 13.1 ) by an independent statistical institute ( department of medical biometry and epidemiology , university medical centre hamburg eppendorf , germany ) ( a.s . , k.w . ) . the first draft of the manuscript was written by the first author , and revised by all co - authors . the authors take full responsibility for the accuracy and completeness of the findings , as well as for the fidelity to the study protocol . the nordic icd trial ( nct01282918 ) was a prospective , randomized , parallel group , multi - centre non - inferiority trial conducted at 48 centres in five european countries , and designed to investigate the effect of df testing at the time of icd implantation on first shock efficacy ( fse ) during follow - up . briefly , patients with an indication for icd implantation according to the current european society of cardiology guidelines were included . patients with hypertrophic or arrhythmogenic right ventricular cardiomyopathy were excluded because of special considerations necessary during icd implantation . one thousand and seventy - seven patients were randomized ( 1 : 1 ) to first time icd implantation with or without df testing during the procedure . patients were followed for at least 12 months through regular on - site visits , and remotely via home monitoring ( hm ; biotronik se & co. kg , berlin , germany ) . all true ventricular tachyarrhythmias treated with an icd shock in any patient were included in the primary analysis . the study was conducted in accordance with the declaration of helsinki , and country - specific regulatory requirements . the protocol was approved by the institutional review board or ethics committee of each participating centre . a standard df testing protocol was used by all participating centres , as described previously . briefly , the initial shock for df was programmed to 15 j. if successful , df testing was terminated . if unsuccessful , a second shock programmed to 24 j was delivered , and had to be confirmed . if the shocks were successful twice , df testing was terminated . if unsuccessful , a system revision was recommended , and the df testing procedure was repeated . the primary end point was the fse to terminate all true episodes of vt / vf during follow - up . a blinded two - member clinical event committee reviewed the diagnostic information of the vt / vf treated with icd shocks . safety end points included procedural serious adverse events , vt / vf conversion rate , and all - cause , cardiac , or arrhythmic mortality during follow - up . all adverse events were reviewed and adjudicated by a data and safety monitoring board ( dsmb ) , consisting of three members . all patient deaths were adjudicated by the dsmb according to a classification originally described by epstein et al . the goal of the nordic icd trial was to demonstrate the non - inferiority of no df testing when compared with df testing with respect to the primary endpoint of fse . this margin was chosen because the variation of fse in published trials was in the range of 8392% . after a pre - specified blinded re - assessment , the sample size was increased to 1080 patients recruited over a period of 28 months , with a follow - up of at least 12 months . the intention - to - treat ( itt ) population which included all randomized patients , the evaluated - for - safety population which included randomized patients who received an icd , and the per protocol ( pps ) population which included randomized patients who received an icd and did not have a major protocol violation . however , for time - dependent outcomes , these patients were included up to the date of the protocol violation . the primary analysis was performed in the per protocol episode data set ( p - pps ) , which included all sufficiently documented shocked episodes in pps patients . baseline characteristics are presented group wise for the itt population as mean ( sd ) or frequency ( % ) . to compensate for the dependence structure induced by recurrent episodes with similar outcome in some of the patients , a random effects logit model with patients as a random factor and group as a fixed factor was used for calculation of a two - sided 95% confidence interval ( ci ) of the difference fsenodftest fsedftest . to state non - inferiority , this ci had to lie completely above the pre - specified non - inferiority margin of 10% . without the use of a model that takes the correlation structure into account , the few patients with an extreme numbers of shocks would have dominated the results . procedural and safety outcomes were compared with a student 's t test , or test or fisher 's exact test , whichever was appropriate . mortality was analysed in the itt population using the kaplan meier approach and the log - rank test . hazard rate reduction was assessed using a cox proportional hazards model . a two - tailed p - value < 0.05 was considered to be statistically significant . data were analysed using sas ( version 9.4 ) and stata ( version 13.1 ) by an independent statistical institute ( department of medical biometry and epidemiology , university medical centre hamburg eppendorf , germany ) ( a.s . , k.w . ) . the first draft of the manuscript was written by the first author , and revised by all co - authors . the authors take full responsibility for the accuracy and completeness of the findings , as well as for the fidelity to the study protocol . the baseline clinical characteristics and cardiovascular medications of the 1077 patients comprising the intention - to - treat population ( without df = 537 and with df = 540 ) are presented in table 1 . seven hundred and one patients ( 65.1% ) had an ischaemic cardiomyopathy and 873 ( 81.8% ) received the icd for primary prevention of sudden cardiac death . ten patients ( six in the df group and four in the without df group ) did not receive an icd , leaving 1067 patients for the safety evaluation ( figure 1 ) . both groups were well matched for baseline demographics and clinical characteristics , as well as for the use of dual ( 51.6% ) and a single coil ( 48.4% ) electrode ( table 2 ) . table 1baseline demographic and clinical characteristics of subjects by treatment group ( intention to 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 ) not done6 ( 0.6)2 ( 0.4)4 ( 0.7)af at enrolment , n ( % ) 85 ( 7.9)45 ( 8.4)40 ( 7.4)indication for 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 / at 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 ) ( other ) 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 ) ( other ) 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 ) other cardiovascular medication , n ( % ) 255 ( 23.7)121 ( 22.5)134 ( 24.8)values are means sd . there were no significant differences at p < 0.05 between groups , except for ace inhibitors p = 0.039 . t - test for metric variables , -test for categorical , or fisher 's exact test ( f ) if is not appropriate.ace inhibitors , angiotensin converting enzyme inhibitors ; af , atrial fibrillation ; at receptor blockers , angiotensin receptor blockers ; bmi , body mass index ; df , defibrillation ; nyha , new york heart association ; itt , intention - to - treat ; lvef , left ventricular ejection fraction . table 2procedural characteristics according to treatment group ( successful primary implantable cardioverter - defibrillator implantation ; evaluated - for - 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 ) at pulse duration of 0.4 ms ( n = 527)0.51 ( 0.20)0.49 ( 0.17)0.53 ( 0.22)0.040pacing threshold ( v ) at pulse duration of 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 at final position ( j ) ( n = 526)16.74 ( 4.47)16.67 ( 4.38)patients with intra - procedural system revisions and icd re - programming , n / total n ( % ) 25/527 ( 4.74)25/520 ( 4.81)patients with electrode repositionings , n / total n ( % ) 8/527 ( 1.52)8/520 ( 1.54)patients with changes of shock polarity , n / total n ( % ) 20/527 ( 3.80)20/520 ( 3.85)patients with modification of lead system ( shock pathway ) , n / total n ( % ) 5/527 ( 0.95)5/520 ( 0.96)patients with other revisions / re - 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 - d , cardiac - resynchronization therapy with defibrillator ; df , defibrillation ; efs , evaluated for safety ; vegm , ventricular electrogram ; icd , implantable cardioverter-defibrillator.one patient was not inducible . itt , full analysis set ( all patients as randomized ) ; efs , evaluated for safety set ( all patients randomized , who had a successful primary icd implantation ) ; pps , per protocol set ( all patients randomized , who are compliant with the study protocol ) ; p - pps , all randomized patients at least one day at risk before protocol deviation ( includes all sufficiently documented shocked episodes until time point of protocol deviation ) . baseline demographic and clinical characteristics of subjects by treatment group ( intention to treat ) values are means sd . there were no significant differences at p < 0.05 between groups , except for ace inhibitors p = 0.039 . t - test for metric variables , -test for categorical , or fisher 's exact test ( f ) if is not appropriate . ace inhibitors , angiotensin converting enzyme inhibitors ; af , atrial fibrillation ; at receptor blockers , angiotensin receptor blockers ; bmi , body mass index ; df , defibrillation ; nyha , new york heart association ; itt , intention - to - treat ; lvef , left ventricular ejection fraction . procedural characteristics according to treatment group ( successful primary implantable cardioverter - defibrillator implantation ; evaluated - for - safety ) crt - d , cardiac - resynchronization therapy with defibrillator ; df , defibrillation ; efs , evaluated for safety ; vegm , ventricular electrogram ; icd , implantable cardioverter - defibrillator . itt , full analysis set ( all patients as randomized ) ; efs , evaluated for safety set ( all patients randomized , who had a successful primary icd implantation ) ; pps , per protocol set ( all patients randomized , who are compliant with the study protocol ) ; p - pps , all randomized patients at least one day at risk before protocol deviation ( includes all sufficiently documented shocked episodes until time point of protocol deviation ) . of the 534 patients randomized to the df arm with a successful first icd implantation ( figure 1 ) , testing was performed in 97.4% . in one patient , vt / vf was not inducible . four hundred and ninety - four of 519 inducible patients ( 95.2% ) passed the test without any system reconfiguration , and 440 patients ( 84.8% ) had their vt / vf terminated with a 15 j shock . in 25 patients , the initial df test was not successful , and system reconfigurations were performed once in 20 and more than once in 5 ( table 2 ) . all patients subsequently passed the test successfully , but still with a higher energy level at the final position ( 22.2 7.4 j vs. 16.4 4.0 j , p < 0.001 ) and with significantly more shocks ( 5.7 3.0 vs. 1.2 0.7 , p < 0.001 ) than the 494 patients who passed the test immediately . in addition , the duration of the procedure ( 90.2 56.2 min vs. 63.5 36.1 min , p < 0.001 ) and fluoroscopy exposure ( 11.6 14.2 min vs. 7.5 10.7 min , p = 0.064 ) was increased . during a median follow - up of 22.7 and 22.9 months , respectively , 211 true vt / vf episodes with at least one appropriate shock occurred in 8.6% of the per protocol patients with df testing , while 218 true vt / vf episodes occurred in 8.8% of patients without df testing ( table 3 , figure 1 ) . all true vt / vf episodes were terminated with any appropriate icd shock in the without df test group , whereas 96.7% of all vt / vf were terminated in the group with df test . thus , the conversion efficacy was 100.0% without df testing and 96.7% with df testing . the model - based shock efficacy was 97.1% without df testing and 94.1% with df testing . the difference in fse was 3.0% ( ci 3.0% , 9.0% ) in favour of the without df test group , and remained above the pre - defined 10% margin for the 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 with at least one delivered appropriate shock for a true vt / vf episode , n / total n ( % ) 91/1046 ( 8.7)46/523 ( 8.8)45/523 ( 8.6)all true vt / vf episodes with at 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 with first appropriate icd shock , n / total n ( % ) 384/429 ( 89.5)204/218 ( 93.6)180/211 ( 85.3)true vt / vf episodes terminated with any appropriate icd shock , n / total n ( % ) 422/429 ( 98.4)218/218 ( 100.0)204/211 ( 96.7)true vt / vf episodes not terminated with any 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 between the groups ( without df testing vs. with 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 between the groups ( without df testing vs. with 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 of 201 serious adverse events related to the study procedure occurred in 168 patients ( 15.7% ) , of which 89 events occurred in 74 patients without df testing and 112 events occurred in 94 patients with df testing within 30 days of follow - up ( p = 0.095 ) . the only significant difference in single serious adverse events was intra - operative hypotension , which manifested more often in patient with df testing ( 1.7% ) than those without df testing ( 0.0% ) ( p = 0.004 ) . lead - related complications occurred in 15 patients without df testing ( 2.8% ) compared with 24 events in 21 patients with df testing ( 3.9% ) ( p = 0.311 ) . one sudden death occurred in each study group within 30 days of follow - up , and the mean length of hospitalization was 4.0 days in both study groups ( table 1 ) . the proportion of patients with inappropriate shocks during follow - up was low at 3.7% , and similar between groups . forty - four and 52 patients without and with df testing died , respectively ( p = 0.377 , table 4 ) . differences with respect to non - cardiac and cardiac mortality were not statistically significant . six deaths ( 5 with df and 1 without df testing ) were adjudicated arrhythmic and due to repetitive ventricular tachyarrhythmias / vt clusters ( p = 0.100 , table 4 ) . table 4mortality ( intention - to - 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 to treat.incidence referring to person - years without df test ( 1001.5 ) and with df test ( 985.9 ) . mortality ( intention - to - treat ) df , defibrillation ; hr , hazard ratio ; itt , intention to treat . incidence referring to person - years without df test ( 1001.5 ) and with df test ( 985.9 ) . the baseline clinical characteristics and cardiovascular medications of the 1077 patients comprising the intention - to - treat population ( without df = 537 and with df = 540 ) are presented in table 1 . seven hundred and one patients ( 65.1% ) had an ischaemic cardiomyopathy and 873 ( 81.8% ) received the icd for primary prevention of sudden cardiac death . ten patients ( six in the df group and four in the without df group ) did not receive an icd , leaving 1067 patients for the safety evaluation ( figure 1 ) . both groups were well matched for baseline demographics and clinical characteristics , as well as for the use of dual ( 51.6% ) and a single coil ( 48.4% ) electrode ( table 2 ) . table 1baseline demographic and clinical characteristics of subjects by treatment group ( intention to 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 ) not done6 ( 0.6)2 ( 0.4)4 ( 0.7)af at enrolment , n ( % ) 85 ( 7.9)45 ( 8.4)40 ( 7.4)indication for 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 / at 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 ) ( other ) 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 ) ( other ) 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 ) other cardiovascular medication , n ( % ) 255 ( 23.7)121 ( 22.5)134 ( 24.8)values are means sd . there were no significant differences at p < 0.05 between groups , except for ace inhibitors p = 0.039 . t - test for metric variables , -test for categorical , or fisher 's exact test ( f ) if is not appropriate.ace inhibitors , angiotensin converting enzyme inhibitors ; af , atrial fibrillation ; at receptor blockers , angiotensin receptor blockers ; bmi , body mass index ; df , defibrillation ; nyha , new york heart association ; itt , intention - to - treat ; lvef , left ventricular ejection fraction . table 2procedural characteristics according to treatment group ( successful primary implantable cardioverter - defibrillator implantation ; evaluated - for - 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 ) at pulse duration of 0.4 ms ( n = 527)0.51 ( 0.20)0.49 ( 0.17)0.53 ( 0.22)0.040pacing threshold ( v ) at pulse duration of 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 at final position ( j ) ( n = 526)16.74 ( 4.47)16.67 ( 4.38)patients with intra - procedural system revisions and icd re - programming , n / total n ( % ) 25/527 ( 4.74)25/520 ( 4.81)patients with electrode repositionings , n / total n ( % ) 8/527 ( 1.52)8/520 ( 1.54)patients with changes of shock polarity , n / total n ( % ) 20/527 ( 3.80)20/520 ( 3.85)patients with modification of lead system ( shock pathway ) , n / total n ( % ) 5/527 ( 0.95)5/520 ( 0.96)patients with other revisions / re - 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 - d , cardiac - resynchronization therapy with defibrillator ; df , defibrillation ; efs , evaluated for safety ; vegm , ventricular electrogram ; icd , implantable cardioverter-defibrillator.one patient was not inducible . itt , full analysis set ( all patients as randomized ) ; efs , evaluated for safety set ( all patients randomized , who had a successful primary icd implantation ) ; pps , per protocol set ( all patients randomized , who are compliant with the study protocol ) ; p - pps , all randomized patients at least one day at risk before protocol deviation ( includes all sufficiently documented shocked episodes until time point of protocol deviation ) . baseline demographic and clinical characteristics of subjects by treatment group ( intention to treat ) values are means sd . there were no significant differences at p < 0.05 between groups , except for ace inhibitors p = 0.039 . t - test for metric variables , -test for categorical , or fisher 's exact test ( f ) if is not appropriate . ace inhibitors , angiotensin converting enzyme inhibitors ; af , atrial fibrillation ; at receptor blockers , angiotensin receptor blockers ; bmi , body mass index ; df , defibrillation ; nyha , new york heart association ; itt , intention - to - treat ; lvef , left ventricular ejection fraction . procedural characteristics according to treatment group ( successful primary implantable cardioverter - defibrillator implantation ; evaluated - for - safety ) crt - d , cardiac - resynchronization therapy with defibrillator ; df , defibrillation ; efs , evaluated for safety ; vegm , ventricular electrogram ; icd , implantable cardioverter - defibrillator . itt , full analysis set ( all patients as randomized ) ; efs , evaluated for safety set ( all patients randomized , who had a successful primary icd implantation ) ; pps , per protocol set ( all patients randomized , who are compliant with the study protocol ) ; p - pps , all randomized patients at least one day at risk before protocol deviation ( includes all sufficiently documented shocked episodes until time point of protocol deviation ) . of the 534 patients randomized to the df arm with a successful first icd implantation ( figure 1 ) , testing was performed in 97.4% . in one patient , vt / vf was not inducible . four hundred and ninety - four of 519 inducible patients ( 95.2% ) passed the test without any system reconfiguration , and 440 patients ( 84.8% ) had their vt / vf terminated with a 15 j shock . in 25 patients , the initial df test was not successful , and system reconfigurations were performed once in 20 and more than once in 5 ( table 2 ) . all patients subsequently passed the test successfully , but still with a higher energy level at the final position ( 22.2 7.4 j vs. 16.4 4.0 j , p < 0.001 ) and with significantly more shocks ( 5.7 3.0 vs. 1.2 0.7 , p < 0.001 ) than the 494 patients who passed the test immediately . in addition , the duration of the procedure ( 90.2 56.2 min vs. 63.5 36.1 min , p < 0.001 ) and fluoroscopy exposure ( 11.6 14.2 min vs. 7.5 10.7 min , p = 0.064 ) was increased . during a median follow - up of 22.7 and 22.9 months , respectively , 211 true vt / vf episodes with at least one appropriate shock occurred in 8.6% of the per protocol patients with df testing , while 218 true vt / vf episodes occurred in 8.8% of patients without df testing ( table 3 , figure 1 ) . all true vt / vf episodes were terminated with any appropriate icd shock in the without df test group , whereas 96.7% of all vt / vf were terminated in the group with df test . thus , the conversion efficacy was 100.0% without df testing and 96.7% with df testing . the model - based shock efficacy was 97.1% without df testing and 94.1% with df testing . the difference in fse was 3.0% ( ci 3.0% , 9.0% ) in favour of the without df test group , and remained above the pre - defined 10% margin for the 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 with at least one delivered appropriate shock for a true vt / vf episode , n / total n ( % ) 91/1046 ( 8.7)46/523 ( 8.8)45/523 ( 8.6)all true vt / vf episodes with at 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 with first appropriate icd shock , n / total n ( % ) 384/429 ( 89.5)204/218 ( 93.6)180/211 ( 85.3)true vt / vf episodes terminated with any appropriate icd shock , n / total n ( % ) 422/429 ( 98.4)218/218 ( 100.0)204/211 ( 96.7)true vt / vf episodes not terminated with any 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 between the groups ( without df testing vs. with 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 in the per protocol population . primary analysis shock efficacy ( per protocol episode data set ) difference between the groups ( without df testing vs. with 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 of 201 serious adverse events related to the study procedure occurred in 168 patients ( 15.7% ) , of which 89 events occurred in 74 patients without df testing and 112 events occurred in 94 patients with df testing within 30 days of follow - up ( p = 0.095 ) . the only significant difference in single serious adverse events was intra - operative hypotension , which manifested more often in patient with df testing ( 1.7% ) than those without df testing ( 0.0% ) ( p = 0.004 ) . lead - related complications occurred in 15 patients without df testing ( 2.8% ) compared with 24 events in 21 patients with df testing ( 3.9% ) ( p = 0.311 ) . one sudden death occurred in each study group within 30 days of follow - up , and the mean length of hospitalization was 4.0 days in both study groups ( table 1 ) . the proportion of patients with inappropriate shocks during follow - up was low at 3.7% , and similar between groups . forty - four and 52 patients without and with df testing died , respectively ( p = 0.377 , table 4 ) . differences with respect to non - cardiac and cardiac mortality were not statistically significant . six deaths ( 5 with df and 1 without df testing ) were adjudicated arrhythmic and due to repetitive ventricular tachyarrhythmias / vt clusters ( p = 0.100 , table 4 ) . table 4mortality ( intention - to - 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 to treat.incidence referring to person - years without df test ( 1001.5 ) and with df test ( 985.9 ) . mortality ( intention - to - treat ) df , defibrillation ; hr , hazard ratio ; itt , intention to treat . incidence referring to person - years without df test ( 1001.5 ) and with df test ( 985.9 ) . defibrillation testing during icd implantation has been a matter of debate for the last two decades . to the best of our knowledge , the nordic icd trial is the first prospective randomized trial , which investigated the effect of df testing on the actual fse during follow - up as the single primary end point . this is in contrast with the recently published simple trial , which used the composite outcome of arrhythmic death or failed appropriate shock to terminate a spontaneous episode of vt / vf to determine the non - inferiority of the no testing approach . nordic icd also provides detailed information about intra - operative df testing outcome . in 9196% of icd implants , system modifications were reported in 2.212.0% of patients . with the high - energy devices used in the nordic icd study , only 4.8% of all inducible patients had an inadequate safety margin , which required one or two system reconfigurations in 3.8 and 1.0% , respectively . this is in agreement with the results of the sudden cardiac death in heart failure trial ( scd - heft ) , in which 97.8% of patients had a df threshold 20 j. following the system revisions , all patients passed the test successfully , but with a significantly higher energy level than for those who did not require revision ( 22.2 j vs. 16.4 j , p < 0.001 ) . this is in agreement with a canadian survey in which an insufficient safety margin with the initial system configuration could be improved in only 56% of patients . while the device revisions provided no benefit to our patients during follow - up , they significantly increased the total procedure time by 30 min and the fluoroscopy exposure by 40% . the mean number of shocks needed to find an acceptable safety margin was also increased more than four - fold . however , in a trial with variable use of devices , the shock efficacy is similar to that observed in the nordic icd trial . the observed lower rate of arrhythmic death for patients without df testing ( 0.2 vs. 0.9% ) corresponds to the sudden cardiac death rate reported in another trial ( 0.9 and 1.2% in patients without and with df testing ) during a similar follow - up period . because the simple trial did not use a specific model for multiple shock events in the same patient , the shock efficacy can only be compared at the level of raw shock efficacy . in the nordic icd and the simple trials , a higher raw shock efficacy was observed in the group without df vs. with df testing ( nordic icd : 93.6 vs. 85.3% and simple : 92.0 vs. 88.5% ) . on the other hand , the higher first shock energy we used ( 40 j vs. 31 j in simple ) was not associated with a higher fse irrespective of testing . the high level of fse is consistent with the relatively low mean df energy ( 16.7 j ) during testing , with > 95% of the inducible patients undergoing successful testing without any system reconfiguration . a 15.7% serious adverse events rate ( 17.6% with testing and 13.9% without testing , p = 0.095 ) was found within 30 days following successful icd implantation . this is much higher than what has been described in recent reports , in which the complication rates ranged between 2.3 and 2.7% in patients with and without an adequate safety margins during df testing or compared with the simple trial results regarding adverse events included in the pre - defined as the primary safety outcome ( 5.6% without df testing vs. 6.5% with df testing ) . it is noteworthy that the nordic icd trial not only collected data on protocol - specified complications but also on all procedure- and patient - related adverse events which might explain our higher event rate . the 30-day procedure - related mortality rate was 0.2% , and consisted of two sudden deaths which is in the range of recent reports . the stroke rate was 0.8% for patients without and 0.2% for patients with df testing ( p = 0.217 ) , which is in agreement with the 0.4% rate reported in the credit registry . the two types of adverse events that were increased in patients with df testing were intra - operative hypotension ( 1.7% vs. 0.0% , p = 0.004 ) and lead - related complications ( 3.9 vs. 2.8% , p = 0.311 ) . a recent article summarized implant - related complications that were directly or indirectly related to the df testing procedure . the rate of haemodynamic complications was 1% , and lead dislodgement was listed as the most frequent single complication with rates between 1 and 6% . both the nordic icd and simple trials showed a trend towards lower procedural complication rates in patients without df testing despite the different definitions and methods used for analysing these events ( nordic icd : 13.9 vs.17.6% , p = 0.095 , simple : 5.6 vs. 6.5% , p = 0.33 ) . the rates of all - cause , cardiac , and non - cardiac mortality were similar between patients with and without df testing , which is in agreement with most non - randomized trials reported . in accordance with the simple trial , no statistical difference in all - cause mortality between patients with and without df testing however , the annual mortality rates of nordic icd were lower when compared with other large randomized icd trials , and also lower than those of the simple trial . the nordic icd trial supports the hypothesis that if 40 j devices are used , df testing does not improve df efficacy during follow - up . it suggests that df testing or the various measures to improve df efficacy lengthen the procedure , and may even be harmful . the nordic icd trial is one of two large prospective randomized trials , which do not support the routine use of df testing during left - sided first time icd implantation . this work was supported by biotronik se & co. kg ( berlin , germany ) . funding to pay reports receiving grants and personal fees form medtronic , st jude medical , biosense , biotronik , and boston scientific during the conduct of the study , and grants and personal fees from biosense and biotronik outside of the submitted work . reports receiving grants , personal fees , and non - financial support from biotronik during the conduct of the study , and personal fees and non - financial support from boehringer ingelheim outside the submitted work . reports personal fees from biotronik during the conduct of the study , and grants from st jude medical and personal fees from boston scientific outside the 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 .

Introduction Methods Trial design and oversight Intra-operative testing End points Statistical analysis Results Study population Intra-operative testing Shock efficacy Serious adverse events Mortality Discussion Conclusion Supplementary material Funding

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

INTRODUCTION History of incontinent urinary diversion Current attempts in artificial conduit construction Future perspectives CONCLUSIONS

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

Introduction Methods Results Discussion Conclusion

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

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 .

Introduction Biomarkers in CSF Structural Neuroimaging Markers Functional Neuroimaging Markers Molecular Neuroimaging Markers Conclusion Conflict of Interest Statement

the cardinal clinical characteristics of sca3 include gait and stance unsteadiness , limb ataxia , dysarthria , oculomotor dysfunction , sensory disorder , pyramidal and extrapyramidal dysfunction , and so on . sca3 is a slowly progressive and unremitting disease , in which patients generally will become wheelchair - bound and bedridden in the end stage , and the median survival time after disease onset is approximately 21 years . the resulting loss of working ability and reduced survival confer significant disease burden to the patients , their families , and the society . thus , it is of vital importance to explore effective therapeutic options in order to alleviate the symptoms or retard the disease progression in sca3 . nerve growth factor ( ngf ) is the founding member of the neurotrophin family , and is essential for the proper development , patterning , and maintenance of the mammalian nervous system . previous studies have revealed that ngf specifically targets sensory and sympathetic neurons in the peripheral nervous system , as well as basal forebrain cholinergic neurons in the central nervous system . there is also growing evidence to support the role of ngf in the development , differentiation , and maintenance of the human cerebellar connectivity . in this context , ngf and its high - affinity receptor tachykinin receptor antagonist ( tkra ) these data imply that ngf may have neuroprotective effects on cerebellar neurons and hence might serve as a therapeutic candidate of sca3 . therefore , this clinical pilot study was set forth to examine the efficacy of ngf in patients with sca3 . this study was an open - label clinical trial assessing the efficacy of ngf in patients with sca3 ; it was conducted at the first affiliated hospital of zhengzhou university from november 2011 to november 2012 . this study was approved by the ethics committee of first affiliated hospital of zhengzhou university and registered at the chinese clinical trial registry ( www.chictr.org ; chictr - onc-11001954 ) . all study procedures were in accordance with the declaration of helsinki and all recruited subjects have provided written informed consents . ataxia patients with family history were screened at the department of neurology , first affiliated hospital of zhengzhou university and referred for genetic testing at the department of genetic diagnosis . patients who fulfilled the following inclusion criteria : ( 1 ) ataxia patients with family history were checked and diagnosed by two independent doctors , then the genotype sca3 was confirmed by genetic test ; ( 2 ) older than 18 years ; and ( 3 ) willing to give informed consent , will be recruited . the exclusion criteria were as follows : ( 1 ) allergy to neurotrophin ; ( 2 ) with concomitant severe systematic diseases or psychiatric disorders ; ( 3 ) unable to finish the scale for assessment and rating of ataxia ( sara ) score ; ( 4 ) refuse to attend the study , and ( 5 ) ataxias attributed to secondary causes ( such as alcohol or drug abuse and toxic exposure ) . all enrolled patients underwent standard neurological , electrophysiological and neuroimaging examinations , and the sca3 subtype was classified according to these clinical findings . china ) used in this study was extracted and purified from the submandibular gland of the male mouse and has high homology in the amino acid sequence with human ngf . the mngf has been safely used in a series of clinical studies . , and has been approved by china food and drug administration . mngf was administered peripherally by intramuscular injection at the dose of 18 g once daily for 4 weeks consecutively . it has been proven to have good reliability and validity among chinese patients with degenerative cerebellar . the sara evaluates axial ( gait , stance , sitting ) , speech , and appendicular ( finger chase , nose - finger test , fast alternating hand movements ( fahms ) , and heel - shin slide ) functions . the sara sum score ranges from 0 to 40 with 0 indicating no ataxia and 40 the most severe ataxia , thus deterioration or improvement of disease severity is , respectively , represented by an increase or decrease of the sara score . for each patient , chinese version of sara was performed at the baseline , 2 weeks ( midpoint ) and 4 weeks ( endpoint ) after treatment . these sara evaluations were all videotaped and reviewed independently by two other investigators who did not attend the original assessment in a random order . the average of score rated by these two evaluators was denoted as the final sara score . the primary outcome measure was the change of sara score after treatment compared with that at baseline . continuous variables were expressed as mean standard deviation ( sd ) , and categorical variables were expressed as frequencies or proportions where appropriate . the observed changes of sara score after treatment from baseline were analyzed with nonparametric wilcoxon signed - rank test . continuous variables were expressed as mean standard deviation ( sd ) , and categorical variables were expressed as frequencies or proportions where appropriate . the observed changes of sara score after treatment from baseline were analyzed with nonparametric wilcoxon signed - rank test . the mean age was 39.14 7.81 years , the mean age of onset was 35.00 6.53 years , the mean disease duration was 4.14 1.90 years , and the mean cag repeats number was 77.57 2.27 . the mean sara score dropped from 8.48 2.40 to 6.94 2.34 ( p < 0.001 ) and 6.30 1.87 ( p < 0.001 ) after 2 and 4 weeks of treatment , respectively [ table 2 ] . significant decrease in subsections sara scores was also observed in stance ( p = 0.008 and 0.003 ) , speech ( p = 0.046 and 0.023 ) , finger - chase ( p = 0.026 and 0.015 ) , fahms ( 0.015 and 0.009 ) , and heel - shin slide ( p = 0.006 and 0.001 ) at 2 and 4 weeks after therapy , respectively . the mean improvement in total sara score was 2.18 1.30 ( ranging from 0 to 5.75 ) in our study . scores of sara and its subsections at baseline , 2 and 4 weeks after ngf therapy * comparisons between midpoint of therapy and baseline ; comparisons between endpoint of therapy and baseline . sara : scale of the assessment of rating of ataxia ; fahm : fast alternating hand movements ; ngf : nerve growth factor . one previous study has shown that insulin - like growth factor-1 , one of the neurotrophic factors , may be effective in reducing the disease progression of sca3 . previous studies have reported that ngf can improve cognitive decline in patients with alzheimer 's disease and may also have potential therapeutic roles in other neurodegenerative diseases . postmortem histopathological study in patients with sca3 has revealed considerable neuronal loss at the cerebellar purkinje cell layer and the four deep cerebellar nuclei . ngf can prevent neuronal death or age - related atrophy in the adult brain by inhibiting apoptosis of cholinergic neurons in the basal forebrain , and it would be reasonable to postulate that it may also inhibit the apoptosis in the cerebellum neurons expressing tyrosine kinase a ( trka ) and serve as a potential therapy for sca3 . such treatment effect is observed as early as 2 weeks after therapy and sustained after 4 weeks . to our knowledge , this study is the first to investigate the efficacy of ngf in sca3 . postmortem study has shown that ngf and its high - affinity receptor tkra are distributed in the neurons of the human cerebellum cortex and its deep nuclei throughout life . these findings support the involvement of ngf in the development , differentiation and maintenance of the cerebellar connectivity . although the blood - brain barrier ( bbb ) has low permeability to large proteins , some autoradiography studies suggested ed that blood - borne ngf and its subunit -ngf can cross the bbb of mice and arrive at the brain parenchyma by direct permeation . second , the therapeutic effect of ngf might also be mediated via the proprioceptive sensation system . it has been reported that most ( 87% ) of the sca3 patients had somatosensory evoked potential abnormalities , especially in the lower limbs , which was due to degenerative lesions in the dorsal column of the spinal cord . hence , ngf therapy may improve stance and heel - knee - shin slide due to improved proprioception . these two proposed mechanisms may explain the observed improvement after therapy and substantiate the use ngf to treat a patient with sca3 . first , it was an open - label study , in which the observed therapeutic efficacy might be contributed by placebo effects . however , in one randomized , double - blind , and placebo - controlled study to evaluate the efficacy of varenicline in sca3 patients , the mean improvements of sara score in the therapeutic group , and the placebo group were 1.97 and 0.86 , respectively . the sara score improvement of 2.18 in our current study is unlikely to be accounted for by placebo effect alone . furthermore , sara is a reliable and valid scale to linearly assess the ataxia symptoms , and changes of sara scores exceeding 1.1 points are considered clinically relevant . nevertheless , our pilot data suggest that ngf may be a promising treatment for patients with sca3 . a large - scale randomized , double - blind placebo - controlled trial would be worthwhile to evaluate the efficacy and tolerability of ngf in 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 .

I M Statistical analysis R D

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

Introduction Materials and Methods Preparation of Nanopores Characterization of the Pore Opening Modification with DNA Recordings of CurrentVoltage Curves Results and Discussion Conclusions

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

Introduction Results Discussion Experimental Procedures Author Contributions

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

1. Introduction 2. Signal Transduction in ER Stress 3. ER Stress and Autophagy 4. ER Stress and Autophagy in Anticancer Therapy: A Double-Edged Sword 5. Conclusions

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

What is already known about this subject? What does this study add? How might this impact on clinical practice? Background Patients and methods Patients and study design End points Statistical analysis Results Discussion

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

Saliva research Challenges and opportunities Biomedical ontology A dental research ontology consortium Saliva ontology BioMart SDxMarta BioMart portal for salivaomics data Summary

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

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 .

I M R D

acute myeloid leukemia ( aml ) is a clinically , morphologically and genetically heterogeneous disease characterized by clonal expansion of myeloid blasts in peripheral bone marrow ( bm ) , blood or other tissues . the incidence of aml increases with age , particularly after 65 years old , and the median age at diagnosis is around 70 years . age in itself is one of the most powerful prognostic factors for survival in aml , , . elderly patients not only present more comorbidities and worse performance status but also have more adverse molecular features compared to younger patients , , . due to the presence of comorbidities and poor performance status , intensive chemotherapy ( ic ) or allogeneic stem cell transplant is often unsuitable for a large proportion of elderly patients with aml , resulting in early mortality and prolonged hospitalization , . however , when feasible , treatment is associated with better survival when compared to best supportive care only but the median overall survival achieved with ic in the elderly is only 513 months , . therefore , there is an increasing need for new treatment options for elderly patients with aml . the aza-001 phase iii trial has demonstrated effectiveness of azacitidine ( aza ) in a dose of 75 mg / m2 daily for 7 consecutive days in patients with high - risk myelodysplastic syndromes . a subset analysis of the aza-001 trial restricted to patients with 2030% bm blasts , demonstrated superior efficacy of aza when compared with conventional care regimens , leading to aza international approval for the treatment of low bm blast count ( 2030% ) who - defined aml . these results raise the question of the potential use of aza in aml irrespectively of the bm blast count . this is supported by the identification of somatic mutations of genes involved in epigenetic regulation in approximately 30% of aml patients . consequently several groups have assessed and reported the efficacy of aza in patients with aml , , , . more recently a randomized phase iii trial comparing first - line aza to conventional therapies , including intensive chemotherapy , in elderly patients with aml revealed that aza achieved clinically meaningful survival benefit compared to conventional treatments . we present herein a multi - centre retrospective analysis of 77 aml patients who were treated with aza in first line or due to relapsed / refractory disease . the main aim was the characterization of the efficacy and safety of aza treatment in aml patients in portugal . we also conducted an exploratory comparison of overall survival between patients receiving 1st line treatment with aza and elderly aml patients treated with ic alone . patients with aml who received at least 1 cycle of aza were selected for inclusion . the comparator group comprised 50 matched patients from a historical cohort of aml patients from a single institute . patient data was collected retrospectively from patient files from 4 portuguese institutions following written informed consent . the study was approved by the ethical committee of instituto portugus de oncologia de lisboa , francisco gentil , epe and all data treated according to the declaration of helsinki . overall survival ( os ) was defined as time from start of treatment with aza ( either 1st or 2nd line ) or ict ( 1st line ) to death from any cause or last follow - up . progression - free survival ( pfs ) was defined as time from start of treatment until disease relapse / progression or death from any cause . survival curves of the different treatment groups were compared using the log - rank test ( univariate analysis ) . all tests were two - tailed and p - values less than 0.05 were considered to be statistically significant . seventy - seven patients treated with aza and 50 elderly aml patients treated with ict were included . 1line n=51ict n ( % ) n=50p - value aza1st line vs ictage at 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 at start of 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 than higher age in the ict control group , there was no significant difference between patients treated with aza or ict in first line . table 2treatment details.table 2aza n ( % ) n=77aza 1line n ( % ) n=51ict n ( % ) n=50p - valueaza1st line vs icttime from diagnosis to start of 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 no of cyclesmedian [ min - max]6 [ 128]1.7 ( 13)n / aaza = azacitidine ; ict = intensive chemotherapy ; dauno 90+arac 100=daunorubicin 90 mg / m d1 - 3 , cytarabine 100 mg / m d1 - 7 ; dauno 60+arac 100=daunorubicin 60 mg / m d1 - 3 , cytarabine 100 mg / m d1 - 7 ; ida 12 + arac 100=idarubicin 12 mg / m d1 - 3 , cytarabine 100 mg / m d1 - 7 . aza = azacitidine ; ict = intensive chemotherapy ; dauno 90+arac 100=daunorubicin 90 mg / m d1 - 3 , cytarabine 100 mg / m d1 - 7 ; dauno 60+arac 100=daunorubicin 60 mg / m d1 - 3 , cytarabine 100 mg / m d1 - 7 ; ida 12 + arac 100=idarubicin 12 mg / m d1 - 3 , cytarabine 100 mg / m d1 - 7 . between start of aza and table 3response to 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 / an / aunknown3 ( 4% ) four ( 3.3% ) patients treated with aza were not evaluable for survival . median follow - up time in patients was 12.4 months in patients receiving aza as first line treatment and 6.9 months in patients treated in second or subsequent lines . during this period 1 . survival estimates for patients treated with aza according to line of treatment . fig . median age at diagnosis of the comparator group was 74.5 years - old ( range : 6686 ) and 44% ( 22 patients ) were female ( table 1 ) . the median time from diagnosis to start of ic in this comparator group was 0.13 months ( range : 09.2 months ) ( table 2 ) . all patients treated with oct alone received only supportive care prior to induction chemotherapy . fig . 4survival of aml patients treated with aza first line compared to those treated with ict alone.fig . 4 . survival of aml patients treated with aza first line compared to those treated with ict alone . table 3response to 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 / an / aunknown3 ( 4% ) four ( 3.3% ) patients treated with aza were not evaluable for survival . median follow - up time in patients was 12.4 months in patients receiving aza as first line treatment and 6.9 months in patients treated in second or subsequent lines . during this period 1 . survival estimates for patients treated with aza according to line of treatment . fig . median age at diagnosis of the comparator group was 74.5 years - old ( range : 6686 ) and 44% ( 22 patients ) were female ( table 1 ) . the median time from diagnosis to start of ic in this comparator group was 0.13 months ( range : 09.2 months ) ( table 2 ) . 4survival of aml patients treated with aza first line compared to those treated with ict alone.fig . 4 . survival of aml patients treated with aza first line compared to those treated with ict alone . their frailty imposes limitations upon the aggressiveness of the treatment which can be administered , . in addition , the poor prognostic disease features often seen in this population mean that refractoriness and relapse are more frequent . treatments for this population require an optimal balance of tolerability and efficacy . the main aim in most cases is to prolong survival and reduce the impact of cytopenias without undue toxicity . aza has demonstrated efficacy in high risk mds , reducing transfusion requirements and prolonging survival . in this setting it had proven to be well tolerated including by those more elderly . based on this experience , several groups have tested the efficacy and safety of aza in elderly patients with aml with promising results , , , . our results are in line with previous reports that aza is efficacious in the treatment of aml regardless of the blast count . the response rates and the overall survival observed in our study are in consonance to those reported by other groups , , , . it is noteworthy that the rates of cr obtained with aza are equivalent to those obtained in some published series of elderly patients treated with ic . improved survival in our cohort was significantly associated with any quality of response , including maintenance of stable disease . this suggests that aza is capable of delaying progression , and thus prolong survival , even if no hematological or marrow improvement are obtained . this supports its use in this population until frank progression is detected or until the patient receives another more definitive treatment , such as allogeneic bone marrow transplant . another significant finding of our analysis is the efficacy of aza in those who are refractory or relapsed following ic . the fact that survival from start of aza is similar in first and second line patients indicates that relapsed / refractory patients benefit equally . the comparison of the cohort treated with aza first line with the comparator group treated only with ic highlights the advantages brought by the former treatment modality . it is important to note that this finding has not been confirmed in a randomized trial . however , 41% of the trial patients were treated with aza following failure with ict whereas our comparator group never received aza . as aza has demonstrated efficacy as second line therapy , this could explain the difference in our findings . the significantly lower survival of the ic cohort indicates that this population benefits from less intensive treatment options which target other pathophysiological processes . the retrospective nature and the limited numbers of patients in the collaborative nature of the study helps reduce bias which may be introduced by single centre data . however our study adds new evidence to the use of aza in elderly patients with aml and indicates that it is at least not inferior to conventional ic . the response rates and survival data here presented support its use in first line in those who are not candidates for ic and in second line in those who are refractory or relapsed following ic . this and future collaborative studies will add to trial data to better identify those patients who benefit most from new treatment modalities , thus improving the management of elderly patients with 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 .

Introduction Methods Results Treatment outcomes Comparative survival analysis Discussion

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

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 .

INTRODUCTION METHODS Study setting Study design and target population Checklist design Statistical analysis Ethical consideration RESULTS DISCUSSION CONCLUSION

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

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS

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

Introduction Background Problem statement Theory and discussion The machine model thinking Complex adaptive systems (CASs) Characteristics of a CAS Management and control in a CAS Criticism of the CAS approach Conclusion Reviewers

we will introduce recent advances on the study of post - translational processing , modification , and targeting of cathepsins and cystatins . almost all the intracellular proteins are passed through principally similar processes from the synthesis to their degradation in general . therefore , i would like to introduce the general fate of intracellular proteins , from the post - translational processing , modification , and targeting to the ordered particles . as figure 1 shows , the intracellular proteins are synthesized as pre - promature complex in polysomes and prepart is removed cotranslationally , and then the promature parts are translocated into golgi - apparatus , and then glycosylated by mannose - rich sugar . the glycosylated mature part is translocated into target organelles and the degradation was started by the splitting from the ordered nicked bonds to make hydrophobic peptides . these hydrophobic peptides are secreted to cytoplasm and are incorporated into the phagosomes or proteosomes after ubiquitination . biological merit of post - translational processing and modifications of proteins are possible considerations . the capability to take variable forms on the way of biosynthesis is important to keep adaptability to the changing of biological requirements and intracellular translocation during the maturation must be regulated . pro - parts or bound sugar are the targeting signals in some cases . in the cases of carbamoyl phosphate synthetase ( cps ) and ornithine transcarbamylase ( otc ) , their pro - parts play the role of signals to be recognized by their receptors located on target organella membrane , such as lysosomes , as shown in figure 4 . the glycosylated cathepsins are targeted into lysosomes mediated by mannose-6 phosphate receptors which are located on the lysosomal . as figures 4(a ) and 4(b ) show , the lysosomes in which cathepsin h or b is located are attached to the cell - membrane . on the contrary , cathepsin l is located in the lysosomes which are distributed diffusely in liver cells . as figure 4(a ) shows , using immunodouble gold - particle staining , cathepsin b ( small gold particle ) and cathepsin h ( large gold particle ) are located clearly in different lysosomes . cathepsin l is translated as 17 amino acids of prepart , 96 amino acids of pro - part and 221 amino acids of mature - part [ 9 , 10 ] . the prepart is removed cotranslationally and formed procathepsin is translocated into golgi - apparatus , and then the 108-asn and the 155-asn in mature parts are glycosylated by high - mannose - type sugar , as shown in figure 2(a ) . the initiation of the degradation is started from the nicking bond ( 178 bond ) cleavage by some cysteine protease . cathepsin b is translated as 17 amino acids of prepart , 62 amino acids of pro - part , and 252 amino acids of mature part [ 12 , 13 ] . the prepart is also removed cotranslationally and the formed procathepsins are translocated into golgi - apparatus and then the 38th - asn in pro part and the 111th - asn in the mature - part are glycosylated by high - mannose - type sugar . then the mannose-6-phosphate - moities play a role as the targeting marker to the lysosomes . cathepsin h is translated as 21 amino acids of prepart , 114 amino acids of pro - part , and 217 amino acids of mature part . the pro - part has two carbohydrate chains at the 70th - asn and the 90th - asn and the mature part consisted of 217 amino acids , and one carbohydrate chain is bound at the 99th - asn [ 8 , 15 ] . the initiation of degradation is started from the 177th nicking bond by cysteine protease . intracellular protein degradation (= autophagosome formation ) is regulated by nutritional and hormonal conditions . fasting or insulin enrichment caused stimulation of the phagosome formation and cathepsin l amount are increased ; on the contrary , refeeding and glucagon enrichment resulted in the suppression of phagosome formation and decreasing of cathepsin l amount . as you see in figure 2 , cathepsins b , l , and h have individual ordered nicking bond as the initiation of their degradations ; these bonds are cleaved by cysteine protease , therefore as table 1 shows , the half - lives ( t1/2 ) of cathepsins and the contents in lysosomes clearly increased by treatment by e-64 ( inhibitor for all cysteine proteases ) . the initiations of degradation of cathepsins are started from the ordered limited proteolysis by cysteine protease in the lysosomes , as figure 2 shows . the formed hydrophobic products were translocated into autophagosome or proteasome ( after ubiquitinated ) to degrade to amino acids . procathepsins or mature cathepsins are secreted from the various cells and play individual physiological , or pathological roles . as figure 5 shows , bone metabolism is consisted of functional balances between osteoblastic cell function and osteoclastic cell function . bone collagen is degraded mainly by cathepsin l and k , therefore they are called collagenolytic cathepsins . the secretion of these cathepsins from osteoclasts is stimulated by pht ( parathyroid hormone ) and suppressed by vitamin d3 , e-64 , ca-148 eta . therefore , bone metastasis of cancers is inhibited by cathepsin inhibitors , such as by e-64 or clik-148 . as pit formation test in table 4 shows , bone - matrix degradation is catalyzed by cathepsin l , but not by cathepsin b. endogenous low - molecular weight cathepsin inhibitors are consisted of two big groups , one is only located in skin and the other is ubiquitously located in all organs . cystatin ( a ) is only located in epidermis , cystatin ( b ) is located ubiquitously in all cells ( organs ) . the secretary formed cystatins are cystatin c in eggwhite and cystatin s in the saliva . another secretary group having high - molecular weight ( repeated low molecular cystatins ) is kininogen family in serum . each cystatin shows different inhibitory specificity for individual cathepsins , for example , cystatin does not inhibit cathepsin b activity but strongly inhibit cathepsin l activity and cathepsin h activities [ 7 , 19 ] . the amino - acid sequences of low - molecular weight cystatin family have strong homology , however , their post - translational processing are quite different . because their localizations and biological functions is quite different . when skin was stained by immunohistochemistry using anticystatin antibody , only the cornified - envelope of skin was stained . the sphingosine treatment to new born skin resulted in the strong suppression of the targeting of cystatin into the cornified envelope . the sphingosine is a powerful inhibitor for protein kinase c. as shown in figures 7(a ) and 7(b ) , threonine residue in near c - terminus ( the 92th threonine in figure 9 shows ) of cystatin is phosphorylated by protein kinase c , and the phosphorglated cystatin is incorporated into cornified envelope . the participation of protein kinase c in the phosphorylation of cystatin was also confirmed using specific inhibitor of protein kinase c , ( hidaka h-7 ) inhibited the incorporation of p into cystatin . the phosphorylated cystatin was incorporated into skin cornified envelop . in the envelop , the phosphorylated cystatin was conjugated with filaggrin - linker sediment peptide which is rich in glutamine residues , mediated by epidermal transglutaminase in the presence of calcium , and yield a high - molecular weight protein ( skin fiber ) , as shown in figure 10 . the phosphorylated cystatin showed strong inhibition to cathepsin l , but not to the cathepsin b and h , it also showed strong inhibition to the bacterial cysteine proteases and virus cathepsins . therefore , cystatin and phosphorylated cystatin showed strong protection from infections of bacteria or viruses in skin . post - translational covalent modification of cystatin and the changing of their inhibitory activities . the third position of the n - termius - cysteine residue of cystatin reacts with glutathione to form a mixed disulfate complex and also is able to make dimmer by the disulfate bond . as figure 8 shows , n - terminus of cystatins was inserted into the substrate binding pocket of cathepsins , the glutathionated or the dimmer form were unable to insert into the binding pocket of cathepsins . the coefficient of oxidized or reduced forms of glutathiones regulates the inhibitory activity of cystatin . therefore , the activities of cathepsins are regulated by the intracellular redox potentials in spite of the changes of the inhibitory activities of cystatin . as figure 7(b ) shows , using -cell of pancreas , the cystatin is located in insulin secretion particles , therefore , cystatin is secreted with insulin in the 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 .

1. Introduction 2. Cathepsins 3. Cystatins

endocrine disorders may complicate , cause or mimic otolaryngologic disorders , some of which may be anatomical , due to an enlargement of the thyroid gland , while others are physiological , resulting from increased or decreased glandular activity.1 hypothyroidism is characterized by the slowing of mental and motor activity , depression , constipation , cold intolerance menorrhagia , stiff muscles , carpal tunnel syndrome , sleep apnea , dry hair and skin , weight gain , snoring and a hoarse voice.1 less common symptoms involve the heart , muscles , joints , and blood.2 dysarthria as the presenting symptom of hypothyroidism has only been reported once before.3 here , we present an unusual case of hypo - thyroidism presenting with dysarthria . a 39-year - old female presented to the clinic with dysarthria of six months duration . , it was revealed that there had been episodes of dysphonea , snoring , sleep apnea , dysphagia and choking during eating or drinking . her past medical history was normal apart from one occasion of delayed recovery from anaesthesia during surgery for a fractured femur the year before . clinical examination , including the central nervous system , was normal apart from a slightly puffy face . there was no abnormality in the movement of the tongue or pharygeal / palate muscles . pmol / l ( normal 9.2 - 23.9 pmol / l ) , a thyroid stimulating hormone of 82.7 miu / l ( normal 0.32 - 5.00 miu / l ) , antithyroglobulin antibodies were 1:320 u / ml ( normal 1:40 u / ml ) , antiperoxidase autoantibodies of 1:1600 u / ml ( normal 1:40 u / ml ) . a complete blood count film was consistent with iron deficiency , a haemoglobin of 7grams ( normal 11 - 16 grams ) , low serum iron of 2 umol / l ( normal 10 - 28 umol / l ) , increased red cell distribution width ( rdw ) of 17.2 ( normal 11.6 - 13.7% ) . other biochemical abnormalities were high serum cholesterol of 6.9 mmol / l ( normal 3.6 - 6.8 , hypo - thyroidism was diagnosed . in the light of the patient 's diagnosis , a second history was taken which showed that she suffered from other symptoms of hypothyroidism such as , dry skin , generalised weakness , excessive sleeping , hoarse voice , and menorrhagia . two months after the initiation of therapy , the patient had no more dysarthria or other associated symptoms . dysarthria is a disturbance of articulation that may be caused by a neuromuscular lesion , or an abnormality of the vocal cords . the first may result from damage to the central or peripheral nervous system such as head trauma , brain stem infarction , bulbar palsy , motor neuron disease , peripheral neuropathy , huntington 's chorea , parkinson 's disease , multiple sclerosis , myasthenia gravis , or muscle disease.4 the second may be attributable to congenital , traumatic , inflammatory , tumors , or post - operative lesions of the vocal cords . these causes were unlikely in this patient , because she showed no associated clinical features of these diseases besides the normal neurological examination and investigations . other causes such as congenital or aquired storage disorders such as amyloidosis , and such endocrine disorders as acromegaly or hypo - thyroidism,3 as in the presented case , may lead to an enlargement of one or more of the components of the vocal cords.56 the most likely cause for the dysarthria in this patient was hypothyroidism . this was supported by the abnormal thyroid functions and the response of the dysarthria to thyroxin . dysarthria due to hypothyroidism had been reported only once previous to this case.3 the pathophysiology of dysarthria in hypothyroidism can be explained by edematous swelling of laryngeal and hypopharyngeal structures in combination with macroglossia.3 it has been shown that macroglossia in hypothyroidism is caused by a thickening of the epithelial tissue.6 these changes can also explain the choking and the dysphagia which this patient experienced . there have been a few reports of hypothyroidism responsible for secondary dysphagia.1012 her sleep apnea may also be a manifestation of hypothyroidism , most likely caused by edema and myopathy.7 sleep apnea attributable to hypothyroidism is reversible with thyroxin replacement therapy.8 the episodic hoarseness of voice can also be explained by hypothyroidism,1 as well as the delayed recovery from anaethesia the year before , most probably the result of undiagnosed hypothyroidism.91314 unfortunately , thyroid function tests had not been performed on our patient at that time . iron deficiency anemia in this patient was due to menorrhagia , which is one of the characteristic features of the disease.1 hyperlipidaemia may also be due to hypothyroidism , a known association.1 clinical implication of the presented patient was that dysarthria may be the presenting symptom of hypothyroidism , even if other symptoms had been present for a long time . hypothyroidism as the cause of dysarthria was confirmed with the discovery of additional symptoms in the patient 's history . otolaryngeal symptoms should therefore be considered possible symptoms of hypothyroidism.15 prompt recovery of dysarthria is expected after 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 .

INTRODUCTION CASE REPORT DISCUSSION

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

1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Clinical Significance of BG Morphology 6. Conclusions

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

background : 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 .

Introduction Materials and Methods Source of Data Data Analysis Results Injuries Distribution by Year Injuries Distribution by Geographic Region Injuries by Industry Severity of injury Discussion Conclusion Ethical considerations

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

MATERIALS AND METHODS Strains and growth conditions Construction of a disruption cassette Nucleic acid preparation, Northern blotting, and Southern blotting RESULTS AND DISCUSSION Effect of -COP depletion on growth and stress response Effect of -COP depletion on development Suppression of

the diagnosis and extent of resection in the management of hd depend on the sensitive and specific identification of ganglion cells.[13 ] however , documenting aganglionosis is often difficult and tedious on routine hematoxylin - eosin ( h and e ) stained sections . acetylcholinesterase ( ache ) has evolved as the gold standard in diagnosing hd ; however , this histochemical analysis is technically challenging , and to date , has not gained worldwide utilization and applicability . the aim of this study was to evaluate the efficacy of calretinin immunostaining in the ganglionic and aganglionic hd colon biopsy specimens and correlate with the h and e , thereby exploring its utility in suspicious cases of hd . our standard protocol of care for a child presenting with hd is barium enema study at presentation followed by a laparotomy where multiple biopsies are taken from the spastic aganglionic segment , transition zone , and the normal colon . a colostomy is sited at the level of the junction between the normal colon and the transition zone . the definitive surgery of choice is duhamel 's pull - through after 6 months of age . thirty - six were full - thickness rectal biopsies ( for suspected hd ) and 24 were bowel segments resected during the definitive pull - through surgery . calretinin ( monoclonal mouse antihuman antibody ( dako ) , ( clone : dak calret 1 , code : ir627 ) immunohistochemistry ( ihc ) staining was done on all paraffin - embedded blocks after routine h and e examination . the age of patients ranged from 1 day to 14 years ( mean 8.2 months ) , and there were 46 boys and 14 girls ( m : f = 3.2 - 3.3:1 ; normal ratio 3:1 - 4:1 ) . twenty - three patients ( 63.8% ) presented at less than 1 month of age . five ( 8.3% ) had long segment disease , 6 ( 10% ) had total colonic aganglionosis . during the analysis of 36 initial full - thickness colon biopsy specimens , h and e staining revealed absence of ganglion cells ( negative ) in 19 cases ( 52.7% ) , presence of ganglion cells ( positive ) in 2 cases ( 0.05% ) , and suspected presence of ganglion cells in 15 cases ( 41.6% ) . of the 19 cases reported negative through h and e staining , 17 ( 47.22% ) were reported negative , and 2 ( 0.05% ) positive for calretinin histochemistry for the ganglion cells and nerve fibers [ table 1 ] . hematoxylin and eosin staining versus calretinin in rectal biopsies in 15 patients , the h and e sections were suspicious of a presence of ganglion cell . calretinin ihc showed immunopositivity in 3 slides , whereas 5 slides showed immunopositivity in nerve fibers . among the 5 slides positive for nerve fibers , in 2 of the cases ( 2/60 ) , calretinin gave a slight positive staining of nerve fibers , but with no staining of other areas . the slight calretinin positivity in the 2 specimens was observed in some large bundles with no staining in other areas , and thus indicating the beginning of transition zone . in the 24 patients where resected specimens from the definitive surgery were sent , serial biopsies were taken from the aganglionic segment , transition zone , and the ganglionic segment [ table 2 ] . calretinin was not expressed in the ganglion cells and nerve fibers of submucosal and myenteric plexus of 24 aganglionic ( spastic ) segments . in the transition zone , calretinin staining was positive in the ganglion cells in 20 cases ( 83.3% ) and was focally positive in the nerve fibers of 22 cases ( 91.6% ) , both in the submucosal and myenteric plexus . in ganglionic bowel segments of hd , calretinin showed immunopositivity in > 90% of ganglion cells and nerve fibers of submucosal and myenteric plexus . the histological diagnosis of hd is challenging , requiring the expertise of a senior pathologist and access to specialized techniques for handling frozen specimens for ache staining . this makes the diagnosis of hd difficult in centers where cases are infrequent , causing a delay in the treatment of the child . calretinin is proven to be highly sensitive for the presence of ganglion cells and nerve fibers . this protein is involved in calcium transport ; and its absence allows accumulation of cytoplasmic calcium , causing excess neuroexcitability and ultimate neurodegeneration . barshack , et al . , procured the colons of 10 patients with proven hd . calretinin ihc was performed on sections from the aganglionic zone , ganglionic zone , and transition zone of these colons . they identified calretinin staining in interstitial nerve fibres ( inf ) and ganglion cells in normal colon [ figures 1 and 2 ] and focal inf staining in 92% of transition zones . in contrast , there was no inf or ganglion cell staining in the aganglionic zone . it can be interpreted as positive or negative for immune reactivity reducing the ambiguity in diagnosis . in our study , in the rectal biopsy specimens , diagnosis was ambiguous in 15 patients on h and e examination . by using calretinin , immune positivity was demonstrated in 3 patients ( 3 for ganglion cells and 5 for nerve fibers ) ; thereby , ruling out hd in these patients . hematoxylin and eosin section with ganglion cell ( arrow ) ( h and e , 10 ) calretinin staining of ganglion cells ( h and e , 40 ) guinard - samuel , et al . , have studied the largest number of suction biopsies using calretinin . they have concluded that calretinin is accurate in proving the absence of ganglion cells , it is easy to interpret , and can replace ache to diagnose hd . they , however , recommend that it should be used as an aid along with h and e examination , especially in ultrashort segment disease and transition zone specimens . in our study , we found that calretinin correlated with h and e examination in both rectal biopsies and the resected bowel specimens . in the rectal biopsy specimens , calretinin also aided in the diagnosis of 15 patients with ambiguous findings . calretinin ihc is accurate in detecting the presence or absence of ganglion cells and holds several advantages such as follows : ( 1 ) it can be carried out on paraffin - embedded tissue sections ; ( 2 ) staining pattern is simple ; ( 3 ) binary pattern of interpretation ( negative or positive ) ; ( 4 ) it is cost effective . in the present study it can serve as a valuable cost - effective diagnostic aid in the centers where ache enzyme histochemistry is not 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 .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS

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

INTRODUCTION METHOD HOW TO DEVELOP QUESTIONNAIRE FROM THE BEGGINING Ethical considerations RESULTS DISCUSSION CONCLUSIONS

q fever is a zoonotic disease that is caused by coxiella burnetii , a microorganism that frequently infects domestic ungulates , as well as wild mammals in many genera ( 1 ) . in animals , the disease is transmitted to humans incidentally by inhalation of aerosols from infected cattle and sheep ( 1 , 2 ) . in humans , c. burnetii infection may be asymptomatic , acute , or chronic . chronic q fever is rare , with endocarditis presenting as the most common complication ( 1 , 3 ) . q fever has been reported in almost every country , except new zealand ( 4 ) . in canada , burnetii antibodies ( 5 ) . in japan , 60 to 84% of cattle with reproductive disorders are seropositive ( 6 ) . a study conducted in southern france showed that 5 to 8% of endocarditis cases in humans were due to c. burnetii , and the prevalence of acute q fever was 50 cases per 100,000 inhabitants ( 7 ) . researchers have suggested that the incidence of q fever is chronically underestimated because clinical manifestations of the disease are often nonspecific or even absent . therefore , concerns with the disease focus on the importance of detection ( 1 , 3 , 8) . in korea , there is a little information concerning the epidemiology of c. burnetii infection in either animals or humans . a few cases of acute q fever in humans have been reported ( 9 , 10 ) . we examined the prevalence of antibodies to c. burnetii in dairy cattle nationwide and in people for health screening in a rural area of korea , and used the data to evaluate the impact of q fever in both animals and humans in korea . upon agreement with dairy owners , serum samples from 414 dairy cattle were collected on 31 farms from march to june , 2001 . serum was collected from people who visited kangwon national university hospital for health examinations between april and december 2002 . the subjects were interviewed to confirm the absence of symptoms of respiratory tract infection during the preceding two weeks . informed consent was obtained from all people for health screening and the animals are treated by the ethical guidelines of kangwon national univesity . coxiella burnetii phase ii antigen ( nine mile whole - cell antigen ) was prepared as previously described at the national institute of infectious diseases ( niid ) in tokyo , japan ( 12 ) , and dotted onto teflon - printed glass slides . each serum sample was diluted 1:16 with phosphate - buffered saline ( pbs ) , overlaid on the antigen dots , and incubated for 45 min at 37 in a moist chamber . the slides were subsequently washed twice for 5 min in pbs plus 0.05% tween-20 and then incubated with a 1:1,400 dilution of fluorescein isothiocyanate ( fitc)-conjugated rabbit anti - bovine igg ( sigma - aldrich , st . louis , mo , u.s.a . ) or fitc - conjugated rabbit anti - human igg ( dakocytomation , glostrup , denmark ) for 45 min at 37 in a moist chamber . the slides were again washed twice using the same method and examined using fluorescence microscopy ( axioskop 2 , zeiss , germany ) at 200 magnification . all sera that produced positive or equivocal reactions at 1:32 were further analyzed using 2-fold serial dilutions up to 1:4,096 . approximately 10% of the sera were divided and tested concurrently at kangwon national university and at the niid for quality control of test reproducibility ; there was greater than 95% concordance between the results from the two laboratories . upon agreement with dairy owners , serum samples from 414 dairy cattle were collected on 31 farms from march to june , 2001 . serum was collected from people who visited kangwon national university hospital for health examinations between april and december 2002 . the subjects were interviewed to confirm the absence of symptoms of respiratory tract infection during the preceding two weeks . informed consent was obtained from all people for health screening and the animals are treated by the ethical guidelines of kangwon national univesity . coxiella burnetii phase ii antigen ( nine mile whole - cell antigen ) was prepared as previously described at the national institute of infectious diseases ( niid ) in tokyo , japan ( 12 ) , and dotted onto teflon - printed glass slides . each serum sample was diluted 1:16 with phosphate - buffered saline ( pbs ) , overlaid on the antigen dots , and incubated for 45 min at 37 in a moist chamber . the slides were subsequently washed twice for 5 min in pbs plus 0.05% tween-20 and then incubated with a 1:1,400 dilution of fluorescein isothiocyanate ( fitc)-conjugated rabbit anti - bovine igg ( sigma - aldrich , st . louis , mo , u.s.a . ) or fitc - conjugated rabbit anti - human igg ( dakocytomation , glostrup , denmark ) for 45 min at 37 in a moist chamber . the slides were again washed twice using the same method and examined using fluorescence microscopy ( axioskop 2 , zeiss , germany ) at 200 magnification . all sera that produced positive or equivocal reactions at 1:32 were further analyzed using 2-fold serial dilutions up to 1:4,096 . approximately 10% of the sera were divided and tested concurrently at kangwon national university and at the niid for quality control of test reproducibility ; there was greater than 95% concordance between the results from the two laboratories . each province had a relatively high prevalence , ranging from 8.9 to 59.3% ; the overall national prevalence was 25.6% . of the positive sera ( n=106 ) , 80 ( 75.5% ) had high antibody titers ( 1:256 ) . the mean age of the subjects was 43.7 yr ( standard deviation 15.9 , range 19 - 82 ) , and the male - to - female ratio was 1:0.86 ( n=110:95 ) . of these , one had a serum sample with a high antibody titer ( 1:512 ) , and two had samples with low antibody titers ( 1:64 ) ( table 2 ) . this study examined the seroprevalence of c. burnetii infection in cattle and people for health screening in korea . the sera of cattle showed a high prevalence ( 25.6% ) of anti - c . burnetii antibodies , with 75.5% of the positive sera having high titers ( 1:256 ) . by contrast , the sera of healthy people showed a relatively low prevalence ( 1.5% ) of anti - c . the sera of dairy cattle were collected regardless of the disease status of each animal , which is a limitation of this study . since we could not find an association between a history of reproductive failure and seropositivity of the cows because of the absence of the data on the disease status , no prediction concerning seropositive status and reproductive problems can be made . however , c. burnetii infection is prevalent in all areas of korea ; every region had a seroprevalence above 8% , and the national prevalence was 25.6% . moreover , the high prevalence of high - titer sera provides evidence that the disease might be very active in this country . in neighboring japan , the seroprevalence of c. burnetii in healthy cattle ranges from 2 to 46% ; in cattle with reproductive disorders , the range was 60 to 84% ( 6 ) . reported that bovine placentitis was highly associated with the presence of c. burnetti ( 13 ) . in addition , 9% of abortions in goats are reported to be caused by this microorganism ( 14 ) . this is the first report to provide data that suggest that c. burnetii infection might be one of the important causes of reproductive problems in cattle in korea . further studies based on the isolation of c. burnetii are needed to elucidate the etiologic role of this microorganism in the reproductive problems of cattle in this country . furthermore , the high seroprevalence of c. burnetii among cattle suggests the possibility of contamination of the environment around farms . since c. burnetii is widely distributed in wild animals and ticks and causes q fever to humans ( 1 , 3 ) , it is necessary to evaluate the environmental hazards associated with c. burnetii infection that may threaten public health in the near future . in addition , the survey on the high - risk group such as farmers and butchers is strongly needed . the finding that people in a rural area demonstrated a relatively low seroprevalence of c. burnetii , as well as lower titers , suggests that infection with this microorganism seems to be relatively low in the rural area of korea . in a previously published report ( 11 ) , less than 1% of both healthy people and patients with fever of unknown origin tested positive for anti - c . another report showed none of the sera from 70 healthy people was reactive for c. burnetii antigen ( 15 ) . recent data showed that 11 out of 448 healthy people were reactive ( 16 ) . on the other hand , our group previously reported that only one of 88 patients with community - acquired pneumonia was diagnosed with q fever with an ifa assay using both phase i and ii antigens ( 18 ) . despite the low seroprevalence of c. burnetii in humans , we can not exclude the risk of q fever to individuals with high - risk occupations , such as farmers , veterinarians , and meat - processing workers , considering the high rate of infection observed in cattle in this study . previous korean report showed two of 46 stock breeders had an antibody titer of 1:20 ( 15 ) , and 5 sera from 202 abattoir workers reacted with phase ii antigen ( 16 ) . in conclusion , c. burnetii appears to be a highly prevalent pathogen in cattle in korea and , accordingly , the studies on the high - risk groups are needed to evaluate the seroprevalence for this organism in 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 .

INTRODUCTION MATERIALS AND METHODS Subjects Indirect microimmunofluorescence antibody (IFA) assay RESULTS DISCUSSION